# Patent

## Title
Oligomeric compounds and compositions for use in modulation of small non-coding RNAs

## Abstract

Compounds, compositions and methods are provided for modulating the expression and function of small non-coding RNAs. The compositions comprise oligomeric compounds, targeted to small non-coding RNAs. Methods of using these compounds for modulation of small non-coding RNAs as well as downstream targets of these RNAs and for diagnosis and treatment of disease associated with small non-coding RNAs are also provided.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 14/245,535, filed Apr. 4, 2014, which is a divisional of U.S. application Ser. No. 13/540,097, filed Jul. 2, 2012, now U.S. Pat. No. 8,809,294, which is a divisional of U.S. application Ser. No. 13/359,271, filed Jan. 26, 2012, now U.S. Pat. No. 8,546,350, which is a continuation of U.S. application Ser. No. 12/346,940, filed Dec. 31, 2008, now U.S. Pat. No. 8,106,025, which is a continuation of U.S. application Ser. No. 10/909,125, filed Jul. 30, 2004, now U.S. Pat. No. 7,683,036, which claims priority to U.S. Provisional Application Nos. 60/492,056, filed Jul. 31, 2003; 60/516,303, filed Oct. 31, 2003; 60/531,596, filed Dec. 19, 2003; and 60/562,417, filed Apr. 14, 2004; each which is incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
The present invention provides compositions and methods for modulation of small non-coding RNAs. In particular, this invention relates to compounds, particularly oligomeric compounds, which, in some embodiments, hybridize with or sterically interfere with nucleic acid molecules comprising or encoding small non-coding RNA targets. Such compounds are shown herein to modulate the levels of small non-coding RNAs. The oligomeric compounds of the invention may include one or more modifications thereon resulting in differences in physical or chemical properties compared to unmodified nucleic acids. These modified oligomeric compounds are used as single compounds or in compositions to modulate or mimic the targeted nucleic acid comprising or encoding the small non-coding RNA. In some embodiments of the invention, modifications include chemical modifications that improve activity of the oligomeric compound. In some embodiments, the modifications include moieties that modify or enhance the pharmacokinetic or pharmacodynamic properties, stability or nuclease resistance of the oligomeric compound. In some embodiments, the modifications render the oligomeric compounds capable of sterically interfering with the natural processing of the nucleic acids comprising or encoding the small non-coding RNA targets.
BACKGROUND OF THE INVENTION
RNA genes were once considered relics of a primordial “RNA world” that was largely replaced by more efficient proteins. More recently, however, it has become clear that non-coding RNA genes produce functional RNA molecules with important roles in regulation of gene expression, developmental timing, viral surveillance, and immunity. Not only the classic transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), but also small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small interfering RNAs (siRNAs), tiny non-coding RNAs (tncRNAs), repeat-associated small interfering RNAs (rasiRNAs) and microRNAs (miRNAs) are now believed to act in diverse cellular processes such as chromosome maintenance, gene imprinting, pre-mRNA splicing, guiding RNA modifications, transcriptional regulation, and the control of mRNA translation (Eddy, Nat. Rev. Genet., 2001, 2, 919-929; Kawasaki and Taira, Nature, 2003, 423, 838-842; Aravin, et al., Dev. Cell, 2003, 5, 337-350). RNA-mediated processes are now also believed to direct heterochromatin formation, genome rearrangements, and DNA elimination (Cerutti, Trends Genet., 2003, 19, 39-46; Couzin, Science, 2002, 298, 2296-2297).
The recently described phenomenon known as RNA interference (RNAi) is involves the processing of double stranded RNA into siRNAs by an RNase III-like dsRNA-specific enzyme known as Dicer (also known as helicase-moi) which are then incorporated into a ribonucleoprotein complex, the RNA-induced silencing complex (RISC). RISC is believed to use the siRNA molecules as a guide to identify complementary RNAs, and an endoribonuclease (to date unidentified) cleaves these target RNAs, resulting in their degradation (Cerutti, Trends Genet., 2003, 19, 39-46; Grishok et al., Cell, 2001, 106, 23-34). In addition to the siRNAs, a large class of small non-coding RNAs known as microRNAs (miRNAs, originally termed stRNA for “short temporal RNAs”) is believed to play a role in regulation of gene expression employing some of the same players involved in the RNAi pathway (Novina and Sharp, Nature, 2004, 430, 161-164).
Like siRNAs, miRNAs are believed to be processed endogenously by the Dicer enzyme, and are approximately the same length, and possess the characteristic 5′-phosphate and 3′-hydroxyl termini. The miRNAs are also incorporated into a ribonucleoprotein complex, the miRNP, which is similar, and may be identical to the RISC (Bartel and Bartel, Plant Physiol., 2003, 132, 709-717). More than 200 different miRNAs have been identified in plants and animals (Ambros et al., Curr. Biol., 2003, 13, 807-818).
In spite of their biochemical and mechanistic similarities, there are also some differences between siRNAs and miRNAs, based on unique aspects of their biogenesis. siRNAs are generated from the cleavage of long exogenous or possibly endogenous dsRNA molecules, such as very long hairpins or bimolecular duplexed dsRNA, and numerous siRNAs accumulate from both strands of dsRNA precursors. In contrast, mature miRNAs appear to originate from long endogenous primary miRNA transcripts (also known as pri-miRNAs, pri-mirs or pri-pre-miRNAs) that are often hundreds of nucleotides in length (Lee, et al., EMBO J., 2002, 21(17), 4663-4670).
The current model of miRNA processing involves primary miRNA transcripts being processed by a nuclear enzyme in the RNase III family known as Drosha, into approximately 70 nucleotide-long pre-miRNAs (also known as stem-loop structures, hairpins, pre-mirs or foldback miRNA precursors) which are subsequently processed by the Dicer RNase into mature miRNAs, approximately 21-25 nucleotides in length. It is believed that, in processing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts the pri-miRNA at the base of the mature miRNA, leaving a 2-nt 3′ overhang (Ambros et al., RNA, 2003, 9, 277-279; Bartel and Bartel, Plant Physiol., 2003, 132, 709-717; Shi, Trends Genet., 2003, 19, 9-12; Lee, et al., EMBO J., 2002, 21(17), 4663-4670; Lee, et al., Nature, 2003, 425, 415-419). The 3′ two-nucleotide overhang structure, a signature of RNaseIII cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway (Murchison, et al., Curr. Opin. Cell Biol., 2004, 16, 223-9). Both the primary RNA transcripts (pri-miRNAs) and foldback miRNA precursors (pre-miRNAs) are believed to be single-stranded RNA molecules with at least partial double-stranded character, often containing smaller, local internal hairpin structures. Primary miRNA transcripts may be processed such that one single-stranded mature miRNA molecule is generated from one arm of the hairpin-like structure of the pri-miRNA. Alternatively, a polycistronic pri-miRNA may contain multiple pre-miRNAs, each processed into a different, single-stranded mature miRNA.
Naturally occurring miRNAs are characterized by imperfect complementarity to their target sequences. Artificially modified miRNAs with sequences completely complementary to their target RNAs have been designed and found to function as double stranded siRNAs that inhibit gene expression by reducing RNA transcript levels. Synthetic hairpin RNAs that mimic siRNAs and miRNA precursor molecules were demonstrated to target genes for silencing by degradation and not translational repression (McManus et al., RNA, 2002, 8, 842-850).
Tiny non-coding RNA (tncRNA), one class of small non-coding RNAs (Ambros et al., Curr. Biol., 2003, 13, 807-818) produce transcripts similar in length (20-21 nucleotides) to miRNAs, and are also thought to be developmentally regulated but, unlike miRNAs, tncRNAs are reportedly not processed from short hairpin precursors and are not phylogenetically conserved. Although none of these tncRNAs are reported to originate from miRNA hairpin precursors, some are predicted to form potential foldback structures reminiscent of pre-miRNAs; these putative tncRNA precursor structures deviate significantly from those of pre-miRNAs in key characteristics, i.e., they exhibit excessive numbers of bulged nucleotides in the stem or have fewer than 16 base pairs involving the small RNA (Ambros et al., Curr. Biol., 2003, 13, 807-818).
Recently, another class of small non-coding RNAs, the repeat-associated small interfering RNAs (rasiRNAs) has been isolated from Drosophila melanogaster. The rasiRNAs are associated with repeated sequences, transposable elements, satellite and microsatellite DNA, and Suppressor of Stellate repeats, suggesting that small RNAs may participate in defining chromatin structure (Aravin, et al., Dev. Cell, 2003, 5, 337-350).
A total of 201 different expressed RNA sequences potentially encoding novel small non-messenger species (smnRNAs) has been identified from mouse brain cDNA libraries. Based on sequence and structural motifs, several of these have been assigned to the snoRNA class of nucleolar localized molecules known to act as guide RNAs for rRNA modification, whereas others are predicted to direct modification within the U2, U4, or U6 small nuclear RNAs (snRNAs). Some of these newly identified smnRNAs remained unclassified and have no identified RNA targets. It was suggested that some of these RNA species may have novel functions previously unknown for snoRNAs, namely the regulation of gene expression by binding to and/or modifying mRNAs or their precursors via their antisense elements (Huttenhofer et al., Embo J., 2001, 20, 2943-2953).
To date, the binding and regulatory sites within nucleic acid targets of the small non-coding RNAs are largely unknown, although a few putative motifs have been suggested to exist in the 3′UTR of certain genes (Lai and Posakony, Development, 1997, 124, 4847-4856; Lai, et al., Development, 2000, 127, 291-306; Lai, Nat Genet. 2002, 30(4), 363-364).
One miRNA is also believed to act as a cell death regulator, implicating it in mechanisms of human disease such as cancer. Recently, the Drosophila mir-14 miRNA was identified as a suppressor of apoptotic cell death and is required for normal fat metabolism. (Xu et al., Curr. Biol., 2003, 13, 790-795).
Downregulation or deletion of other miRNAs has been associated with B-cell chronic lymphocytic leukemia (B-CLL) (Calin et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 15524-15529), and human homologues of the murine mir-143 and mir-145 mature miRNAs were recently reported to be expressed and processed at reduced steady-state levels at the adenomatous and cancerous stages of colorectal neoplasia (Michael, et al., Mol. Cancer Res., 2003, 1, 882-891).
Expression of the human mir-30 miRNA specifically blocked the translation in human cells of an mRNA containing artificial mir-30 target sites. In these studies, putative miRNAs were excised from transcripts encompassing artificial miRNA precursors and could inhibit the expression of mRNAs containing a complementary target site. These data indicate that novel miRNAs can be readily produced in vivo and can be designed to specifically inactivate the expression of selected target genes in human cells (Zeng et al., Mol. Cell, 2002, 9, 1327-1333).
Disclosed and claimed in PCT Publication WO 03/029459 are miRNAs from several species, or a precursor thereof; a nucleotide sequence which is the complement of said nucleotide sequence which has an identity of at least 80% to said sequence; and a nucleotide sequence which hybridizes under stringent conditions to said sequence. Also claimed is a pharmaceutical composition containing as an active agent at least one of said nucleic acid and optionally a pharmaceutically acceptable carrier, and a method of identifying microRNA molecules or precursor molecules thereof comprising ligating 5′- and 3′-adapter molecules to the ends of a size-fractionated RNA population, reverse transcribing said adapter containing RNA population and characterizing the reverse transcription products (Tuschl et al., Genes Dev., 1999, 13, 3191-3197).
Small non-coding RNA-mediated regulation of gene expression is an attractive approach to the treatment of diseases as well as infection by pathogens such as bacteria, viruses and prions and other disorders associated with RNA expression or processing.
Consequently, there remains a long-felt need for agents that regulate gene expression via the mechanisms mediated by small non-coding RNAs. Identification of modified miRNAs or miRNA mimics that can increase or decrease gene expression or activity is therefore desirable.
The present invention therefore provides oligomeric compounds and methods useful for modulating gene levels, expression, function or pathways, including those relying on mechanisms of action such as RNA interference and dsRNA enzymes, as well as antisense and non-antisense mechanisms. One having skill in the art, once armed with this disclosure will be able, without undue experimentation, to identify compounds, compositions and methods for these uses.
SUMMARY OF THE INVENTION
The present invention provides oligomeric compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to or mimic nucleic acids comprising or encoding small non-coding RNAs, and which act to modulate the levels of small non-coding RNAs, or interfere with their function.
The present invention also provides oligomeric compounds comprising a first strand and a second strand wherein at least one strand contains a modification and wherein a portion of one of the oligomeric compound strands is capable of hybridizing to a small non-coding RNA target nucleic acid.
The present invention also provides oligomeric compounds comprising a first region and a second region and optionally a third region wherein at least one region contains a modification and wherein a portion of the oligomeric compound is capable of hybridizing to a small non-coding RNA target nucleic acid.
The present invention also provides oligomeric compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding human Dicer, and which act to modulate the levels of the human Dicer RNase III enzyme and interfere with its function, as well as modulating the levels of small non-coding RNAs.
Pharmaceutical and other compositions comprising the compounds of the invention are also provided.
Also provided are methods of screening for modulators of small non-coding RNAs and methods of modulating the levels of small non-coding RNAs in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention.
Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of small non-coding RNAs are also set forth herein. Such methods comprise optionally identifying such an animal, and administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the animal or person.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the interaction of the mir-143 miRNA with three novel binding sites in the ERK5 mRNA coding sequence (GenBank Accession NM—139032.1) identified herein, along with their bimolecular hybridization free energies.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides oligomeric compounds useful in, for example, the modulation of expression, endogenous levels or the function of small non-coding RNAs. As used herein, the term “small non-coding RNA” is used to encompass, without limitation, a polynucleotide molecule ranging from about 17 to about 450 nucleotides in length, which can be endogenously transcribed or produced exogenously (chemically or synthetically), but is not translated into a protein. Small non-coding RNAs may include isolated single-, double-, or multiple-stranded molecules, any of which may include regions of intrastrand nucleobase complementarity, said regions capable of folding and forming a molecule with fully or partially double-stranded or multiple-stranded character based on regions of perfect or imperfect complementarity. Examples of small non-coding RNAs include, but are not limited to, primary miRNA transcripts (also known as pri-pre-miRNAs, pri-mirs and pri-miRNAs, which range from around 70 nucleotides to about 450 nucleotides in length and often taking the form of a hairpin structure); pre-miRNAs (also known as pre-mirs and foldback miRNA precursors, which range from around 50 nucleotides to around 110 nucleotides in length); miRNAs (also known as microRNAs, Mirs, miRs, mirs, and mature miRNAs, and generally refer either to double-stranded intermediate molecules around 17 to about 25 nucleotides in length, or to single-stranded miRNAs, which may comprise a bulged structure upon hybridization with a partially complementary target nucleic acid molecule); or mimics of pri-miRNAs, pre-miRNAs or miRNAs. Small non-coding RNAs can be endogenously transcribed in cells, or can be synthetic oligonucleotides, in vitro transcribed polynucleotides or nucleic acid oligomeric compounds expressed from vectors. Pri-miRNAs and pre-miRNAs, or mimics thereof, may be processed into smaller molecules.
As used herein, the term “miRNA precursor” is used to encompass, without limitation, primary RNA transcripts, pri-miRNAs and pre-miRNAs.
In some embodiments, pri-miRNAs, or mimics thereof, are 70 to 450 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449 or 450 nucleobases in length, or any range therewithin.
In some embodiments, pri-miRNAs, or mimics thereof, are 110 to 430 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429 or 430 nucleobases in length, or any range therewithin.
In some embodiments, pri-miRNAs, or mimics thereof, are 110 to 280 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279 or 280 nucleobases in length, or any range therewithin.
In some embodiments, pre-miRNAs, or mimics thereof, are 50 to 110 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 nucleobases in length, or any range therewithin. In some embodiments, pre-miRNAs, or mimics thereof, are 60 to 80 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length, or any range therewithin.
In some embodiments, miRNAs, or mimics thereof, are 15 to 49 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 nucleobases in length, or any range therewithin. In some embodiments, miRNAs, or mimics thereof, are 17 to 25 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleobases in length, or any range therewithin.
Oligomeric compounds of the invention modulate the levels, expression or function of small non-coding RNAs by hybridizing to a nucleic acid comprising or encoding a small non-coding RNA nucleic acid target resulting in alteration of normal function by, for example, facilitating destruction of the small non-coding RNA through cleavage, by sequestration, or by sterically occluding the function of the small non-coding RNA. Further, modified synthetic oligomeric compounds of the present invention may be designed to mimic endogenous small non-coding RNAs. These modifications include, but are not limited to, improved pharmacokinetic or pharmacodynamic properties, binding affinity, stability, charge, localization or uptake. Synthetic mimics can therefore act as replacements for small non-coding RNAs, as competitive inhibitors of naturally occurring small non-coding RNAs or as delivery systems wherein the mimic construct contains one or more functional components.
As used herein, the terms “target nucleic acid,” “target RNA,” “target RNA transcript” or “nucleic acid target” are used to encompass any nucleic acid capable of being targeted including, without limitation, RNA (including microRNAs, stRNAs, small nuclear RNAs, small nucleolar RNAs, small ribosomal RNAs, small hairpin RNAs, endogenous antisense RNAs, guide RNAs, tiny noncoding RNAs, small single or double stranded RNAs that are encoded by heterochromatic repeats at centromeres or other chromosomal origin, and any precursors thereof). These nucleic acid targets can be coding or non-coding sequences; pre-mRNAs or mRNAs; single- or double-stranded, or single-stranded with partial double-stranded character; may occur naturally within introns or exons of messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), or transfer RNAs (tRNAs); and can be endogenously transcribed or exogenously produced.
In some embodiments of this invention, modulation of small non-coding RNA levels, expression or function is achieved via oligomeric compounds which target a further RNA associated with the particular small non-coding RNA. This association can be a physical association between that RNA and the particular small non-coding RNA such as, but not limited to, in an RNA or ribonucleoprotein complex. This association can also be within the context of a biological pathway, such as but not limited to, the regulation of levels, expression or function of a protein-encoding mRNA or its precursor by a small non-coding RNA. As such, the invention provides for modulation of the levels, expression or function of a target nucleic acid where the target nucleic acid is a messenger RNA whose expression levels and/or function are associated with one or more small non-coding RNAs. The messenger RNA function or processing may be disrupted by degradation through an antisense mechanism, including but not limited to, RNA interference, or RNase H, as well as other mechanisms wherein double stranded nucleic acid structures are recognized and degraded, cleaved, sterically occluded, sequestered or otherwise rendered inoperable.
The compounds or compositions of the present invention may also interfere with the function of endogenous RNA molecules. The functions of RNA to be interfered with can include, for example, nuclear events such as replication or transcription as the compounds of the present invention could target or mimic small non-coding RNAs in these cellular processes. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include cytoplasmic events such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, RNA signaling and regulatory activities, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA as the compounds of the present invention could target or mimic small non-coding RNAs in these cellular processes.
In the context of the present invention, “modulation” and “modulation of expression” mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a small non-coding RNA, nucleic acid target, an RNA or protein associated with a small non-coding RNA, or a downstream target of the small non-coding RNA (e.g., a mRNA representing a protein-coding nucleic acid that is regulated by a small non-coding RNA) Inhibition is a suitable form of modulation and small non-coding RNA is a suitable target nucleic acid.
In the context of the present invention, “modulation of function” means an alteration in the function of the small non-coding RNA or an alteration in the function of any cellular component with which the small non-coding RNA has an association or downstream effect.
The present invention provides, inter alia, oligomeric compounds and compositions containing the same wherein the oligomeric compound includes one or more modifications that render the compound capable of supporting modulation of the levels, expression or function of the small non-coding RNA by a degradation or cleavage mechanism.
The present invention also provides methods of maintaining a pluripotent stem cell comprising contacting the cell with an effective amount of an oligomeric compound targeting human Dicer. The pluripotent stem cell can be present in a sample of cord blood or bone marrow, or may be present as part of a cell line. In addition, the pluripotent stem cell can be an embryonic stem cell.
The present invention also provides oligomeric compounds and compositions containing the same wherein the oligomeric compound includes one or more modifications that render the compound capable of blocking or interfering with the levels, expression or function of one or more small non-coding RNAs by steric occlusion.
The present invention also provides oligomeric compounds and compositions containing the same wherein the oligomeric compound includes one or more modifications or structural elements or motifs that render the compound capable of mimicking or replacing one or more small non-coding RNAs.
Oligomeric Compounds
In the context of the present invention, the term “oligomeric compound(s)” refers to polymeric structures which are capable of hybridizing to at least a region of a small non-coding RNA molecule or a target of small non-coding RNAs, or polymeric structures which are capable of mimicking small non-coding RNAs. The term “oligomeric compound” includes, but is not limited to, compounds comprising oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and combinations of these. Oligomeric compounds also include, but are not limited to, antisense oligomeric compounds, antisense oligonucleotides, siRNAs, alternate splicers, primers, probes and other compounds that hybridize to at least a portion of the target nucleic acid. Oligomeric compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular and may also include branching. Separate oligomeric compounds can hybridize to form double stranded compounds that can be blunt-ended or may include overhangs on one or both termini. In general, an oligomeric compound comprises a backbone of linked monomeric subunits where each linked monomeric subunit is directly or indirectly attached to a heterocyclic base moiety. The linkages joining the monomeric subunits, the sugar moieties or sugar surrogates and the heterocyclic base moieties can be independently modified giving rise to a plurality of motifs for the resulting oligomeric compounds including hemimers, gapmers and chimeras.
As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base moiety. The two most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′,3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. The respective ends of this linear polymeric structure can be joined to form a circular structure by hybridization or by formation of a covalent bond. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded structure. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide. The normal internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage.
In the context of this invention, the term “oligonucleotide” refers generally to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside linkages. The term “oligonucleotide analog” refers to oligonucleotides that have one or more non-naturally occurring portions which function in a similar manner to oligonucleotides. Such non-naturally occurring oligonucleotides are often selected over naturally occurring forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets and increased stability in the presence of nucleases.
In the context of this invention, the term “oligonucleoside” refers to nucleosides that are joined by internucleoside linkages that do not have phosphorus atoms. Internucleoside linkages of this type include short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one or more short chain heteroatomic and one or more short chain heterocyclic. These internucleoside linkages include but are not limited to siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl, sulfamate; methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide and others having mixed N, O, S and CH2 component parts. In addition to the modifications described above, the nucleosides of the oligomeric compounds of the invention can have a variety of other modifications. Additional nucleosides amenable to the present invention having altered base moieties and or altered sugar moieties are disclosed in U.S. Pat. No. 3,687,808 and PCT application PCT/US89/02323.
For nucleotides that are incorporated into oligonucleotides of the invention, these nucleotides can have sugar portions that correspond to naturally occurring sugars or modified sugars. Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at one or more of their 2′,3′ or 4′ positions and sugars having substituents in place of one or more hydrogen atoms of the sugar.
Altered base moieties or altered sugar moieties also include other modifications consistent with the spirit of this invention. Such oligomeric compounds are best described as being structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified oligonucleotides. All such oligomeric compounds are comprehended by this invention so long as they function effectively to mimic the structure or function of a desired RNA or DNA oligonucleotide strand.
A class of representative base modifications include tricyclic cytosine analog, termed “G clamp” (Lin, et al., J. Am. Chem. Soc. 1998, 120, 8531). This analog can form four hydrogen bonds with a complementary guanine (G) by simultaneously recognizing the Watson-Crick and Hoogsteen faces of the targeted G. This G clamp modification when incorporated into phosphorothioate oligomeric compounds, dramatically enhances potencies as measured by target reduction in cell culture. The oligomeric compounds of the invention also can include phenoxazine-substituted bases of the type disclosed by Flanagan, et al., Nat. Biotechnol. 1999, 17(1), 48-52.
The oligomeric compounds in accordance with this invention comprise from about 8 to about 80 monomeric subunits (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies oligomeric compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits in length, or any range therewithin.
In one embodiment, the oligomeric compounds of the invention are 12 to 50 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 subunits in length, or any range therewithin.
In one embodiment, the oligomeric compounds of the invention are 13 to 80 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits in length, or any range therewithin.
In one embodiment, the oligomeric compounds of the invention are 15 to 30 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 subunits in length, or any range therewithin.
In one embodiment, the oligomeric compounds of the invention are 70 to 450 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449 or 450 subunits in length, or any range therewithin.
In one embodiment, the oligomeric compounds of the invention are 110 to 430 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429 or 430 subunits in length, or any range therewithin.
In one embodiment, the oligomeric compounds of the invention are 110 to 280 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279 or 280 subunits in length, or any range therewithin.
In one embodiment, the oligomeric compounds of the invention are 50 to 110 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 subunits in length, or any range therewithin.
In one embodiment, the oligomeric compounds of the invention are 60 to 80 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 subunits in length, or any range therewithin.
In one embodiment, the oligomeric compounds of the invention are 15 to 49 monomeric subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 subunits in length, or any range therewithin.
In one embodiment, the oligomeric compounds of the invention are 17 to 25 subunits in length. One having ordinary skill in the art will appreciate that this embodies oligomeric compounds of 17, 18, 19, 20, 21, 22, 23, 24 or 25 subunits in length, or any range therewithin.
In accordance with the present invention, oligomeric compounds designed to mimic pri-miRNAs are from about 70 to about 450 monomeric subunits in length, or from about 110 to 430 subunits in length. Oligomeric compounds of the invention designed to mimic pre-miRNAs are from about 50 to about 110 monomeric subunits in length, or from about 60 to about 80 subunits in length. Oligomeric compounds of the invention designed to mimic mature miRNAs are from about 17 to about 25 monomeric subunits in length, and can be single- or double-stranded with either or both strands comprising from about 17 to about 25 subunits.
As used herein, the term “about” means±5% of the variable thereafter.
The size or length of any oligomeric compound of the present invention, within any range cited herein, can be determined as follows:
Let R(n, n+m−1) be a region from a target nucleobase sequence, where “n” is the 5′-most nucleobase position of the region, where “n+m−1” is the 3′-most nucleobase position of the region and where “m” is the length of the region. A set “S(m)”, of regions of length “m” is defined as the regions where n ranges from 1 to L−m+1, where L is the length of the target nucleic acid sequence and L>m. A set, “A”, of all regions can be constructed as a union of the sets of regions for each length from where m is greater than or equal to a lower limit of any recited range (8 in this example) and is less than or equal to the upper limit of any recited range (80 in this example).
This set of regions can be represented using the following mathematical notation:




  A
  =
  
    
      ⋃
      m
    
    ⁢
    
      S
      ⁡
      
        (
        m
        )
      
    
  




where mεN|8≦m≦80

and

S(m)={Rn,n+m−1|nε{1,2,3, . . . , L−m+1}}

where the mathematical operator | indicates “such that”,
where the mathematical operator ε indicates “a member of a set” (e.g. yεZ indicates that element y is a member of set Z),
where x is a variable,
where N indicates all natural numbers, defined as positive integers,
and where the mathematical operator ∪ indicates “the union of sets”.
For example, the set of regions for m equal to 8, 20 and 80 can be constructed in the following manner. The set of regions, each 8 monomeric subunits in length, S(m=8), in a target nucleic acid sequence 100 subunits in length (L=100), beginning at position 1 (n=1) of the target nucleic acid sequence, can be created using the following expression:

S(8)={R1,8|nε{1,2,3, . . . , 93}}

and describes the set of regions comprising nucleobases 1-8, 2-9, 3-10, 4-11, 5-12, 6-13, 7-14, 8-15, 9-16, 10-17, 11-18, 12-19, 13-20, 14-21, 15-22, 16-23, 17-24, 18-25, 19-26, 20-27, 21-28, 22-29, 23-30, 24-31, 25-32, 26-33, 27-34, 28-35, 29-36, 30-37, 31-38, 32-39, 33-40, 34-41, 35-42, 36-43, 37-44, 38-45, 39-46, 40-47, 41-48, 42-49, 43-50, 44-51, 45-52, 46-53, 47-54, 48-55, 49-56, 50-57, 51-58, 52-59, 53-60, 54-61, 55-62, 56-63, 57-64, 58-65, 59-66, 60-67, 61-68, 62-69, 63-70, 64-71, 65-72, 66-73, 67-74, 68-75, 69-76, 70-77, 71-78, 72-79, 73-80, 74-81, 75-82, 76-83, 77-84, 78-85, 79-86, 80-87, 81-88, 82-89, 83-90, 84-91, 85-92, 86-93, 87-94, 88-95, 89-96, 90-97, 91-98, 92-99, 93-100.

An additional set for regions 20 monomeric subunits in length, in a target sequence 100 subunits in length, beginning at position 1 of the target nucleic acid sequence, can be described using the following expression:

S(20)={R1,20|nε{1,2,3, . . . , 81}}

and describes the set of regions comprising nucleobases 1-20, 2-21, 3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32, 14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42, 24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52, 34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59, 41-60, 42-61, 43-62, 44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72, 54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82, 64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92, 74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100.

An additional set for regions 80 monomeric subunits in length, in a target sequence 100 subunits in length, beginning at position 1 of the target nucleic acid sequence, can be described using the following expression:

S(80)={R1,80|nε{1,2,3, . . . , 21}}

and describes the set of regions comprising nucleobases 1-80, 2-81, 3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91, 13-92, 14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.

The union of these aforementioned example sets and other sets for lengths from 10 to 19 and 21 to 79 can be described using the mathematical expression




  A
  =
  
    
      ⋃
      m
    
    ⁢
    
      S
      ⁡
      
        (
        m
        )
      
    
  




where ∪ represents the union of the sets obtained by combining all members of all sets.
Thus, in this example, A would include regions 1-8, 2-9, 3-10 . . . 93-100, 1-20, 2-21, 3-22 . . . 81-100, 1-80, 2-81, 3-82 . . . 21-100.
The mathematical expressions described herein define all possible target regions in a target nucleic acid sequence of any length L, where the region is of length m, and where m is greater than or equal to the lower limit and less than or equal to the upper limit of monomeric units, and where m is less than L, and where n is less than L−m+1.
In the context of this invention, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the present invention, the mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
An oligomeric compound of the invention is “specifically hybridizable” when association of the compound with the target nucleic acid interferes with the normal function of the target nucleic acid to alter the activity, disrupt the function, or modulate the level of the target nucleic acid, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target nucleic acid sequences under conditions in which specific hybridization is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under standard assay conditions in the case of in vitro assays.
In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which an oligomeric compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will vary with different circumstances and in the context of this invention; “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated. One having ordinary skill in the art will understand variability in the experimental protocols and be able to determine when conditions are optimal for stringent hybridization with minimal non-specific hybridization events.
“Complementary,” as used herein, refers to the capacity for precise pairing of two monomeric subunits regardless of where in the oligomeric compound or target nucleic acid the two are located. For example, if a monomeric subunit at a certain position of an oligomeric compound is capable of hydrogen bonding with a monomeric subunit at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligomeric compound and the target nucleic acid is considered to be a complementary position. The oligomeric compound and the target nucleic acid are “substantially complementary” to each other when a sufficient number of complementary positions in each molecule are occupied by monomeric subunits that can hydrogen bond with each other. Thus, the term “substantially complementary” is used to indicate a sufficient degree of precise pairing over a sufficient number of monomeric subunits such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.
Generally, an oligomeric compound is “antisense” to a target nucleic acid when, written in the 5′ to 3′ direction, it comprises the reverse complement of the corresponding region of the target nucleic acid. “Antisense compounds” are also often defined in the art to comprise the further limitation of, once hybridized to a target, being able to induce or trigger a reduction in target gene expression.
It is understood in the art that the sequence of the oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligomeric compound may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization (e.g., a bulge, a loop structure or a hairpin structure).
In some embodiments of the invention, the oligomeric compounds comprise at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 85% sequence complementarity to a target region within the target nucleic acid. In other embodiments of the invention, the oligomeric compounds comprise at least 90% sequence complementarity to a target region within the target nucleic acid. In other embodiments of the invention, the oligomeric compounds comprise at least 95% or at least 99% sequence complementarity to a target region within the target nucleic acid. For example, an oligomeric compound in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target sequence would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an oligomeric compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
In some embodiments of the invention, the oligomeric compounds act as mimics or replacements for small non-coding RNAs. In this case, the oligomeric compounds of the invention can comprise at least 70% sequence identity to a small non-coding RNA or a region thereof. In some embodiments the oligomeric compounds of the invention can comprise at least 90% sequence identity and in some embodiments can comprise at least 95% sequence identity to to a small non-coding RNA or a region thereof.
“Targeting” an oligomeric compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose levels, expression or function is to be modulated. This target nucleic acid may be, for example, a mRNA transcribed from a cellular gene whose expression is associated with a particular disorder or disease state, a small non-coding RNA or its precursor, or a nucleic acid molecule from an infectious agent.
The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the interaction to occur such that the desired effect, e.g., modulation of levels, expression or function, will result. Within the context of the present invention, the term “region” is defined as a portion of the target nucleic acid having at least one identifiable sequence, structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as specific positions within a target nucleic acid. The terms region, segment, and site can also be used to describe an oligomeric compound of the invention such as for example a gapped oligomeric compound having three separate segments.
Targets of the present invention include both coding and non-coding nucleic acid sequences. For coding nucleic acid sequences, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon.” A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding a nucleic acid target, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).
The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with the oligomeric compounds of the present invention.
The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a further suitable region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.
Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA (or corresponding nucleotides on the gene). The 5′ cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also suitable to target the 5′ cap region.
Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts.” It is also known that introns can be effectively targeted using oligomeric compounds targeted to, precursor molecules for example, pre-mRNA.
It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants.” More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequences.
Upon excision of one or more exon or intron regions, or portions thereof, during splicing, pre-mRNA variants produce smaller “mRNA variants.” Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants.” If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.
It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also target nucleic acids.
Certain non-coding RNA genes are known to produce functional RNA molecules with important roles in diverse cellular processes. Such non-translated, non-coding RNA molecules can include ribosomal RNAs, tRNAs, snRNAs, snoRNAs, tncRNAs, rasiRNAs, short hairpin RNAs (shRNAs), short temporal RNAs (stRNAs), short hairpin RNAs (shRNAs), siRNAs, miRNAs and smnRNAs. These non-coding RNA genes and their products are also suitable targets of the compounds of the invention. Such cellular processes include transcriptional regulation, translational regulation, developmental timing, viral surveillance, immunity, chromosome maintenance, ribosomal structure and function, gene imprinting, subcellular compartmentalization, pre-mRNA splicing, and guidance of RNA modifications. RNA-mediated processes are now also believed to direct heterochromatin formation, genome rearrangements, cellular differentiation and DNA elimination.
A total of 201 different expressed RNA sequences potentially encoding novel small non-messenger species (smnRNAs) has been identified from mouse brain cDNA libraries. Based on sequence and structural motifs, several of these have been assigned to the snoRNA class of nucleolar localized molecules known to act as guide RNAs for rRNA modification, whereas others are predicted to direct modification within the U2, U4, or U6 small nuclear RNAs (snRNAs). Some of these newly identified smnRNAs remained unclassified and have no identified RNA targets. It was suggested that some of these RNA species may have novel functions previously unknown for snoRNAs, namely the regulation of gene expression by binding to and/or modifying mRNAs or their precursors via their antisense elements (Huttenhofer et al., Embo J., 2001, 20, 2943-2953). Therefore, these smnRNAs are also suitable targets for the compounds of the present invention.
The locations on the target nucleic acid to which compounds and compositions of the invention hybridize are herein referred to as “suitable target segments.” As used herein the term “suitable target segment” is defined as at least an 8-nucleobase portion of a target region to which oligomeric compound is targeted.
Once one or more targets, target regions, segments or sites have been identified, oligomeric compounds are designed to be sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. The desired effect may include, but is not limited to modulation of the levels, expression or function of the target.
In accordance with the present invention, a series of single stranded oligomeric compounds can be designed to target or mimic one or more specific small non-coding RNAs. These oligomeric compounds can be of a specified length, for example from 8 to 80, 12 to 50, 13 to 80, 15 to 30, 70 to 450, 110 to 430, 110 to 280, 50 to 110, 60 to 80, 15 to 49, 17 to 25 or 19 to 23 nucleotides long and have one or more modifications.
In accordance with one embodiment of the invention, a series of double-stranded oligomeric compounds (duplexes) comprising, as the antisense strand, the single-stranded oligomeric compounds of the present invention, and the fully or partially complementary sense strand, can be designed to modulate the levels, expression or function of one or more small non-coding RNAs or small non-coding RNA targets. One or both termini of the duplex strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the duplex may be designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the duplex would be complementary over the central region of the duplex, each having overhangs at one or both termini.
For the purposes of this invention, the combination of an antisense strand and a sense strand, each of which can be of a specified length, for example from 8 to 80, 12 to 50, 13 to 80, 15 to 30, 15 to 49, 17 to 25 or 19 to 23 subunits long, is identified as a complementary pair of oligomeric compounds. This complementary pair of oligonucleotides can include additional nucleotides on either of their 5′ or 3′ ends. They can include other molecules or molecular structures on their 3′ or 5′ ends, such as a phosphate group on the 5′ end, or non-nucleic acid moieties conjugated to either terminus of either strand or both strands. One group of compounds of the invention includes a phosphate group on the 5′ end of the antisense strand compound. Other compounds also include a phosphate group on the 5′ end of the sense strand compound. Some compounds include additional nucleotides such as a two base overhang on the 3′ end as well as those lacking overhangs.
For example, a complementary pair of oligomeric compounds may comprise an antisense strand oligomeric compound having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:2181), having a two-nucleobase overhang of deoxythymidine (dT) and its complement sense strand. This complementary pair of oligomeric compounds would have the following structure:







  cgagaggcggacgggaccgTT Antisense Strand


  |||||||||||||||||||   (SEQ ID NO: 2182)


TTgctctccgcctgccctggc   Complement Sense Strand


                        (SEQ ID NO: 2183)






In some embodiments, a single-stranded oligomeric compound may be designed comprising the antisense portion as a first region and the sense portion as a second region. The first and second regions can be linked together by either a nucleotide linker (a string of one or more nucleotides that are linked together in a sequence) or by a non-nucleotide linker region or by a combination of both a nucleotide and non-nucleotide structure. In any of these structures, the oligomeric compound, when folded back on itself, would form at least a partially complementary structure at least between a portion of the first region, the antisense portion, and a portion of the second region, the sense portion.
In one embodiment, the invention includes an oligomeric compound/protein composition. This composition has both an oligomeric compound component and a protein component. The oligomeric compound component comprises at least one oligomeric compound, either the antisense or the sense oligomeric compound but preferably the antisense oligomeric compound (the oligomeric compound that is antisense to the target nucleic acid). The protein component of the composition comprises at least one protein that forms a portion of the RNA-induced silencing complex, i.e., the RISC complex. The oligomeric compound component can also comprise both antisense and sense strand oligomeric compounds.
RISC is a ribonucleoprotein complex that contains proteins of the Argonaute family of proteins. While not wishing to be bound by theory, it is believed that the Argonaute proteins are a class of proteins, some of which have been shown to contain a PAZ and/or a Piwi domain and that have been implicated in processes previously linked to posttranscriptional silencing. The Argonaute family of proteins includes, but depending on species, is not necessary limited to e1F2C1 and e1F2C2. It is also believed that at least the antisense strand of double-stranded compounds shown to act as siRNAs is bound to one of the protein components that form the RISC complex, and that the RISC complex interacts with the ribosomes or polyribosome complexes which may contain small non-coding RNA molecules amenable to targeting with the oligomeric compounds of the present invention. Consequently, one embodiment of the invention includes oligomeric compounds that mimic RNA components of the RISC complex.
In one embodiment, the oligomeric compounds of the invention are designed to exert their modulatory effects via mimicking or targeting small non-coding RNAs associated with cellular factors such as transporters or chaperones. These cellular factors can be protein, lipid or carbohydrate based and can have structural or enzymatic functions that may or may not require the complexation of one or more metal ions.
Furthermore, the oligomeric compounds of the invention can have one or more moieties bound or conjugated, which facilitates the active or passive transport, localization, or compartmentalization of the oligomeric compound. Cellular localization includes, but is not limited to, localization to within the nucleus, the nucleolus, or the cytoplasm. Compartmentalization includes, but is not limited to, any directed movement of the oligonucleotides of the invention to a cellular compartment including the nucleus, nucleolus, mitochondrion, or imbedding into a cellular membrane.
In some embodiments of the invention, the oligomeric compounds are designed to exert their modulatory effects via mimicking or targeting small non-coding RNAs associated with cellular factors that affect gene expression, more specifically those involved in RNA or DNA modifications. These modifications include, but are not limited to, posttranscriptional or chromosomal modifications such as methylation, acetylation, pseudouridylation or amination.
Furthermore, the oligomeric compounds of the invention comprise one or more conjugate moieties which facilitate posttranscriptional modification.
The oligomeric compounds of the invention may be in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the oligomeric compounds of the invention may elicit the action of one or more enzymes or proteins to effect modulation of the levels, expression or function of the target nucleic acid.
One non-limiting example of such a protein is the Drosha RNase III enzyme. Drosha is a nuclear enzyme that processes long primary RNA transcripts (pri-miRNAs) from approximately 70 to 450 nucleotides in length into pre-miRNAs (from about 50 to about 80 nucleotides in length) which are exported from the nucleus to encounter the human Dicer enzyme which then processes pre-miRNAs into miRNAs. It is believed that, in processing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts the pri-miRNA at the base of the mature miRNA, leaving a 2-nt 3′overhang (Lee, et al., Nature, 2003, 425, 415-419). The 3′ two-nucleotide overhang structure, a signature of RNaseIII enzymatic cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway (Murchison, et al., Curr. Opin. Cell Biol., 2004, 16, 223-9).
A further non-limiting example involves the enzymes of the RISC complex. Use of the RISC complex to effect cleavage of RNA targets thereby greatly enhances the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
Oligomeric compounds or compositions of the invention are used to induce potent and specific modulation of gene function through interactions with or mimicry of small non-coding RNAs that are processed by the RISC complex. These compounds include single-stranded oligomeric compounds that bind in a RISC complex, double-stranded antisense/sense pairs of oligomeric compounds, or single-stranded oligomeric compounds that include both an antisense portion and a sense portion.
General Oligomer Synthesis:
Oligomerization of modified and unmodified nucleosides is performed according to literature procedures for DNA like compounds (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA like compounds (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA:Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713) synthesis as appropriate. In addition, specific protocols for the synthesis of oligomeric compounds of the invention are illustrated in the examples below.
RNA oligomers can be synthesized by methods disclosed herein or purchased from various RNA synthesis companies such as for example Dharmacon Research Inc., (Lafayette, Colo.).
Irrespective of the particular protocol used, the oligomeric compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed.
Synthesis of Nucleoside Phosphoramidites:
The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N4-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidine penultimate intermediate, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N6-benzoyladenosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4-isobutyrylguanosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O2-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-((2-phthalimidoxy)ethyl)-5′-t-butyldiphenylsilyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-((2-formadoximinooxy)ethyl)-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O—(N,N dimethylaminooxyethyl)-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite), 2′-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite), 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.
Oligonucleotide and Oligonucleoside Synthesis:
Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.
Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH4OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.
Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.
3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.
Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.
Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.
3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.
Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.
Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.
Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone oligomeric compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.
Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.
Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.
RNA Synthesis:
In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl.
Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.
RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.
Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S2Na2) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.
The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.
Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).
The present invention is also useful for the preparation of oligomeric compounds incorporating at least one 2′-O-protected nucleoside. After incorporation and appropriate deprotection the 2′-O-protected nucleoside will be converted to a ribonucleoside at the position of incorporation. The number and position of the 2-ribonucleoside units in the final oligomeric compound can vary from one at any site or the strategy can be used to prepare up to a full 2′-OH modified oligomeric compound. All 2′-O-protecting groups amenable to the synthesis of oligomeric compounds are included in the present invention.
In general a protected nucleoside is attached to a solid support by for example a succinate linker. Then the oligonucleotide is elongated by repeated cycles of deprotecting the 5′-terminal hydroxyl group, coupling of a further nucleoside unit, capping and oxidation (alternatively sulfurization). In a more frequently used method of synthesis the completed oligonucleotide is cleaved from the solid support with the removal of phosphate protecting groups and exocyclic amino protecting groups by treatment with an ammonia solution. Then a further deprotection step is normally required for the more specialized protecting groups used for the protection of 2′-hydroxyl groups which will give the fully deprotected oligonucleotide.
A large number of 2′-O-protecting groups have been used for the synthesis of oligoribonucleotides but over the years more effective groups have been discovered. The key to an effective 2′-O-protecting group is that it is capable of selectively being introduced at the 2′-O-position and that it can be removed easily after synthesis without the formation of unwanted side products. The protecting group also needs to be inert to the normal deprotecting, coupling, and capping steps required for oligoribonucleotide synthesis. Some of the protecting groups used initially for oligoribonucleotide synthesis included tetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. These two groups are not compatible with all 5′-O-protecting groups so modified versions were used with 5′-DMT groups such as 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp). Reeseiii has identified a number of piperidine derivatives (like Fpmp) that are useful in the synthesis of oligoribonucleotides including 1-((chloro-4-methyl)phenyl)-4′-methoxypiperidin-4-yl (Reese et al., Tetrahedron Lett., 1986, (27), 2291). Another approach was to replace the standard 5′-DMT (dimethoxytrityl) group with protecting groups that were removed under non-acidic conditions such as levulinyl and 9-fluorenylmethoxycarbonyl. Such groups enable the use of acid labile 2′-protecting groups for oligoribonucleotide synthesis. Another more widely used protecting group initially used for the synthesis of oligoribonucleotides was the t-butyldimethylsilyl group (Ogilvie et al., Tetrahedron Lett., 1974, 2861; Hakimelahi et al., Tetrahedron Lett., 1981, (22), 2543; and Jones et al., J. Chem. Soc. Perkin I., 2762). The 2′-O-protecting groups can require special reagents for their removal such as for example the t-butyldimethylsilyl group is normally removed after all other cleaving/deprotecting steps by treatment of the oligomeric compound with tetrabutylammonium fluoride (TBAF).
One group of researchers examined a number of 2′-O-protecting groups (Pitsch, S., Chimia, 2001, (55), 320-324.) The group examined fluoride labile and photolabile protecting groups that are removed using moderate conditions. One photolabile group that was examined was the (2-(nitrobenzyl)oxy)methyl (nbm) protecting group (Schwartz et al., Bioorg. Med. Chem. Lett., 1992, (2), 1019.) Other groups examined included a number structurally related formaldehyde acetal-derived, 2′-O-protecting groups. Also prepared were a number of related protecting groups for preparing 2′-O-alkylated nucleoside phosphoramidites including 2′-O-((triisopropylsilyl)oxy)methyl (2′-O—CH2—O-Si(iPr)3, TOM). One 2′-O-protecting group that was prepared to be used orthogonally to the TOM group was 2′-O—((R)-1-(2-nitrophenyl)ethyloxy)methyl) ((R)-mnbm).
Another strategy using a fluoride labile 5′-O-protecting group (non-acid labile) and an acid labile 2′-O-protecting group has been reported (Scaringe, Stephen A., Methods, 2001, (23) 206-217). A number of possible silyl ethers were examined for 5′-O-protection and a number of acetals and orthoesters were examined for 2′-O-protection. The protection scheme that gave the best results was 5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses a modified phosphoramidite synthesis approach in that some different reagents are required that are not routinely used for RNA/DNA synthesis.
Although a lot of research has focused on the synthesis of oligoribonucleotides the main RNA synthesis strategies that are presently being used commercially include 5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS), 5′-O-DMT-2′-O-(1(2-fluorophenyl)-4-methoxypiperidin-4-yl) (FPMP), 2′-O-((triisopropylsilyl)oxy)methyl (2′-O—CH2—O-Si(iPr)3 (TOM), and the 5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). A current list of some of the major companies currently offering RNA products include Pierce Nucleic Acid Technologies, Dharmacon Research Inc., Amen Biotechnologies Inc., and Integrated DNA Technologies, Inc. One company, Princeton Separations, is marketing an RNA synthesis activator advertised to reduce coupling times especially with TOM and TBDMS chemistries. Such an activator would also be amenable to the present invention.
The structures corresponding to these protecting groups are shown below.
TBDMS=5′-O-DMT-2′-O-t-butyldimethylsilyl;
TOM=2′-O-((triisopropylsilyl)oxy)methyl;
DOD/ACE=(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether-2′-O-bis(2-acetoxyethoxy)methyl
FPMP=5′-O-DMT-2′-O-(1(2-fluorophenyl)-4-methoxypiperidin-4-yl)







All of the aforementioned RNA synthesis strategies are amenable to the present invention. Strategies that would be a hybrid of the above e.g. using a 5′-protecting group from one strategy with a 2′-O-protecting from another strategy is also amenable to the present invention.
The preparation of ribonucleotides and oligomeric compounds having at least one ribonucleoside incorporated and all the possible configurations falling in between these two extremes are encompassed by the present invention. The corresponding oligomeric compounds can be hybridized to further oligomeric compounds including oligoribonucleotides having regions of complementarity to form double-stranded (duplexed) oligomeric compounds.
The methods of preparing oligomeric compounds of the present invention can also be applied in the areas of drug discovery and target validation.
Oligonucleotide Isolation:
After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH4OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32 +/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.
Oligonucleotide Synthesis—96 Well Plate Format:
Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.
Oligonucleotides were cleaved from support and deprotected with concentrated NH4OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.
Oligonucleotide Analysis—96-Well Plate Format:
The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACETM MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACETM 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the oligomeric compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the oligomeric compounds on the plate were at least 85% full length.
For double-stranded compounds of the invention, once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 μM. Once diluted, 30 μL of each strand is combined with 15 μL of a 5× solution of annealing buffer. The final concentration of the buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the double-stranded compounds are used in experimentation. The final concentration of the duplexed compound is 20 μM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.
Once prepared, the double-stranded compounds are evaluated for their ability to modulate target levels, expression or function. When cells reach 80% confluency, they are treated with synthetic double-stranded compounds comprising at least one oligomeric compound of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEMTM-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEMTM-1 containing 12 μg/mL LIPOFECTINTM (Invitrogen Corporation, Carlsbad, Calif.) and the desired double stranded compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by real-time RT-PCR.
Specific examples of oligomeric compounds useful in this invention include oligonucleotides containing modified e.g. non-naturally occurring internucleoside linkages. As defined in this specification, oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
In the C. elegans system, modification of the internucleotide linkage (phosphorothioate) did not significantly interfere with RNAi activity. Based on this observation, it is suggested that certain oligomeric compounds of the invention can also have one or more modified internucleoside linkages. A suitable phosphorus-containing modified internucleoside linkage is the phosphorothioate internucleoside linkage.
Modified oligonucleotide backbones (internucleoside linkages) containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′,5′ to 5′ or 2′ to 2′ linkage. Oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
In other embodiments of the invention, oligomeric compounds have one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— (known as a methylene (methylimino) or MMI backbone), —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —O—N(CH3)—CH2—CH2— (wherein the native phosphodiester internucleotide linkage is represented as —O—P(═O)(OH)—O—CH2—). The MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Amide internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,602,240.
Modified oligonucleotide backbones (internucleoside linkages) that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.
Another group of oligomeric compounds amenable to the present invention includes oligonucleotide mimetics. The term mimetic as it is applied to oligonucleotides is intended to include oligomeric compounds wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with novel groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA oligomeric compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA oligomeric compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Teaching of PNA oligomeric compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
PNA has been modified to incorporate numerous modifications since the basic PNA structure was first prepared. The basic structure is shown below:




wherein

Bx is a heterocyclic base moiety;
T4 is hydrogen, an amino protecting group, —C(O)R5, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group, a reporter group, a conjugate group, a D or L α-amino acid linked via the α-carboxyl group or optionally through the ω-carboxyl group when the amino acid is aspartic acid or glutamic acid or a peptide derived from D, L or mixed D and L amino acids linked through a carboxyl group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;
T5 is —OH, —N(Z1)Z2, R5, D or L α-amino acid linked via the α-amino group or optionally through the ω-amino group when the amino acid is lysine or ornithine or a peptide derived from D, L or mixed D and L amino acids linked through an amino group, a chemical functional group, a reporter group or a conjugate group;
Z1 is hydrogen, C1-C6 alkyl, or an amino protecting group;
Z2 is hydrogen, C1-C6 alkyl, an amino protecting group, —C(═O)—(CH2)n-J-Z3, a D or L α-amino acid linked via the α-carboxyl group or optionally through the ω-carboxyl group when the amino acid is aspartic acid or glutamic acid or a peptide derived from D, L or mixed D and L amino acids linked through a carboxyl group;
Z3 is hydrogen, an amino protecting group, —C1-C6 alkyl, —C(═O)—CH3, benzyl, benzoyl, or —(CH2)n—N(H)Z1;
each J is O, S or NH;
R5 is a carbonyl protecting group; and
n is from 2 to about 450.
Another class of oligonucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. A suitable class of linking groups have been selected to give a non-ionic oligomeric compound. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based oligomeric compounds are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. The morpholino class of oligomeric compounds have been prepared having a variety of different linking groups joining the monomeric subunits.
Morpholino nucleic acids have been prepared having a variety of different linking groups (L2) joining the monomeric subunits. The basic formula is shown below:




wherein

T1 is hydroxyl or a protected hydroxyl;
T5 is hydrogen or a phosphate or phosphate derivative;
L2 is a linking group; and
n is from 2 to about 450.
Another class of oligonucleotide mimetic is referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in an DNA/RNA molecule is replaced with a cyclohenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation. Furthermore the incorporation of CeNA into a sequence targeting RNA was stable to serum and able to activate E. coli RNase resulting in cleavage of the target RNA strand.
The general formula of CeNA is shown below:




wherein

each Bx is a heterocyclic base moiety;
T1 is hydroxyl or a protected hydroxyl;
T2 is hydroxyl or a protected hydroxyl;
L3 is a linking group; and
n is from 2 to about 450.
Another class of oligonucleotide mimetic (anhydrohexitol nucleic acid) can be prepared from one or more anhydrohexitol nucleosides (see, Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) and would have the general formula:




Another group of modifications includes nucleosides having sugar moieties that are bicyclic thereby locking the sugar conformational geometry. The most studied of these nucleosides is a bicyclic sugar moiety having a 4′-CH2—O-2′ bridge. As can be seen in the structure below the 2′-O— has been linked via a methylene group to the 4′ carbon. This bridge attaches under the sugar as shown forcing the sugar ring into a locked 3′-endo conformation geometry. The ∀-L nucleoside has also been reported wherein the linkage is above the ring and the heterocyclic base is in the V rather than the ∃-conformation (see U.S. Patent Application Publication No.: Application 2003/0087230). The xylo analog has also been prepared (see U.S. Patent Application Publication No.: 2003/0082807). The preferred bridge for a locked nucleic acid (LNA) is 4′-(—CH2—)n—O-2′ wherein n is 1 or 2. The literature is confusing when the term locked nucleic acid is used but in general locked nucleic acids refers to n=1, ENATM refers to n=2 (Kaneko et al., U.S. Patent Application Publication No.: US 2002/0147332, Singh et al., Chem. Commun, 1998, 4, 455-456, also see U.S. Pat. Nos. 6,268,490 and 6,670,461 and U.S. Patent Application Publication No.: US 2003/0207841). However the term locked nucleic acids can also be used in a more general sense to describe any bicyclic sugar moiety that has a locked conformation.
ENATM along with LNA (n=1) have been studied more than the myriad of other analogs. Oligomeric compounds incorporating LNA and ENA analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10 C), stability towards 3′-exonucleolytic degradation and good solubility properties.
The basic structure of LNA showing the bicyclic ring system is shown below:




wherein

each Bx is a heterocyclic base moiety;
each L1 is an internucleoside linking group;
T1 is hydroxyl or a protected hydroxyl;
T2 is hydroxyl or a protected hydroxyl, and
n is from 1 to about 80.
The conformations of LNAs determined by 2D NMR spectroscopy have shown that the locked orientation of the LNA nucleotides, both in single-stranded LNA and in duplexes, constrains the phosphate backbone in such a way as to introduce a higher population of the N-type conformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53). These conformations are associated with improved stacking of the nucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18, 1365-1370).
LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNA hybridization was shown to be the most thermally stable nucleic acid type duplex system, and the RNA-mimicking character of LNA was established at the duplex level. Introduction of 3 LNA monomers (T or A) significantly increased melting points (Tm=+15/+11) toward DNA complements. The universality of LNA-mediated hybridization has been stressed by the formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking of LNA was reflected with regard to the N-type conformational restriction of the monomers and to the secondary structure of the LNA:RNA duplex.
LNAs also form duplexes with complementary DNA, RNA or LNA with high thermal affinities. Circular dichroism (CD) spectra show that duplexes involving fully modified LNA (esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination of an LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer. Recognition of double-stranded DNA has also been demonstrated suggesting strand invasion by LNA. Studies of mismatched sequences show that LNAs obey the Watson-Crick base pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands.
Novel types of LNA-oligomeric compounds, as well as the LNAs, are useful in a wide range of diagnostic and therapeutic applications. Among these are antisense applications, PCR applications, strand-displacement oligomers, substrates for nucleic acid polymerases and generally as nucleotide based drugs.
Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638.) The authors have demonstrated that LNAs confer several desired properties to antisense agents. LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. LNA/DNA copolymers exhibited potent antisense activity in assay systems as disparate as G-protein-coupled receptor signaling in living rat brain and detection of reporter genes in Escherichia coli. LIPOFECTINTM-mediated efficient delivery of LNA into living human breast cancer cells has also been accomplished.
The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., PCT International Application WO 98-DK393 19980914). Furthermore, synthesis of 2′-amino-LNA, a novel conformationally restricted high-affinity oligonucleotide analog with a handle has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-Amino- and 2′-methylamino-LNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.
Some oligonucleotide mimetics have been prepared to include bicyclic and tricyclic nucleoside analogs having the formulas (amidite monomers shown):




(see Steffens et al., Helv. Chim Acta, 1997, 80, 2426-2439; Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberg et al., J. Am. Chem. Soc., 2002, 124, 5993-6002). These modified nucleoside analogs have been oligomerized using the phosphoramidite approach and the resulting oligomeric compounds containing tricyclic nucleoside analogs have shown increased thermal stabilities (Tm's) when hybridized to DNA, RNA and itself. Oligomeric compounds containing bicyclic nucleoside analogs have shown thermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to as phosphonomonoester nucleic acid and incorporates a phosphorus group in the backbone. This class of olignucleotide mimetic is reported to have useful physical and biological and pharmacological properties in the areas of inhibiting gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex-forming oligonucleotides), as probes for the detection of nucleic acids and as auxiliaries for use in molecular biology.
The general formula (for definitions of Markush variables see: U.S. Pat. Nos. 5,874,553 and 6,127,346 herein incorporated by reference in their entirety) is shown below.




Another oligonucleotide mimetic has been reported wherein the furanosyl ring has been replaced by a cyclobutyl moiety.
Modified Sugars
Oligomeric compounds of the invention may also contain one or more substituted sugar moieties. These oligomeric compounds comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Some oligonucleotides comprise a sugar substituent group selected from: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. One modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. One modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH3)2.
Other sugar substituent groups include methoxy (—O—CH3), aminopropoxy (—OCH2CH2CH2NH2), allyl (—CH2—CH═CH2), —O-allyl (—O—CH2—CH═CH2) and fluoro (F). 2′-Sugar substituent groups may be in the arabino (up) position or ribo (down) position. One 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
Representative sugar substituent groups include groups of formula Ia or IIa:




wherein:

Rb is O, S or NH;
Rd is a single bond, O, S or C(═O);
Re is C1-C10 alkyl, N(Rk)(Rm), N(Rk)(Rn), N═C(Rp)(Rq), N═C(Rp)(Rr) or has formula IIIa;




Rp and Rq are each independently hydrogen or C1-C10 alkyl;
Rr is —Rx—Ry;
each Rs, Rt, Ru and Rv is, independently, hydrogen, C(O)Rw, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;
or optionally, Ru and Rv, together form a phthalimido moiety with the nitrogen atom to which they are attached;
each Rw is, independently, substituted or unsubstituted C1-C10 alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;
Rk is hydrogen, a nitrogen protecting group or —Rx—Ry;
Rp is hydrogen, a nitrogen protecting group or —Rx—Ry;
Rx is a bond or a linking moiety;
Ry is a chemical functional group, a conjugate group or a solid support medium;
each Rm and Rn is, independently, H, a nitrogen protecting group, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, alkynyl; NH3+, N(Ru)(Rv), guanidino and acyl where said acyl is an acid amide or an ester;
or Rm and Rn, together, are a nitrogen protecting group, are joined in a ring structure that optionally includes an additional heteroatom selected from N and O or are a chemical functional group;
Ri is ORz, SRz, or N(Rz)2;
each Rz is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(═NH)N(H)Ru, C(═O)N(H)Ru or OC(═O)N(H)Ru;
Rf, Rg and Rh comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;
Rj is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(Rk)(Rm) ORk, halo, SRk or CN;
ma is 1 to about 10;
each mb is, independently, 0 or 1;
mc is 0 or an integer from 1 to 10;
md is an integer from 1 to 10;
me is from 0, 1 or 2; and
provided that when mc is 0, md is greater than 1.
Representative substituents groups are disclosed in U.S. patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled “Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in its entirety.
Representative cyclic substituent groups are disclosed in U.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998, entitled “RNA Targeted 2′-Oligomeric compounds that are Conformationally Preorganized,” hereby incorporated by reference in its entirety.
Particular sugar substituent groups include O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3))2, where n and m are from 1 to about 10.
Representative guanidino substituent groups are disclosed in U.S. patent application Ser. No. 09/349,040, entitled “Functionalized Oligomers,” filed Jul. 7, 1999, hereby incorporated by reference in its entirety.
Representative acetamido substituent groups are disclosed in U.S. Pat. No. 6,147,200 which is hereby incorporated by reference in its entirety.
Representative dimethylaminoethyloxyethyl substituent groups are disclosed in International Patent Application PCT/US99/17895, entitled “2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6, 1999, hereby incorporated by reference in its entirety.
Synthesis of Chimeric Oligonucleotides:
Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers.”
(2′-O-Me)-(2′-deoxy)-(2′-O-Me) Chimeric Phosphorothioate Oligonucleotides
Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH4OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.
(2′-O-(2-Methoxyethyl))-(2′-deoxy)-(2′-O-(Methoxyethyl)) Chimeric Phosphorothioate Oligonucleotides
(2′-O-(2-methoxyethyl))-(2′-deoxy)-(-2′-O-(methoxyethyl)) chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.
(2′-O-(2-Methoxyethyl)Phosphodiester)-(2′-deoxy Phosphorothioate)-(2′-O-(2-Methoxyethyl) Phosphodiester) Chimeric Oligonucleotides
(2′-O-(2-methoxyethyl phosphodiester)-(2′-deoxy phosphorothioate)-(2′-O-(methoxyethyl) phosphodiester) chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.
Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.
The terms used to describe the conformational geometry of homoduplex nucleic acids are “A Form” for RNA and “B Form” for DNA. The respective conformational geometry for RNA and DNA duplexes was determined from X-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins, Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNA duplexes are more stable and have higher melting temperatures (Tm's) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNA biases the sugar toward a C3′ endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry. In addition, the 2′ hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleic acids prefer a C2′ endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.). As used herein, B-form geometry is inclusive of both C2′-endo pucker and O4′-endo pucker. This is consistent with Berger, et. al., Nucleic Acids Research, 1998, 26, 2473-2480, who pointed out that in considering the furanose conformations which give rise to B-form duplexes consideration should also be given to a O4′-endo pucker contribution.
DNA:RNA hybrid duplexes, however, are usually less stable than pure RNA:RNA duplexes, and depending on their sequence may be either more or less stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533). The stability of the duplex formed between a target RNA and a synthetic sequence is central to therapies such as, but not limited to, antisense mechanisms, including RNase H-mediated and RNA interference mechanisms, as these mechanisms involved the hybridization of a synthetic sequence strand to an RNA target strand. In the case of RNase H, effective inhibition of the mRNA requires that the antisense sequence achieve at least a threshold of hybridization.
One routinely used method of modifying the sugar puckering is the substitution of the sugar at the 2′-position with a substituent group that influences the sugar geometry. The influence on ring conformation is dependent on the nature of the substituent at the 2′-position. A number of different substituents have been studied to determine their sugar puckering effect. For example, 2′-halogens have been studied showing that the 2′-fluoro derivative exhibits the largest population (65%) of the C3′-endo form, and the 2′-iodo exhibits the lowest population (7%). The populations of adenosine (2′-OH) versus deoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, the effect of the 2′-fluoro group of adenosine dimers (2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is also correlated to the stabilization of the stacked conformation.
As expected, the relative duplex stability can be enhanced by replacement of 2′-OH groups with 2′-F groups thereby increasing the C3′-endo population. It is assumed that the highly polar nature of the 2′-F bond and the extreme preference for C3′-endo puckering may stabilize the stacked conformation in an A-form duplex. Data from UV hypochromicity, circular dichroism, and 1H NMR also indicate that the degree of stacking decreases as the electronegativity of the halo substituent decreases. Furthermore, steric bulk at the 2′-position of the sugar moiety is better accommodated in an A-form duplex than a B-form duplex. Thus, a 2′-substituent on the 3′-terminus of a dinucleoside monophosphate is thought to exert a number of effects on the stacking conformation: steric repulsion, furanose puckering preference, electrostatic repulsion, hydrophobic attraction, and hydrogen bonding capabilities. These substituent effects are thought to be determined by the molecular size, electronegativity, and hydrophobicity of the substituent. Melting temperatures of complementary strands is also increased with the 2′-substituted adenosine diphosphates. It is not clear whether the 3′-endo preference of the conformation or the presence of the substituent is responsible for the increased binding. However, greater overlap of adjacent bases (stacking) can be achieved with the 3′-endo conformation.
Nucleoside conformation is influenced by various factors including substitution at the 2′,3′ or 4′-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2′ position to favor the 3′-endo conformation can be achieved while maintaining the 2′-OH as a recognition element, as illustrated in FIG. 2, below (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference for the 3′-endo conformation can be achieved by deletion of the 2′-OH as exemplified by 2′deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3′-endo conformation positioning the electronegative fluorine atom in the axial position. Other modifications of the ribose ring, for example substitution at the 4′-position to give 4′-F modified nucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example modification to yield methanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) also induce preference for the 3′-endo conformation.
In one aspect of the present invention oligomeric compounds include nucleosides synthetically modified to induce a 3′-endo sugar conformation. A nucleoside can incorporate synthetic modifications of the heterocyclic base, the sugar moiety or both to induce a desired 3′-endo sugar conformation. These modified nucleosides are used to mimic RNA-like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3′-endo conformational geometry (see Scheme 1). There is an apparent preference for an RNA type duplex (A form helix, predominantly 3′-endo) as a requirement (e.g. trigger) of RNA interference which is supported in part by the fact that duplexes composed of 2′-deoxy-2′-F-nucleosides appears efficient in triggering RNAi response in the C. elegans system. Properties that are enhanced by using more stable 3′-endo nucleosides include but aren't limited to modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage. The present invention provides oligomeric compounds designed to act as triggers of RNAi having one or more nucleosides modified in such a way as to favor a C3′-endo type conformation.




Along similar lines, oligomeric triggers of RNAi response might be composed of one or more nucleosides modified in such a way that conformation is locked into a C3′-endo type conformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun (1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.) Examples of modified nucleosides amenable to the present invention are shown below. These examples are meant to be representative and not exhaustive.





Oligomeric compounds may also include nucleobase (often referred to in the art simply as “base” or “heterocyclic base moiety”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases also referred herein as heterocyclic base moieties include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Some nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
In one aspect of the present invention oligomeric compounds are prepared having polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G-clamps or cytidine analogs. Many of these polycyclic heterocyclic compounds have the general formula:




Representative cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand include 1,3-diazaphenoxazine-2-one (R10=O, R11−R14=H) (Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16, 1837-1846), 1,3-diazaphenothiazine-2-one (R10=S, R11−R14=H), (Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R10=O, R11−R14=F) (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388). When incorporated into oligonucleotides, these base modifications were shown to hybridize with complementary guanine and the latter was also shown to hybridize with adenine and to enhance helical thermal stability by extended stacking interactions (also see U.S. Patent Application Publication 20030207804 and U.S. Patent Application Publication 20030175906, both of which are incorporated herein by reference in their entirety).
Helix-stabilizing properties have been observed when a cytosine analog/substitute has an aminoethoxy moiety attached to the rigid 1,3-diazaphenoxazine-2-one scaffold (R10=O, R11=—O— (CH2)2—NH2, R12-14=H) (Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). Binding studies demonstrated that a single incorporation could enhance the binding affinity of a model oligonucleotide to its complementary target DNA or RNA with a ΔTm of up to 18° relative to 5-methyl cytosine (dC5me), which is the highest known affinity enhancement for a single modification. On the other hand, the gain in helical stability does not compromise the specificity of the oligonucleotides. The Tm data indicate an even greater discrimination between the perfect match and mismatched sequences compared to dC5me. It was suggested that the tethered amino group serves as an additional hydrogen bond donor to interact with the Hoogsteen face, namely the O6, of a complementary guanine thereby forming 4 hydrogen bonds. This means that the increased affinity of G-clamp is mediated by the combination of extended base stacking and additional specific hydrogen bonding.
Tricyclic heterocyclic compounds and methods of using them that are amenable to the present invention are disclosed in U.S. Pat. No. 6,028,183, and U.S. Pat. No. 6,007,992, the contents of both are incorporated herein in their entirety.
The enhanced binding affinity of the phenoxazine derivatives together with their sequence specificity makes them valuable nucleobase analogs for the development of more potent antisense-based drugs. In fact, promising data have been derived from in vitro experiments demonstrating that heptanucleotides containing phenoxazine substitutions can activate RNaseH, enhance cellular uptake and exhibit an increased antisense activity (Lin, K-Y; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). The activity enhancement was even more pronounced in case of G-clamp, as a single substitution was shown to significantly improve the in vitro potency of a 20mer 2′-deoxyphosphorothioate oligonucleotides (Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518).
Modified polycyclic heterocyclic compounds useful as heterocyclic bases are disclosed in but not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. Patent Application Publication 20030158403, each of which is incorporated herein by reference in its entirety.
One substitution that can be appended to the oligomeric compounds of the invention involves the linkage of one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting oligomeric compounds. In one embodiment such modified oligomeric compounds are prepared by covalently attaching conjugate groups to functional groups such as hydroxyl or amino groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, carbohydrates, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
The oligomeric compounds of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.
Representative U.S. patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.
Oligomeric compounds used in the compositions of the present invention can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of oligomeric compounds to enhance properties such as for example nuclease stability. Included in stabilizing groups are cap structures. By “cap structure or terminal cap moiety” is meant chemical modifications, which have been incorporated at either terminus of oligonucleotides (see for example Wincott et al., WO 97/26270, incorporated by reference herein). These terminal modifications protect the oligomeric compounds having terminal nucleic acid molecules from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present on both termini. For double-stranded oligomeric compounds, the cap may be present at either or both termini of either strand. In non-limiting examples, the 5′-cap includes inverted abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl riucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (see Wincott et al., International PCT publication No. WO 97/26270, incorporated by reference herein).
Particularly preferred 3′-cap structures of the present invention include, for example 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).
Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an oligomeric compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.
It is not necessary for all positions in an oligomeric compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligomeric compound or even at a single monomeric subunit such as a nucleoside within a oligomeric compound. The present invention also includes oligomeric compounds which are chimeric oligomeric compounds. “Chimeric” oligomeric compounds or “chimeras,” in the context of this invention, are oligomeric compounds that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a nucleic acid based oligomer.
Chimeric oligomeric compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligomeric compound may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, an oligomeric compound may be designed to comprise a region that serves as a substrate for
RNase H. RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H by an oligomeric compound having a cleavage region, therefore, results in cleavage of the RNA target, thereby enhancing the efficiency of the oligomeric compound. Consequently, comparable results can often be obtained with shorter oligomeric compounds having substrate regions when chimeras are used, compared to for example phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
Chimeric oligomeric compounds of the invention may be formed as composite structures of two or more oligonucleotides, oligonucleotide mimics, oligonucleotide analogs, oligonucleosides and/or oligonucleotide mimetics as described above. Such oligomeric compounds have also been referred to in the art as hybrids, hemimers, gapmers or inverted gapmers. Representative U.S. patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.
The conformation of modified nucleosides and their oligomers can be estimated by various methods such as molecular dynamics calculations, nuclear magnetic resonance spectroscopy and CD measurements. Hence, modifications predicted to induce RNA-like conformations (A-form duplex geometry in an oligomeric context), are useful in the oligomeric compounds of the present invention. The synthesis of modified nucleosides amenable to the present invention are known in the art (see for example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988, Plenum Press.)
In one aspect, the present invention is directed to oligomeric compounds that are designed to have enhanced properties compared to native RNA. One method to design optimized or enhanced oligomeric compounds involves each nucleoside of the selected sequence being scrutinized for possible enhancing modifications. One modification would be the replacement of one or more RNA nucleosides with nucleosides that have the same 3′-endo conformational geometry. Such modifications can enhance chemical and nuclease stability relative to native RNA while at the same time being much cheaper and easier to synthesize and/or incorporate into an oligonucleotide. The sequence can be further divided into regions and the nucleosides of each region evaluated for enhancing modifications that can be the result of a chimeric configuration. Consideration is also given to the 5′ and 3′-termini as there are often advantageous modifications that can be made to one or more of the terminal nucleosides. The oligomeric compounds of the present invention may include at least one 5′-modified phosphate group on a single strand or on at least one 5′-position of a double-stranded sequence or sequences. Other modifications considered are internucleoside linkages, conjugate groups, substitute sugars or bases, substitution of one or more nucleosides with nucleoside mimetics and any other modification that can enhance the desired property of the oligomeric compound.
One synthetic 2′-modification that imparts increased nuclease resistance and a very high binding affinity to nucleotides is the 2-methoxyethoxy (2′-MOE, 2′-OCH2CH2OCH3) side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). One of the immediate advantages of the 2′-MOE substitution is the improvement in binding affinity, which is greater than many similar 2′ modifications such as O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-O-methoxyethyl substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926). Relative to DNA, the oligonucleotides having the 2′-MOE modification displayed improved RNA affinity and higher nuclease resistance. Chimeric oligonucleotides having 2′-MOE substituents in the wing nucleosides and an internal region of deoxy-phosphorothioate nucleotides (also termed a gapped oligonucleotide or gapmer) have shown effective reduction in the growth of tumors in animal models at low doses. 2′-MOE substituted oligonucleotides have also shown outstanding promise as antisense agents in several disease states. One such MOE substituted oligonucleotide is presently being investigated in clinical trials for the treatment of CMV retinitis.
Unless otherwise defined herein, alkyl means C1-C12, C1-C8, or C1-C6, straight or (where possible) branched chain aliphatic hydrocarbyl.
Unless otherwise defined herein, heteroalkyl means C1-C12, C1-C8, or C1-C6, straight or (where possible) branched chain aliphatic hydrocarbyl containing at least one, or about 1 to about 3 hetero atoms in the chain, including the terminal portion of the chain. Suitable heteroatoms include N, O and S.
Unless otherwise defined herein, cycloalkyl means C3-C12, C3-C8, or C3-C6, aliphatic hydrocarbyl ring.
Unless otherwise defined herein, alkenyl means C2-C12, C2-C8, or C2-C6 alkenyl, which may be straight or (where possible) branched hydrocarbyl moiety, which contains at least one carbon-carbon double bond.
Unless otherwise defined herein, alkynyl means C2-C12, C2-C8, or C2-C6 alkynyl, which may be straight or (where possible) branched hydrocarbyl moiety, which contains at least one carbon-carbon triple bond.
Unless otherwise defined herein, heterocycloalkyl means a ring moiety containing at least three ring members, at least one of which is carbon, and of which 1, 2 or three ring members are other than carbon. The number of carbon atoms can vary from 1 to about 12, from 1 to about 6, and the total number of ring members varies from three to about 15, or from about 3 to about 8. Suitable ring heteroatoms are N, O and S. Suitable heterocycloalkyl groups include, but are not limited to, morpholino, thiomorpholino, piperidinyl, piperazinyl, homopiperidinyl, homopiperazinyl, homomorpholino, homothiomorpholino, pyrrolodinyl, tetrahydrooxazolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydroisoxazolyl, tetrahydropyrrazolyl, furanyl, pyranyl, and tetrahydroisothiazolyl.
Unless otherwise defined herein, aryl means any hydrocarbon ring structure containing at least one aryl ring. Suitable aryl rings have about 6 to about 20 ring carbons. Especially suitable aryl rings include phenyl, napthyl, anthracenyl, and phenanthrenyl.
Unless otherwise defined herein, hetaryl means a ring moiety containing at least one fully unsaturated ring, the ring consisting of carbon and non-carbon atoms. The ring system can contain about 1 to about 4 rings. The number of carbon atoms can vary from 1 to about 12, from 1 to about 6, and the total number of ring members varies from three to about 15, or from about 3 to about 8. Suitable ring heteroatoms are N, O and S. Suitable hetaryl moieties include, but are not limited to, pyrazolyl, thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl, pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl, benzothiophenyl, etc.
Unless otherwise defined herein, where a moiety is defined as a compound moiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl and alkyl), etc., each of the sub-moieties is as defined herein.
Unless otherwise defined herein, an electron withdrawing group is a group, such as the cyano or isocyanato group that draws electronic charge away from the carbon to which it is attached. Other electron withdrawing groups of note include those whose electronegativities exceed that of carbon, for example halogen, nitro, or phenyl substituted in the ortho- or para-position with one or more cyano, isothiocyanato, nitro or halo groups.
Unless otherwise defined herein, the terms halogen and halo have their ordinary meanings. Suitable halo (halogen) substituents are Cl, Br, and I.
The aforementioned optional substituents are, unless otherwise herein defined, suitable substituents depending upon desired properties. Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties, NO2, NH3 (substituted and unsubstituted), acid moieties (e.g. —CO2H, —OSO3H2, etc.), heterocycloalkyl moieties, hetaryl moieties, aryl moieties, etc. In all the preceding formulae, the squiggle ( ̃) indicates a bond to an oxygen or sulfur of the 5′-phosphate.
Phosphate protecting groups include those described in U.S. Pat. No. 5,760,209, U.S. Pat. No. 5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat. No. 6,020,475, U.S. Pat. No. 6,326,478, U.S. Pat. No. 6,169,177, U.S. Pat. No. 6,121,437, U.S. Pat. No. 6,465,628 each of which is expressly incorporated herein by reference in its entirety.
Screening methods for the identification of effective modulators of small non-coding RNAs are also comprehended by the instant invention and comprise the steps of contacting a small non-coding RNA, or portion thereof, with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the levels, expression or alter the function of the small non-coding RNA. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the levels, expression or altering the function of the small non-coding RNA, the modulator may then be employed in further investigative studies, or for use as a target validation, research, diagnostic, or therapeutic agent in accordance with the present invention.
Screening methods for the identification of small non-coding RNA mimics are also within the scope of the invention. Screening for small non-coding RNA modulators or mimics can also be performed in vitro, ex vivo, or in vivo by contacting samples, tissues, cells or organisms with candidate modulators or mimics and selecting for one or more candidate modulators which show modulatory effects.
Design and Screening of Duplexed Oligomeric Compounds:
In screening and target validation studies, oligomeric compounds of the invention can be used in combination with their respective complementary strand oligomeric compound to form stabilized double-stranded (duplexed) oligonucleotides. In accordance with the present invention, a series of duplexes comprising the oligomeric compounds of the present invention and their complements can be designed to target a small non-coding RNA. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in some embodiments, both strands of the duplex would be complementary over the central nucleobases, each having overhangs at one or both termini, as described supra.
In some embodiments, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:2181) may be prepared with blunt ends (no single stranded overhang) as shown:







cgagaggcggacgggaccg  Antisense Strand


||||||||||||||||||| (SEQ ID NO: 2181)


gctctccgcctgccctggc Complement (SEQ ID NO: 2184)






In other embodiments, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG, having a two-nucleobase overhang of deoxythymidine (dT) and its complement sense strand may be prepared with overhangs as shown:







  cgagaggcggacgggaccgTT  Antisense Strand


  |||||||||||||||||||   (SEQ ID NO: 2182)


TTgctctccgcctgccctggc    Complement Sense Strand


                         (SEQ ID NO: 2183)






RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.).
For use in drug discovery, oligomeric compounds of the present invention are used to elucidate relationships that exist between small non-coding RNAs, genes or proteins and a disease state, phenotype, or condition. These methods include detecting or modulating a target comprising contacting a sample, tissue, cell, or organism with the oligomeric compounds and compositions of the present invention, measuring the levels of the target and/or the levels of downstream gene products including mRNA or proteins encoded thereby, a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to an untreated sample, a positive control or a negative control. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a disease.
The oligomeric compounds and compositions of the present invention can additionally be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Such uses allows for those of ordinary skill to elucidate the function of particular non-coding or coding nucleic acids or to distinguish between functions of various members of a biological pathway.
For use in kits and diagnostics, the oligomeric compounds and compositions of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of non-coding or coding nucleic acids expressed within cells and tissues.
As one non-limiting example, expression patterns within cells or tissues treated with one or more oligomeric compounds or compositions of the invention are compared to control cells or tissues not treated with the compounds or compositions and the patterns produced are analyzed for differential levels of nucleic acid expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds that affect expression patterns.
Cell Culture and Oligonucleotide Treatment:
The effects of oligomeric compounds on target nucleic acid expression or function can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or real-time RT-PCR. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is present in the cell type chosen.
T-24 Cells:
The human transitional cell bladder carcinoma cell line T-24 is obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. For Northern blotting or other analyses, cells harvested when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in real-time RT-PCR analysis.
A549 Cells:
The human lung carcinoma cell line A549 is obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.
HMECs:
Normal human mammary epithelial cells (HMECs) are obtained from American Type Culture Collection (Manassas, Va.). HMECs are routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. HMECs are plated in 24-well plates (Falcon-Primaria #353047, BD Biosciences, Bedford, Mass.) at a density of 50,000-60,000 cells per well, and allowed to attach overnight prior to treatment with oligomeric compounds. HMECs are plated in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 10,000 cells per well and allowed to attach overnight prior to treatment with oligomeric compounds.
MCF7 Cells:
The breast carcinoma cell line MCF7 is obtained from American Type Culture Collection (Manassas, Va.). MCF7 cells are routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. MCF7 cells are plated in 24-well plates (Falcon-Primaria #353047, BD Biosciences, Bedford, Mass.) at a density of approximately 140,000 cells per well, and allowed to attach overnight prior to treatment with oligomeric compounds. MCF7 cells are plated in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 20,000 cells per well and allowed to attach overnight prior to treatment with oligomeric compounds.
T47D Cells:
The breast carcinoma cell line T47D is obtained from American Type Culture Collection (Manassas, Va.). T47D cells are deficient in expression of the tumor suppressor gene p53. T47D cells are cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. T47D cells are plated in 24-well plates (Falcon-Primaria #353047, BD Biosciences, Bedford, Mass.) at a density of approximately 170,000 cells per well, and allowed to attach overnight prior to treatment with oligomeric compounds. T47D cells are plated in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 20,000 cells per well and allowed to attach overnight prior to treatment with oligomeric compounds.
BJ Cells:
The normal human foreskin fibroblast BJ cell line was obtained from American Type Culture Collection (Manassas, Va.). BJ cells were routinely cultured in MEM high glucose with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate (all media and supplements from Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 80% confluence. Cells were plated on collagen-coated 24-well plates (Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately 50,000 cells per well, and allowed to attach to wells overnight.
B16-F10 Cells:
The mouse melanoma cell line B16-F10 was obtained from American Type Culture Collection (Manassas, Va.). B16-F10 cells were routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 80% confluence. Cells were seeded into collagen-coated 24-well plates (Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately 50,000 cells per well and allowed to attach overnight.
HUVECs:
Human vascular endothelial cells (HUVECs) are obtained from American Type Culture Collection (Manassas, Va.). HUVECs are routinely cultured in EBM (Clonetics Corporation, Walkersville, Md.) supplemented with SingleQuots supplements (Clonetics Corporation, Walkersville, Md.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence and are maintained for up to 15 passages. HUVECs are plated at approximately 3000 cells/well in 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) and treated with oligomeric compounds one day later.
NHDF Cells:
Human neonatal dermal fibroblast (NHDF) cells are obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.
HEK Cells:
Human embryonic keratinocytes (HEK) are obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.
293T Cells:
The human 293T cell line is obtained from American Type Culture Collection (Manassas, Va.). 293T cells are a highly transfectable cell line constitutively expressing the simian virus 40 (SV40) large T antigen. 293T cells were maintained in Dulbeccos' Modified Medium (DMEM) (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum and antibiotics (Life Technologies).
HepG2 Cells:
The human hepatoblastoma cell line HepG2 is obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). HepG2 cells are routinely cultured in Eagle's MEM supplemented with 10% fetal bovine serum, 1 mM non-essential amino acids, and 1 mM sodium pyruvate (medium and all supplements from Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. For treatment with oligomeric compounds, cells are seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 7000 cells/well prior to treatment with oligomeric compounds. For the caspase assay, cells are seeded into collagen coated 96-well plates (BIOCOAT cellware, Collagen type I, B-D #354407/356407, Becton Dickinson, Bedford, Mass.) at a density of 7500 cells/well.
Preadipocytes:
Human preadipocytes are obtained from Zen-Bio, Inc. (Research Triangle Park, NC). Preadipocytes were routinely maintained in Preadipocyte Medium (ZenBio, Inc., Research Triangle Park, NC) supplemented with antibiotics as recommended by the supplier. Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were routinely maintained for up to 5 passages as recommended by the supplier. To induce differentiation of preadipocytes, cells are then incubated with differentiation media consisting of Preadipocyte Medium further supplemented with 2% more fetal bovine serum (final total of 12%), amino acids, 100 nM insulin, 0.5 mM IBMX, 1 μM dexamethasone and 1 μM BRL49653. Cells are left in differentiation media for 3-5 days and then re-fed with adipocyte media consisting of Preadipocyte Medium supplemented with 33 μM biotin, 17 μM pantothenate, 100 nM insulin and 1 μM dexamethasone. Cells differentiate within one week. At this point cells are ready for treatment with the oligomeric compounds of the invention. One day prior to transfection, 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) are seeded with approximately 3000 cells/well prior to treatment with oligomeric compounds.
Differentiated Adipocytes:
Human adipocytes are obtained from Zen-Bio, Inc. (Research Triangle Park, NC). Adipocytes were routinely maintained in Adipocyte Medium (ZenBio, Inc., Research Triangle Park, NC) supplemented with antibiotics as recommended by the supplier. Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were routinely maintained for up to 5 passages as recommended by the supplier.
NT2 Cells:
The NT2 cell line is obtained from the American Type Culture Collection (ATCC; Manassa, Va.). The NT2 cell line, which has the ATCC designation NTERA-2 cl.D1, is a pluripotent human testicular embryonal carcinoma cell line derived by cloning the NTERA-2 cell line. The parental NTERA-2 line was established in 1980 from a nude mouse xenograft of the Tera-2 cell line (ATCC HTB-106). NT2 cells were routinely cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. For Northern blotting or other analyses, cells harvested when they reached 90% confluence.
HeLa Cells:
The human epitheloid carcinoma cell line HeLa is obtained from the American Tissue Type Culture Collection (Manassas, Va.). HeLa cells were routinely cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. For Northern blotting or other analyses, cells were harvested when they reached 90% confluence.
For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.
Treatment with Antisense Oligomeric Compounds:
In general, when cells reach approximately 80% confluency, they are treated with oligomeric compounds of the invention. Oligomeric compounds are introduced into cells using the cationic lipid transfection reagent LIPOFECTINTM (Invitrogen Life Technologies, Carlsbad, Calif.). Oligomeric compounds are mixed with LIPOFECTINTM in OPTI-MEMTM (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired final concentration of oligomeric compound and LIPOFECTINTM. Before adding to cells, the oligomeric compound, LIPOFECTINTM and OPTI-MEMTM are mixed thoroughly and incubated for approximately 0.5 hrs. The medium is removed from the plates and the plates are tapped on sterile gauze. Each well of a 96-well plate is washed with 150 μl of phosphate-buffered saline or Hank's balanced salt solution. Each well of a 24-well plate is washed with 250 μL of phosphate-buffered saline or Hank's balanced salt solution. The wash buffer in each well is replaced with 100 μL or 250 μL of the oligomeric compound/OPTI-MEMTM/LIPOFECTINTM cocktail for 96-well or 24-well plates, respectively. Untreated control cells receive LIPOFECTINTM only. The plates are incubated for approximately 4 to 7 hours at 37° C., after which the medium is removed and the plates are tapped on sterile gauze. 100 μl or 1 mL of full growth medium is added to each well of a 96-well plate or a 24-well plate, respectively. Cells are harvested 16-24 hours after oligonucleotide treatment, at which time RNA can be isolated and target reduction measured by real-time RT-PCR, or other phenotypic assays performed. In general, data from treated cells are obtained in triplicate, and results presented as an average of the three trials.
In some embodiments, cells are transiently transfected with oligomeric compounds of the instant invention. In some embodiments, cells are transfected and selected for stable expression of an oligomeric compound of the instant invention.
The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide may be selected from ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2) or another suitable positive control. Controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone or having chemical modifications similar to the oligonucleotides being tested. For mouse or rat cells the positive control oligonucleotide may be ISIS 15770 (ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3), a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) or other suitable control target RNA may then be utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of target expression or function is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. The concentrations of oligonucleotides used herein can range from 10 nM to 300 nM.
Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904), mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41) and real-time quantitative RT-PCR (Heid, et al., Genome Res., 1996, 6(10), 986-94).
Analysis of Oligonucleotide Inhibition of a Target Levels or Expression:
Modulation of target levels or expression can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time quantitative RT-PCR (also known as RT-PCR). Real-time quantitative RT-PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative RT-PCR can be conveniently accomplished using the commercially available ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
RNA Isolation:
Poly(A)+mRNA Isolation
Poly(A)+mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold phosphate-buffered saline (PBS). 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.
Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.
Total RNA Isolation
Total RNA was isolated using an RNEASY 96TM kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96TM well plate attached to a QIAVACTM manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96TM plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96TM plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96TM plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVACTM manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVACTM manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.
The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.
Real-Time Quantitative PCR Analysis of a Target RNA Levels:
Quantitation of a target RNA levels was accomplished by real-time quantitative PCR using the ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISMTM Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of RNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after oligonucleotide treatment of test samples.
Prior to quantitative PCR analysis, primer/probe sets specific to the target gene (or RNA) being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene (or RNA) and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, RNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer/probe sets specific for GAPDH only, target gene (or RNA) only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target RNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer/probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.
PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl2, 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
Gene (or RNA) target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreenTM (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreenTM are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).
In this assay, 170 μL of RiboGreenTM working reagent (RiboGreenTM reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.
Probes and primers are designed to hybridize to the target sequence.
Northern Blot Analysis of Target RNA Levels:
Eighteen hours after treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOLTM (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBONDTM-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKERTM UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYBTM hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.
To detect a target, a target specific primer/probe set is prepared for analysis by PCR. To normalize for variations in loading and transfer efficiency, membranes can be stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).
Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGERTM and IMAGEQUANTTM Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data can be normalized to GAPDH levels in untreated controls.
The compounds and compositions of the invention are useful for research and diagnostics, because these compounds and compositions hybridize to nucleic acids or interfere with the normal function of these nucleic acids. Hybridization of the compounds and compositions of the invention with a nucleic acid can be detected by means known in the art. Such means may include conjugation of an enzyme to the compound or composition, radiolabeling or any other suitable detection means. Kits using such detection means for detecting the level of selected proteins in a sample may also be prepared.
The specificity and sensitivity of compounds and compositions can also be harnessed by those of skill in the art for therapeutic uses. Antisense oligomeric compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligomeric compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
For therapeutics, an animal, preferably a human, suspected of having a disease or disorder presenting conditions that can be treated, ameliorated, or improved by modulating the expression of a selected small non-coding target nucleic acid is treated by administering the compounds and compositions. For example, in one non-limiting embodiment, the methods comprise the step of administering to or contacting the animal, an effective amount of a modulator or mimic to treat, ameliorate or improve the conditions associated with the disease or disorder. The compounds of the present invention effectively modulate the activity or function of the small non-coding RNA target or inhibit the expression or levels of the small non-coding RNA target. In one embodiment, the activity or expression of the target in an animal is inhibited by about 10%. In another embodiment the activity or expression of a target in an animal is inhibited by about 30%. Further, the activity or expression of a target in an animal is inhibited by 50% or more, by 60% or more, by 70% or more, by 80% or more, by 90% or more, or by 95% or more. In another embodiment, the present invention provides for the use of a compound of the invention in the manufacture of a medicament for the treatment of any and all conditions disclosed herein.
The reduction of target levels may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal known to contain the small non-coding RNA or its precursor. Further, the cells contained within the fluids, tissues or organs being analyzed contain a nucleic acid molecule of a downstream target regulated or modulated by the small non-coding RNA target itself.
The oligomeric compounds and compositions of the invention can be utilized in pharmaceutical compositions by adding an effective amount of the compound or composition to a suitable pharmaceutically acceptable diluent or carrier. Use of the oligomeric compounds and methods of the invention may also be useful prophylactically.
The oligomeric compounds and compositions of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative U.S. patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.
The oligomeric compounds and compositions of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the oligomeric compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligomeric compounds of the invention can be prepared as SATE ((S-acetyl-2-thioethyl) phosphate) derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al. Larger oligomeric compounds that are processed to supply, as cleavage products, compounds capable of modulating the function or expression of small non-coding RNAs or their downstream targets are also considered prodrugs.
The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds and compositions of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Suitable examples include, but are not limited to, sodium and postassium salts. For oligonucleotides, examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
The present invention also includes pharmaceutical compositions and formulations that include the oligomeric compounds and compositions of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
Oligomeric compounds may be formulated for delivery in vivo in an acceptable dosage form, e.g. as parenteral or non-parenteral formulations. Parenteral formulations include intravenous (IV), subcutaneous (SC), intraperitoneal (IP), intravitreal and intramuscular (IM) formulations, as well as formulations for delivery via pulmonary inhalation, intranasal administration, topical administration, etc. Non-parenteral formulations include formulations for delivery via the alimentary canal, e.g. oral administration, rectal administration, intrajejunal instillation, etc. Rectal administration includes administration as an enema or a suppository. Oral administration includes administration as a capsule, a gel capsule, a pill, an elixir, etc.
In some embodiments, an oligomeric compound can be administered to a subject via an oral route of administration. The subject may be an animal or a human (man). An animal subject may be a mammal, such as a mouse, a rat, a dog, a guinea pig, a monkey, a non-human primate, a cat or a pig. Non-human primates include monkeys and chimpanzees. A suitable animal subject may be an experimental animal, such as a mouse, rat, mouse, a rat, a dog, a monkey, a non-human primate, a cat or a pig.
In some embodiments, the subject may be a human. In certain embodiments, the subject may be a human patient. In certain embodiments, the subject may be in need of modulation of expression of one or more genes as discussed in more detail herein. In some particular embodiments, the subject may be in need of inhibition of expression of one or more genes as discussed in more detail herein. In particular embodiments, the subject may be in need of modulation, i.e. inhibition or enhancement, of a nucleic acid target in order to obtain therapeutic indications discussed in more detail herein.
In some embodiments, non-parenteral (e.g. oral) oligomeric compound formulations according to the present invention result in enhanced bioavailability of the compound. In this context, the term “bioavailability” refers to a measurement of that portion of an administered drug which reaches the circulatory system (e.g. blood, especially blood plasma) when a particular mode of administration is used to deliver the drug. Enhanced bioavailability refers to a particular mode of administration's ability to deliver oligonucleotide to the peripheral blood plasma of a subject relative to another mode of administration. For example, when a non-parenteral mode of administration (e.g. an oral mode) is used to introduce the drug into a subject, the bioavailability for that mode of administration may be compared to a different mode of administration, e.g. an IV mode of administration. In some embodiments, the area under a compound's blood plasma concentration curve (AUC0) after non-parenteral (e.g. oral, rectal, intrajejunal) administration may be divided by the area under the drug's plasma concentration curve after intravenous (i.v) administration (AUCiv) to provide a dimensionless quotient (relative bioavailability, RB) that represents the fraction of compound absorbed via the non-parenteral route as compared to the IV route. A composition's bioavailability is said to be enhanced in comparison to another composition's bioavailability when the first composition's relative bioavailability (RB1) is greater than the second composition's relative bioavailability (RB2).
In general, bioavailability correlates with therapeutic efficacy when a compound's therapeutic efficacy is related to the blood concentration achieved, even if the drug's ultimate site of action is intracellular (van Berge-Henegouwen et al., Gastroenterol., 1977, 73, 300). Bioavailability studies have been used to determine the degree of intestinal absorption of a drug by measuring the change in peripheral blood levels of the drug after an oral dose (DiSanto, Chapter 76 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 1451-1458).
In general, an oral composition's bioavailability is said to be “enhanced” when its relative bioavailability is greater than the bioavailability of a composition substantially consisting of pure oligonucleotide, i.e. oligonucleotide in the absence of a penetration enhancer.
Organ bioavailability refers to the concentration of compound in an organ. Organ bioavailability may be measured in test subjects by a number of means, such as by whole-body radiography. Organ bioavailability may be modified, e.g. enhanced, by one or more modifications to the oligomeric compound, by use of one or more carrier compounds or excipients. In general, an increase in bioavailability will result in an increase in organ bioavailability.
Oral oligomeric compound compositions according to the present invention may comprise one or more “mucosal penetration enhancers,” also known as “absorption enhancers” or simply as “penetration enhancers.” Accordingly, some embodiments of the invention comprise at least one oligomeric compound in combination with at least one penetration enhancer. In general, a penetration enhancer is a substance that facilitates the transport of a drug across mucous membrane(s) associated with the desired mode of administration, e.g. intestinal epithelial membranes. Accordingly it is desirable to select one or more penetration enhancers that facilitate the uptake of one or more oligomeric compounds, without interfering with the activity of the compounds, and in such a manner the compounds can be introduced into the body of an animal without unacceptable side-effects such as toxicity, irritation or allergic response.
Embodiments of the present invention provide compositions comprising one or more pharmaceutically acceptable penetration enhancers, and methods of using such compositions, which result in the improved bioavailability of oligomeric compounds administered via non-parenteral modes of administration. Heretofore, certain penetration enhancers have been used to improve the bioavailability of certain drugs. See Muranishi, Crit. Rev. Ther. Drug Carrier Systems, 1990, 7, 1 and Lee et al., Crit. Rev. Ther. Drug Carrier Systems, 1991, 8, 91. It has been found that the uptake and delivery of oligonucleotides can be greatly improved even when administered by non-parenteral means through the use of a number of different classes of penetration enhancers.
In some embodiments, compositions for non-parenteral administration include one or more modifications from naturally-occurring oligonucleotides (i.e. full-phosphodiester deoxyribosyl or full-phosphodiester ribosyl oligonucleotides). Such modifications may increase binding affinity, nuclease stability, cell or tissue permeability, tissue distribution, or other biological or pharmacokinetic property. Modifications may be made to the base, the linker, or the sugar, in general, as discussed in more detail herein with regards to oligonucleotide chemistry. In some embodiments of the invention, compositions for administration to a subject, and in particular oral compositions for administration to an animal or human subject, will comprise modified oligonucleotides having one or more modifications for enhancing affinity, stability, tissue distribution, or other biological property.
Suitable modified linkers include phosphorothioate linkers. In some embodiments according to the invention, the oligomeric compound has at least one phosphorothioate linker Phosphorothioate linkers provide nuclease stability as well as plasma protein binding characteristics to the compound. Nuclease stability is useful for increasing the in vivo lifetime of oligomeric compounds, while plasma protein binding decreases the rate of first pass clearance of oligomeric compound via renal excretion. In some embodiments according to the present invention, the oligomeric compound has at least two phosphorothioate linkers. In some embodiments, wherein the oligomeric compound has exactly n nucleosides, the oligomeric compound has from one to n−1 phosphorothioate linkages. In some embodiments, wherein the oligomeric compound has exactly n nucleosides, the oligomeric compound has n−1 phosphorothioate linkages. In other embodiments wherein the oligomeric compound has exactly n nucleoside, and n is even, the oligomeric compound has from 1 to n/2 phosphorothioate linkages, or, when n is odd, from 1 to (n−1)/2 phosphorothioate linkages. In some embodiments, the oligomeric compound has alternating phosphodiester (PO) and phosphorothioate (PS) linkages. In other embodiments, the oligomeric compound has at least one stretch of two or more consecutive PO linkages and at least one stretch of two or more PS linkages. In other embodiments, the oligomeric compound has at least two stretches of PO linkages interrupted by at least one PS linkage.
In some embodiments, at least one of the nucleosides is modified on the ribosyl sugar unit by a modification that imparts nuclease stability, binding affinity or some other beneficial biological property to the sugar. In some cases, the sugar modification includes a 2′-modification, e.g. the 2′-OH of the ribosyl sugar is replaced or substituted. Suitable replacements for 2′-OH include 2′-F and 2′-arabino-F. Suitable substitutions for OH include 2′-O-alkyl, e.g. 2′-O-methyl, and 2′-O-substituted alkyl, e.g. 2′-O-methoxyethyl, 2′-O-aminopropyl, etc. In some embodiments, the oligomeric compound contains at least one 2′-modification. In some embodiments, the oligomeric compound contains at least 2 2′-modifications. In some embodiments, the oligomeric compound has at least one 2′-modification at each of the termini (i.e. the 3′- and 5′-terminal nucleosides each have the same or different 2′-modifications). In some embodiments, the oligomeric compound has at least two sequential 2′-modifications at each end of the compound. In some embodiments, oligomeric compounds further comprise at least one deoxynucleoside. In particular embodiments, oligomeric compounds comprise a stretch of deoxynucleosides such that the stretch is capable of activating RNase (e.g. RNase H) cleavage of an RNA to which the oligomeric compound is capable of hybridizing. In some embodiments, a stretch of deoxynucleosides capable of activating RNase-mediated cleavage of RNA comprises about 8 to about 16, e.g. about 8 to about 16 consecutive deoxynucleosides. In further embodiments, oligomeric compounds are capable of eliciting cleaveage by dsRNAse enzymes.
Oral compositions for administration of non-parenteral oligomeric compounds and compositions of the present invention may be formulated in various dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The term “alimentary delivery” encompasses e.g. oral, rectal, endoscopic and sublingual/buccal administration. A common requirement for these modes of administration is absorption over some portion or all of the alimentary tract and a need for efficient mucosal penetration of the nucleic acid(s) so administered.
Delivery of a drug via the oral mucosa, as in the case of buccal and sublingual administration, has several desirable features, including, in many instances, a more rapid rise in plasma concentration of the drug than via oral delivery (Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711).
Endoscopy may be used for delivery directly to an interior portion of the alimentary tract. For example, endoscopic retrograde cystopancreatography (ERCP) takes advantage of extended gastroscopy and permits selective access to the biliary tract and the pancreatic duct (Hirahata et al., Gan To Kagaku Ryoho, 1992, 19(10 Suppl.), 1591). Pharmaceutical compositions, including liposomal formulations, can be delivered directly into portions of the alimentary canal, such as, e.g., the duodenum (Somogyi et al., Pharm. Res., 1995, 12, 149) or the gastric submucosa (Akamo et al., Japanese J. Cancer Res., 1994, 85, 652) via endoscopic means. Gastric lavage devices (Inoue et al., Artif. Organs, 1997, 21, 28) and percutaneous endoscopic feeding devices (Pennington et al., Ailment Pharmacol. Ther., 1995, 9, 471) can also be used for direct alimentary delivery of pharmaceutical compositions.
In some embodiments, oligomeric compound formulations may be administered through the anus into the rectum or lower intestine. Rectal suppositories, retention enemas or rectal catheters can be used for this purpose and may be preferred when patient compliance might otherwise be difficult to achieve (e.g., in pediatric and geriatric applications, or when the patient is vomiting or unconscious). Rectal administration can result in more prompt and higher blood levels than the oral route. (Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711). Because about 50% of the drug that is absorbed from the rectum will bypass the liver, administration by this route significantly reduces the potential for first-pass metabolism (Benet et al., Chapter 1 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).
Some embodiments of the present invention employ various penetration enhancers in order to effect transport of oligomeric compounds and compositions across mucosal and epithelial membranes. Penetration enhancers may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Penetration enhancers and their uses are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Accordingly, some embodiments comprise oral oligomeric compound compositions comprising at least one member of the group consisting of surfactants, fatty acids, bile salts, chelating agents, and non-chelating surfactants. Further embodiments comprise oral oligomeric compound comprising at least one fatty acid, e.g. capric or lauric acid, or combinations or salts thereof. Other embodiments comprise methods of enhancing the oral bioavailability of an oligomeric compound, the method comprising co-administering the oligomeric compound and at least one penetration enhancer.
Other excipients that may be added to oral oligomeric compound compositions include surfactants (or “surface-active agents”), which are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligomeric compounds through the alimentary mucosa and other epithelial membranes is enhanced. In addition to bile salts and fatty acids, surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and perfluorohemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Phamacol., 1988, 40, 252).
Fatty acids and their derivatives which act as penetration enhancers and may be used in compositions of the present invention include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines and mono- and di-glycerides thereof and/or physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J. Pharm. Phamacol., 1992, 44, 651).
In some embodiments, oligomeric compound compositions for oral delivery comprise at least two discrete phases, which phases may comprise particles, capsules, gel-capsules, microspheres, etc. Each phase may contain one or more oligomeric compounds, penetration enhancers, surfactants, bioadhesives, effervescent agents, or other adjuvant, excipient or diluent. In some embodiments, one phase comprises at least one oligomeric compound and at least one penetration enhancer. In some embodiments, a first phase comprises at least one oligomeric compound and at least one penetration enhancer, while a second phase comprises at least one penetration enhancer. In some embodiments, a first phase comprises at least one oligomeric compound and at least one penetration enhancer, while a second phase comprises at least one penetration enhancer and substantially no oligomeric compound. In some embodiments, at least one phase is compounded with at least one degradation retardant, such as a coating or a matrix, which delays release of the contents of that phase. In some embodiments, a first phase comprises at least one oligomeric compound, at least one penetration enhancer, while a second phase comprises at least one penetration enhancer and a release-retardant. In particular embodiments, an oral oligomeric compound comprises a first phase comprising particles containing an oligomeric compound and a penetration enhancer, and a second phase comprising particles coated with a release-retarding agent and containing penetration enhancer.
A variety of bile salts also function as penetration enhancers to facilitate the uptake and bioavailability of drugs. The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term “bile salt” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579).
In some embodiments, penetration enhancers useful in some embodiments of present invention are mixtures of penetration enhancing compounds. One such penetration enhancer is a mixture of UDCA (and/or CDCA) with capric and/or lauric acids or salts thereof e.g. sodium. Such mixtures are useful for enhancing the delivery of biologically active substances across mucosal membranes, in particular intestinal mucosa. Other penetration enhancer mixtures comprise about 5-95% of bile acid or salt(s) UDCA and/or CDCA with 5-95% capric and/or lauric acid. Particular penetration enhancers are mixtures of the sodium salts of UDCA, capric acid and lauric acid in a ratio of about 1:2:2 respectively. Anther such penetration enhancer is a mixture of capric and lauric acid (or salts thereof) in a 0.01:1 to 1:0.01 ratio (mole basis). In particular embodiments capric acid and lauric acid are present in molar ratios of e.g. about 0.1:1 to about 1:0.1, in particular about 0.5:1 to about 1:0.5.
Other excipients include chelating agents, i.e. compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligomeric compounds through the alimentary and other mucosa is enhanced. With regard to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315). Chelating agents of the invention include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J. Control Rel., 1990, 14, 43).
As used herein, non-chelating non-surfactant penetration enhancers may be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligomeric compounds through the alimentary and other mucosal membranes (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1). This class of penetration enhancers includes, but is not limited to, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621).
Agents that enhance uptake of oligomeric compounds at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), can be used.
Some oral oligomeric compound compositions also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which may be inert (i.e., does not possess biological activity per se) or may be necessary for transport, recognition or pathway activation or mediation, or is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of an oligomeric compound having biological activity by, for example, degrading the biologically active oligomeric compound or promoting its removal from circulation. The coadministration of a oligomeric compound and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of oligomeric compound recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the oligomeric compound for a common receptor. For example, the recovery of a partially phosphorothioate oligomeric compound in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177).
A “pharmaceutical carrier” or “excipient” may be a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more oligomeric compounds to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with an oligomeric compound and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, EXPLOTAB); and wetting agents (e.g., sodium lauryl sulphate, etc.).
Oral oligomeric compound compositions may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipuritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The oligomeric compounds and compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations.
Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug that may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged nucleic acid molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap nucleic acids rather than complex with it. Both cationic and noncationic liposomes have been used to deliver nucleic acids and oligomeric compounds to cells.
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.
Formulations for topical administration include those in which the oligomeric compounds of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
For topical or other administration, oligomeric compounds and compositions of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, they may be complexed to lipids, in particular to cationic lipids. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Oral formulations are those in which oligomeric compounds of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. A particularly suitable combination is the sodium salt of lauric acid, capric acid and UDCA. Penetration enhancers also include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Compounds and compositions of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Certain oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and U.S. Application Publication 20030027780, each of which is incorporated herein by reference in their entirety.
Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Certain embodiments of the invention provide pharmaceutical compositions containing one or more of the compounds and compositions of the invention and one or more other chemotherapeutic agents that function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the oligomeric compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of oligomeric compounds and compositions of the invention and other drugs are also within the scope of this invention. Two or more combined compounds such as two oligomeric compounds or one oligomeric compound combined with further compounds may be used together or sequentially.
In another embodiment, compositions of the invention may contain one or more of the compounds and compositions of the invention targeted to a first nucleic acid target and one or more additional oligomeric compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more oligomeric compounds and compositions targeted to different regions, segments or sites of the same target. Two or more combined compounds may be used together or sequentially.
The formulation of therapeutic compounds and compositions of the invention and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligomeric compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1.0 μg to 1 g per kg of body weight, from 10.0 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, or from 1 mg to 5 mg per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily determine repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligomeric compound is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1 μg to 1 g per kg of body weight, from 10 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, or from 100 μg to 1 mg per kg of body weight, once or more daily, to once every 20 years. The effects of treatments with therapeutic compositions can be assessed following collection of tissues or fluids from a patient or subject receiving said treatments. It is known in the art that a biopsy sample can be procured from certain tissues without resulting in detrimental effects to a patient or subject. In certain embodiments, a tissue and its constituent cells comprise, but are not limited to, blood (e.g., hematopoietic cells, such as human hematopoietic progenitor cells, human hematopoietic stem cells, CD34+ cells CD4+ cells), lymphocytes and other blood lineage cells, bone marrow, breast, cervix, colon, esophagus, lymph node, muscle, peripheral blood, oral mucosa and skin. In other embodiments, a fluid and its constituent cells comprise, but are not limited to, blood, urine, semen, synovial fluid, lymphatic fluid and cerebro-spinal fluid. Tissues or fluids procured from patients can be evaluated for expression levels of a target small non-coding RNA, mRNA or protein. Additionally, the mRNA or protein expression levels of other genes known or suspected to be associated with the specific disease state, condition or phenotype can be assessed. mRNA levels can be measured or evaluated by real-time PCR, Northern blot, in situ hybridization or DNA array analysis.
Protein levels of a downstream target modulated or regulated by a small non-coding RNA can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.
Western blot analysis of protein levels:
When small non-coding RNAs have effects on expression of downstream genes or proteins encoded by genes, it is advantageous to measure the protein levels of those gene products. To do this, western blot analysis may be employed.
Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligomeric compound treatment, washed once with PBS, suspended in Laemmli buffer (100 μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gradient gels (4-20%) may also be used for the separation of proteins, as is known in the art. Gels are typically run for 1.5 hours at 150 V, and transferred to a membrane, such as PVDF, for western blotting. Appropriate primary antibody directed to a target is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGERTM (Molecular Dynamics, Sunnyvale Calif.).
Furthermore, the effects of treatment can be assessed by measuring biomarkers associated with the disease or condition in the aforementioned tissues and fluids, collected from a patient or subject receiving treatment, by routine clinical methods known in the art. These biomarkers include but are not limited to: glucose, cholesterol, lipoproteins, triglycerides, free fatty acids and other markers of glucose and lipid metabolism; liver transaminases, bilirubin, albumin, blood urea nitrogen, creatine and other markers of kidney and liver function; interleukins, tumor necrosis factors, intracellular adhesion molecules, C-reactive protein and other markers of inflammation; testosterone, estrogen and other hormones; tumor markers; vitamins, minerals and electrolytes.
In Vitro and In Vivo Assays:
Phenotypic Assays
Once modulators are designed or identified by the methods disclosed herein, the oligomeric compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive or suggestive of efficacy in the treatment, amelioration or improvement of physiologic conditions associated with a particular disease state or condition.
Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of a target in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).
In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with an oligomeric compound identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.
Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.
Analysis of the genotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the oligomeric compound. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.
Cell Proliferation and Survival Assays:
In some embodiments, cell proliferation and survival assays are used. Cell cycle regulation is the basis for many cancer therapeutic agents. Unregulated cell proliferation is a characteristic of cancer cells, thus most current chemotherapy agents target dividing cells, for example, by blocking the synthesis of new DNA required for cell division. However, cells in healthy tissues are often also affected by agents that modulate cell proliferation.
In some cases, a cell cycle inhibitor will cause apoptosis in cancer cells, but allow normal cells to undergo growth arrest and therefore remain unaffected (Blagosklonny, Bioessays, 1999, 21, 704-709; Chen et al., Cancer Res., 1997, 57, 2013-2019; Evan and Littlewood, Science, 1998, 281, 1317-1322; Lees and Weinberg, Proc. Natl. Acad. Sci. USA, 1999, 96, 4221-4223). An example of sensitization to anti-cancer agents is observed in cells that have reduced or absent expression of the tumor suppressor genes p53 (Bunz et al., Science, 1998, 282, 1497-1501; Bunz et al., J. Clin. Invest., 1999, 104, 263-269; Stewart et al., Cancer Res., 1999, 59, 3831-3837; Wahl et al., Nat. Med., 1996, 2, 72-79). However, cancer cells often escape apoptosis (Lowe and Lin, Carcinogenesis, 2000, 21, 485-495; Reed, Cancer J. Sci. Am., 1998, 4 Suppl l, S8-14). Further disruption of cell cycle checkpoints in cancer cells can increase sensitivity to chemotherapy while allowing normal cells to take refuge in G1 and remain unaffected.
Cell Cycle Assay:
A cell cycle assay is employed to identify genes whose modulation affects cell cycle progression. In addition to normal cells, cells lacking functional p53 are utilized to identify genes whose modulation will sensitize p53-deficient cells to anti-cancer agents. Oligomeric compounds of the invention are tested for their effects on the cell cycle in normal human mammary epithelial cells (HMECs) as well as the breast carcinoma cell lines MCF7 and T47D. The latter two cell lines express similar genes but MCF7 cells express the tumor suppressor p53, while T47D cells are deficient in p53. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of cell cycle progression. An oligomeric compound targeting kinesin-like 1 is known to inhibit cell cycle progression and may be used as a positive control.
Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINTM in OPTI-MEMTM to achieve a final concentration of 200 nM of oligomeric compound and 6 μg/mL LIPOFECTINTM. Compounds of the invention and the positive control are tested in triplicate. The negative control is tested in up to six replicate wells. Untreated control cells receive LIPOFECTINTM only. Approximately 24, 48 or 72 hours following transfection, routine procedures are used to prepare cells for flow cytometry analysis and cells are stained with propidium iodide to generate a cell cycle profile using a flow cytometer. The cell cycle profile is analyzed with the ModFit program (Verity Software House, Inc., Topsham Me.).
Fragmentation of nuclear DNA is a hallmark of apoptosis and produces an increase in cells with a hypodiploid DNA content, which are categorized as “subG1.” An increase in cells in G1 phase is indicative of a cell cycle arrest prior to entry into S phase; an increase in cells in S phase is indicative of cell cycle arrest during DNA synthesis; and an increase in cells in the G2/M phase is indicative of cell cycle arrest just prior to or during mitosis. Cell cycle profiles of cells treated with oligomeric compounds can be normalized to those of untreated control cells, and values above or below 100% are considered to indicate an increase or decrease, respectively, in the proportion of cells in a particular phase of the cell cycle.
Oligomeric compounds that prevent cell cycle progression are candidate therapeutic agents for the treatment of hyperproliferative disorders, such as cancer or inflammation.
Caspase Assay:
Programmed cell death, or apoptosis, is an important aspect of various biological processes, including normal cell turnover, immune system development and embryonic development. Apoptosis involves the activation of caspases, a family of intracellular proteases through which a cascade of events leads to the cleavage of a select set of proteins. The caspase family can be divided into two groups: the initiator caspases, such as caspase-8 and -9, and the executioner caspases, such as caspase-3, -6 and -7, which are activated by the initiator caspases. The caspase family contains at least 14 members, with differing substrate preferences (Thornberry and Lazebnik, Science, 1998, 281, 1312-1316). A caspase assay is utilized to identify genes whose modulation causes apoptosis. The chemotherapeutic drugs taxol, cisplatin, etoposide, gemcitabine, camptothecin, aphidicolin and 5-fluorouracil all have been shown to induce apoptosis in a caspase-dependent manner.
In a further embodiment, a caspase assay is employed to identify genes or targets whose modulation affects apoptosis. In addition to normal cells, cells lacking functional p53 are utilized to identify genes or targets whose modulation will sensitize p53-deficient cells to agents that induce apoptosis. Oligomeric compounds of the invention are assayed for their affects on apoptosis in normal HMECs as well as the breast carcinoma cell lines MCF7 and T47D. HMECs and MCF7 cells express p53, whereas T47D cells do not express this tumor suppressor gene. Cells are cultured in 96-well plates with black sides and flat, transparent bottoms (Corning Incorporated, Corning, N.Y.). DMEM medium, with and without phenol red, is obtained from Invitrogen Life Technologies (Carlsbad, Calif.). MEGM medium, with and without phenol red, is obtained from Cambrex Bioscience (Walkersville, Md.). A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of caspase activity. An oligomeric compound targeted to human Jagged2 or human Notch1, both of which are known to induce caspase activity, may be used as a positive control for caspase activation.
Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINTM in OPTI-MEMTM to achieve a final concentration of 200 nM of oligomeric compound and 6 μg/mL LIPOFECTINTM. Compounds of the invention and the positive controls are tested in triplicate, and the negative control is tested in up to six replicate wells. Untreated control cells receive LIPOFECTINTM only.
Caspase-3 activity is evaluated with a fluorometric HTS Caspase-3 assay (Catalog # HTS02; EMD Biosciences, San Diego, Calif.) that detects cleavage after aspartate residues in the peptide sequence DEVD. The DEVD substrate is labeled with a fluorescent molecule, which exhibits a blue to green shift in fluorescence upon cleavage by caspase-3. Active caspase-3 in the oligomeric compound-treated cells is measured by this assay according to the manufacturer's instructions. Approximately 48 hours following treatment, 50 μL of assay buffer containing 10 μM dithiothreitol is added to each well, followed by addition 20 μL of the caspase-3 fluorescent substrate conjugate. Fluorescence in wells is immediately detected (excitation/emission 400/505 nm) using a fluorescent plate reader (SpectraMAX GeminiXS, Molecular Devices, Sunnyvale, Calif.). The plate is covered and incubated at 37° C. for an additional three hours, after which the fluorescence is again measured (excitation/emission 400/505 nm). The value at time zero is subtracted from the measurement obtained at 3 hours. The measurement obtained from the untreated control cells is designated as 100% activity. Caspase-3 activity in cells treated with oligomeric compounds is normalized to that in untreated control cells. Values for caspase activity above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit caspase activity, respectively.
Oligomeric compounds that cause a significant induction in apoptosis are candidate therapeutic agents with applications in the treatment of conditions in which the induction of apoptosis is desirable, for example, in hyperproliferative disorders. Oligomeric compounds that inhibit apoptosis are candidate therapeutic agents with applications in the treatment of conditions where the reduction of apoptosis is useful, for example, in neurodegenerative disorders.
Angiogenesis Assays:
In some embodiments, angiogenesis assays are used. Angiogenesis is the growth of new blood vessels (veins and arteries) by endothelial cells. This process is important in the development of a number of human diseases, and is believed to be particularly important in regulating the growth of solid tumors. Without new vessel formation it is believed that tumors will not grow beyond a few millimeters in size. In addition to their use as anti-cancer agents, inhibitors of angiogenesis have potential for the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis (Carmeliet and Jain, Nature, 2000, 407, 249-257; Freedman and Isner, J. Mol. Cell. Cardiol., 2001, 33, 379-393; Jackson et al., Faseb J., 1997, 11, 457-465; Saaristo et al., Oncogene, 2000, 19, 6122-6129; Weber and De Bandt, Joint Bone Spine, 2000, 67, 366-383; Yoshida et al., Histol. Histopathol., 1999, 14, 1287-1294).
Expression of Angiogenic Genes as a Measure of Angiogenesis:
During the process of angiogenesis, endothelial cells perform several distinct functions, including the degradation of the extracellular matrix (ECM), migration, proliferation and the formation of tube-like structures (Liekens et al., Biochem. Pharmacol., 2001, 61, 253-270). Endothelial cells must regulate the expression of many genes in order to perform the functions necessary for angiogenesis. This gene regulation has been the subject of intense scrutiny, and many genes have been identified as being important for the angiogenic phenotype. Genes highly expressed in angiogenic endothelial cells include integrin β3, endoglin/CD105, TEM5 and MMP-14/MT-MMP1.
Integrin β3 is part of a family of heterodimeric transmembrane receptors that consist of alpha and beta subunits (Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822). Each subunit recognizes a unique set of ECM ligands, thereby allowing cells to transmit angiogenic signals from the extracellular matrix. Integrin β3 is prominently expressed on proliferating vascular endothelial cells, and it plays roles in allowing new blood vessels to form at tumor sites as well as allowing the epithelial cells of breast tumors to spread (Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822; Drake et al., J. Cell Sci., 1995, 108 (Pt 7), 2655-2661). Blockage of integrin β3 with monoclonal antibodies or low molecular weight antagonists inhibits blood vessel formation in a variety of in-vivo models, including tumor angiogenesis and neovascularization during oxygen-induced retinopathy (Brooks et al., Science, 1994, 264, 569-571; Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822; Hammes et al., Nat. Med., 1996, 2, 529-533).
Endoglin is a transforming growth factor receptor-associated protein highly expressed on endothelial cells, and present on some leukemia cells and minor subsets of bone marrow cells (Burrows et al., Clin. Cancer Res., 1995, 1, 1623-1634; Haruta and Seon, Proc. Natl. Acad. Sci. USA, 1986, 83, 7898-7902). Its expression is upregulated in endothelial cells of angiogenic tissues and is therefore used as a prognostic indicator in various tumors (Burrows et al., Clin. Cancer Res., 1995, 1, 1623-1634). Endoglin functions as an ancillary receptor influencing binding of the transforming growth factor beta (TGF-beta) family of ligands to signaling receptors, thus mediating cell survival (Massague and Chen, Genes Dev., 2000, 14, 627-644).
Tumor endothelial marker 5 (TEM5) is a putative 7-pass transmembrane protein (GPCR) (Carson-Walter et al., Cancer Res., 2001, 61, 6649-6655). The mRNA transcript, designated KIAA1531, encodes one of many tumor endothelium markers (TEMs) that display elevated expression (greater than 10-fold) during tumor angiogenesis (St Croix et al., Science, 2000, 289, 1197-1202). TEM5 is coordinately expressed with other TEMs on tumor endothelium in humans and mice.
Matrix metalloproteinase 14 (MMP-14), a membrane-type MMP covalently linked to the cell membrane, is involved in matrix detachment and migration. MMP-14 is thought to promote tumor angiogenesis; antibodies directed against the catalytic domain of MMP-14 block endothelial-cell migration, invasion and capillary tube formation in vitro (Galvez et al., J. Biol. Chem., 2001, 276, 37491-37500). MMP-14 can degrade the fibrin matrix that surrounds newly formed vessels potentially allowing the endothelial cells to invade further into the tumor tissue (Hotary et al., J. Exp. Med., 2002, 195, 295-308). MMP-14 null mice have impaired angiogenesis during development, further demonstrating the role of MMP-14 in angiogenesis (Vu and Werb, Genes Dev., 2000, 14, 2123-2133; Zhou et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 4052-4057).
In some embodiments, HUVECs are used to measure the effects of oligomeric compounds of the invention on the activity of endothelial cells stimulated with human vascular endothelial growth factor (VEGF). A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of HUVEC activity.
Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINTM in OPTI-MEMTM to achieve a final concentration of 75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTINTM. Compounds of the invention are tested in triplicate, and the negative control is tested in up to six replicate wells. Untreated control cells receive LIPOFECTINTM only.
Approximately twenty hours after transfection, cells are induced to express angiogenic genes with recombinant VEGF. Total RNA is harvested approximately 52 hours following transfection, and the amount of total RNA from each sample is determined using a Ribogreen Assay (Invitrogen Life Technologies, Carlsbad, Calif.). Real-time RT-PCR is performed on the total RNA using primer/probe sets for four angiogenic hallmark genes described herein: integrin β3, endoglin, TEM5 and MMP14. Expression levels for each gene are normalized to total RNA. Gene expression in cells treated with oligomeric compounds is normalized to that in untreated control cells. A value above or below 100% is considered to indicated an increase or decrease in gene expression, respectively.
Oligomeric compounds resulting in a decrease in the expression of angiogenic hallmark genes are candidate therapeutic agents for the inhibition of angiogenesis where such activity is desired, for example, in the treatment of cancer, diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis. Oligomeric compounds that increase the expression of angiogenic hallmark genes are candidate therapeutic agents with applications where the stimulation of angiogenesis is desired, for example, in wound healing.
Endothelial Tube Formation Assay as a Measure of Angiogenesis:
Angiogenesis is stimulated by numerous factors that promote interaction of endothelial cells with each other and with extracellular matrix molecules, resulting in the formation of capillary tubes. This morphogenic process is necessary for the delivery of oxygen to nearby tissues and plays an essential role in embryonic development, wound healing, and tumor growth (Carmeliet and Jain, Nature, 2000, 407, 249-257). Moreover, this process can be reproduced in a tissue culture assay that evaluated the formation of tube-like structures by endothelial cells. There are several different variations of the assay that use different matrices, such as collagen I (Kanayasu et al., Lipids, 1991, 26, 271-276), Matrigel (Yamagishi et al., J. Biol. Chem., 1997, 272, 8723-8730) and fibrin (Bach et al., Exp. Cell Res., 1998, 238, 324-334), as growth substrates for the cells. In this assay, HUVECs are plated on a matrix derived from the Engelbreth-Holm-Swarm mouse tumor, which is very similar to Matrigel (Kleinman et al., Biochemistry, 1986, 25, 312-318; Madri and Pratt, J. Histochem. Cytochem., 1986, 34, 85-91). Untreated HUVECs form tube-like structures when grown on this substrate. Loss of tube formation in vitro has been correlated with the inhibition of angiogenesis in vivo (Carmeliet and Jain, Nature, 2000, 407, 249-257; Zhang et al., Cancer Res., 2002, 62, 2034-2042), which supports the use of in vitro tube formation as an endpoint for angiogenesis.
In some embodiments, HUVECs are used to measure the effects of oligomeric compounds of the invention on endothelial tube formation activity. The tube formation assay is performed using an in vitro Angiogenesis Assay Kit (Chemicon International, Temecula, Calif.). A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of endothelial tube formation.
Oligomeric compounds are mixed with LIPOFECTINTM in OPTI-MEMTM to achieve a final concentration of 75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTINTM. Untreated control cells receive LIPOFECTINTM only. Compounds of the invention are tested in triplicate, and the negative control is tested in up to six replicates.
Approximately fifty hours after transfection, cells are transferred to 96-well plates coated with ECMatrixTM (Chemicon International). Under these conditions, untreated HUVECs form tube-like structures. After an overnight incubation at 37° C., treated and untreated cells are inspected by light microscopy. Tube formation in cells treated with oligomeric compounds is compared to that in untreated control cells. Individual wells are assigned discrete scores from 1 to 5 depending on the extent of tube formation. A score of 1 refers to a well with no tube formation while a score of 5 is given to wells where all cells are forming an extensive tubular network.
Oligomeric compounds resulting in a decrease in tube formation are candidate therapeutic agents for the inhibition of angiogenesis where such activity is desired, for example, in the treatment of cancer, diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis. Oligomeric compounds that promote endothelial tube formation are candidate therapeutic agents with applications where the stimulation of angiogenesis is desired, for example, in wound healing.
Matrix Metalloproteinase Activity:
During angiogenesis, endothelial cells must degrade the extracellular matrix (ECM) and thus secrete matrix metalloproteinases (MMPs) in order to accomplish this degradation. MMPs are a family of zinc-dependent endopeptidases that fall into eight distinct classes: five are secreted and three are membrane-type MMPs (MT-MMPs) (Egeblad and Werb, J. Cell Science, 2002, 2, 161-174). MMPs exert their effects by cleaving a diverse group of substrates, which include not only structural components of the extracellular matrix, but also growth-factor-binding proteins, growth-factor pre-cursors, receptor tyrosine-kinases, cell-adhesion molecules and other proteinases (Xu et al., J. Cell Biol., 2002, 154, 1069-1080).
In some embodiments, oligomeric compounds of the invention are evaluated for their effects on MMP activity in the medium above cultured HUVECs. MMP activity is measured using the EnzChek Gelatinase/Collagenase Assay Kit (Molecular Probes, Eugene, Oreg.). In this assay, HUVECs are plated at approximately 4000 cells per well in 96-well plates and transfected one day later. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of MMP activity. An oligomeric compound targeted to integrin β3 is known to inhibit MMP activity and may be used as a positive control.
Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINTM in OPTI-MEMTM to achieve a final concentration of 75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTINTM. Compounds of the invention and the positive control are tested in triplicate, and the negative control is tested in up to six replicates. Untreated control cells receive LIPOFECTINTM only.
Approximately 50 hours after transfection, a p-aminophenylmercuric acetate (APMA, Sigma-Aldrich, St. Louis, Mo.) solution is added to each well of a Corning-Costar 96-well clear bottom plate (VWR International, Brisbane, Calif.). The APMA solution is used to promote cleavage of inactive MMP precursor proteins. Medium above the HUVECs is then transferred to the wells in the 96-well plate. After approximately 30 minutes, the quenched, fluorogenic MMP cleavage substrate is added, and baseline fluorescence is read immediately at 485 nm excitation/530 nm emission. Following an overnight incubation at 37° C. in the dark, plates are read again to determine the amount of fluorescence, which corresponds to MMP activity. Total protein from HUVEC lysates is used to normalize the readings, and MMP activity from cells treated with oligomeric compounds is normalized to that of untreated control cells. MMP activities above or below 100% are considered to indicate a stimulation or inhibition, respectively, of MMP activity.
Oligomeric compounds resulting in a decrease in MMP activity are candidate therapeutic agents for the inhibition of angiogenesis where such activity is desired, for example, in the treatment of cancer, diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis. Oligomeric compounds that increase the expression of angiogenic hallmark genes are candidate therapeutic agents with applications in conditions requiring angiogenesis, for example, in wound healing.
Adipocyte Assays:
In some embodiments, adipocytes assays are used. Insulin is an essential signaling molecule throughout the body, but its major target organs are the liver, skeletal muscle and adipose tissue. Insulin is the primary modulator of glucose homeostasis and helps maintain a balance of peripheral glucose utilization and hepatic glucose production. The reduced ability of normal circulating concentrations of insulin to maintain glucose homeostasis manifests in insulin resistance which is often associated with diabetes, central obesity, hypertension, polycystic ovarian syndrome, dyslipidemia and atherosclerosis (Saltiel, Cell, 2001, 104, 517-529; Saltiel and Kahn, Nature, 2001, 414, 799-806).
Response of Undifferentiated Adipocytes to Insulin:
Insulin promotes the differentiation of preadipocytes into adipocytes. The condition of obesity, which results in increases in fat cell number, occurs even in insulin-resistant states in which glucose transport is impaired due to the antilipolytic effect of insulin Inhibition of triglyceride breakdown requires much lower insulin concentrations than stimulation of glucose transport, resulting in maintenance or expansion of adipose stores (Kitamura et al., Mol. Cell. Biol., 1999, 19, 6286-6296; Kitamura et al., Mol. Cell. Biol., 1998, 18, 3708-3717).
One of the hallmarks of cellular differentiation is the upregulation of gene expression. During adipocyte differentiation, the gene expression patterns in adipocytes change considerably. Some genes known to be upregulated during adipocyte differentiation include hormone-sensitive lipase (HSL), adipocyte lipid binding protein (aP2), glucose transporter 4 (Glut4), and peroxisome proliferator-activated receptor gamma (PPAR-γ). Insulin signaling is improved by compounds that bind and inactivate PPAR-γ, a key regulator of adipocyte differentiation (Olefsky, J. Clin. Invest., 2000, 106, 467-472). Insulin induces the translocation of GLUT4 to the adipocyte cell surface, where it transports glucose into the cell, an activity necessary for triglyceride synthesis. In all forms of obesity and diabetes, a major factor contributing to the impaired insulin-stimulated glucose transport in adipocytes is the downregulation of GLUT4. Insulin also induces hormone sensitive lipase (HSL), which is the predominant lipase in adipocytes that functions to promote fatty acid synthesis and lipogenesis (Fredrikson et al., J. Biol. Chem., 1981, 256, 6311-6320). Adipocyte fatty acid binding protein (aP2) belongs to a multi-gene family of fatty acid and retinoid transport proteins. aP2 is postulated to serve as a lipid shuttle, solubilizing hydrophobic fatty acids and delivering them to the appropriate metabolic system for utilization (Fu et al., J. Lipid Res., 2000, 41, 2017-2023; Pelton et al., Biochem. Biophys. Res. Commun., 1999, 261, 456-458). Together, these genes play important roles in the uptake of glucose and the metabolism and utilization of fats.
Leptin secretion and an increase in triglyceride content are also well-established markers of adipocyte differentiation. In addition to its role in adipocytes differentiation, leptin also regulates glucose homeostasis through mechanisms (autocrine, paracrine, endocrine and neural) independent of the adipocyte's role in energy storage and release. As adipocytes differentiate, insulin increases triglyceride accumulation by both promoting triglyceride synthesis and inhibiting triglyceride breakdown (Spiegelman and Flier, Cell, 2001, 104, 531-543). As triglyceride accumulation correlates tightly with cell size and cell number, it is an excellent indicator of differentiated adipocytes.
Oligomeric compounds of the invention are tested for their effects on preadipocyte differentiation. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of adipocyte differentiation. Tumor necrosis factor alpha (TNF-α) is known to inhibit adipocyte differentiation and may be used as a positive control for the inhibition of adipocyte differentiation as evaluated by leptin secretion. For the other adipocyte differentiation markers assayed, an oligomeric compound targeted to PPAR-γ, also known to inhibit adipocyte differentiation, may be used as a positive control.
Cells are transfected as described herein. Oligomeric componds are mixed with LIPOFECTINTM in OPTI-MEMTM to achieve a final concentration of 250 nM of oligomeric compound and 7.5 μg/mL LIPOFECTINTM. Untreated control cells receive LIPOFECTINTM only. Compounds of the invention and the positive control are tested in triplicate, and the negative control is tested in up to six replicate wells.
After the cells have reach confluence (approximately three days), they are exposed for an additional three days to differentiation medium (Zen-Bio, Inc., Research Triangle Park, NC) containing a PPAR-γ agonist, IBMX, dexamethasone, and insulin. Cells are then fed adipocyte medium (Zen-Bio, Inc.), which is replaced at 2 or 3 day intervals.
Leptin secretion into the medium in which adipocytes are cultured is measured by protein
ELISA. On day nine post-transfection, 96-well plates are coated with a monoclonal antibody to human leptin (R&D Systems, Minneapolis, Minn.) and left at 4° C. overnight. The plates are blocked with bovine serum albumin (BSA), and a dilution of the treated adipoctye medium is incubated in the plate at room temperature for approximately 2 hours. After washing to remove unbound components, a second monoclonal antibody to human leptin (conjugated with biotin) is added. The plate is then incubated with strepavidin-conjugated horse radish peroxidase (HRP) and enzyme levels are determined by incubation with 3,3′,5,5′-tetramethlybenzidine, which turns blue when cleaved by HRP. The OD450 is read for each well, where the dye absorbance is proportional to the leptin concentration in the cell lysate. Leptin secretion from cells treated with oligomeric compounds is normalized to that from untreated control cells. With respect to leptin secretion, values above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit leptin secretion, respectively.
The triglyceride accumulation assay measures the synthesis of triglyceride by adipocytes. Triglyceride accumulation is measured using the InfinityTM Triglyceride reagent kit (Sigma-Aldrich, St. Louis, Mo.). On day nine post-transfection, cells are washed and lysed at room temperature, and the triglyceride assay reagent is added. Triglyceride accumulation is measured based on the amount of glycerol liberated from triglycerides by the enzyme lipoprotein lipase. Liberated glycerol is phosphorylated by glycerol kinase, and hydrogen peroxide is generated during the oxidation of glycerol-1-phosphate to dihydroxyacetone phosphate by glycerol phosphate oxidase. Horseradish peroxidase (HRP) uses H2O2 to oxidize 4-aminoantipyrine and 3,5 dichloro-2-hydroxybenzene sulfonate to produce a red-colored dye. Dye absorbance, which is proportional to the concentration of glycerol, is measured at 515 nm using an UV spectrophotometer. Glycerol concentration is calculated from a standard curve for each assay, and data are normalized to total cellular protein as determined by a Bradford assay (Bio-Rad Laboratories, Hercules, Calif.). Triglyceride accumulation in cells treated with oligomeric compounds is normalized to that in untreated control cells. Values for triglyceride accumulation above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit triglyceride accumulation, respectively.
Expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, is also measured in adipocytes transfected with oligomeric compounds of the invention. Cells are lysed on day nine post-transfection and total RNA is harvested. The amount of total RNA in each sample is determined using a Ribogreen Assay (Invitrogen Life Technologies, Carlsbad, Calif.). Real-time PCR is performed on the total RNA using primer/probe sets for the adipocyte differentiation hallmark genes Glut4, HSL, aP2, and PPAR-γ. Gene expression in cells treated with oligomeric compounds is normalized to that in untreated control cells. With respect to the four adipocyte differentiation hallmark genes, values above or below 100% are considered to indicate that the compound has the ability to stimulate or inhibit adipocyte differentiation, respectively.
Oligomeric compounds that reduce the expression levels of markers of adipocyte differentiation are candidate therapeutic agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells. Oligomeric compounds of the invention resulting in a significant increase in leptin secretion are potentially useful for the treatment of obesity.
Response of Liver-Derived Cells to Insulin:
Insulin mediates its effects by suppressing the RNA expression levels of enzymes important for gluconeogenesis and glycogenolysis, and also by controlling the activities of some metabolic enzymes through post-translational mechanisms (Hall and Granner, J. Basic Clin. Physiol. Pharmacol., 1999, 10, 119-133; Moller, Nature, 2001, 414, 821-827; Saltiel and Kahn, Nature, 2001, 414, 799-806). In liver cells, genes involved in regulating glucose metabolism can be identified by monitoring changes in the expression of selective insulin-responsive genes in a cell culture model. However, primary human hepatocytes are difficult to obtain and work with in culture. Therefore, the insulin signaling assay described herein is performed in the hepatocellular carcinoma cell line HepG2, the most widely used cell culture model for hepatocytes. The insulin responsive genes evaluate in this assay are phosphoenolpyruvate carboxykinase (PEPCK), insulin-like growth factor binding protein 1 (IGFBP-1) and follistatin.
IGFBP-1 is one of a family of six secreted proteins that bind insulin-like growth factor (IGF) with high affinity and thereby modulate IGFs action in vivo (Baxter, Am. J. Physiol. Endocrinol. Metab., 2000, 278, E967-976; Lee et al., Proc. Soc. Exp. Biol. Med., 1997, 216, 319-357). IGFBP-1 is characterized by dynamic variability of levels in circulation due to the regulation of its hepatic secretion (Lee et al., Proc. Soc. Exp. Biol. Med., 1997, 216, 319-357). The multi-hormonal regulation of PEPCK and IGFBP-1 are similar. Glucocorticoids and cyclic AMP (cAMP) stimulate transcription of the IGFBP-1 gene expression whereas insulin acts in a dominant manner to suppress both basal and cAMP or glucocorticoid-stimulated IGFBP-1 gene transcription (O'Brien and Granner, Physiol. Rev., 1996, 76, 1109-1161). PEPCK catalyzes the rate-limiting step in gluconeogenesis, and thereby contributes to hepatic glucose output (Hall and Granner, J. Basic Clin. Physiol. Pharmacol., 1999, 10, 119-133; Moller, Nature, 2001, 414, 821-827; Saltiel and Kahn, Nature, 2001, 414, 799-806). In hepatoma cells, studies have shown that the expression of PEPCK is stimulated by glucocorticoids, glucagon (via cAMP), and retinoic acid. Insulin acts in a dominant manner to suppress these stimulations as well as basal transcription (O'Brien and Granner, Physiol. Rev., 1996, 76, 1109-1161). In HepG2 cells, prolonged serum starvation induces the expression of PEPCK and subsequent insulin stimulation significantly reduces the PEPCK mRNA level.
Follistatin is significantly stimulated by insulin in HepG2 cells. Interestingly, follistatin levels have been shown to be higher in women with polycystic ovary syndrome (PCOS) (Norman et al., Hum. Reprod., 2001, 16, 668-672). PCOS is a metabolic as well as a reproductive disorder, and an important cause of type 2 diabetes mellitus in women. It is often associated with profound insulin resistance and hyperinsulinemia as well as with a defect in insulin secretion (Dunaif, Endocr. Rev., 1997, 18, 774-800; Nestler et al., Fertil. Steril., 2002, 77, 209-215). PCOS is the most common cause of female infertility in the U.S. and affects 5%-10% of women of child-bearing age (Dunaif, Endocr. Rev., 1997, 18, 774-800; Nestler et al., Fertil. Steril., 2002, 77, 209-215).
In some embodiments, HepG2 cells are used to measure the effects of compounds of the invention on hepatic gene expression following insulin stimulation. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of hepatic gene expression. Insulin at a concentration of 100 nM may be used as a positive control for the stimulation of hepatic gene expression. An oligomeric compound targeted to human forkhead is known to inhibit hepatic gene expression and may be used as a positive control for the inhibition of gene expression in the presence of insulin.
Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINTM in OPTI-MEMTM to achieve a final concentration of 100 nM of oligomeric compound and 3 μg/mL LIPOFECTINTM. Untreated control cells receive LIPOFECTINTM only. Compounds of the invention and the positive controls are tested in triplicate, and the negative control is tested in up to six replicate wells.
Approximately 28 hours after transfection, the cells are subjected to serum starvation for a period of 12 to 16 hours, using serum-free growth medium. Following serum starvation, cells are treated with 1 nM insulin (insulin-treated) or are left untreated (basal conditions) for approximately four hours. At the same time, untreated control cells in both plates are treated with 100 nM insulin to determine the maximal insulin response. Following insulin treatment (forty-eight hours after transfection), total RNA is harvested from all samples, and the amount of total RNA from each sample is determined using a Ribogreen assay (Invitrogen Corporation, Carlsbad, Calif.). Real-time PCR is performed on the total RNA samples using primer/probe sets for three insulin responsive genes: insulin-like growth factor binding protein-1 (IGFBP-1), cytosolic PEPCK (PEPCK-C), and follistatin. Gene expression levels obtained by real-time PCR are normalized for total RNA content in the samples. Gene expression in cells treated with oligomeric compounds is normalized to that from untreated control cells. Values above or below 100% are considered to indicate an increase or decrease in gene expression, respectively.
Oligomeric compounds that interfere with the expression of genes involved in glucose metabolism are candidate therapeutic agents for the treatment of conditions associated with abnormal glucose metabolism, for example, obesity and diabetes.
Inflammation Assays:
In some embodiments, inflammation assays are used. Inflammation assays are designed to identify genes that regulate the activation and effector phases of the adaptive immune response. During the activation phase, T lymphocytes (also known as T-cells) receiving signals from the appropriate antigens undergo clonal expansion, secrete cytokines, and up-regulate their receptors for soluble growth factors, cytokines and co-stimulatory molecules (Cantrell, Annu. Rev. Immunol., 1996, 14, 259-274). These changes drive T-cell differentiation and effector function. Response to cytokines by non-immune effector cells controls the production of inflammatory mediators that can do extensive damage to host tissues. The cells of the adaptive immune systems, their products, as well as their interactions with various enzyme cascades involved in inflammation (e.g., the complement, clotting, fibrinolytic and kinin cascades) all represent potential points for intervention in inflammatory disease.
Dendritic cells treated with oligomeric compounds targeting different genes are used to identify regulators of dendritic cell-mediated T-cell co-stimulation. The level of interleukin-2 (IL-2) production by T-cells, a critical consequence of T-cell activation (DeSilva et al., J. Immunol., 1991, 147, 3261-3267; Salomon and Bluestone, Annu. Rev. Immunol., 2001, 19, 225-252), is used as an endpoint for T-cell activation. T lymphocytes are important immunoregulatory cells that mediate pathological inflammatory responses. Optimal activation of T lymphocytes requires both primary antigen recognition events as well as secondary or co-stimulatory signals from antigen presenting cells (APC). Dendritic cells are the most efficient APCs known and are principally responsible for antigen presentation to T-cells, expression of high levels of co-stimulatory molecules during infection and disease, and the induction and maintenance of immunological memory (Banchereau and Steinman, Nature, 1998, 392, 245-252). While a number of co-stimulatory ligand-receptor pairs have been shown to influence T-cell activation, a principal signal is delivered by engagement of CD28 on T-cells by CD80 (B7-1) and CD86 (B7-2) on APCs (Boussiotis et al., Curr. Opin. Immunol., 1994, 6, 797-807; Lenschow et al., Annu. Rev. Immunol., 1996, 14, 233-258). In contrast, a B7 counter-receptor, CTLA-4, has been shown to negatively regulate T-cell activation, maintain immunological homeostasis and promote immune tolerance (Walunas and Bluestone, J. Immunol., 1998, 160, 3855-3860) Inhibition of T-cell co-stimulation by APCs holds promise for novel and more specific strategies of immune suppression. In addition, blocking co-stimulatory signals may lead to the development of long-term immunological anergy (unresponsiveness or tolerance) that would offer utility for promoting transplantation or dampening autoimmunity. T-cell anergy is the direct consequence of failure of T-cells to produce the growth factor interleukin-2 (DeSilva et al., J. Immunol., 1991, 147, 3261-3267; Salomon and Bluestone, Annu. Rev. Immunol., 2001, 19, 225-252). Dendritic cell cytokine production as a measure of the activation phase of the immune response:
In some embodiments, the effects of the oligomeric compounds of the invention are examined on the dendritic cell-mediated costimulation of T-cells. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of dendritic cell-mediated T-cell costimulation. An oligomeric compound targeted to human CD86 is known to inhibit dendritic cell-mediated T-cell stimulation and may be used as a positive control.
Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINTM in OPTI-MEMTM to achieve a final concentration of 200 nM of oligomeric compound and 6 μg/mL LIPOFECTINTM. Untreated control cells receive LIPOFECTINTM only. Compounds of the invention and the positive control are tested in triplicate, and the negative control is tested in up to six replicates. Following incubation with the oligomeric compounds and LIPOFECTINTM, fresh growth medium with cytokines is added and DC culture is continued for an additional 48 hours. DCs are then co-cultured with Jurkat T-cells in RPMI medium (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Chemical Company, St. Louis, Mo.). Culture supernatants are collected 24 hours later and assayed for IL-2 levels (IL-2 DuoSet, R&D Systems, Minneapolis, Minn.). IL-2 levels in cells treated with oligomeric compounds are normalized to those from untreated control cells. A value greater than 100% indicates an induction of the inflammatory response, whereas a value less than 100% demonstrates a reduction in the inflammatory response.
Oligomeric compounds that inhibit T-cell co-stimulation are candidate therapeutic compounds with applications in the prevention, treatment or attenuation of conditions associated with hyperstimulation of the immune system, including rheumatoid arthritis, irritable bowel disease, asthma, lupus and multiple sclerosis. Oligomeric compounds that induce T-cell co-stimulation are candidate therapeutic agents for the treatment of immunodeficient conditions.
Cytokine Signaling as a Measure of the Effector Phase of the Inflammatory Response:
The cytokine signaling assay further identifies genes that regulate inflammatory responses of non-immune effector cells (initially endothelial cells) to stimulation with cytokines such as interferon-gamma (IFN-γ). Response to IFN-γ is assessed by measuring the expression levels of three genes: intercellular adhesion molecule-1 (ICAM-1), interferon regulatory factor 1 (IRF1) and small inducible cytokine subfamily B (Cys-X-Cys), member 11 (SCYB11). The cytokine signaling assay further identifies genes that regulate inflammatory responses of non-immune effector cells (initially endothelial cells) to stimulation with IL-1β or TNF-α (Heyninck et al., J Cell Biol, 1999, 145, 1471-1482; Zetoune et al., Cytokine, 2001, 15, 282-298). Response to IL-1β or TNF-α stimulation is monitored by measuring the expression levels of four genes: A20, intracellular adhesion molecule 1 (ICAM-1), interleukin-9 (IL-8) and macrophage-inflammatory protein 2 (MIP2α). As described below, all of these genes regulate numerous parameters of the inflammatory response.
ICAM-1 is an adhesion molecule expressed at low levels on resting endothelial cells that is markedly up-regulated in response to inflammatory mediators like tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interferon-γ (IFN-γ) (Springer, Nature, 1990, 346, 425-434). ICAM-1 expression serves to attract circulating leukocytes into the inflammatory site.
IRF-1 binds to upstream cis-regulatory elements of interferon-inducible genes and functions as a transcriptional activator. IRF-1 directly binds to a functional IFN-γ-stimulated response element in the cathepsin S promoter and mediates IFN-γ dependent transcriptional activation (Storm van's Gravesande et al., J Immunol, 2002, 168, 4488-4494).
SCYB11 is essential for mediating normal leukocyte recruitment and trafficking during inflammation. SCYB11 induces a chemotactic response in IL-2 activated T-cells, monocytes and granulocytes (Mohan et al., J Immunol, 2002, 168, 6420-6428).
A20 is a zinc-finger protein that limits the transcription of pro-inflammatory genes by blocking TRAF2-stimulated NK-κB signaling Studies in mice show that TNF-α dramatically increases A20 expression in mice, and that A20 expression is crucial for their survival (Lee et al., Science, 2000, 289, 2350-2354).
IL-8 is a member of the chemokine gene superfamily, members of which promote the pro-inflammatory phenotype of macrophages, vascular smooth muscle cells and endothelial cells (Koch et al., Science, 1992, 258, 1798-1801). IL-8 has been known as one of the major inducible chemokines with the ability to attract neutrophils to the site of inflammation. More recently, IL-8 has been implicated as a major mediator of acute neutrophil-mediated inflammation, and is therefore a potential anti-inflammatory target (Mukaida et al., Cytokine Growth Factor Rev, 1998, 9, 9-23).
MIP2α, another chemokine known to play a central role in leukocyte extravasation, has more recently been shown to be involved in acute inflammation (Lukacs et al., Chem Immunol, 1999, 72, 102-120). MIP2α is expressed in response to microbial infection, to injection of lipopolysaccharides (LPS), and to stimulation of cells with pro-inflammatory mediators such as IL-1β and TNF-α (Kopydlowski et al., J Immunol, 1999, 163, 1537-1544). Endothelial cells are one of several cell types that are sources of MIP2α (Rudner et al., J Immunol, 2000, 164, 6576-6582).
In some embodiments, the effects of the oligomeric compounds of the invention on the cellular response to cytokines may be examined in HUVECs. A 20-nucleotide oligomeric compound with a randomized sequence may be used as a negative control, as it does not target modulators of cytokine signaling.
Cells are transfected as described herein. Oligomeric compounds are mixed with LIPOFECTINTM in OPTI-MEMTM to achieve a final concentration of 75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTINTM. Untreated control cells receive LIPOFECTINTM only. Compounds of the invention are tested in triplicate, and the negative control is tested in up to six replicate wells.
For IFN-γ stimulation, following transfection, fresh growth medium is added and DC culture is continued for an additional 44 hours, after which HUVECS are stimulated with 10 ng/ml of IFN-γ for a period of 4 hours. For stimulation with IL-1β or TNF-α, fresh growth medium is added and DC culture is continued for an additional 46 hours, after which HUVECs are stimulated with 0.1 ng/mL of IL-1β or 1 ng/mL of TNF-α for a period of 2 hours. Total RNA is harvested 48 hours following transfection, and real time PCR is performed using primer/probe sets to detect ICAM-1, IRF-1 and SCYB11 in IFN-γ-stimulated cells, or ICAM-1, A20, IL-8 and MIP2α in IL-1β-stimulated and TNF-α-stimulated cells. Expression levels of each gene are normalized to total RNA. Gene expression levels from cells treated with oligomeric compounds are normalized to those from untreated control cells. A value greater than 100% indicates an induction of the inflammatory response, whereas a value less than 100% demonstrates a reduction in the inflammatory response.
Oligomeric compounds that inhibit the inflammatory response are candidate therapeutic compounds with applications in the prevention, treatment or attenuation of conditions associated with hyperstimulation of the immune system, including rheumatoid arthritis, irritable bowel disease, asthma, lupus and multiple sclerosis.
In Vivo Studies
The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.
Mouse Model of Tumorigenesis:
Animal models of tumorigenesis are used in some embodiments of the present invention. In this model, tumorigenic cells are injected into immunocompromised mice (i.e. nude mice), and subsequent growth of a tumor is measured.
Serially transplanted MDA-MB-231 (a human breast carcinoma cell line, American Type Culture Collection, Manassas, Va.) tumors are established subcutaneously in nude mice. Beginning two weeks later, one or more of the oligomeric compounds of the invention are administered intravenously daily for 14 days at dosages of 15 mg/kg or 30 mg/kg. Control compounds are also administered at these doses, and a saline control is also given. Tumor growth rates are monitored for the two-week period of oligonucleotide administration. Activity of the oligomeric compounds of the invention is measured by a reduction in tumor growth. Activity is measured by reduced tumor volume compared to saline or control compound. Following death or sacrifice of mice, tumor tissue is fixed in 4% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Tumor tissue sections are evaluated for tumor morphology and size.
Human A549 lung tumor cells are also injected into nude mouse to produce tumors. 200 μl of A549 cells (5×106 cells) are implanted subcutaneously in the inner thigh of nude mice. Oligomeric compounds of the invention are administered twice weekly for four weeks, beginning one week following tumor cell inoculation. Oligomeric compounds are formulated with cationic lipids (LIPOFECTINTM, Invitrogen Corporation, Carlsbad, Calif.) and given subcutaneously in the vicinity of the tumor. Oligomeric compound dosage is 5 mg/kg with 60 mg/kg cationic lipid. Tumor size is recorded weekly. Activity of the oligomeric compounds of the invention is measured by reduction in tumor size compared to controls.
Xenograft studies are also performed using the U-87 human glioblastoma cell line (American Type Culture Collection, Manassas, Va.). Nude mice are injected subcutaneously with 2×107 U-87 cells. Mice are injected intraperitoneally with one or more of the oligomeric compounds of the invention or a control compound at dosages of either 15 mg/kg or 30 mg/kg for 21 consecutive days beginning 7 days after xenografts are implanted. Saline-injected animals serve as a control. Tumor volumes are measured on days 14, 21, 24, 31 and 35. Activity is measured by reduced tumor volume compared to saline or control compound. Following death or sacrifice of mice, tumor tissue is fixed in 4% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Tumor tissue sections are evaluated for tumor morphology and size.
Alternatively, intracerebral U-87 xenografts are generated by implanting U-87 glioblastoma cells into the brains of nude mice. Mice are treated via continuous intraperitoneal administration with one or more of the oligomeric compounds of the invention at 20 mg/kg, control compound at 20 mg/kg or saline beginning on day 7 after xenograft implantation. Activity of the oligomeric compounds of the invention is measured by an increase in survival time compared to controls. Following death or sacrifice, brain tissue is fixed in 4% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Brain tissue sections are evaluated for tumor growth.
Leptin-Deficient Mice (a Model of Obesity and Diabetes (ob/ob Mice)):
Leptin is a hormone produced by fat cells that regulates appetite. Deficiencies in this hormone in both humans and non-human animals leads to obesity. ob/ob mice have a mutation in the leptin gene which results in obesity and hyperglycemia. As such, these mice are a useful model for the investigation of obesity and diabetes and treatments designed to treat these conditions. ob/ob mice have higher circulating levels of insulin and are less hyperglycemic than db/db mice, which harbor a mutation in the leptin receptor. In accordance with the present invention, the oligomeric compounds of the invention are tested in the ob/ob model of obesity and diabetes.
Seven-week old male C57B1/6J-Lep ob/ob mice (Jackson Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of 10-15% and are subcutaneously injected with the oligomeric compounds of the invention or a control compound at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals, leptin wildtype littermates (i.e. lean littermates) and ob/ob mice fed a standard rodent diet serve as controls. After the treatment period, mice are sacrificed and target levels are evaluated in liver, brown adipose tissue (BAT) and white adipose tissue (WAT). RNA isolation and target RNA expression level quantitation are performed as described by other examples herein.
To assess the physiological effects resulting from modulation of target, the ob/ob mice are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis, or clearing of lipids from the liver, is assessed by measuring the liver triglyceride content. Hepatic steatosis is assessed by routine histological analysis of frozen liver tissue sections stained with oil red O stain, which is commonly used to visualize lipid deposits, and counterstained with hematoxylin and eosin, to visualize nuclei and cytoplasm, respectively.
The effects of target modulation on glucose and insulin metabolism are evaluated in the ob/ob mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following at 2 weeks and at 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.
To assess the metabolic rate of ob/ob mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice are also measured.
The ob/ob mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism. These genes include, but are not limited to, HMG-CoA reductase, acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2, carnitine palmitoyltransferase I and glycogen phosphorylase, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1, lipoprotein lipase and hormone sensitive lipase. mRNA levels in liver and white and brown adipose tissue are quantitated by real-time PCR as described in other examples herein, employing primer/probe sets that are generated using published sequences of each gene of interest.
Leptin Receptor-Deficient Mice (a Model of Obesity and Diabetes (db/db Mice)):
db/db mice have a mutation in the leptin receptor gene which results in obesity and hyperglycemia. As such, these mice are a useful model for the investigation of obesity and diabetes and treatments designed to treat these conditions. db/db mice, which have lower circulating levels of insulin and are more hyperglycemic than ob/ob mice which harbor a mutation in the leptin gene, are often used as a rodent model of type 2 diabetes. In accordance with the present invention, oligomeric compounds of the present invention are tested in the db/db model of obesity and diabetes.
Seven-week old male C57B1/6J-Lepr db/db mice (Jackson Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of 15-20% and are subcutaneously injected with one or more of the oligomeric compounds of the invention or a control compound at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals, leptin receptor wildtype littermates (i.e. lean littermates) and db/db mice fed a standard rodent diet serve as controls. After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.
To assess the physiological effects resulting from modulation of target, the db/db mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.
The effects of target modulation on glucose and insulin metabolism are also evaluated in the db/db mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment.
Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose levels are measured before the insulin or glucose challenge and 15, 30, 60, 90 and 120 minutes following the injection.
To assess the metabolic rate of db/db mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is also measured.
The db/db mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.
Lean Mice on a Standard Rodent Diet:
C57B1/6 mice are maintained on a standard rodent diet and are used as control (lean) animals. In one embodiment of the present invention, the oligomeric compounds of the invention are tested in normal, lean animals.
Seven-week old male C57B1/6 mice are fed a diet with a fat content of 4% and are subcutaneously injected with one or more of the oligomeric compounds of the invention or control compounds at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals serve as a control. After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.
To assess the physiological effects resulting from modulation of the target, the lean mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.
The effects of target modulation on glucose and insulin metabolism are also evaluated in the lean mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose levels are measured before the insulin or glucose challenge and 15, 30, 60, 90 and 120 minutes following the injection.
To assess the metabolic rate of lean mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is also measured.
The lean mice that received treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.
Levin Model of Diet-Induced Obesity in Rats:
The Levin Model is a polygenic model of rats selectively bred to develop diet-induced obesity (DIO) associated with impaired glucose tolerance, dyslipidemia and insulin resistance when fed a high-fat diet (Levin, et al., Am. J. Physiol, 1997, 273, R725-30). The advantage of this model is that it displays traits more similar to human obesity and glucose intolerance than in animals that are obese/hyperinsulinemic due to genetic defects e.g. defects in leptin signaling. This model is useful in investigating the oligomeric compounds of the present invention for their ability to affect obesity and related complications, such as impaired glucose tolerance, dyslipidemia and insulin resistance. In accordance with the present invention, the oligomeric compounds of the invention are tested in the Levin model of diet-induced obesity.
Eight-week old male Levin rats (Charles River Laboratories, Wilmington, Mass.), weighing  ̃500 g, are fed a diet with a fat content of 60% for eight weeks, after which they are subcutaneously injected with one or more of the oligomeric compounds of the invention at a dose of 25 mg/kg×2 per week for 8 weeks. Control groups consist of animals injected with saline or a control compound and lean littermates fed a standard rodent diet. The control compound is injected at the same dose as the target-specific compound.
Throughout the treatment period, the rats are evaluated for food consumption, weight gain, as well as serum levels of glucose, insulin, cholesterol, free fatty acids, triglycerides and liver enzymes.
The effects of target modulation on glucose and insulin metabolism are also evaluated in the Levin rats treated with the oligomeric compounds of the invention. Plasma glucose and insulin are monitored throughout the treatment by analyzing blood samples. Glucose and tolerance are assessed in fed or fasted rats. After blood is collected for baseline glucose and insulin levels, a glucose challenge is administered, after which blood glucose and insulin levels are measured at 15, 20 or 30 minute intervals for up to 3 hours. Insulin tolerance is similarly analyzed, beginning with blood collection for baseline glucose and insulin levels, followed by an insulin challenge, after which blood glucose levels are measured at 15, 20 or 30 minute intervals for up to 3 hours. Plasma insulin and glucose are also measured at study termination.
At the end of the treatment period, the rats are sacrificed. Organs are removed and weighed, including liver, white adipose tissue, brown adipose tissue and spleen. Target RNA expression levels are measured in all tissues that are isolated, using quantitative real-time PCR. Target protein levels are also evaluated by immunoblot analysis using antibodies that specifically recognize the target protein.
Also evaluated at the end of the treatment period are serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.
The Levin rats that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.
C57BL/6 on a high-fat diet (a model of diet-induced obesity (DIO)):
The C57BL/6 mouse strain is reported to be susceptible to hyperlipidemia-induced atherosclerotic plaque formation. Consequently, when these mice are fed a high-fat diet, they develop diet-induced obesity. Accordingly these mice are a useful model for the investigation of obesity and treatments designed to treat these conditions. In one embodiment of the present invention, the oligomeric compounds of the invention are tested in a model of diet-induced obesity.
Male C57BL/6 mice (7-weeks old) receive a 60% fat diet for 8 weeks, after which mice are subcutaneously injected with one or more of the oligomeric compounds of the invention at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected or control compound-injected animals serve as a control. After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.
To assess the physiological effects resulting from modulation of target, the diet-induced obese mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.
The effects of target modulation on glucose and insulin metabolism are also evaluated in the diet-induced obese mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.
To assess the metabolic rate of diet-induced obese mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is also measured.
The diet-induced obese mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.
P-407 Mouse Model of Hyperlipidemia:
Poloxamer 407 (P-407), an inert block copolymer comprising a hydrophobic core flanked by hydrophilic polyoxyethelene units has been shown to induce hyperlipidemia in rodents. In the mouse, one injection, intraperitoneally, of P-407 (0.5 g/kg) produced hypercholesterolemia that peaked at 24 hours and returned to control levels by 96 hours following treatment (Palmer, et al., Atherosclerosis, 1998, 136, 115-123). Consequently, these mice are a useful model for the investigation of compounds that modulate hyperlipidemia. In accordance with the present invention, the oligomeric compounds of the invention are tested in the P-407 model of hyperlipidemia.
Seven-week old male C57B1/6 mice are divided into two groups; (1) control and (2) P-407 injected animals (0.5 g/kg every 3 days, following an overnight fast). Animals in each group receive either a saline injection or injection with one or more of the oligomeric compounds of the invention or control compounds at 25 mg/kg three times per week or 50 mg/kg two times per week. All injections are administered intraperitoneally.
After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.
To assess the physiological effects resulting from modulation of target, the P-407 injected animals that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.
The effects of target modulation on glucose and insulin metabolism are evaluated in the P-407 injected animals treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.
To assess the metabolic rate of P-407 injected animals treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice is measured.
The P-407 injected animals that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.
ApoE Knockout Mice (a Model of Dyslipidemia and Obesity):
B6.129P-ApoEtm1Unc knockout mice (herein referred to as ApoE knockout mice) obtained from The Jackson Laboratory (Bar Harbor, Me.), are homozygous for the Apoetm1Unc mutation and show a marked increase in total plasma cholesterol levels that are unaffected by age or sex. These animals present with fatty streaks in the proximal aorta at 3 months of age. These lesions increase with age and progress to lesions with less lipid but more elongated cells, typical of a more advanced stage of pre-atherosclerotic lesion.
The mutation in these mice resides in the apolipoprotein E (ApoE) gene. The primary role of the ApoE protein is to transport cholesterol and triglycerides throughout the body. It stabilizes lipoprotein structure, binds to the low density lipoprotein receptor (LDLR) and related proteins, and is present in a subclass of HDLs, providing them the ability to bind to LDLR. ApoE is expressed most abundantly in the liver and brain. In one embodiment of the present invention, female B6.129P-Apoetm1Unc knockout mice (ApoE knockout mice) are used in the following studies to evaluate the oligomeric compounds of the invention as potential lipid lowering compounds.
Female ApoE knockout mice range in age from 5 to 7 weeks and are placed on a normal diet for 2 weeks before study initiation. ApoE knockout mice are then fed ad libitum a 60% fat diet, with 0.15% added cholesterol to induce dyslipidemia and obesity. Control animals include ApoE knockout mice and ApoE wildtype mice (i.e. lean littermates) maintained on a high-fat diet with no added cholesterol. After overnight fasting, mice from each group are dosed intraperitoneally every three days with saline, 50 mg/kg of a control compound or 5, 25 or 50 mg/kg of one or more of the oligomeric compounds of the invention.
After the treatment period, mice are sacrificed and target levels are evaluated in liver, BAT and WAT as described supra.
To assess the physiological effects resulting from modulation of target, the ApoE knockout mice that receive treatment are evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis is assessed by measuring the liver triglyceride content and oil red O staining, as described supra.
The effects of target modulation on glucose and insulin metabolism are also evaluated in the ApoE knockout mice treated with the oligomeric compounds of the invention. Plasma glucose is measured at the start of the treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly measured at the beginning of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.
To assess the metabolic rate of ApoE knockout mice treated with the oligomeric compounds of the invention, the respiratory quotient and oxygen consumption of the mice are measured.
The ApoE knockout mice that receive treatment are evaluated at the end of the treatment period for the effects of target modulation on the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, as described supra.
In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner. Throughout these examples, molecular cloning reactions, and other standard recombinant DNA techniques, were carried out according to methods described in Maniatis et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, except where otherwise noted.
EXAMPLES
Example 1
Oligomeric Compounds Targeting Small Non-Coding RNAs
In accordance with the present invention, a series of oligomeric compounds are designed to target different regions of small non-coding target RNAs. The oligomeric compounds can be investigated for their effect on small non-coding RNA levels by quantitative real-time PCR. The target regions to which these sequences are complementary are herein referred to as “suitable target regions”.
Example 2
Oligomeric Compounds that Mimic or Replace Small Non-Coding RNAs
In accordance with the present invention, a series of oligomeric compounds are designed to mimic the structure and/or function of small non-coding RNAs. These mimics may include isolated single-, double-, or multiple-stranded compounds, any of which may include regions of intrastrand nucleobase complementarity, said regions capable of folding and forming a molecule with fully or partially double-stranded or multiple-stranded character based on regions of precise or imperfect complementarity. The oligomeric compound mimics can then be investigated for their effects on a cell, tissue or organism system lacking endogenous small non-coding RNAs or systems with aberrant expression of small non-coding RNAs using the screening methods disclosed herein or those commonly used in the art. Changes in levels, expression or function of the small non-coding RNA or its downstream target nucleic acid levels can be analyzed by quantitative real-time PCR as described, supra.
Example 3
Pri-miRNAs Targeted by Compounds of the Present Invention
In accordance with the present invention, oligomeric compounds were designed to target one or more microRNA (miRNA) genes or gene products. Certain pri-miRNAs have been reported by Lim et al. Science, 2003, 299; 1540; in Brevia (detailed in the supplemental online materials; www.sciencemag.org/cgi/content/full/299/5612/1540/DC1) and these were used as starting targets.
A list of pri-miRNAs targeted is shown in Table 1. The gene name for each of the 188 targets (assigned by Lim et al.) is given in the table. For those pri-miRNAs that did not produce an identifiable miRNA detectable by PCR in the Lim publication, the position and sequence of the miRNAs were identified herein and are referred to as novel or hypothetical miRNAs. Also shown is the Genbank Accession number of the source sequence from which the pri-miRNA was extracted. The sequence is set forth in the Sequence Listing and is written in the 5′ to 3′ direction and is represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.







TABLE 1







pri-miRNAs












Genbank





Accession #





of source
SEQ



pri-miRNA
sequence
ID NO















mir-140
NT_037896.1
4



mir-30a
NT_007299.11
5



mir-34
NT_028054.10
6



mir-29b-1
NT_021877.13
7



mir-29b-2
NT_007933.10
8



mir-16-3
NT_005612.11
9



mir-203
NT_026437.9
10



mir-7-1
NT_023935.13
11



mir-10b
NT_037537.1
12



mir-128a
NT_034487.2
13



mir-153-1
NT_005403.11
14



mir-153-2
NT_007741.10
15



hypothetical miRNA-013
NT_010194.13
16



mir-27b
NT_008476.13
17



mir-96
NT_007933.10
18



mir-17as/mir-91
NT_009952.11
19



mir-123/mir-126as
NT_024000.13
20



mir-132
NT_010692.9
21



mir-108-1
NT_010799.11
22



mir-23b
NT_008476.13
23



let-7i
NT_009711.13
24



mir-212
NT_010692.9
25



hypothetical miRNA-023
NT_004658.12
26



mir-131-2
NT_029973.6
27



let-7b
NT_011523.8
28



mir-1d
NT_035608.1
29



mir-122a
NT_033907.3
30



mir-22
NT_010692.9
31



mir-92-1
NT_009952.11
32



hypothetical miRNA-030
NT_007933.10
33



mir-142
NT_010783.11
34



mir-183
NT_007933.10
35



hypothetical miRNA-033
NT_011588.11
36



mir-214
NT_029874.7
37



mir-143
NT_006859.11
38



mir-192-1
NT_033241.3
39



mir-192-2
NT_033241.3
39



mir-192-3
NT_033241.3
39



hypothetical miRNA-039
NT_028392.4
42



hypothetical miRNA-040
NT_023148.9
43



hypothetical miRNA-041
NT_023089.11
44



let-7a-3
NT_011523.8
45



hypothetical miRNA-043
NT_004902.12
46



hypothetical miRNA-044
NT_009952.11
47



mir-181a
NT_017568.11
48



let-7a-1
NT_008476.13
49



mir-205
NT_021877.13
50



mir-103-1
NT_037665.1
51



mir-26a
NT_005580.13
52



mir-33a
NT_011520.8
53



mir-196-2
NT_009458.12
54



mir-107
NT_033890.3
55



mir-106
NT_011786.11
56



let-7f-1
NT_008476.13
57



hypothetical miRNA-055
NT_006713.11
58



mir-29c
NT_021877.13
59



mir-130a
NT_033903.3
60



hypothetical miRNA-058
NT_037537.1
61



mir-218-1
NT_006316.13
62



mir-124a-2
NT_008183.13
63



mir-21
NT_035426.2
64



mir-16-1
NT_033922.3
65



mir-144
NT_010799.11
66



mir-221
NT_011568.10
67



mir-222
NT_011568.10
68



mir-30d
NT_028251.8
69



mir-19b-2
NT_011786.11
70



mir-128b
NT_005580.13
71



hypothetical miRNA-069
NT_017568.11
72



hypothetical miRNA-070
NT_005375.11
73



hypothetical miRNA-071
NT_011512.7
74



mir-29b-3
NT_007933.10
75



mir-129-2
NT_009237.13
76



mir-133b
NT_007592.11
77



hypothetical miRNA-075
NT_006044.8
78



let-7d
NT_008476.13
79



mir-15b
NT_005612.11
80



mir-29a-1
NT_007933.10
81



hypothetical miRNA-079
NT_021907.13
82



mir-199b
NT_017568.11
83



mir-129-1
NT_007933.10
84



let-7e
NT_011109.13
85



hypothetical miRNA-083
NT_024524.11
86



let-7c
NT_011512.7
87



mir-204
NT_008580.11
88



mir-145
NT_006859.11
89



mir-124a-1
NT_019483.13
90



hypothetical miRNA-088
NT_011519.9
91



mir-213
NT_029862.8
92



hypothetical miRNA-090
NT_006171.13
93



mir-20
NT_009952.11
94



mir-133a-1
NT_011044.11
95



mir-138-2
NT_010498.11
96



mir-98
NT_011799.10
97



mir-196-1
NT_010783.11
98



mir-125b-1
NT_033899.3
99



mir-199a-2
NT_029874.7
100



mir-29a-2
NT_007933.10
101



hypothetical miRNA-099
NT_016297.12
102



mir-181b
NT_029862.8
103



hypothetical miRNA-101
NT_030828.7
104



mir-141
NT_035206.1
105



mir-131-1
NT_004858.13
106



mir-133a-2
NT_035608.1
107



hypothetical miRNA-105
NT_017795.13
108



hypothetical miRNA-106
NT_017795.13
109



hypothetical miRNA-107
NT_008583.13
110



mir-1b
NT_011044.11
111



mir-18
NT_009952.11
112



mir-220
NT_011588.11
113



hypothetical miRNA-111
NT_004525.13
114



mir-7-3
NT_011255.11
115



mir-218-2
NT_023132.10
116



mir-24-2
NT_031915.4
117



mir-24-1
NT_008476.13
118



mir-103-2
NT_011387.8
119



mir-211
NT_010363.13
120



mir-101-3
NT_008413.13
121



mir-30b
NT_028251.8
122



hypothetical miRNA-120
NT_009952.11
123



let-7a-4
NT_033899.3
124



mir-10a
NT_010783.11
125



mir-19a
NT_009952.11
126



let-7f-2
NT_011799.10
127



mir-15a-1
NT_010393.11
128



mir-108-2
NT_034392.2
129



mir-137
NT_033951.3
130



mir-219
NT_007592.11
131



mir-148b
NT_009458.12
132



mir-130b
NT_011520.8
133



mir-19b-1
NT_009952.11
134



let-7a-2
NT_033899.3
135



mir-216
NT_005375.11
136



mir-100-1
NT_033899.3
137



mir-100-2
NT_033899.3
137



mir-187
NT_010966.11
139



hypothetical miRNA-137
NT_011387.8
140



hypothetical miRNA-138
NT_008902.13
141



hypothetical miRNA-139
NT_008902.13
142



mir-124a-3
NT_011333.5
143



mir-7-2
NT_033276.3
144



hypothetical miRNA-142
NT_033317.3
145



hypothetical miRNA-143
NT_007819.11
146



hypothetical miRNA-144
NT_010783.11
147



mir-210
NT_035113.2
148



mir-215
NT_021953.13
149



mir-223
NT_011669.11
150



mir-131-3
NT_033276.3
151



mir-199a-1
NT_011176.13
152



mir-30c
NT_007299.11
153



mir-101-1
NT_029865.8
154



mir-101-2
NT_029865.8
154



hypothetical miRNA-153
NT_005332.11
156



hypothetical miRNA-154
NT_030828.7
157



mir-26b
NT_005403.11
158



hypothetical miRNA-156
NT_029289.7
159



mir-152
NT_010783.11
160



mir-135-1
NT_005986.13
161



mir-135-2
NT_009681.13
162



mir-217
NT_005375.11
163



hypothetical miRNA-161
NT_004658.12
164



mir-15a-2
NT_033922.3
165



let-7g
NT_005986.13
166



hypothetical miRNA-164
NT_010783.11
167



mir-33b
NT_030843.4
168



hypothetical miRNA-166
NT_011588.11
169



mir-16-2
NT_033922.3
170



hypothetical miRNA-168
NT_011520.8
171



hypothetical miRNA-169
NT_007933.10
172



hypothetical miRNA-170
NT_005151.11
173



hypothetical miRNA-171
NT_006171.13
174



hypothetical miRNA-172
NT_037752.1
175



hypothetical miRNA-173
NT_008413.13
176



mir-182
NT_007933.10
177



hypothetical miRNA-175
NT_006258.12
178



hypothetical miRNA-176
NT_025004.11
179



hypothetical miRNA-177
NT_023098.7
180



hypothetical miRNA-178
NT_037537.1
181



hypothetical miRNA-179
NT_010194.13
182



hypothetical miRNA-180
NT_010363.13
183



hypothetical miRNA-181
NT_033899.3
184



mir-148a
NT_007819.11
185



hypothetical miRNA-183
NT_010363.13
186



mir-23a
NT_031915.4
187



hypothetical miRNA-185
NT_007592.11
188



hypothetical miRNA-186
NT_008705.13
189



mir-181c
NT_031915.4
190



hypothetical miRNA-188
NT_023148.9
191










Example 4
miRNAs within pri-miRNAs
miRNAs found within the pri-miRNA structures disclosed above were used in certain embodiments of the present invention. These miRNAs represent target nucleic acids to which the oligomeric compounds of the present invention were designed. The oligomeric compounds of the present invention can also be designed to mimic the miRNA while incorporating certain chemical modifications that alter one or more properties of the mimic, thereby creating a construct with superior properties over the endogenous miRNA. The miRNA target sequences are shown in Table 2.







TABLE 2







miRNAs found within pri-miRNAs










miRNA sequence
SEQ ID


Pri-miRNA
(DNA form; where T replaces U in RNA)
NO





mir-140
AGTGGTTTTACCCTATGGTAG
192


mir-30a
CTTTCAGTCGGATGTTTGCAGC
193


mir-34
TGGCAGTGTCTTAGCTGGTTGT
194


mir-29b-1
TAGCACCATTTGAAATCAGTGTT
195


mir-29b-2
TAGCACCATTTGAAATCAGTGTT
195


mir-16-3
TAGCAGCACGTAAATATTGGCG
196


mir-203
GTGAAATGTTTAGGACCACTAG
197


mir-7-1
TGGAAGACTAGTGATTTTGTT
198


mir-10b
TACCCTGTAGAACCGAATTTGT
199


mir-128a
TCACAGTGAACCGGTCTCTTTT
200


mir-153-1
TTGCATAGTCACAAAAGTGA
201


mir-153-2
TTGCATAGTCACAAAAGTGA
201


mir-27b
TTCACAGTGGCTAAGTTCTG
202


mir-96
TTTGGCACTAGCACATTTTTGC
203


mir-17as/mir-91
CAAAGTGCTTACAGTGCAGGTAGT
204


mir-123/mir-126as
CATTATTACTTTTGGTACGCG
205


mir-132
TAACAGTCTACAGCCATGGTCGC
206


mir-108-1
ATAAGGATTTTTAGGGGCATT
207


mir-23b
ATCACATTGCCAGGGATTACCAC
208


let-7i
TGAGGTAGTAGTTTGTGCT
209


mir-212
TAACAGTCTCCAGTCACGGCC
210


mir-131-2
TAAAGCTAGATAACCGAAAGT
211


let-7b
TGAGGTAGTAGGTTGTGTGGTT
212


mir-1d
TGGAATGTAAAGAAGTATGTAT
213


mir-122a
TGGAGTGTGACAATGGTGTTTGT
214


mir-22
AAGCTGCCAGTTGAAGAACTGT
215


mir-92-1
TATTGCACTTGTCCCGGCCTGT
216


mir-142
CATAAAGTAGAAAGCACTAC
217


mir-183
TATGGCACTGGTAGAATTCACTG
218


mir-214
ACAGCAGGCACAGACAGGCAG
219


mir-143
TGAGATGAAGCACTGTAGCTCA
220


mir-192-1
CTGACCTATGAATTGACAGCC
221


mir-192-2
CTGACCTATGAATTGACAGCC
221


mir-192-3
CTGACCTATGAATTGACAGCC
221


let-7a-3
TGAGGTAGTAGGTTGTATAGTT
222


mir-181a
AACATTCAACGCTGTCGGTGAGT
223


let-7a-1
TGAGGTAGTAGGTTGTATAGTT
222


mir-205
TCCTTCATTCCACCGGAGTCTG
224


mir-103-1
AGCAGCATTGTACAGGGCTATGA
225


mir-26a
TTCAAGTAATCCAGGATAGGCT
226


mir-33a
GTGCATTGTAGTTGCATTG
227


mir-196-2
TAGGTAGTTTCATGTTGTTGGG
228


mir-107
AGCAGCATTGTACAGGGCTATCA
229


mir-106
AAAAGTGCTTACAGTGCAGGTAGC
230


let-7f-1
TGAGGTAGTAGATTGTATAGTT
231


mir-29c
CTAGCACCATTTGAAATCGGTT
232


mir-130a
CAGTGCAATGTTAAAAGGGC
233


mir-218-1
TTGTGCTTGATCTAACCATGT
234


mir-124a-2
TTAAGGCACGCGGTGAATGCCA
235


mir-21
TAGCTTATCAGACTGATGTTGA
236


mir-16-1
TAGCAGCACGTAAATATTGGCG
196


mir-144
TACAGTATAGATGATGTACTAG
237


mir-221
AGCTACATTGTCTGCTGGGTTTC
238


mir-222
AGCTACATCTGGCTACTGGGTCTC
239


mir-30d
TGTAAACATCCCCGACTGGAAG
240


mir-19b-2
TGTGCAAATCCATGCAAAACTGA
241


mir-128b
TCACAGTGAACCGGTCTCTTTC
242


mir-29b-3
TAGCACCATTTGAAATCAGTGTT
195


mir-129-2
CTTTTTGCGGTCTGGGCTTGC
243


mir-133b
TTGGTCCCCTTCAACCAGCTA
244


let-7d
AGAGGTAGTAGGTTGCATAGT
245


mir-15b
TAGCAGCACATCATGGTTTACA
246


mir-29a-1
CTAGCACCATCTGAAATCGGTT
247


mir-199b
CCCAGTGTTTAGACTATCTGTTC
248


mir-129-1
CTTTTTGCGGTCTGGGCTTGC
243


let-7e
TGAGGTAGGAGGTTGTATAGT
249


let-7c
TGAGGTAGTAGGTTGTATGGTT
250


mir-204
TTCCCTTTGTCATCCTATGCCT
251


mir-145
GTCCAGTTTTCCCAGGAATCCCTT
252


mir-124a-1
TTAAGGCACGCGGTGAATGCCA
235


mir-213
ACCATCGACCGTTGATTGTACC
253


mir-20
TAAAGTGCTTATAGTGCAGGTAG
254


mir-133a-1
TTGGTCCCCTTCAACCAGCTGT
255


mir-138-2
AGCTGGTGTTGTGAATC
256


mir-98
TGAGGTAGTAAGTTGTATTGTT
257


mir-196-1
TAGGTAGTTTCATGTTGTTGGG
228


mir-125b-1
TCCCTGAGACCCTAACTTGTGA
258


mir-199a-2
CCCAGTGTTCAGACTACCTGTTC
259


mir-29a-2
CTAGCACCATCTGAAATCGGTT
247


mir-181b
AACATTCATTGCTGTCGGTGGGTT
260


mir-141
AACACTGTCTGGTAAAGATGG
261


mir-131-1
TAAAGCTAGATAACCGAAAGT
211


mir-133a-2
TTGGTCCCCTTCAACCAGCTGT
255


mir-1b
TGGAATGTAAAGAAGTATGTAT
213


mir-18
TAAGGTGCATCTAGTGCAGATA
262


mir-220
CCACACCGTATCTGACACTTT
263


mir-7-3
TGGAAGACTAGTGATTTTGTT
198


mir-218-2
TTGTGCTTGATCTAACCATGT
234


mir-24-2
TGGCTCAGTTCAGCAGGAACAG
264


mir-24-1
TGGCTCAGTTCAGCAGGAACAG
264


mir-103-2
AGCAGCATTGTACAGGGCTATGA
225


mir-211
TTCCCTTTGTCATCCTTCGCCT
264


mir-101-3
TACAGTACTGTGATAACTGA
265


mir-30b
TGTAAACATCCTACACTCAGC
266


let-7a-4
TGAGGTAGTAGGTTGTATAGTT
222


mir-10a
TACCCTGTAGATCCGAATTTGTG
267


mir-19a
TGTGCAAATCTATGCAAAACTGA
268


let-7f-2
TGAGGTAGTAGATTGTATAGTT
231


mir-15a-1
TAGCAGCACATAATGGTTTGTG
269


mir-108-2
ATAAGGATTTTTAGGGGCATT
207


mir-137
TATTGCTTAAGAATACGCGTAG
270


mir-219
TGATTGTCCAAACGCAATTCT
271


mir-148b
TCAGTGCATCACAGAACTTTGT
272


mir-130b
CAGTGCAATGATGAAAGGGC
273


mir-19b-1
TGTGCAAATCCATGCAAAACTGA
241


let-7a-2
TGAGGTAGTAGGTTGTATAGTT
222


mir-216
TAATCTCAGCTGGCAACTGTG
274


mir-100-1
AACCCGTAGATCCGAACTTGTG
275


mir-100-2
AACCCGTAGATCCGAACTTGTG
275


mir-187
TCGTGTCTTGTGTTGCAGCCGG
276


mir-124a-3
TTAAGGCACGCGGTGAATGCCA
235


mir-7-2
TGGAAGACTAGTGATTTTGTT
198


mir-210
CTGTGCGTGTGACAGCGGCTG
277


mir-215
ATGACCTATGAATTGACAGAC
278


mir-223
TGTCAGTTTGTCAAATACCCC
279


mir-131-3
TAAAGCTAGATAACCGAAAGT
211


mir-199a-1
CCCAGTGTTCAGACTACCTGTTC
259


mir-30c
TGTAAACATCCTACACTCTCAGC
280


mir-101-1
TACAGTACTGTGATAACTGA
265


mir-101-2
TACAGTACTGTGATAACTGA
265


mir-26b
TTCAAGTAATTCAGGATAGGTT
281


mir-152
TCAGTGCATGACAGAACTTGG
282


mir-135-1
TATGGCTTTTTATTCCTATGTGAT
283


mir-135-2
TATGGCTTTTTATTCCTATGTGAT
283


mir-217
TACTGCATCAGGAACTGATTGGAT
284


mir-15a-2
TAGCAGCACATAATGGTTTGTG
269


let-7g
TGAGGTAGTAGTTTGTACAGT
285


mir-33b
GTGCATTGCTGTTGCATTG
286


mir-16-2
TAGCAGCACGTAAATATTGGCG
196


mir-182
TTTGGCAATGGTAGAACTCACA
287


mir-148a
TCAGTGCACTACAGAACTTTGT
288


mir-23a
ATCACATTGCCAGGGATTTCC
289


mir-181c
AACATTCAACCTGTCGGTGAGT
290









Example 5
Uniform 2′-MOE Phosphorothioate (PS) Oligomeric Compounds Targeting miRNAs
In accordance with the present invention, a series of oligomeric compounds were designed and synthesized to target miRNA sequences disclosed by Lim et al. Science, 2003, 299, 1540. The compounds are shown in Table 3. “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 3 are composed of 2′-methoxyethoxy (2′-MOE) nucleotides throughout and the internucleoside (backbone) linkages are phosphorothioate (P═S) throughout. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or the function of targets downstream of miRNAs.







TABLE 3







Uniform 2′-MOE PS Compounds targeting miRNAs










ISIS Number
SEQ ID NO
Sequence
Pri-miRNA





327873
291
CTACCATAGGGTAAAACCACT
mir-140


327874
292
GCTGCAAACATCCGACTGAAAG
mir-30a


327875
293
ACAACCAGCTAAGACACTGCCA
mir-34


327876
294
AACACTGATTTCAAATGGTGCTA
mir-29b-1


327877
295
CGCCAATATTTACGTGCTGCTA
mir-16-3


327878
296
CTAGTGGTCCTAAACATTTCAC
mir-203


327879
297
AACAAAATCACTAGTCTTCCA
mir-7-1


327880
298
ACAAATTCGGTTCTACAGGGTA
mir-10b


327881
299
AAAAGAGACCGGTTCACTGTGA
mir-128a


327882
300
TCACTTTTGTGACTATGCAA
mir-153-1


327883
301
CAGAACTTAGCCACTGTGAA
mir-27b


327884
302
GCAAAAATGTGCTAGTGCCAAA
mir-96


327885
303
ACTACCTGCACTGTAAGCACTTTG
mir-17as/mir-91


327886
304
CGCGTACCAAAAGTAATAATG
mir-123/mir-126as


327887
305
GCGACCATGGCTGTAGACTGTTA
mir-132


327888
306
AATGCCCCTAAAAATCCTTAT
mir-108-1


327889
307
GTGGTAATCCCTGGCAATGTGAT
mir-23b


327890
308
AGCACAAACTACTACCTCA
let-7i


327891
309
GGCCGTGACTGGAGACTGTTA
mir-212


327892
310
ACTTTCGGTTATCTAGCTTTA
mir-131-2/mir-9


327893
311
AACCACACAACCTACTACCTCA
let-7b


327894
312
ATACATACTTCTTTACATTCCA
mir-1d


327895
313
ACAAACACCATTGTCACACTCCA
mir-122a


327896
314
ACAGTTCTTCAACTGGCAGCTT
mir-22


327897
315
ACAGGCCGGGACAAGTGCAATA
mir-92-1


327898
316
GTAGTGCTTTCTACTTTATG
mir-142


327899
317
CAGTGAATTCTACCAGTGCCATA
mir-183


327900
318
CTGCCTGTCTGTGCCTGCTGT
mir-214


327901
319
TGAGCTACAGTGCTTCATCTCA
mir-143


327902
320
GGCTGTCAATTCATAGGTCAG
mir-192-1


327903
321
AACTATACAACCTACTACCTCA
let-7a-3


327904
322
ACTCACCGACAGCGTTGAATGTT
mir-181a


327905
323
CAGACTCCGGTGGAATGAAGGA
mir-205


327906
324
TCATAGCCCTGTACAATGCTGCT
mir-103-1


327907
325
AGCCTATCCTGGATTACTTGAA
mir-26a


327908
326
CAATGCAACTACAATGCAC
mir-33a


327909
327
CCCAACAACATGAAACTACCTA
mir-196-2


327910
328
TGATAGCCCTGTACAATGCTGCT
mir-107


327911
329
GCTACCTGCACTGTAAGCACTTTT
mir-106


327912
330
AACTATACAATCTACTACCTCA
let-7f-1


327913
331
AACCGATTTCAAATGGTGCTAG
mir-29c


327914
332
GCCCTTTTAACATTGCACTG
mir-130a


327915
333
ACATGGTTAGATCAAGCACAA
mir-218-1


327916
334
TGGCATTCACCGCGTGCCTTAA
mir-124a-2


327917
335
TCAACATCAGTCTGATAAGCTA
mir-21


327918
336
CTAGTACATCATCTATACTGTA
mir-144


327919
337
GAAACCCAGCAGACAATGTAGCT
mir-221


327920
338
GAGACCCAGTAGCCAGATGTAGCT
mir-222


327921
339
CTTCCAGTCGGGGATGTTTACA
mir-30d


327922
340
TCAGTTTTGCATGGATTTGCACA
mir-19b-2


327923
341
GAAAGAGACCGGTTCACTGTGA
mir-128b


327924
342
GCAAGCCCAGACCGCAAAAAG
mir-129-2


327925
343
TAGCTGGTTGAAGGGGACCAA
mir-133b


327926
344
ACTATGCAACCTACTACCTCT
let-7d


327927
345
TGTAAACCATGATGTGCTGCTA
mir-15b


327928
346
AACCGATTTCAGATGGTGCTAG
mir-29a-1


327929
347
GAACAGATAGTCTAAACACTGGG
mir-199b


327930
348
ACTATACAACCTCCTACCTCA
let-7e


327931
349
AACCATACAACCTACTACCTCA
let-7c


327932
350
AGGCATAGGATGACAAAGGGAA
mir-204


327933
351
AAGGGATTCCTGGGAAAACTGGAC
mir-145


327934
352
GGTACAATCAACGGTCGATGGT
mir-213


327935
353
CTACCTGCACTATAAGCACTTTA
mir-20


327936
354
ACAGCTGGTTGAAGGGGACCAA
mir-133a-1


327937
355
GATTCACAACACCAGCT
mir-138-2


327938
356
AACAATACAACTTACTACCTCA
mir-98


327939
357
TCACAAGTTAGGGTCTCAGGGA
mir-125b-1


327940
358
GAACAGGTAGTCTGAACACTGGG
mir-199a-2


327941
359
AACCCACCGACAGCAATGAATGTT
mir-181b


327942
360
CCATCTTTACCAGACAGTGTT
mir-141


327943
361
TATCTGCACTAGATGCACCTTA
mir-18


327944
362
AAAGTGTCAGATACGGTGTGG
mir-220


327945
363
CTGTTCCTGCTGAACTGAGCCA
mir-24-2


327946
364
AGGCGAAGGATGACAAAGGGAA
mir-211


327947
365
TCAGTTATCACAGTACTGTA
mir-101-3


327948
366
GCTGAGTGTAGGATGTTTACA
mir-30b


327949
367
CACAAATTCGGATCTACAGGGTA
mir-10a


327950
368
TCAGTTTTGCATAGATTTGCACA
mir-19a


327951
369
CACAAACCATTATGTGCTGCTA
mir-15a-1


327952
370
CTACGCGTATTCTTAAGCAATA
mir-137


327953
371
AGAATTGCGTTTGGACAATCA
mir-219


327954
372
ACAAAGTTCTGTGATGCACTGA
mir-148b


327955
373
GCCCTTTCATCATTGCACTG
mir-130b


327956
374
CACAGTTGCCAGCTGAGATTA
mir-216


327957
375
CACAAGTTCGGATCTACGGGTT
mir-100-1


327958
376
CCGGCTGCAACACAAGACACGA
mir-187


327959
377
CAGCCGCTGTCACACGCACAG
mir-210


327960
378
GTCTGTCAATTCATAGGTCAT
mir-215


327961
379
GGGGTATTTGACAAACTGACA
mir-223


327962
380
GCTGAGAGTGTAGGATGTTTACA
mir-30c


327963
381
AACCTATCCTGAATTACTTGAA
mir-26b


327964
382
CCAAGTTCTGTCATGCACTGA
mir-152


327965
383
ATCACATAGGAATAAAAAGCCATA
mir-135-1


327966
384
ATCCAATCAGTTCCTGATGCAGTA
mir-217


327967
385
ACTGTACAAACTACTACCTCA
let-7g


327968
386
CAATGCAACAGCAATGCAC
mir-33b


327969
387
TGTGAGTTCTACCATTGCCAAA
mir-182


327970
388
ACAAAGTTCTGTAGTGCACTGA
mir-148a


327971
389
GGAAATCCCTGGCAATGTGAT
mir-23a


327972
390
ACTCACCGACAGGTTGAATGTT
mir-181c









Example 6
Uniform 2′-MOE Phosphorothioate (PS) Oligomeric Compounds Targeting Novel miRNAs
In accordance with the present invention, a series of oligomeric compounds were designed and synthesized to target novel miRNAs. The compounds are shown in Table 4. “Pri-miRNA” indicates the particular pri-miRNA defined herein which contains the miRNA that the oligomeric compound was designed to target. The sequence of the compounds represent the full complement of the novel miRNA defined herein. All compounds in Table 4 are composed of 2′-methoxyethoxy (2′-MOE) nucleotides throughout and the internucleoside (backbone) linkages are phosphorothioate (P═S) throughout. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or downstream targets of miRNAs.







TABLE 4







Uniform 2′-MOE PS Compounds targeting novel pri-miRNAs










ISIS

Sequence



Number
SEQ ID NO
(5′-3′)
Pri-miRNA





328089
391
ACTGTAGGAATATGTTTGATA
hypothetical miRNA-013


328090
392
ATTAAAAAGTCCTCTTGCCCA
hypothetical miRNA-023


328091
393
GCTGCCGTATATGTGATGTCA
hypothetical miRNA-030


328092
394
GGTAGGTGGAATACTATAACA
hypothetical miRNA-033


328093
395
TAAACATCACTGCAAGTCTTA
hypothetical miRNA-039


328094
396
TTGTAAGCAGTTTTGTTGACA
hypothetical miRNA-040


328095
397
TCACAGAGAAAACAACTGGTA
hypothetical miRNA-041


328096
398
CCTCTCAAAGATTTCCTGTCA
hypothetical miRNA-043


328097
399
TGTCAGATAAACAGAGTGGAA
hypothetical miRNA-044


328098
400
GAGAATCAATAGGGCATGCAA
hypothetical miRNA-055


328099
401
AAGAACATTAAGCATCTGACA
hypothetical miRNA-058


328100
402
AATCTCTGCAGGCAAATGTGA
hypothetical miRNA-070


328101
403
AAACCCCTATCACGATTAGCA
hypothetical miRNA-071


328102
404
GCCCCATTAATATTTTAACCA
hypothetical miRNA-075


328103
405
CCCAATATCAAACATATCA
hypothetical miRNA-079


328104
406
TATGATAGCTTCCCCATGTAA
hypothetical miRNA-083


328105
407
CCTCAATTATTGGAAATCACA
hypothetical miRNA-088


328106
408
ATTGATGCGCCATTTGGCCTA
hypothetical miRNA-090


328107
409
CTGTGACTTCTCTATCTGCCT
hypothetical miRNA-099


328108
410
AAACTTGTTAATTGACTGTCA
hypothetical miRNA-101


328109
411
AAAGAAGTATATGCATAGGAA
hypothetical miRNA-105


328110
412
GATAAAGCCAATAAACTGTCA
hypothetical miRNA-107


328111
413
TCCGAGTCGGAGGAGGAGGAA
hypothetical miRNA-111


328112
414
ATCATTACTGGATTGCTGTAA
hypothetical miRNA-120


328113
415
CAAAAATTATCAGCCAGTTTA
hypothetical miRNA-137


328114
416
AATCTCATTTTCATACTTGCA
hypothetical miRNA-138


328115
417
AGAAGGTGGGGAGCAGCGTCA
hypothetical miRNA-142


328116
418
CAAAATTGCAAGCAAATTGCA
hypothetical miRNA-143


328117
419
TCCACAAAGCTGAACATGTCT
hypothetical miRNA-144


328118
420
TATTATCAGCATCTGCTTGCA
hypothetical miRNA-153


328119
421
AATAACACACATCCACTTTAA
hypothetical miRNA-154


328120
422
AAGAAGGAAGGAGGGAAAGCA
hypothetical miRNA-156


328121
423
ATGACTACAAGTTTATGGCCA
hypothetical miRNA-161


328122
424
CAAAACATAAAAATCCTTGCA
hypothetical miRNA-164


328123
425
TTACAGGTGCTGCAACTGGAA
hypothetical miRNA-166


328124
426
AGCAGGTGAAGGCACCTGGCT
hypothetical miRNA-168


328125
427
TATGAAATGCCAGAGCTGCCA
hypothetical miRNA-169


328126
428
CCAAGTGTTAGAGCAAGATCA
hypothetical miRNA-170


328127
429
AACGATAAAACATACTTGTCA
hypothetical miRNA-171


328128
430
AGTAACTTCTTGCAGTTGGA
hypothetical miRNA-172


328129
431
AGCCTCCTTCTTCTCGTACTA
hypothetical miRNA-173


328130
432
ACCTCAGGTGGTTGAAGGAGA
hypothetical miRNA-175


328131
433
ATATGTCATATCAAACTCCTA
hypothetical miRNA-176


328132
434
GTGAGAGTAGCATGTTTGTCT
hypothetical miRNA-177


328133
435
TGAAGGTTCGGAGATAGGCTA
hypothetical miRNA-178


328134
436
AATTGGACAAAGTGCCTTTCA
hypothetical miRNA-179


328135
437
ACCGAACAAAGTCTGACAGGA
hypothetical miRNA-180


328136
438
AACTACTTCCAGAGCAGGTGA
hypothetical miRNA-181


328137
439
GTAAGCGCAGCTCCACAGGCT
hypothetical miRNA-183


328138
440
GAGCTGCTCAGCTGGCCATCA
hypothetical miRNA-185


328139
441
TACTTTTCATTCCCCTCACCA
hypothetical miRNA-188









Example 7
Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and a Deoxy Gap Targeting Pri-miRNAs
In accordance with the present invention, a series of oligomeric compounds were designed and synthesized to target different regions of pri-miRNA structures. The compounds are shown in Table 5. “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 5 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets.







TABLE 5







Chimeric phosphorothioate oligomeric compounds having


2′-MOE wings and a deoxy gap targeting pri-miRNAs










ISIS





Number
SEQ ID NO
Sequence
pri-miRNA





328333
442
AGAACAGCATGACGTAACCT
mir-140


328334
443
GCCCATCTGTGGCTTCACAG
mir-30a


328335
444
GAAGTCCGAGGCAGTAGGCA
mir-30a


328336
445
CTTCCTTACTATTGCTCACA
mir-34


328337
446
GCTAGATACAAAGATGGAAA
mir-29b-1


328338
447
CTAGACAATCACTATTTAAA
mir-29b-2


328339
448
GCAGCGCAGCTGGTCTCCCC
mir-29b-2


328340
449
TAATATATATTTCACTACGC
mir-16-3


328341
450
TGCTGTATCCCTGTCACACT
mir-16-3


328342
451
CAATTGCGCTACAGAACTGT
mir-203


328343
452
TCGATTTAGTTATCTAAAAA
mir-7-1


328344
453
CTGTAGAGGCATGGCCTGTG
mir-7-1


328345
454
TGACTATACGGATACCACAC
mir-10b


328346
455
GGAACAAGGCCAATTATTGC
mir-128a


328347
456
AGAAATGTAAACCTCTCAGA
mir-128a


328348
457
AGCTGTGAGGGAGAGAGAGA
mir-153-1


328349
458
CTGGAGTGAGAATACTAGCT
mir-153-1


328350
459
ACTGGGCTCATATTACTAGC
mir-153-2


328351
460
TTGGATTAAATAACAACCTA
hypothetical miRNA-013


328352
461
CCCGGAGACAGGGCAAGACA
hypothetical miRNA-013


328353
462
AAAGCGGAAACCAATCACTG
mir-27b


328354
463
GTCCCCATCTCACCTTCTCT
mir-27b


328355
464
TCAGAGCGGAGAGACACAAG
mir-96


328356
465
TAGATGCACATATCACTACC
mir-17as/mir-91


328357
466
CTTGGCTTCCCGAGGCAGCT
mir-17as/mir-91


328358
467
AGTTTGAAGTGTCACAGCGC
mir-123/mir-126as


328359
468
GCGTTTTCGATGCGGTGCCG
mir-123/mir-126as


328360
469
GAGACGCGGGGGCGGGGCGC
mir-132


328361
470
TACCTCCAGTTCCCACAGTA
mir-132


328362
471
TGTGTTTTCTGACTCAGTCA
mir-108-1


328363
472
AGAGCACCTGAGAGCAGCGC
mir-23b


328364
473
TCTTAAGTCACAAATCAGCA
mir-23b


328365
474
TCTCCACAGCGGGCAATGTC
let-7i


328366
475
GGCGCGCTGTCCGGGCGGGG
mir-212


328367
476
ACTGAGGGCGGCCCGGGCAG
mir-212


328368
477
GTCCTCTTGCCCAAGCAACA
hypothetical miRNA-023


328369
478
GAAGACCAATACACTCATAC
mir-131-2


328370
479
CCGAGGGGCAACATCACTGC
let-7b


328371
480
TCCATAGCTTAGCAGGTCCA
mir-1d


328372
481
TTTGATAGTTTAGACACAAA
mir-122a


328373
482
GGGAAGGATTGCCTAGCAGT
mir-122a


328374
483
AGCTTTAGCTGGGTCAGGAC
mir-22


328375
484
TACCATACAGAAACACAGCA
mir-92-1


328376
485
TCACAATCCCCACCAAACTC
mir-92-1


328377
486
TCACTCCTAAAGGTTCAAGT
hypothetical miRNA-030


328378
487
CACCCTCCAGTGCTGTTAGT
mir-142


328379
488
CTGACTGAGACTGTTCACAG
mir-183


328380
489
CCTTTAGGGGTTGCCACACC
hypothetical miRNA-033


328381
490
ACAGGTGAGCGGATGTTCTG
mir-214


328382
491
CAGACTCCCAACTGACCAGA
mir-143


328383
492
AGAGGGGAGACGAGAGCACT
mir-192-1


328384
493
TCACGTGGAGAGGAGTTAAA
hypothetical miRNA-039


328385
494
AGTGCTAATACTTCTTTCAT
hypothetical miRNA-040


328386
495
ACCTGTGTAACAGCCGTGTA
hypothetical miRNA-041


328387
496
TTATCGGAACTTCACAGAGA
hypothetical miRNA-041


328388
497
TCCCATAGCAGGGCAGAGCC
let-7a-3


328389
498
GGCACTTCATTGCTGCTGCC
hypothetical miRNA-043


328390
499
GGAGCCTTGCGCTCAGCATT
hypothetical miRNA-043


328391
500
ATGGTAATTTCATTTCAGGC
hypothetical miRNA-044


328392
501
GATTGCACATCCACACTGTC
hypothetical miRNA-044


328393
502
GCTGGCCTGATAGCCCTTCT
mir-181a


328394
503
GTTTTTTCAAATCCCAAACT
mir-181a


328395
504
CCCAGTGGTGGGTGTGACCC
let-7a-1


328396
505
CTGGTTGGGTATGAGACAGA
mir-205


328397
506
TTGATCCATATGCAACAAGG
mir-103-1


328398
507
GCCATTGGGACCTGCACAGC
mir-26a


328399
508
ATGGGTACCACCAGAACATG
mir-33a


328400
509
AGTTCAAAACTCAATCCCAA
mir-196-2


328401
510
GCCCTCGACGAAAACCGACT
mir-196-2


328402
511
TTGAACTCCATGCCACAAGG
mir-107


328403
512
AGGCCTATTCCTGTAGCAAA
mir-106


328404
513
GTAGATCTCAAAAAGCTACC
mir-106


328405
514
CTGAACAGGGTAAAATCACT
let-7f-1


328406
515
AGCAAGTCTACTCCTCAGGG
let-7f-1


328407
516
AATGGAGCCAAGGTGCTGCC
hypothetical miRNA-055


328408
517
TAGACAAAAACAGACTCTGA
mir-29c


328409
518
GCTAGTGACAGGTGCAGACA
mir-130a


328410
519
GGGCCTATCCAAAGTGACAG
hypothetical miRNA-058


328411
520
TACCTCTGCAGTATTCTACA
hypothetical miRNA-058


328412
521
TTTACTCATACCTCGCAACC
mir-218-1


328413
522
AATTGTATGACATTAAATCA
mir-124a-2


328414
523
CTTCAAGTGCAGCCGTAGGC
mir-124a-2


328415
524
TGCCATGAGATTCAACAGTC
mir-21


328416
525
ACATTGCTATCATAAGAGCT
mir-16-1


328417
526
TAATTTTAGAATCTTAACGC
mir-16-1


328418
527
AGTGTCTCATCGCAAACTTA
mir-144


328419
528
TGTTGCCTAACGAACACAGA
mir-221


328420
529
GCTGATTACGAAAGACAGGA
mir-222


328421
530
GCTTAGCTGTGTCTTACAGC
mir-30d


328422
531
GAGGATGTCTGTGAATAGCC
mir-30d


328423
532
CCACATATACATATATACGC
mir-19b-2


328424
533
AGGAAGCACACATTATCACA
mir-19b-2


328425
534
GACCTGCTACTCACTCTCGT
mir-128b


328426
535
GGTTGGCCGCAGACTCGTAC
hypothetical miRNA-069


328427
536
GATGTCACTGAGGAAATCAC
hypothetical miRNA-070


328428
537
TCAGTTGGAGGCAAAAACCC
hypothetical miRNA-071


328429
538
GGTAGTGCAGCGCAGCTGGT
mir-29b-3


328430
539
CCGGCTATTGAGTTATGTAC
mir-129-2


328431
540
ACCTCTCAGGAAGACGGACT
mir-133b


328432
541
GAGCATGCAACACTCTGTGC
hypothetical miRNA-075


328433
542
CCTCCTTGTGGGCAAAATCC
let-7d


328434
543
CGCATCTTGACTGTAGCATG
mir-15b


328435
544
TCTAAGGGGTCACAGAAGGT
mir-29a-1


328436
545
GAAAATTATATTGACTCTGA
mir-29a-1









Example 8
Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and a Deoxy Gap Targeting Pri-miRNAs
In accordance with the present invention, a second series of oligomeric compounds were designed and synthesized to target different regions of pri-miRNA structures. The compounds are shown in Table 6. “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 6 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets.







TABLE 6







Chimeric phosphorothioate oligomeric compounds having


2′-MOE wings and a deoxy gap targeting pri-miRNAs










ISIS





Number
SEQ ID NO
Sequence
pri-miRNA





328637
546
GGTTCCTAATTAAACAACCC
hypothetical miRNA-079


328638
547
CCGAGGGTCTAACCCAGCCC
mir-199b


328639
548
GACTACTGTTGAGAGGAACA
mir-129-1


328640
549
TCTCCTTGGGTGTCCTCCTC
let-7e


328641
550
TGCTGACTGCTCGCCCTTGC
hypothetical miRNA-083


328642
551
ACTCCCAGGGTGTAACTCTA
let-7c


328643
552
CATGAAGAAAGACTGTAGCC
mir-204


328644
553
GACAAGGTGGGAGCGAGTGG
mir-145


328645
554
TGCTCAGCCAGCCCCATTCT
mir-124a-1


328646
555
GCTTTTAGAACCACTGCCTC
hypothetical miRNA-088


328647
556
GGAGTAGATGATGGTTAGCC
mir-213


328648
557
ACTGATTCAAGAGCTTTGTA
hypothetical miRNA-090


328649
558
GTAGATAACTAAACACTACC
mir-20


328650
559
AATCCATTGAAGAGGCGATT
mir-133a-1


328651
560
GGTAAGAGGATGCGCTGCTC
mir-138-2


328652
561
GGCCTAATATCCCTACCCCA
mir-98


328653
562
GTGTTCAGAAACCCAGGCCC
mir-196-1


328654
563
TCCAGGATGCAAAAGCACGA
mir-125b-1


328655
564
TACAACGGCATTGTCCTGAA
mir-199a-2


328656
565
TTTCAGGCTCACCTCCCCAG
hypothetical miRNA-099


328657
566
AAAAATAATCTCTGCACAGG
mir-181b


328658
567
AGAATGAGTTGACATACCAA
hypothetical miRNA-101


328659
568
GCTTCACAATTAGACCATCC
mir-141


328660
569
AGACTCCACACCACTCATAC
mir-131-1


328661
570
ATCCATTGGACAGTCGATTT
mir-133a-2


328662
571
GGCGGGCGGCTCTGAGGCGG
hypothetical miRNA-105


328663
572
CTCTTTAGGCCAGATCCTCA
hypothetical miRNA-106


328664
573
TAATGGTATGTGTGGTGATA
hypothetical miRNA-107


328665
574
ATTACTAAGTTGTTAGCTGT
mir-1b


328666
575
GATGCTAATCTACTTCACTA
mir-18


328667
576
TCAGCATGGTGCCCTCGCCC
mir-220


328668
577
TCCGCGGGGGCGGGGAGGCT
hypothetical miRNA-111


328669
578
AGACCACAGCCACTCTAATC
mir-7-3


328670
579
TCCGTTTCCATCGTTCCACC
mir-218-2


328671
580
GCCAGTGTACACAAACCAAC
mir-24-2


328672
581
AAGGCTTTTTGCTCAAGGGC
mir-24-1


328673
582
TTGACCTGAATGCTACAAGG
mir-103-2


328674
583
TGCCCTGCTCAGAGCCCTAG
mir-211


328675
584
TCAATGTGATGGCACCACCA
mir-101-3


328676
585
ACCTCCCAGCCAATCCATGT
mir-30b


328677
586
TCCTGGATGATATCTACCTC
hypothetical miRNA-120


328678
587
TCTCCCTTGATGTAATTCTA
let-7a-4


328679
588
AGAGCGGAGTGTTTATGTCA
mir-10a


328680
589
TCATTCATTTGAAGGAAATA
mir-19a


328681
590
TCCAAGATGGGGTATGACCC
let-7f-2


328682
591
TTTTTAAACACACATTCGCG
mir-15a-1


328683
592
AGATGTGTTTCCATTCCACT
mir-108-2


328684
593
CCCCCTGCCGCTGGTACTCT
mir-137


328685
594
CGGCCGGAGCCATAGACTCG
mir-219


328686
595
CTTTCAGAGAGCCACAGCCT
mir-148b


328687
596
GCTTCCCAGCGGCCTATAGT
mir-130b


328688
597
CAGCAGAATATCACACAGCT
mir-19b-1


328689
598
TACAATTTGGGAGTCCTGAA
mir-199b


328690
599
GCCTCCTTCATATATTCTCA
mir-204


328691
600
CCCCATCTTAGCATCTAAGG
mir-145


328692
601
TTGTATGGACATTTAAATCA
mir-124a-1


328693
602
TTTGATTTTAATTCCAAACT
mir-213


328694
603
CAAACGGTAAGATTTGCAGA
hypothetical miRNA-090


328695
604
GGATTTAAACGGTAAACATC
mir-125b-1


328696
605
CTCTAGCTCCCTCACCAGTG
hypothetical miRNA-099


328697
606
GCTTGTCCACACAGTTCAAC
mir-181b


328698
607
GCATTGTATGTTCATATGGG
mir-1b


328699
608
TGTCGTAGTACATCAGAACA
mir-7-3


328700
609
AGCCAGTGTGTAAAATGAGA
mir-24-1


328701
610
TTCAGATATACAGCATCGGT
mir-101-3


328702
611
TGACCACAAAATTCCTTACA
mir-10a


328703
612
ACAACTACATTCTTCTTGTA
mir-19a


328704
613
TGCACCTTTTCAAAATCCAC
mir-15a-1


328705
614
AACGTAATCCGTATTATCCA
mir-137









Example 9
Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and a Deoxy Gap Targeting Pri-miRNAs
In accordance with the present invention, a third series of oligomeric compounds were designed and synthesized to target different pri-miRNA structures. The compounds are shown in Table 7. “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds in Table 7 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets.







TABLE 7







Chimeric phosphorothioate oligomeric compounds having


2′-MOE wings and a deoxy gap targeting pri-miRNAs










ISIS





Number
SEQ ID NO
Sequence
pri-miRNA





328706
615
CGTGAGGGCTAGGAAATTGC
mir-216


328707
616
GCAACAGGCCTCAATATCTT
mir-100-1


328708
617
ACGAGGGGTCAGAGCAGCGC
mir-187


328709
618
GGCAGACGAAAGGCTGACAG
hypothetical miRNA-137


328710
619
CTGCACCATGTTCGGCTCCC
hypothetical miRNA-138


328711
620
GGGGCCCTCAGGGCTGGGGC
mir-124a-3


328712
621
CCGGTCCACTCTGTATCCAG
mir-7-2


328713
622
GCTGGGAAAGAGAGGGCAGA
hypothetical miRNA-142


328714
623
TCAGATTGCCAACATTGTGA
hypothetical miRNA-143


328715
624
CTGGGGAGGGGGTTAGCGTC
hypothetical miRNA-144


328716
625
TGGGTCTGGGGCAGCGCAGT
mir-210


328717
626
TTGAAGTAGCACAGTCATAC
mir-215


328718
627
TCTACCACATGGAGTGTCCA
mir-124a-3


328719
628
AGTGCCGCTGCCGCGCCGTG
mir-7-2


328720
629
ACACATTGAGAGCCTCCTGA
hypothetical miRNA-142


328721
630
GTCGCTCAGTGCTCTCTAGG
hypothetical miRNA-143


328722
631
AGGCTCCTCTGATGGAAGGT
hypothetical miRNA-144


328723
632
GCTGTGACTTCTGATATTAT
hypothetical miRNA-153


328724
633
GACATCATGTGATTTGCTCA
hypothetical miRNA-154


328725
634
CACCCCAAGGCTGCAGGGCA
mir-26b


328726
635
TGTCAAGCCTGGTACCACCA
hypothetical miRNA-156


328727
636
CTGCTCCAGAGCCCGAGTCG
mir-152


328728
637
ACCCTCCGCTGGCTGTCCCC
mir-135-1


328729
638
TAGAGTGAATTTATCTTGGT
mir-135-2


328730
639
TGGTGACTGATTCTTATCCA
mir-217


328731
640
CAATATGATTGGATAGAGGA
hypothetical miRNA-161


328732
641
TTTAAACACACATTCGCGCC
mir-15a-2


328733
642
ACCGGGTGGTATCATAGACC
let-7g


328734
643
TGCATACCTGTTCAGTTGGA
hypothetical miRNA-164


328735
644
GCCCGCCTCTCTCGGCCCCC
mir-33b


328736
645
TCGCCCCCTCCCAGGCCTCT
hypothetical miRNA-166


328737
646
ACAACTGTAGAGTATGGTCA
mir-16-2


328738
647
GCTGACCATCAGTACTTTCC
hypothetical miRNA-168


328739
648
TTATAGAACAGCCTCCAGTG
hypothetical miRNA-169


328740
649
TTCAGGCACTAGCAGTGGGT
hypothetical miRNA-170


328741
650
AGTACTGCGAGGTTAACCGC
hypothetical miRNA-171


328742
651
GGACCTTTAAGATGCAAAGT
hypothetical miRNA-172


328743
652
TTCATATTATCCACCCAGGT
hypothetical miRNA-173


328744
653
CGGATCCTGTTACCTCACCA
mir-182


328745
654
TGGTGCCTGCCACATCTTTG
hypothetical miRNA-175


328746
655
TGGGAGGCTGAATCAAGGAC
hypothetical miRNA-176


328747
656
TGACAACCAGGAAGCTTGTG
hypothetical miRNA-177


328748
657
GCCAGGCAGCGAGCTTTTGA
hypothetical miRNA-178


328749
658
CAGCCTGCCACCGCCGCTTT
hypothetical miRNA-179


328750
659
CTGCCCCCGTGGACCGAACA
hypothetical miRNA-180


328751
660
TCGTGCACCTGAGGAGTCTG
hypothetical miRNA-181


328752
661
CAAACGTGCTGTCTTCCTCC
mir-148a


328753
662
AAGGACTCAGCAGTGTTTCA
hypothetical miRNA-183


328754
663
TCCTCGGTGGCAGAGCTCAG
mir-23a


328755
664
AGACAATGAGTACACAGTTC
hypothetical miRNA-185


328756
665
CTGCAAGCACTGGTTCCCAT
hypothetical miRNA-186


328757
666
TTGCCTGAGCTGCCCAAACT
mir-181c


328758
667
TCCATCACACTGTCCTATGA
hypothetical miRNA-188


328759
668
GAGGGATTGTATGAACATCT
mir-216


328760
669
GCTTGTGCGGACTAATACCA
mir-100-1


328761
670
GCAGGCTAAAAGAAATAAGC
hypothetical miRNA-138


328762
671
ATTGTATAGACATTAAATCA
mir-124a-3


328763
672
GTTGAGCGCAGTAAGACAAC
mir-7-2


328764
673
AGATGTTTCTGGCCTGCGAG
hypothetical miRNA-142


328765
674
GACAAACTCAGCTATATTGT
mir-215


328766
675
ACGGCTCTGTGGCACTCATA
mir-131-3


328767
676
GCTTTCTTACTTTCCACAGC
mir-30c


328768
677
TACCTTTAGAATAGACAGCA
mir-101-1


328769
678
AGGCTGGACAGCACACAACC
mir-26b


328770
679
AGCAGGAGCCTTATCTCTCC
hypothetical miRNA-156


328771
680
ATGAGTGAGCAGTAGAATCA
mir-135-1


328772
681
TGAGACTTTATTACTATCAC
mir-135-2


328773
682
TACTTTACTCCAAGGTTTTA
mir-15a-2


328774
683
GCACCCGCCTCACACACGTG
mir-33b


328775
684
TTCCCGACCTGCCTTTACCT
hypothetical miRNA-166


328776
685
TCCTGTAATTATAGGCTAGC
hypothetical miRNA-169


328777
686
GGATCATATCAATAATACCA
hypothetical miRNA-172


328778
687
TGCTGAGACACACAATATGT
hypothetical miRNA-176


328779
688
TGTTTGTCTCCAAGAAACGT
hypothetical miRNA-177


328780
689
TGTCATGGACAGGATGAATA
hypothetical miRNA-179


328781
690
TCTATCATACTCAGAGTCGG
mir-148a


328782
691
TTGTGACAGGAAGCAAATCC
mir-23a


328783
692
CATCAGAGTCACCAACCCCA
hypothetical miRNA-185


328784
693
CAAGAGATGTCTCGTTTTGC
hypothetical miRNA-186









Example 10
Chimeric Phosphorothioate Compounds Having 2′-MOE Wings and a Deoxy Gap Targeted to the Stem Loop of Pri-miRNA Structures
In accordance with the present invention, a fourth series of oligomeric compounds were designed to target the stem loop of different pri-miRNA structures. In some cases, these oligomeric compounds contain mismatches, and thus hybridize with partial complementarity to the stemloop structure of the pri-miRNA targeted. The compounds are shown in Table 8. “Pri-miRNA” indicates the particular pri-miRNA that the oligomeric compound was designed to target. All compounds in Table 8 are chimeric oligonucleotides (“gapmers”), composed of a central “gap” region consisting of 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR as described, supra, or they can be used in other assays to investigate the role of miRNAs or downstream nucleic acid targets.







TABLE 8







Chimeric phosphorothioate compounds having


2′-MOE wings and a deoxy gap targeted


to the stem loop of pri-miRNA structures










Compound
SEQ ID




Number
NO.
Sequence
Pri-miRNA





RG1
694
GTGGTAGAACAGCATGACGTC
mir-140


RG2
695
AGCTGTGAAGCCACGATGGGC
mir-30a


RG3
696
AGATACAAAGATGGAAAAATC
mir-29b-1


RG4
697
CTTCCTTACTATTGCTCACAA
mir-34


RG5
698
TGTTTAATATATATTTCACTC
mir-16-3


RG6
699
TGTCAAGACATCGCGTTAACA
mir-203


RG7
700
TGTCGATTTAGTTATCCAACA
mir-7-1


RG8
701
GTGACTATACGGATACCACAC
mir-10b


RG9
702
ACCTCTCCAAATGTAAAGA
mir-128a


RG10
703
CAAAGCGGAAACCAATCACTG
mir-27b


RG11
704
CTGCAGTACATGCACATATCA
mir-91


RG12
705
AACAATGACACCCTTGACCT
mir-132


RG13
706
TTTTAATCTTAAGTCACAAA
mir-23b


RG14
707
ATCTCCACAGCGGGCAATGTC
let-7i


RG15
708
TATGAAGACCAATACACTCCA
mir-131-2


RG16
709
GGGGCAACATCACTGCCC
let-7b


RG17
710
CCATGTTAGCAGGTCCATATG
mir-1d


RG18
711
GTTTGATAGTTTAGACACAAA
mir-122a


RG19
712
TGGGTCAGGACTAAAGCTTC
mir-22


RG20
713
AATACCATACAGAAACACAGC
mir-92-1


RG21
714
TTCGTGATGATTGTCGTGCC
mir-142


RG22
715
ACTGCGAGACTGTTCACAGTT
mir-183


RG23
716
TACAGGTGAGCGGATGTTCTG
mir-214


RG24
717
TCTCAGCTCCCAACTGACCAG
mir-143


RG25
718
ACCGCAGATATTACAGCCACT
let-7a-3


RG26
719
CCTGATAGCCCTTCTTAAGGA
mir-181a


RG27
720
CTTGATCCATATGCAACAAGG
mir-103-1


RG28
721
GCCATTGGGACCTGCACACC
mir-26a


RG29
722
GCATGGGTACCACCCCATGC
mir-33a


RG30
723
CGAGTTCAAAACTCAATCCCA
mir-196-2


RG31
724
CTTGAACTCCATGCCACAAGG
mir-107


RG32
725
GTAGATCTCAAAAAGCTAGC
mir-106


RG33
726
GAACAGGGTAAAATCACTAC
let-7f-1


RG34
727
AGACAAAAACAGACTCTGAA
mir-29c


RG35
728
GCTAGTGACAGGTCCAGACAG
mir-130a


RG36
729
TTTACTCATACCTCGCAACCA
mir-218-1


RG37
730
TTAATTGTATGACATTAAATCA
mir-124a-2


RG38
731
TGCCATGAGATTCAACAGTCA
mir-21


RG39
732
GATAATATTTAGAATCTTAAC
mir-16-1


RG40
733
TAGTGTCTCATCGCAAACTTA
mir-144


RG41
734
CTGTTGCCTAACGAACACAGA
mir-221


RG42
735
TGCTGATTACGAAAGACAGGAT
mir-222


RG43
736
GCTTAGCTGTGTCTTACAGCT
mir-30d









Example 11
Effects of Oligomeric Compounds Targeting miRNAs on Apoptosis in Caspase Assay 
Programmed cell death or apoptosis involves the activation of proteases, a family of intracellular proteases, through a cascade which leads to the cleavage of a select set of proteins. The caspase family contains at least 14 caspases, with differing substrate preferences. The caspase activity assay uses a DEVD peptide to detect activated caspases in cell culture samples. The peptide is labeled with a fluorescent molecule, 7-amino-4-trifluoromethyl coumarin (AFC). Activated caspases cleave the DEVD peptide resulting in a fluorescence shift of the AFC. Increased fluorescence is indicative of increased caspase activity and consequently increased cell death. The chemotherapeutic drugs taxol, cisplatin, etoposide, gemcitabine, camptothecin, aphidicolin and 5-fluorouracil all have been shown to induce apoptosis in a caspase-dependent manner.
The effect of several oligomeric compounds of the present invention was examined in cells expressing miRNA targets. The cells expressing the targets used in these experiments were T47D, a breast carcinoma cell line. Other cell lines can also be employed in this assay and these include normal human mammary epithelial cells (HMECs) as well as two breast carcinoma cell lines, MCF7 and T47D. All of the cell lines were obtained from the American Type Culture Collection (Manassas, Va.). The latter two cell lines express similar genes but MCF7 cells express the tumor suppressor p53, while T47D cells are deficient in p53. MCF-7 cells are routinely cultured in DMEM low glucose (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. T47D cells were cultured in Gibco DMEM High glucose media supplemented with 10% Fetal Bovine Serum (FBS).
Cells were plated at 10,000 cells per well for HMEC cells or 20,000 cells per well for MCF7 and T47D cells, and allowed to attach to wells overnight. Plates used were 96 well Costar plate 1603 (black sides, transparent bottom). DMEM high glucose medium, with and without phenol red, were obtained from Invitrogen (San Diego, Calif.). MEGM medium, with and without phenol red, were obtained from Biowhittaker (Walkersville, Md.). The caspase-3 activity assay kit was obtained from Calbiochem (Cat. #HTS02) (EMD Biosciences, San Diego, Calif.).
Before adding to cells, the oligomeric compound cocktail was mixed thoroughly and incubated for 0.5 hrs. The oligomeric compound or the LIPOFECTINTM-only vehicle control was added (generally from a 3 μM stock of oligonucleotide) to a final concentration of 200 nM with 6 μg/ml LIPOFECTINTM. The medium was removed from the plates and the plates were tapped on sterile gauze. Each well was washed in 150 μl of PBS (150 μL HBSS for HMEC cells). The wash buffer in each well was replaced with 100 μL of the oligomeric compound/OPTI-MEMTM/LIPOFECTINTM cocktail (this was T=0 for oligomeric compound treatment). The plates were incubated for 4 hours at 37° C., after which the medium was dumped and the plate was tapped on sterile gauze. 100 μl of full growth medium without phenol red was added to each well. After 48 hours, 50 μl of oncogene buffer (provided with Calbiochem kit) with 10 μM DTT was added to each well. 20 μl of oncogene substrate (DEVD-AFC) was added to each well. The plates were read at 400±25 nm excitation and 508±20 nm emission at t=0 and t=3 time points. The t=0×(0.8) time point was subtracted from the t=3 time point, and the data are shown as percent of LIPOFECTINTM-only (untreated control) treated cells.
Four experiments were performed and the results are shown in Tables 9-12. The concentration of oligomeric compound used was 200 nM. All compounds in Tables 9-12 are chimeric oligomeric compounds (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the compound. All cytidine residues are 5-methylcytidines. As a control a 20-mer oligonucleotide random-mer, ISIS-29848 (NNNNNNNNNNNNNNNNNNNN; where N is A,T,C or G; herein incorporated as SEQ ID NO: 737) was used. In addition, two positive controls targeting expressed genes known to induce apoptosis when inhibited were included. These were ISIS-148715 (TTGTCCCAGTCCCAGGCCTC; herein incorporated as SEQ ID NO: 738) which targets human Jagged2 and ISIS-226844 (GCCCTCCATGCTGGCACAGG; herein incorporated as SEQ ID NO: 739) which targets human Notch1. Both positive controls have the same chemistry and gap structure as the compounds being tested. An increase in fluorescence indicates that the compound, by inhibiting its target, induces apoptosis as compared to untreated controls (UTC).







TABLE 9







Effects of oligomeric compounds targeting


miRNAs on Apoptosis in Caspase Assay











SEQ ID

Fold Increase


ISIS Number
NO.
Pri-miRNA
over UTC





UTC
N/A
N/A
1.0


Untreated control





ISIS-29848
737
N/A
3.5


n-mer





ISIS-148715
738
Jagged2
1.5


Positive control





ISIS-226844
739
Notch1
3.6


Positive control





328371
480
mir-1d
1.2


328400
509
mir-196-2
1.3


328420
529
mir-222
1.0


328692
601
mir-124a-1
1.2


328381
490
mir-214
1.1


328691
600
mir-145
0.9


328391
500
hypothetical miRNA-044
0.8


328415
524
mir-21
1.1


328433
542
let-7d
1.0


328643
552
mir-204
0.9


328377
486
hypothetical miRNA-030
0.7


328405
514
let-7f-1
1.0


328372
481
mir-122a
1.0


328403
512
mir-106
1.0


328424
533
mir-19b-2
0.9


328648
557
hypothetical miRNA-090
1.1


328397
506
mir-103-1
1.2


328656
565
hypothetical miRNA-099
1.1


328392
501
hypothetical miRNA-044
1.0


328421
530
mir-30d
1.2


328417
526
mir-16-1
1.0


328647
556
mir-213
0.9


328378
487
mir-142
1.0


328416
525
mir-16-1
0.9
















TABLE 10







Effects of oligomeric compounds targeting


miRNAs on Apoptosis in Caspase Assay











SEQ ID

Fold Increase


ISIS Number
NO.
Pri-miRNA
over UTC





UTC
N/A
N/A
0.9


Untreated control





ISIS-29848
737
N/A
3.0


n-mer





ISIS-148715
738
Jagged2
1.0


Positive control





ISIS-226844
739
Notch1
3.1


Positive control





328375
484
mir-92-1
0.9


328382
491
mir-143
0.9


328383
492
mir-192-1
1.2


328385
494
hypothetical miRNA-040
0.9


328395
504
let-7a-1
1.0


328398
507
mir-26a
0.9


328399
508
mir-33a
1.0


328402
511
mir-107
1.2


328408
517
mir-29c
0.9


328409
518
mir-130a
0.7


328422
531
mir-30d
1.0


328423
532
mir-19b-2
0.6


328425
534
mir-128b
0.8


328431
540
mir-133b
0.9


328436
545
mir-29a-1
0.9


328646
555
hypothetical miRNA-088
1.1


328649
558
mir-20
1.0


328651
560
mir-138-2
0.9


328652
561
mir-98
1.2


328657
566
mir-181b
0.8


328672
581
mir-24-1
0.9


328694
603
hypothetical miRNA-090
0.8


328696
605
hypothetical miRNA-099
1.5


328700
609
mir-24-1
0.8
















TABLE 11







Effects of oligomeric compounds targeting


miRNAs on Apoptosis in Caspase Assay











SEQ ID

Fold Increase


ISIS Number
NO.
Pri-miRNA
over UTC





UTC
N/A
N/A
0.9


Untreated control





ISIS-29848
737
N/A
3.2


n-mer





ISIS-148715
738
Jagged2
1.1


Positive control





ISIS-226844
739
Notch1
3.1


Positive control





328374
483
mir-22
1.1


328376
485
mir-92-1
0.7


328384
493
hypothetical miRNA-039
1.0


328386
495
hypothetical miRNA-041
0.7


328390
499
hypothetical miRNA-043
0.9


328393
502
mir-181a
1.5


328404
513
mir-106
0.9


328406
515
let-7f-1
1.0


328407
516
hypothetical miRNA-055
1.2


328410
519
hypothetical miRNA-058
1.5


328411
520
hypothetical miRNA-058
0.8


328413
522
mir-124a-2
0.8


328426
535
hypothetical miRNA-069
1.3


328427
536
hypothetical miRNA-070
0.8


328435
544
mir-29a-1
1.3


328637
546
hypothetical miRNA-079
1.0


328638
547
mir-199b
0.8


328639
548
mir-129-1
0.8


328645
554
mir-124a-1
2.2


328653
562
mir-196-1
1.1


328654
563
mir-125b-1
1.0


328655
564
mir-199a-2
0.7


328689
598
mir-199b
0.8


328695
604
mir-125b-1
0.8
















TABLE 12







Effects of oligomeric compounds targeting


miRNAs on Apoptosis in Caspase Assay











SEQ ID

Fold Increase


ISIS Number
NO.
Pri-miRNA
over UTC





UTC
N/A
N/A
1.0


Untreated control





ISIS-29848
737
N/A
3.5


n-mer





ISIS-148715
738
Jagged2
1.3


Positive control





ISIS-226844
739
Notch1
3.5


Positive control





328373
482
mir-122a
0.9


328379
488
mir-183
1.1


328387
496
hypothetical miRNA-041
1.4


328388
497
let-7a-3
0.9


328389
498
hypothetical miRNA-043
1.1


328394
503
mir-181a
0.8


328396
505
mir-205
0.8


328401
510
mir-196-2
0.8


328412
521
mir-218-1
1.2


328414
523
mir-124a-2
0.9


328418
527
mir-144
1.0


328419
528
mir-221
0.7


328430
539
mir-129-2
1.3


328432
541
hypothetical miRNA-075
0.6


328434
543
mir-15b
0.8


328640
549
let-7e
0.9


328641
550
hypothetical miRNA-083
1.1


328642
551
let-7c
1.0


328644
553
mir-145
0.7


328650
559
mir-133a-1
0.8


328658
567
hypothetical miRNA-101
1.2


328690
599
mir-204
0.8


328693
602
mir-213
1.0


328697
606
mir-181b
1.0









From these data, it is evident that SEQ ID NOs. 480, 509, 601, 490, 524, 557, 506, 565, 530, 605, 492, 561, 511, 555, 483, 502, 535, 562, 544, 519, 516, 554, 496, 567, 521, 539, 488, 498, and 550 induce apoptosis in T47D cells, while SEQ ID NOs. 500, 486, 518, 532, 534, 566, 603, 609, 485, 495, 520, 522, 536, 547, 548, 564, 598, 604, 503, 505, 510, 528, 541, 543, 553, 559, and 599 prevent or have a protective effect from apoptosis in the same system.
Example 12
Oligomeric Compounds Targeting the mir-30a Pri-miRNA Structure
In one embodiment of the invention, oligomeric compounds targeting the hairpin structure of mir-30a pri-miRNA were designed and tested for their effects on miRNA signaling in 293T cells (American Type Culture Collection (Manassas, Va.)).
A synthetic DNA fragment comprised of four tandem repeats of the target site for mir-30a was cloned into the vector pGL3-C (purchased from Promega Corp., Madison Wis.) at the unique XbaI site (pGL3C-M30-4X). This places the target site in the 3′UTR of the luciferase reporter vector. An oligomeric compound mimicking the mir-30a pri-miRNA (AATTTAATACGACTCACTATAGGGCGACTGTAAACATCCTCGACTGGAAGCTGTGAAG CCACAGATGGGCTTTCAGTCGGATGTTTGCAGCTGC, herein incorporated as SEQ ID NO: 1749) was in vitro transcribed using T7 RNA polymerase and a DNA template produced by PCR (the T7 promoter is shown in bold).
On the day prior to the experiment 24-well plates were seeded with 293T cells at 50% confluency. The following morning cells were treated with oligomeric compounds targeted to the mir-30a pri-miRNA mimic. The oligomeric compounds used in this study are shown in Table 13. All of the compounds are 20 nucleobases in length having either a phosphorothioate backbone throughout (PS) or a phosphodiester backbone throughout (PO). As designated in the table, ISIS 328076, 328078, 328081, 328084, 328086, 328088 are chimeric oligomeric compounds (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. All cytidine residues are 5-methylcytidines. The remaining compounds in the table have 2′-methoxyethoxy (MOE) nucleotides throughout with either a phosphorothioate (PS) or phosphodiester (PO) internucleoside linkages.
If the compound targeted the pre-loop of the mir-30a pri-miRNA structure, that designation is also noted in the table.







TABLE 13







Oligomeric compounds targeting the mir-30a pri-miRNA










Isis


SEQ ID


Number
Sequence
Chemistry
NO





328075
GCTTCACAGCTTCCAGTCGA
(PS/MOE)
740


328076
GCTTCACAGCTTCCAGTCGA
(PS/MOE 5-10-5 gapmer)
740


328077
CCCATCTGTGGCTTCACAGC
(PS/MOE); pre-loop
741


328078
CCCATCTGTGGCTTCACAGC
(PS/MOE 5-10-5 gapmer); pre-loop
741


328079
CCCATCTGTGGCTTCACAGC
(PO/MOE); pre-loop
741


328080
TGAAAGCCCATCTGTGGCTT
(PS/MOE); pre-loop
742


328081
TGAAAGCCCATCTGTGGCTT
(PS/MOE 5-10-5 gapmer); pre-loop
742


328082
TGAAAGCCCATCTGTGGCTT
(PO/MOE); pre-loop
742


328083
GCAGCTGCAAACATCCGACT
(PS/MOE)
743


328084
GCAGCTGCAAACATCCGACT
(PS/MOE 5-10-5 gapmer)
743


328085
CATCTGTGGCTTCACAGCTT
(PS/MOE)
744


328086
CATCTGTGGCTTCACAGCTT
(PS/MOE 5-10-5 gapmer)
744


328087
AAGCCCATCTGTGGCTTCAC
(PS/MOE)
745


328088
AAGCCCATCTGTGGCTTCAC
(PS/MOE 5-10-5 gapmer)
745









Cells were washed once with PBS then oligomeric compounds were added to triplicate wells at 150 nM in OPTI-MEMTM media and 4.5 μl/ml LIPOFECTINTM reagent (Invitrogen Corporation, Carlsbad, Calif.). After 3 hours, the media was removed, and the cells were treated with the mir-30a pri-miRNA mimic at 200 nM in OPTI-MEMTM with 6 μl/ml LIPOFECTINTM reagent. After 3 hours the media was removed from the cells. The reporter plasmid, pGL3C-M30-4X, was then transfected using SuperFect reagent. 20 μg of pGL3C-M30-4X and 2 μg of pRL-CMV, a plasmid expressing Renilla luciferase, were suspended in 600 μl of serum-free DMEM to which 120 μl of Superfect was added. After a 5 minute incubation, 6 mls of DMEM+10% FCS was added. 125 μl of the plasmid/SuperFect suspension was added to each well. After a 2 hour incubation cells were washed and fresh growth media added. Cells were incubated overnight.
The following morning the media was removed and the cells were lysed in 120 μl passive lysis buffer (PLB; Promega). 40 μl of the lysate was then assayed for Photinus (PL) and Renilla (RL) luciferases using a Dual Luciferase Assay kit (Promega) according to the manufacturer's protocol. The results below are given as percent pGL3C-M30-4X expression (PL) normalized to pRL-CMV expression (RL). The 20-nucleobase oligonucleotide random-mer ISIS Number 29848 was used as a negative control. The data are shown in Table 14.







TABLE 14







Effects of oligomeric compounds targeting the


mir-30a pri-miRNA on reporter gene expression











percent control


SEQ ID

luciferase


NO
ISIS Number
expression












N/A
Untreated control
100


N/A
Mir-30a pri-miRNA only
62


737
29848 control added after
63



mir-30a pri-miRNA



292
327874
66


740
328075
55


740
328076
57


741
328077
70


741
328078
63


742
328080
72


742
328081
80


743
328084
75


744
328085
72


744
328086
95


745
328087
83


745
328088
107









Upon administration of the mir-30a pri-miRNA mimic, the pri-miRNA is believed to be processed in the cell by the endogenous Drosha RNase III enzyme into a pre-miRNA, which is then processed by human Dicer into a mature miRNA, which is then able to hybridize to the target site, thus effectively reducing luciferase reporter expression.
Upon treatment of the system with the oligomeric compounds targeting the mir-30a pri-miRNA, the processing and/or production of the mir-30a mature miRNA is inhibited, and the mir-30a miRNA is no longer able to bind its target site, thus allowing luciferase reporter expression to increase.
Cells treated with mir-30a pri-miRNA mimic show an approximately 38% reduction in luciferase expression compared to the untreated controls. Treatment with ISIS 328086, 328087 and 328088 had the most dramatic effect in reversing the mir-30a miRNA-mediated silencing, restoring luciferase reporter expression to near control levels. Thus, it was demonstrated that the oligomeric compound mimicking the mir-30a pri-miRNA silences luciferase activity from the reporter vector, and that oligomeric compounds targeting the mir-30a pri-miRNA can inhibit its silencing activity, possibly by interfering with its processing into the pre-miRNA or mature miRNA molecules.
ISIS 328085 to ISIS 328088 were designed to target the mir-30a pri-miRNA as pseudo half-knot compounds. Methods for the preparation of pseudo half-knot compounds are disclosed in U.S. Pat. No. 5,512,438 which is incorporated herein by reference. This motif has been used to disrupt the structure of regulatory RNA stem loops in larger viral genomic structures. (Ecker et al, Science. 1992; 257:958-61). However, this is the first example of the pseudo half-knot motif being used to regulate a small non-coding RNA, more specifically a miRNA such as those disclosed herein. It is also the first demonstration of apoptotic modulation in a cell by pseudo half-knot structured oligomeric compounds.
Example 13
Effects of Oligomeric Compounds Targeting miRNAs on Expression of Adipocyte Differentiation Markers
The effect of several oligomeric compounds of the present invention targeting miRNAs on the expression of markers of cellular differentiation was examined in preadipocytes.
One of the hallmarks of cellular differentiation is the upregulation of gene expression. During adipocyte differentiation, the gene expression patterns in adipocytes change considerably. An excessive recruitment and differentiation of preadipocytes into mature adipocytes is a characteristic of human obesity, which is a strong risk factor for type 2 diabetes, hypertension, atherosclerosis, cardiovascular disease, and certain cancers. Some genes known to be upregulated during adipocyte differentiation include hormone-sensitive lipase (HSL), adipocyte lipid binding protein (aP2), glucose transporter 4 (Glut4), and PPARγ (Peroxisome proliferator-activated receptor gamma) These genes play important roles in the uptake of glucose and the metabolism and utilization of fats. For example, HSL is involved in the mobilization of fatty acids from adipose tissue into the bloodstream; studies suggest that increased free fatty acid levels are one of the causative factors in type 2 diabetes. aP2 is believed to play a role in athersclerosis. Glut4 is important in insulin signaling. PPARγ is believed to be involved in adipocyte differentiation, insulin sensitivity, and colonic tumor development.
Leptin is also a marker for differentiated adipocytes. In the adipocyte assay, leptin secretion into the media above the differentiated adipocytes was measured by protein ELISA. Cell growth, transfection and differentiation procedures were carried out as described for the Triglyceride accumulation assay (see below). On day nine post-transfection, 96-well plates were coated with a monoclonal antibody to human leptin (R&D Systems, Minneapolis, Minn.) and left at 4° C. overnight. The plates were blocked with bovine serum albumin (BSA), and a dilution of the media was incubated in the plate at RT for 2 hours. After washing to remove unbound components, a second monoclonal antibody to human leptin (conjugated with biotin) was added. The plate was then incubated with strepavidin-conjugated horseradish peroxidase (HRP) and enzyme levels are determined by incubation with 3,3′,5,5′-Tetramethlybenzidine, which turns blue when cleaved by HRP. The OD450 was read for each well, where the dye absorbance is proportional to the leptin concentration in the cell lysate. Results are expressed as a percent±standard deviation relative to transfectant-only controls.
An increase in triglyceride content is another well-established marker for adipocyte differentiation. The triglyceride accumulation assay measures the synthesis of triglyceride by adipocytes. Triglyceride accumulation was measured using the InfinityTM Triglyceride reagent kit (Sigma-Aldrich, St. Louis, Mo.). Human white preadipocytes (Zen-Bio Inc., Research Triangle Park, NC) were grown in preadipocyte media (ZenBio Inc.). One day before transfection, 96-well plates were seeded with 3000 cells/well. Cells were transfected according to standard published procedures with 250 nM oligomeric compound in LIPOFECTINTM (Invitrogen Corporation, Carlsbad, Calif.) (Monia et al., J. Biol. Chem. 1993 268(19):14514-22). Oligomeric compounds were tested in triplicate on each 96-well plate, and the effects of TNF-α, a positive drug control that inhibits adipocyte differentiation, were also measured in triplicate. Negative and transfectant-only controls may be measured up to six times per plate. After the cells have reached confluence (approximately three days), they were exposed to differentiation media (Zen-Bio, Inc.) containing a PPAR-γ agonist, IBMX, dexamethasone, and insulin for three days. Cells were then fed adipocyte media (Zen-Bio, Inc.), which was replaced at 2 to 3 day intervals. On day nine post-transfection, cells were washed and lysed at room temperature, and the triglyceride assay reagent was added. Triglyceride accumulation was measured based on the amount of glycerol liberated from triglycerides by the enzyme lipoprotein lipase. Liberated glycerol is phosphorylated by glycerol kinase, and hydrogen peroxide is generated during the oxidation of glycerol-1-phosphate to dihydroxyacetone phosphate by glycerol phosphate oxidase. Horseradish peroxidase (HRP) uses H2O2 to oxidize 4-aminoantipyrine and 3,5 dichloro-2-hydroxybenzene sulfonate to produce a red-colored dye. Dye absorbance, which is proportional to the concentration of glycerol, was measured at 515 nm using an UV spectrophotometer. Glycerol concentration was calculated from a standard curve for each assay, and data were normalized to total cellular protein as determined by a Bradford assay (Bio-Rad Laboratories, Hercules, Calif.). Results are expressed as a percent±standard deviation relative to transfectant-only control.
For assaying adipocyte differentiation, expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulation and leptin secretion were measured in adipocytes transfected with the uniform 2′-MOE phosphorothioate (PS) oligomeric compounds previously described. Cells are lysed on day nine post-transfection, in a guanadinium-containing buffer and total RNA is harvested. Real-time PCR is performed (Applied Biosystems, Prism 7700) on the total RNA using the following primer/probe sets for the adipocyte differentiation hallmark genes: (aP2): forward 5′-GGTGGTGGAATGCGTCATG-3′ (SEQ ID NO: 746), reverse 5′-CAACGTCCCTTGGCTTATGC-3′ (SEQ ID NO: 747), probe 5′-FAM-AAGGCGTCACTTCCACGAGAGTTTATGAGA-TAMRA-3′ (SEQ ID NO: 748); (Glut4): forward 5′-GGCCTCCGCAGGTTCTG-3′ (SEQ ID NO: 749), reverse 5′-TTCGGAGCCTATCTGTTGGAA-3′ (SEQ ID NO: 750), probe 5′-FAM-TCCAGGCCGGAGTCAGAGACTCCA-TAMRA-3′ (SEQ ID NO: 751); (HSL): forward 5′-ACCTGCGCACAATGACACA-3′ (SEQ ID NO: 752), reverse 5′-TGGCTCGAGAAGAAGGCTATG-3′ (SEQ ID NO: 753), probe 5′-FAM-CCTCCGCCAGAGTCACCAGCG-TAMRA-3′ (SEQ ID NO: 754); (PPAR-γ): forward 5′-AAATATCAGTGTGAATTACAGCAAACC-3′ (SEQ ID NO: 755), reverse 5′-GGAATCGCTTTCTGGGTCAA-3′ (SEQ ID NO: 756), probe 5′-FAM-TGCTGTTATGGGTGAAACTCTGGGAGATTCT-TAMRA-3′ (SEQ ID NO: 757). The amount of total RNA in each sample is determined using a Ribogreen Assay (Molecular Probes, Eugene, Oreg.), and expression levels of the adipocyte differentiation hallmark genes were normalized to total RNA. Leptin protein and triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed relative to control levels (control=treatment with ISIS-29848 (SEQ ID NO: 737)). Results of two experiments are shown in Tables 15 and 16.







TABLE 15







Effects of oligomeric compounds targeting 


miRNAs on expression of adipocyte differentiation markers













ISIS 
SEQ 




PPAR 


Number
ID NO
TG
AP2
HSL
Glut4 
gamma





327876
294
0.47
0.75
0.47
0.36
0.57


327878
296
0.65
0.85
0.93
0.69
0.97


327880
298
0.52
0.97
0.80
1.11
0.53


327888
306
0.98
1.18
1.38
1.37
1.36


327889
307
0.47
0.69 
0.59
0.55 
0.71


327890
308
0.92
0.91 
0.86
1.10 
1.18


327892
310
0.42
0.31 
0.25
0.07 
0.32


327901
319
0.54
0.42 
0.33
0.19 
0.30


327903
321
1.20
1.15 
1.23
1.72 
1.19


327905
323
0.69
1.14 
1.11
0.84 
0.54


327913
331
0.59
0.99 
0.92
0.84 
0.72


327919
337
0.58
0.79 
0.57
0.32 
0.52


327922
340
1.09
0.99 
0.95
1.75 
1.37


327925
343
0.72
0.77 
0.78
1.99 
0.60


327933
351
1.48
1.46 
1.35
2.52 
1.52


327934
352
0.99
1.20 
1.02
1.22 
0.97


327939
357
0.92
1.08 
1.21
0.87 
0.83


327941
359
1.31
1.78 
1.73
2.07 
0.80


327954
372
0.58
0.95 
1.03
0.92 
0.73
















TABLE 16







Effects of oligomeric compounds targeting 


miRNAs on expression of adipocyte differentiation markers














ISIS
SEQ





PPAR 


Number
ID NO
TG
Leptin
AP2
HSL 
Glut4
gamma





327888
306
0.44
1.38
0.47 
0.50 
0.17
0.66


327889
307
0.46
1.05
0.57 
0.54 
0.46 
0.82


327890
308
0.61
1.36
0.69 
0.67 
0.67 
0.94


327893
311
0.95
1.14
0.97 
0.85 
1.47 
1.03


327901
319
0.53
1.02
0.47 
0.47 
0.29 
0.72


327903
321
0.58
1.61
0.92 
0.80 
1.12 
0.98


327905
323
0.58
1.62
0.68 
0.69 
0.40 
0.83


327919
337
0.40
1.44
0.48 
0.37 
0.18 
0.57


327922
340
0.43
1.25
0.75 
0.72 
0.43 
0.80


327925
343
0.63
1.40
0.77 
0.75 
0.61 
0.83


327926
344
1.06
1.47
0.85 
0.82 
1.10 
0.93


327930
348
0.97
0.95
0.86 
0.89 
1.01 
0.98


327931
349
1.11
1.12
1.00 
0.99 
1.37 
1.56


327934
352
0.62
1.25
0.66 
0.64 
0.44 
0.72


327938
356
1.05
1.35
0.86 
0.85 
0.80 
0.90


327939
357
0.59
2.67
0.69 
0.63 
0.30 
0.70


327941
359
0.42
0.54
0.88 
0.81 
0.44 
0.86


327942
360
0.85
2.03
0.82 
0.79 
0.66 
0.87


327955
373
0.81
1.22
0.74 
0.82 
0.45 
0.92


327967
385
0.90
1.22
0.86 
0.97 
0.56 
0.89









From these data, values above 1.0 for triglyceride accumulation (column “TG” in the tables) indicate that the compound has the ability to stimulate triglyceride accumulation, whereas values at or below 1.0 indicate that the compound inhibits triglyceride accumulation. With respect to leptin secretion (column “Leptin” in the tables), values above 1.0 indicate that the compound has the ability to stimulate secretion of the leptin hormone, and values at or below 1.0 indicate that the compound has the ability to inhibit secretion of leptin. With respect to the four adipocyte differentiation hallmark genes (columns “AP2,” “HSL,” “Glut4,” and “PPAR gamma” in the tables), values above 1.0 indicate induction of cell differentiation, whereas values at or below 1.0 indicate that the compound inhibits differentiation.
Several compounds were found to have remarkable effects. For example, the oligomeric compounds ISIS Number 327889 (SEQ ID NO: 307), targeted to mir-23b; ISIS Number 327892 (SEQ ID NO: 310), targeted to mir-131-1, mir-131-2 and mir-131-3 (also known as mir-9); ISIS Number 327942 (SEQ ID NO: 360) targeted to mir-141 and ISIS Number 327901 (SEQ ID NO: 319), targeted to mir-143 were shown to significantly reduce the expression levels of 5 of 6 markers of adipocyte differentiation (excepting leptin levels), indicating that these oligomeric compounds have the ability to block adipocyte differentiation. Therefore, these oligomeric compounds may be useful as pharmaceutical agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells.
The compound ISIS Number 327939 (SEQ ID NO: 357), targeted to mir-125b-1, for example, produced surprising results in that it demonstrates a significant increase in leptin secretion but a concomitant decrease in triglyeride accumulation and a decrease in the expression of all four adipocyte differentiation hallmark genes, indicating that this oligomeric compound may be useful as a pharmaceutic agent in the treatment of obesity, as well as having applications in other metabolic diseases.
The oligomeric compound ISIS Number 327931 (SEQ ID NO: 349), targeted to let-7c is an example of a compound which demonstrates an increase in four out of six markers of adipocyte differentiation, including a significant increase in the expression of PPAR-γ. This oligomeric compound may be useful as a pharmaceutical agent in the treatment of diseases in which the induction of cell differentiation is desirable.
The oligomeric compound ISIS Number 327933 (SEQ ID NO: 351), targeted to mir-145 is an example of a compound which demonstrates an increase in all six markers of adipocyte differentiation. This oligomeric compound may be useful as a pharmaceutical agent in the treatment of diseases in which the induction of adipocyte differentiation is desirable, such as anorexia, or for conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which regeneration of neural tissue (such as from pluripotent stem cells) would be beneficial. Furthermore, this oligomeric compound may be useful in the treatment, attenuation or prevention of diseases in which it is desirable to induce cellular differentiation and/or quiescence, for example in the treatment of hyperproliferative disorders such as cancer.
In some embodiments, differentiating adipocytes were treated with uniform 2′-MOE phosphorothioate oligomeric compounds according to the methods described above, and the expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulation were measured. TG levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed as a percentage of control levels (control=treatment with ISIS 342673; AGACTAGCGGTATCTTTATCCC; herein incorporated as SEQ ID NO: 758), a uniform 2′-MOE phosphorothioate oligomeric compound containing 15 mismatches with respect to the mature mir-143 miRNA). Undifferentiated adipocytes were also compared as a negative control. As a positive control, differentiating adipocytes were treated with ISIS 105990 (AGCAAAAGATCAATCCGTTA; herein incorporated as SEQ ID NO: 759), a 5-10-5 gapmer oligomeric compound targeting the PPAR-gamma mRNA, previously demonstrated to inhibit adipocyte differentiation. The effects of TNF-α; also known to inhibit adipocyte differentiation, were also measured. Results of these experiments are shown in Tables 17 and 18.







TABLE 17







Effects of oligomeric compounds targeting 


miRNAs on expression of adipocyte differentiation markers













ISIS
SEQ




PPAR 


Number
ID NO
TG
AP2
HSL
Glut4
gamma
















Untreated
N/A
88.5
87.8
88.6
102.7
94.9


control








105990
759
28.2
51.6
49.2
59.5
51.8


342673
758
100.0
100.0
100.0
100.0
100.0


TNF-alpha
N/A
10.0
5.5
0.7
0.5
18.8


Undiff.
N/A
2.7
0.0
0.3
0.1
9.2


adipocytes








328116
418 
82.1
87.7
75.8
75.2
78.4


328117
419 
55.0
65.4
61.7
68.1
64.1


328118
420 
69.3
92.7
85.3
76.6
80.2


328119
421 
90.2
99.9
98.5
95.2
82.7


328120
422 
82.7
81.0
77.7
94.8
70.5


328121
423
134.8
127.0
126.0
140.8
103.6


328122
424
78.9
79.3
72.7
85.9
77.8


328123
425
120.8
106.7
85.4
162.4
74.7


328124
426
99.1
101.8
103.6
122.7
90.4


328125
427
81.7
86.9
75.8
99.5
76.1


328126
428
98.9
90.9
83.2
100.7
75.0


328127
429
74.5
86.9
89.7
80.8
77.6


328128
430
98.7
100.7
94.1
101.9
84.0


328129
431
53.8
67.6
56.5
60.0
71.8


328130
432
122.4
86.6
76.5
83.8
99.4


328131
433
89.1
95.4
81.8
103.6
88.2


328132
434
114.1
90.2
73.7
72.1
90.0


328133
435
61.2
69.5
63.0
91.9
63.8


328134
436
85.7
80.1
74.7
88.3
78.4


328135
437
63.6
80.6
76.7
90.3
70.0


328136
438
47.0
73.0
65.0
66.7
72.7


328137
439
83.2
99.6
86.3
88.5
85.7


328138
440
100.6
85.3
89.8
86.8
83.8


328139
441
89.1
98.3
92.6
106.3
115.0
















TABLE 18







Effects of oligomeric compounds targeting 


miRNAs on expression of adipocyte differentiation markers














SEQ




PPAR 


ISIS #
ID NO
TG
AP2
HSL
Glut4
gamma
















Untreated
N/A
102.2
90.8
94.9
117.8
103.3


control








105990
759 
32.8
49.8
52.0
68.1
60.1


342673
758
100
100
100
100
100


TNF-alpha
N/A
14.5
9.6
3.1
1.9
27.9


Undiff.
N/A
2.8
0.0
1.4
0.3
10.7


adipocytes








327912
330
107.4
90.1
90.6
89.0
76.9


327969
387
46.0
59.8
66.4
60.6
69.2


328099
401
93.9
85.9
88.4
86.8
81.9


328100
402
71.5
61.9
72.0
74.2
66.7


328101
403
108.6
83.2
91.8
84.7
79.3


328102
404
95.9
87.9
97.0
79.2
93.7


328103
405
110.2
83.2
82.5
94.3
74.3


328104
406
122.6
102.2
98.2
119.1
90.4


328105
407
93.1
88.2
94.2
94.2
93.3


328106
408
90.5
88.8
94.9
105.7
90.7


328107
409
66.7
67.5
61.0
72.5
79.3


328108
410
89.6
83.7
90.1
94.9
84.0


328109
411
84.9
84.9
86.9
106.6
96.1


328110
412
97.7
93.3
91.0
104.7
91.2


328111
413
101.9
71.5
69.5
59.6
74.9


328112
414
98.1
99.1
101.2
122.5
102.4


328113
415
80.8
84.5
90.6
99.9
93.8


328114
416
117.3
94.4
93.3
114.9
89.3


328115
417
108.7
80.0
89.0
132.0
95.8


341803
760
85.9
77.3
75.5
86.8
71.2


341804
761
60.9
70.8
71.6
73.6
74.1


341805
762
78.1
81.9
81.8
88.2
80.4


341806
763
83.2
75.8
73.4
69.4
72.6


341807
764
114.1
74.8
96.8
119.5
86.2









Several compounds were found to have remarkable effects. For example, the oligomeric compounds ISIS Number 328117 (SEQ ID NO: 419), targeted to hypothetical miRNA-144, ISIS Number 328129 (SEQ ID NO: 431), targeted to hypothetical miRNA-173, ISIS Number 328136 (SEQ ID NO: 438), targeted to hypothetical miRNA-181, and ISIS Number 327969 (SEQ ID NO: 387), targeted to mir-182, were each shown to reduce the expression levels of triglycerides by at least 50%, and treatment with ISIS 328117, 328129, or 328136 also each resulted in a reduction of expression of the other four hallmark genes, indicating that these oligomeric compounds targeted to hypothetical miRNA-144, hypothetical miRNA-173, hypothetical miRNA-181, and mir-182, may be useful as therapeutic agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases.
The oligomeric compound ISIS Number 328121 (SEQ ID NO: 423), targeted to hypothetical miRNA-161 is an example of a compound which stimulates an increase in all five markers of adipocyte differentiation. This oligomeric compound may be useful as a pharmaceutical agent in the treatment of diseases in which the induction of adipocyte differentiation is desirable, such as anorexia, or for conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which regeneration of neural tissue would be beneficial. Furthermore, this oligomeric compound may be useful in the treatment, attenuation or prevention of diseases in which it is desirable to induce cellular differentiation and/or quiescence, for example in the treatment of hyperproliferative disorders such as cancer.
Example 14
Expression of Mir-143 in Human Tissues and Cell Lines
Total RNA from spleen, kidney, testicle, heart and liver tissues as well as total RNA from human promyelocytic leukemia HL-60 cells, human embryonic kidney 293 (HEK293) cells, and T47D human breast carcinoma cells was purchased from Ambion, Inc. (Austin, Tex.). RNA from preadipocytes and differentiated adipocytes was purchased from Zen-Bio, Inc. (Research Triangle Park, NC). RNA was prepared from the HeLa, NT2, T-24, and A549 cell lines cultured as described above, using the following protocol: cell monolayers were washed twice with cold PBS, and cells were lysed in 1 mL TRIZOLTM (Invitrogen) and total RNA prepared using the manufacturer's recommended protocols.
Fifteen to twenty micrograms of total RNA was fractionated by electrophoresis through 10% acrylamide urea gels using a TBE buffer system (Invitrogen). RNA was transferred from the gel to HYBONDTM-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by electroblotting in an Xcell SureLockTM Minicell (Invitrogen). Membranes were fixed by UV cross-linking using a STRATALINKERTM UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using Rapid Hyb buffer solution (Amersham) using manufacturer's recommendations for oligonucleotide probes.
To detect mir-143, a target specific DNA oligonucleotide probe with the sequence TGAGCTACAGTGCTTCATCTCA (SEQ ID NO: 319) was synthesized by IDT (Coralville, Iowa). The oligo probe was 5′ end-labeled with T4 polynucleotide kinase with (γ-32P) ATP (Promega). To normalize for variations in loading and transfer efficiency membranes can be stripped and probed for U6 RNA. Hybridized membranes were visualized and quantitated using a Storm 860 PHOSPHORIMAGERTM and IMAGEQUANTTM Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.).
Using this probe, the mir-143 miRNA was found to be most highly expressed in human heart, thymus and kidney, and was also expressed to a lesser extent in lung, spleen, liver, and brain tissues. For example, as compared to expression levels in liver, mir-143 was expressed approximately 24-fold higher in heart, 17-fold higher in thymus, and 8-fold higher in kidney.
The mir-143 miRNA was also found to be expressed in adipocytes and preadipocytes, and levels of mir-143 were found to be dramatically upregulated in differentiated adipocytes as compared to preadipocytes, indicating that this miRNA may be important in adipocyte differentiation. These data, taken together with the finding that the oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319), targeted to mir-143, was shown to inhibit the adipocyte differentiation markers (described above, Example 13), supports the conclusion that mir-143 is involved in cellular differentiation pathways.
Example 15
Effects of Oligomeric Compounds Targeting miRNAs on Apoptosis in the Caspase Assay in Preadipocytes
The effect of oligomeric compounds of the present invention targeting miRNAs was examined in preadipocytes (Zen-Bio, Inc., Research Triangle Park, NC) using the fluorometric caspase assay previously described in Example 11. The oligonucleotide random-mer, ISIS-29848 (SEQ ID NO: 737) was used as a negative control, and ISIS-148715 (SEQ ID NO: 738), targeting the human Jagged2 mRNA, known to induce apoptosis when inhibited, was used as a positive control. The measurement obtained from the untreated control cells is designated as 100% activity and was set equal to 1.0. Results are shown in Table 19.







TABLE 19







Effects of targeting miRNAs on apoptosis in preadipocytes











SEQ ID




ISIS Number
NO.
Pri-miRNA
Fold Increase over UTC













UTC
N/A
N/A
1.0


Untreated control





ISIS-29848
737
N/A
1.2


n-mer





ISIS-148715
738
Jagged2
36.9


Positive control





327888
306
mir-108-1
1.1


327889
307
mir-23b
1.1


327890
308
let-7i
1.3


327893
311
let-7b
1.3


327901
319
mir-143
2.0


327903
321
let-7a-3
1.6


327905
323
mir-205
1.5


327919
337
mir-221
1.3


327922
340
mir-19b-2
1.0


327925
343
mir-133b
2.0


327926
344
let-7d
1.8


327930
348
let-7e
1.4


327931
349
let-7c
1.5


327934
352
mir-213
2.0


327938
356
mir-98
1.0


327939
357
mir-125b-1
2.2


327941
359
mir-181b
1.3


327942
360
mir-141
1.0


327955
373
mir-130b
4.3


327967
385
let-7g
1.5









From these data, it is evident that the oligomeric compounds of the present invention generally do not induce the activity of caspases involved in apoptotic pathways in preadipocytes. In particular, the oligomeric compound targeting mir-143, ISIS Number 327901 (SEQ ID NO: 319), does not result in a significant increase in caspase activity as compared to the Jagged2 positive control. Taken together with the results from the adipocyte differentiation assay (Example 13) and the expression analysis of mir-143 (Example 14), these data suggest that the mir-143 miRNA plays a role in stimulating cellular differentiation, employing pathways other than the caspase cascades activated during apoptosis.
It was recently reported that bone marrow cells may contribute to the pathogenesis of vascular diseases, and that cell differentiation appears to be important in models of postangioplasty restenosis, graft vasculopathy, and hyperlipidemia-induced atherosclerosis. Bone marrow cells have the potential to give rise to vascular progenitor cells that home in on damaged vessels and differentiate into smooth muscle cells or endothelial cells, thereby contributing to vascular repair, remodeling, and lesion formation (Sata, M. Trends Cardiovasc Med. 2003 13(6):249-53). Thus, the ability to modulate cell differentiation may provide the basis for the development of new therapeutic strategies for vascular diseases, targeting mobilization, homing, differentiation, and proliferation of circulating vascular progenitor cells.
Example 16
Comparison of Effects of Oligomeric Compounds Targeting the Mir-143 Pri-miRNA or Mature Mir-143 miRNA on Adipocyte Differentiation
Two oligomeric compounds targeting the mature mir-143 miRNA and two oligomeric compounds targeting the 110-nucleotide mir-143 pri-miRNA were compared for their effects on adipocyte differentiation using the same adipocyte differentiation assay as described in Example 13.
The oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319), 22-nucleotides in length, targets the mature mir-143 miRNA and is composed of 2′-methoxyethoxy (2′-MOE) nucleotides and phosphorothioate (P═S) internucleoside (backbone) linkages throughout. The oligomeric compound ISIS Number 338664 (CAGACTCCCAACTGACCAGA; SEQ ID NO: 491) is also a uniform 2′-MOE oligonucleotide, which is designed to target the mir-143 pri-miRNA. Another oligomeric compound targeting the mir-143 pri-miRNA, ISIS Number 328382 (SEQ ID NO: 491) is a chimeric oligonucleotide, 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings” having 2′-MOE substituents in the wing nucleosides (a “5-10-5 gapmer”), and ISIS Number 340927 (TGAGCTACAGTGCTTCATCTCA; SEQ ID NO: 319) is a 5-10-7 gapmer designed to target mature mir-143. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The effect of these oligomeric compounds targeting the mir-143 miRNA and the mir-143 pri-miRNA on expression of the 5 hallmark genes indicating cellular differentiation was examined in preadipocytes using the same methods described in Example 13. Results are shown in Table 20.







TABLE 20







Comparison of uniform 2′-MOE and chimeric oligomeric 


compounds targeting the mir-143 miRNA and pri-miRNAs on


expression of adipocyte differentiation markers













ISIS
SEQ ID




PPAR


Number
NO
TG
AP2
HSL
Glut4
gamma





327901
319
0.54
0.42
0.33
0.19
0.30


328382
491
0.72
0.89
0.75
0.85
0.96


338664
491
1.42
1.01
0.76
1.81
0.86


340927
319
0.65
0.77
0.73
0.54
0.36









From these data, it was observed that while the gapmer oligomeric compound targeting the mature mir-143 (ISIS Number 340972) results in reduced expression of the adipocyte differentiation markers, the uniform 2′-MOE oligomeric compound targeting mature mir-143 (ISIS Number 327901) was more effective. For the oligomeric compounds targeting the mir-143 pri-miRNA, the gapmer compound (ISIS Number 328382) appeared to be more effective in blocking adipocyte differentiation than was the uniform 2′-MOE oligomeric compound (ISIS Number 338664).
Dose Responsiveness:
In one embodiment, the oligomeric compound ISIS Number 327901 (SEQ ID NO: 319) targeting mature mir-143 was selected for additional dose response studies in the adipocyte differentiation assay. Differentiating adipocytes (at day 10 post-induction of differentiation) were treated with 50, 100, 200, and 300 nM ISIS 327901, or the scrambled control ISIS Number 342673 (SEQ ID NO: 758) containing 15 mismatches with respect to the mature mir-143 miRNA. ISIS Numbers 327901 and 342673 are uniform 2′-MOE phosphorothioate oligomeric compounds 22 nucleotides in length. Differentiating adipocytes treated with ISIS Number 29848 (SEQ ID NO: 737) served as the negative control to which the data were normalized. Differentiating adipocytes treated with ISIS 105990 (SEQ ID NO: 759), a 5-10-5 gapmer oligomeric compound targeting the PPAR-gamma mRNA which has been demonstrated previously to inhibit adipocyte differentiation, served as the positive control. Triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes (PPAR-gamma, aP2, HSL, and GLUT4) were measured 24 hours after treatment as described above. Untreated cells were compared to cells treated with oligomeric compounds, and results of these dose response studies are shown in Table 21, where levels of the markers is expressed as a percentage of untreated control (% UTC) levels. Where present, “N.D.” indicates “no data.”







TABLE 21







Effects of oligomeric compounds targeting mir-143


on expression of adipocyte differentiation markers











% UTC


Hallmark

Dose of oligomeric compound












Measured:
Isis #:
50 nM
100 nM
200 nM
300 nM















Triglycer-
342673
94.2
105.3 
98.3
108.2


ides
negative control







105990
N.D.
N.D.
N.D.
16.6



positive control







327901
85.3
68.9
34.0
23.0


PPAR-gamma
342673
77.5
89.9
94.6
85.8


mRNA
negative control







105990
N.D.
N.D.
N.D.
43.9



positive control







327901
74.6
70.8
51.8
39.3


AP2 mRNA
342673
82.4
90.3
81.1
70.9



negative control







105990
N.D.
N.D.
N.D.
17.9



positive control







327901
78.3
64.6
39.0
22.4


HSL mRNA
342673
92.0
95.6
97.3
85.2



negative control







105990
N.D.
N.D.
N.D.
7.4



positive control







327901
89.5
73.5
40.2
11.9


GLUT4 mRNA
342673
94.9
90.7
97.6
102.7



negative control







105990
N.D.
N.D.
N.D.
11.8



positive control







327901
74.2
49.7
32.8
17.4









From these data, it was observed that treatment of differentiating adipocytes with the uniform 2′-MOE oligomeric compound, ISIS Number 327901 targeting mir-143 results in a dose responsive reduction of expression of all five markers of differentiation. Thus, this oligomeric compound may be useful in the treatment of diseases associated with increased expression of these hallmark genes, such as obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells.
Example 17
Human Let 7 Homologs
Let-7 is one of the two miRNAs originally identified in C. elegans as an antisense translational repressor of messenger RNAs encoding key developmental timing regulators in nematode larva. Several genes predicted to encode let-7-like miRNAs have been identified in a wide variety of species, and these let-7-like homologs are believed to control temporal transitions during development across animal phylogeny. Oligomeric compounds of the present invention were designed to target several human let-7-like genes. Additionally, a series of target-specific DNA oligonucleotide probes were synthesized by IDT (Coralville, Iowa) and used in Northern analyses to assess the expression of let-7-like miRNA homologs in various tissues. These let-7 homolog specific probes are shown in Table 22.







TABLE 22







Probes for Northern analyses of


mRNA expression of let-7 homologs











SEQ




ISIS Number
ID NO
Sequence
pri-miRNA





327890
308
AGCACAAACTACTACCTCA
let-7i


327893
311
AACCACACAACCTACTACCTCA
let-7b


327903
321
AACTATACAACCTACTACCTCA
let-7a-3


327926
344
ACTATGCAACCTACTACCTCT
let-7d


327930
348
ACTATACAACCTCCTACCTCA
let-7e


327931
349
AACCATACAACCTACTACCTCA
let-7c


327967
385
ACTGTACAAACTACTACCTCA
let-7g









For Northern analyses with let-7 homolog probes, total RNA from spleen, kidney, testes, heart, and liver tissues as well as total RNA from HEK293, T47D, T-24, MCF7, HepG2, and K-562 Leukemia cell lines was either prepared as described above or purchased from Ambion, Inc. (Austin, Tex.). Northern blotting was performed as described above (Example 14). The let-7c miRNA was observed to be expressed in spleen, kidney, testes, heart and liver tissues, as well as in HEK293 and T47D cell lines. The let-7e miRNA was observed to be expressed in T-24, MCF7, T47D, 293T, HepG2, and K-562 cell lines.
In one embodiment, expression of let-7-like pri-miRNA homologs was detected in total RNA from brain, liver and spleen tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and HeLa, HEK-293, and T-24 cell lines by real-time RT-PCR. Primer/probe sets were designed to distinguish between and amplify specific let-7-like pri-miRNA homologs. These primer/probe sets are shown in Table 23.







TABLE 23







Primer/probe sets for assaying expression of let-7 miRNA homologs












Primer or
Isis
SEQ ID



Pri-miRNA
probe
number
NO.
sequence





let-7b
forward
341672
765
GAGGTAGTAGGTTGTGTGGTTTCA



reverse
341673
766
AGGGAAGGCAGTAGGTTGTATAGTT



probe
341674
767
CAGTGATGTTGCCCCTCGGAAGA


let-7c
forward
341675
768
TGCATCCGGGTTGAGGTA



reverse
341676
769
AGGAAAGCTAGAAGGTTGTACAGTTAA



probe
341677
770
AGGTTGTATGGTTTAGAGTTACACCCTGGGA


let-7d
forward
341678
771
CCTAGGAAGAGGTAGTAGGTTGCA



reverse
341679
772
CAGCAGGTCGTATAGTTACCTCCTT



probe
341680
773
AGTTTTAGGGCAGGGATTTTGCCCA


let-7g
forward
341681
774
TTCCAGGCTGAGGTAGTAGTTTG



reverse
341682
775
TTATCTCCTGTACCGGGTGGT



probe
341683
776
ACAGTTTGAGGGTCTAT


let-7i
forward
341684
777
TGAGGTAGTAGTTTGTGCTGTTGGT



reverse
341685
778
AGGCAGTAGCTTGCGCAGTTA



probe
341686
779
TTGTGACATTGCCCGCTGTGGAG


let-7a-1
forward
341687
780
GGATGAGGTAGTAGGTTGTATAGTTTTAGG



reverse
341688
781
CGTTAGGAAAGACAGTAGATTGTATAGTTATC



probe
341689
782
TCACACCCACCACTGG


let-7a-3
forward
341690
783
GGGTGAGGTAGTAGGTTGTATAGTTTGG



reverse
341691
784
CACTTCAGGAAAGACAGTAGATTGTATAGTT



probe
341692
785
CTCTGCCCTGCTATGG









Using these primer/probe sets, the let-7-like pri-miRNA homologs were found to be expressed in human brain, liver and spleen, as well as preadipocytes, differentiated adipocytes, and HeLa, T-24 and HEK-293 cells lines. In particular, the let-7b pri-miRNA exhibited approximately 100-fold higher expression in differentiated adipocytes as compared to preadipocytes. Furthermore, the let-7b, let-7c, let-7d, let-7i, and let-7a-3 pri-miRNAs were highly expressed in brain and spleen tissues.
In summary, the let-7-like homologs have been found to be widely expressed in various human tissues and several cell lines. Furthermore, some oligomeric compounds targeted to human let-7 pri-miRNAs generally appeared to result in the induction of cell differentiation, consistent with the functional role of let-7 as a regulator of developmental timing in nematode larva. Specifically, the oligomeric compounds targeted to let-7c (ISIS Number 327931; SEQ ID NO: 349) and let-7a-3 (ISIS Number 327903; SEQ ID NO: 321) resulted in an increase in expression levels for several markers of adipocyte differentiation. Furthermore, inhibition of the let-7-like homologs by oligomeric compounds of the present invention did not appear to induce caspases activated in apoptotic pathways (performed in Example 15). Thus, the oligomeric compounds of the present invention targeting let-7-like pri-miRNA homologs appear to stimulate adipocyte differentiation and do not promote cell death by apoptosis. Thus, the oligomeric compounds of the present invention may be useful as pharmaceutical agents in the treatment of anorexia or diseases, conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which neural regeneration would be beneficial.
Example 18
Effects of Oligomeric Compounds Targeting miRNAs on Insulin Signaling in HepG2 Cells
Insulin is secreted from pancreatic n-cells in response to increasing blood glucose levels. Through the regulation of protein expression, localization and activity, insulin ultimately stimulates conversion of excess glucose to glycogen, and results in the restoration of blood glucose levels. Insulin is known to regulate the expression of over 100 gene products in multiple cell types. For example, insulin completely inhibits the expression of hepatic insulin-like growth factor binding protein-1 (IGFBP-1), a protein which can sequester insulin-like growth factors, and phosphoenolpyruvate carboxykinase-cytosolic (PEPCK-c) which is a rate-controlling enzyme of hepatic gluconeogenesis. Levels of the follistatin mRNA are also believed to decrease in response to insulin treatment. IGFBP-1 and PEPCK-c are overexpressed in diabetes, and PEPCK-c overexpression in animals promotes hyperglycemia, impaired glucose tolerance and insulin-resistance. Thus, the IGFBP-1, PEPCK-c and follistatin genes serve as marker genes for which mRNA expression can be monitored and used as an indicator of an insulin-resistant state. Oligomeric compounds with the ability to reduce expression of IGFBP-1, PEPCK-c and follistatin are highly desirable as agents potentially useful in the treatment of diabetes and hypertension.
Oligomeric compounds of the present invention were tested for their effects on insulin signaling in HepG2 cells. HepG2 cells were plated at 7500 cells/well in collagen coated 96-well plates. The following day, cells were transfected with oligomeric compounds targeting miRNAs using 100 nM oligomeric compound in LIPOFECTINTM (Invitrogen Corporation, Carlsbad, Calif.) in two 96-well plates. The oligomeric compounds were tested in triplicate on each 96-well plate, except for positive and negative controls, which were measured up to six times per plate. At the end of transfection, the transfection medium was replaced by regular growth medium. Twenty-eight hours post-transfection, the cells were subjected to overnight (sixteen to eighteen hours) serum starvation using serum free growth medium. Forty-four hours post-transfection, the cells in the transfected wells were treated with either no insulin (“basal” Experiment 1, for identification of insulin-mimetic compounds) or with 1 nM insulin (“insulin treated” Experiment 2, for identification of insulin sensitizers) for four hours. At the same time, in both plates, cells in some of the un-transfected control wells are treated with 100 nM insulin to determine maximal insulin response. At the end of the insulin or no-insulin treatment (forty-eight hours post-transfection), total RNA is isolated from both the basal and insulin treated (1 nM) 96-well plates, and the amount of total RNA from each sample is determined using a Ribogreen assay (Molecular Probes, Eugene, Oreg.). Real-time PCR is performed on all the total RNA samples using primer/probe sets for three insulin responsive genes: PEPCK-c, IGFBP-1 and follistatin. Expression levels for each gene are normalized to total RNA, and values±standard deviation are expressed relative to the transfectant only untreated control (UTC) and negative control compounds. Results of these experiments are shown in Tables 24 and 25.







TABLE 24







Experiment 1: Effects of oligomeric compounds targeting miRNAs


on insulin-repressed gene expression in HepG2 cells













SEQ



Folli-


ISIS
ID

IGFBP-1
PEPCK-c
statin


Number
NO
Pri-miRNA
(% UTC)
(% UTC)
(% UTC)















UTC
N/A
N/A
100
100
100


29848 n-mer
737
N/A
95
87
94


327876
294
mir-29b-1
93
119
104


327878
296
mir-203
162
45
124


327880
298
mir-10b
137
110
107


327889
307
mir-23b
56
137
56


327890
308
let-7I
99
85
78


327892
310
mir-131-2/
108
75
91




mir-9





327901
319
mir-143
133
119
93


327903
321
let-7a-3
71
71
60


327905
323
mir-205
107
129
104


327913
331
mir-29c
123
229
115


327919
337
mir-221
96
71
74


327922
340
mir-19b-2
109
77
57


327925
343
mir-133b
152
145
110


327933
351
mir-145
125
118
112


327934
352
mir-213
231
99
140


327939
357
mir-125b-1
125
125
104


327941
359
mir-181b
83
101
80


327954
372
mir-148b
118
79
100


338664
491
mir-143
90
75
93




pri-miRNA





340927
319
mir-143
201
87
111









Under “basal” conditions (without insulin), treatments of HepG2 cells with oligomeric compounds of the present invention resulting in decreased mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that the oligomeric compounds have an insulin mimetic effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin repression of mRNA expression of these genes.
From these data, it is evident that the oligomeric compounds, ISIS Number 327878 targeting mir-203 and ISIS Number 327922 targeting mir-19b-2, for example, result in a 55% and a 23% decrease, respectively, in PEPCK-c mRNA, a marker widely considered to be insulin-responsive. Thus, these oligomeric compounds may be useful as pharmaceutic agents comprising insulin mimetic properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.
Conversely, the results observed with the oligomeric compound targeting mir-29c (ISIS Number 327913), for example, exhibiting increased expression of the IGFBP-1, PEPCK-c and follistatin marker genes, suggest that the mir-29c miRNA target may be involved in the regulation of these insulin-responsive genes. When the mir-29c miRNA is inactivated by an oligomeric compound, IGFBP-1, PEPCK-c and follistatin gene expression is no longer repressed.







TABLE 25







Experiment 2: Effects of oligomeric compounds targeting miRNAs


on insulin-sensitization of gene expression in HepG2 cells













SEQ



Folli-


ISIS
ID

IGFBP-1
PEPCK-c
statin


Number
NO
Pri-miRNA
(% UTC)
(% UTC)
(% UTC)















UTC(1 nm
N/A
N/A
100
100
100


insulin)







29848 n-mer
737
N/A
92
94
97


327876
294
mir-29b-1
118
176
138


327878
296
mir-203
185
29
150


327880
298
mir-10b
136
125
149


327890
307
let-7i
88
113
115


327892
308
mir-131-2/
139
104
96




mir-9





327901
310
mir-143
135
117
135


327903
319
let-7a-3
81
87
89


327905
321
mir-205
115
147
148


327913
323
mir-29c
147
268
123


327919
331
mir-221
154
105
178


327922
337
mir-19b-2
104
76
61


327925
340
mir-133b
166
182
148


327933
343
mir-145
179
115
185


327934
351
mir-213
244
105
103


327939
352
mir-125b-1
175
153
192


327941
357
mir-181b
80
98
68


327954
359
mir-148b
120
102
105


327889
372
mir-23b
73
202
72


338664
491
mir-143
100
76
84




pri-miRNA





340927
319
mir-143
285
103
128









For HepG2 cells treated with 1 nM insulin, treatments with oligomeric compounds of the present invention resulting in a decrease in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds have an insulin sensitization effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin response of repression of mRNA expression of these genes.
From these data, it is evident that the oligomeric compounds, ISIS Number 327878 targeting mir-203 and ISIS Number 327922 targeting mir-19b-2, for example, were observed to result in a 71% and a 24% reduction, respectively, of PEPCK-c mRNA expression, widely considered to be a marker of insulin-responsiveness. Thus, these oligomeric compounds may be useful as pharmaceutic agents with insulin-sensitizing properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.
Conversely, the results observed with the oligomeric compounds targeting mir-29c (ISIS Number 327913), mir-133b (ISIS Number 327925), and mir-125b-1 (ISIS Number 327939), all exhibiting increased expression of the IGFBP-1, PEPCK-c and follistatin marker genes, support the conclusion that the mir-29c, mir-133b, and mir-125b-1 miRNAs may be involved in the regulation of insulin-responsive genes. When these miRNAs are inactivated by the oligomeric compounds of the present invention, IGFBP-1, PEPCK-c and follistatin gene expression is no longer repressed or insulin-sensitive.
A caspase assay was also performed (as in Example 11 above) in HepG2 cells treated with oligomeric compounds of the present invention, and it was determined that oligomeric compounds targeting the mir-29c, mir-133b, and mir-125b-1 miRNAs were not toxic to the cells and that the observed reduction in mRNA expression levels of insulin-responsive genes was not due to a general toxicity of the compounds or an induction of apoptotic pathways.
Example 19
Analysis of Expression of Mir-143 Pri-miRNA and Mature Mir-143
Ribonuclease Protection Assays:
The ribonuclease protection assay (RPA) is known in the art to be a sensitive and accurate method of measuring and/or following temporal changes in the expression of one or more RNA transcripts in a complex mixture of total RNA. Briefly, this method employs a radioactive probe that specifically hybridizes to a target transcript RNA. The probe is added to a sample of total RNA isolated from tissues or cells of interest, and, upon hybridization to its target, the probe forms a double-stranded RNA region. If the region of hybridization is shorter than the entire length of either the probe or the target RNA molecule, the molecule will be a hybrid molecule with partial double-stranded and partial single-stranded character. These hybrid molecules are then digested with single-strand-specific RNases such as RNase A and/or T1, which remove any non-hybridized single stranded portions of the hybrid molecules, leaving only the “protected” dsRNA fragments. The RNase protected fragments are then electrophoresed on a denaturing gel, causing the strands to dissociate, and the intensity of radioactive probe signal observed is directly proportional to the amount of specific target transcript RNA in the original total RNA sample.
In an embodiment of the present invention, small non-coding RNAs in a sample were detected by RPA using probes that hybridize to pri-miRNAs, pre-miRNAs or mature miRNAs. Probes were in vitro transcribed using the mir VanaTM miRNA Probe Construction Kit (Ambion Inc., Austin, Tex.) according to the manufacturer's protocol, beginning with a DNA oligonucleotide representing sense strand of the mature miRNA to be detected plus four thymidylate residues plus an 8-base sequence complementary to the 3′-end of the T7 promoter primer supplied with the kit. When the T7 primer is annealed to this DNA oligonucleotide, the Klenow DNA polymerase is used to generate a double-stranded DNA, and then in vitro transcription is performed using the T7 RNA polymerase and radiolabeled nucleotides to generate a radioactive RNA probe for detection of the miRNA.
In one embodiment, a probe specifically hybridizing to the murine mir-143 miRNA was used in a RPA of 5 μg total RNA from kidney, liver, heart, lung, brain, spleen, and thymus tissues from mouse as well as adipose tissue from db/db obese mice, total RNA from an 11-day-old embryo, and total RNA from undifferentiated and differentiated 3T3-L1 cells. All signals were normalized to the levels of 5.8S rRNA. Expression levels of mir-143 were highest in lung, heart, spleen, thymus and kidney tissues from wildtype mice. Notably, mir-143 expression levels were significantly elevated in adipose tissue from db/db mice (approximately 4 times higher than expression levels in kidney, 2.4 times higher than levels in heart and 1.6 times higher than levels in lung tissues from wildtype mice).
In one embodiment, a probe hybridizing to the mir-143 pri-miRNA molecule was used in a RPA of 2-5 μg total RNA from human spleen, thymus, testes, heart, liver, kidney, skeletal muscle, brain, lung and adipose tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and HepG2 cells. A probe hybridizing to the β-actin mRNA was used as a control. The highest levels of mir-143 pri-miRNA were observed in heart, kidney, thymus and adipose tissues, as well as in differentiated adipocytes.
In one embodiment, a probe hybridizing to the mature mir-143 miRNA was also used in a RPA of 2 μg total RNA from human spleen, thymus, heart, liver, kidney and brain, tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and total RNA from HepG2, A549, T-24, HEK293, HuVEC (human umblical vein endothelial cells), HL-60 and T47D cell lines. A probe hybridizing to the β-actin mRNA was used as a control, and all signals were normalized to the levels of mir-143 expression in preadipocytes. The results are shown in Table 26.







TABLE 26







RNase protection of mature mir-143 in


total RNA from tissues and cell lines











Fold Increase over



Tissue or cell line
preadipocytes













Spleen
2.6



Thymus
3.8



Heart
8.2



Liver
0



Kidney
10.0



Brain
0.9



Preadipocytes
1.0



Differentiated
2.6



adipocytes




HepG2
0.5



A549
N.D.



T-24
0.4



HEK293
0.5



HuVEC
0.3



HL-60
0.4



T47D
0.3









From these data, the highest levels of expression of the mature mir-143 miRNA were observed in total RNA from kidney and heart tissues. High levels of expression of the mature mir-143 miRNA were also observed in total RNA from lymphoid tissues such as spleen and thymus. Expression of the mature mir-143 miRNA is increased in differentiated adipocytes as compared to levels in preadipocytes. These data also suggest that the mir-143 miRNA plays a role in cellular differentiation.
In one embodiment, a uniform 2′-MOE phosphorothioate oligomeric compound with a sequence antisense to the mature mir-143 miRNA was spiked into the RPA mixture above. This antisense mir-143 compound was found to block the ribonuclease protection expression pattern previously observed, suggesting that this antisense mir-143 oligomeric compound specifically hybridizes to and inhibits the activity of mir-143. This oligomeric compound targeting the mir-143 miRNA is predicted to form a double stranded molecule that blocks endogenous mir-143 miRNA activity when employed in vivo.
It was also noted that, while expression of the mir-143 miRNA can be detected in non-transformed cells, such as HuVECs, in general, transformed cell lines have not been observed to exhibit high levels expression of mir-143. When taken together with the observation that the mir-143 miRNA is upregulated as adipocytes differentiate as well as the observation that oligomeric compounds targeting mir-143 inhibit adipocyte differentiation, these data suggest that mir-143 normally promotes adipocyte differentiation and mir-143 may have an inhibitory effect on cellular transformation that is consistent with its role in promoting cellular differentiation. Lack of expression or downregulation of mir-143 in transformed cell lines may be a cause or consequence of the undifferentiated state. Thus, mir-143 mimics may be useful as pharmaceutical agents in the treatment of hyperproliferative disorders such as cancer.
In one embodiment, the expression of human mir-143 was assessed during adipocyte differentiation. A probe hybridizing to the human mir-143 miRNA was used in a RPA of 5 μg total RNA from pre-adipocytes, and differentiated adipocytes sampled at one, four, and ten days post-differentiation. All signals were normalized to the levels of 5.8S rRNA. mir-143 expression levels were 2.5 to 3-fold higher by day 10 post-differentiation when compared to mir-143 expression levels in pre-adipocytes by ribonuclease protection assay.
Real-Time RT-PCR Analysis of Mir-143 Pri-miRNA Expression:
Expression levels of mir-143 pri-miRNA were compared in total RNAs from various tissues and total RNA from several cell lines. Total RNA from spleen, heart, liver, and brain tissues, as well as total RNA from preadipocytes, differentiated adipocytes, and HepG2, T-24 and HeLa cell lines was purchased or prepared as described supra. 80 ng of total RNA from each source was used to perform real-time RT-PCR using a primer/probe set specific for the mir-143 pri-miRNA molecule. ISIS 339314 (TCCCAGCCTGAGGTGCA; SEQ ID NO: 786) was used as the forward primer, ISIS 342897 (GCTTCATCTCAGACTCCCAACTG; SEQ ID NO: 787) was used as the reverse primer, and ISIS 342898 (TGCTGCATCTCTG; SEQ ID NO: 788) was used as the probe. RNA levels from all sources were compared to RNA levels from preadipocytes. Greater than 32-fold higher levels of mir-143 pri-miRNA were observed in heart tissue as compared to preadipocytes; 19-fold higher levels of mir-143 pri-miRNA were observed in differentiated adipocytes relative to levels in preadipocytes; 5-fold higher levels of mir-143 pri-miRNA were observed in spleen as compared to preadipocytes.
Northern blot analyses were performed in differentiating adipocytes as described in Example 14 using the mir-143-specific DNA oligonucleotide probe (SEQ ID NO: 319) to detect the mir-143 target and a probe for the U6 RNA to normalize for variations in loading and transfer efficiency, and it was confirmed by Northern analysis that expression of mature mir-143 increases from day 1 through day 10 after induction of differentiation.
In human pre-adipocytes and adipocytes sampled one, four, seven and ten days post-differentiation, expression levels of mir-143 pri-miRNA were also assessed using real-time RT-PCR analysis as described herein. 80 ng of total RNA from pre-adipocytes or differentiated adipocytes was used to perform real-time RT-PCR using the same primer/probe set specific for the mir-143 pri-miRNA molecule described supra (ISIS 339314, SEQ ID NO: 786 was used as the forward primer, ISIS 342897, SEQ ID NO: 787 was used as the reverse primer, and ISIS 342898, SEQ ID NO: 788 was used as the probe). RNA levels from all sources were normalized to 5.8S rRNA levels. mir-143 pri-miRNA levels in preadipocytes were 94% of the level of the 5.8S rRNA. At day 1 post-differentiation, mir-143 pri-miRNA levels had decreased to 38% of the level of the 5.8S rRNA. By day 4 post-differentiation, mir-143 pri-miRNA levels had decreased to 26%, by day 7 post-differentiation, mir-143 pri-miRNA levels were at 25%, and by day 10 post-differentiation, mir-143 pri-miRNA levels had dropped to 23% of the level of the 5.8S rRNA. Taken together with the results from RPA analysis, it appears that levels of the mature mir-143 miRNA increases approximately 2- to 3-fold by day 10 post-differentiation in differentiated adipocytes, accompanied by a concomittant approximately 4-fold decrease in the levels of unprocessed mir-143 pri-miRNA, indicating that adipocyte differentiation coincides with either an increase in processing of the mir-143 miRNA from the mir-143 pri-miRNA or an overall decrease in mir-143 pri-miRNA production.
Effects of Oligomeric Compounds on Expression of Pri-miRNAs:
Mature miRNAs originate from long endogenous primary transcripts (pri-miRNAs) that are often hundreds of nucleotides in length. It is believed that a nuclear enzyme in the RNase III family, known as Drosha, processes pri-miRNAs (which can range in size from about 110 nucleotides up to about 450 nucleotides in length) into pre-miRNAs (from about 70 to 110 nucleotides in length) which are subsequently exported from the nucleus to the cytoplasm, where the pre-miRNAs are processed by human Dicer into double-stranded intermediates resembling siRNAs, which are then processed into mature miRNAs. Using the real-time RT-PCR methods described herein, the expression levels of several pri-miRNAs were compared in differentiating adipocytes. Total RNA from preadipocytes and differentiating adipocytes was prepared as described herein.
In one embodiment, modified oligomeric compounds can be transfected into preadipocytes or other undifferentiated cells, which are then induced to differentiate (as described in detail, herein), and it can be determined whether these modified oligomeric compounds act to inhibit or promote cellular differentiation. Real-time RT-PCR methods can then be used to determine whether modified oligomeric compounds targeting miRNAs can affect the expression or processing of the pre-miRNAs from the pri-miRNA (by the Drosha enzyme), the processing of the mature miRNAs from the pre-miRNA molecules (by the Dicer enzyme), or the RISC-mediated binding of a miRNA to its target nucleic acid.
Here, oligomeric compounds targeting mir-143 were transfected into preadipocytes which were then induced to differentiate, in order to assess the effects of these compounds on mir-143 pri-miRNA levels during differentiation. mir-143 pri-miRNA levels were assessed on days 3 and 9 after differentiation.
In addition to the uniform 2′-MOE phosphorothioate oligomeric compound ISIS Number 327901 (SEQ ID NO: 319) targeting mature mir-143, a 5-10-7 gapmer oligomeric compound, ISIS Number 340927 (SEQ ID NO: 319), was designed to target mature mir-143. As negative controls, “scrambled” oligomeric compounds were also designed; ISIS Number 342672 (ATACCGCGATCAGTGCATCTTT; incorporated herein as SEQ ID NO: 789) contains 13 mismatches with respect to the mature mir-143 miRNA, and ISIS Number 342673 (SEQ ID NO: 758) contains 15 mismatches with respect to the mature mir-143 miRNA. ISIS 342672 and ISIS 342673 are uniform 2′-MOE phosphorothioate oligomeric compounds 22 nucleotides in length. ISIS Number 342677 (SEQ ID NO: 789) and ISIS Number 342678 (SEQ ID NO: 758) are the corresponding 5-10-7 scrambled 2′-MOE gapmer oligomeric compounds. All cytidine residues are 5-methylcytidines. Additionally, ISIS Number 342683 (CCTTCCCTGAAGGTTCCTCCTT; herein incorporated as SEQ ID NO: 790), representing the scrambled sequence of an unrelated PTP1B antisense oligonucleotide, was also used as a negative control.
These compounds were transfected into differentiating adipocytes and their effects on levels of the mir-143 pri-miRNA molecule were assessed in pre-adipocytes vs. differentiated adipocytes, by real-time RT-PCR using the primer/probe set specific for the mir-143 pri-miRNA (forward primer=ISIS 339314, SEQ ID NO: 786; reverse primer=ISIS 342897, SEQ ID NO.: 787; probe=ISIS 342898, SEQ ID NO.: 788). Thus, it was observed that in the presence of the oligomeric compound ISIS Number 327901 (SEQ ID NO: 319), levels of the mir-143 pri-miRNA are enhanced approximately 4-fold in differentiated adipocytes 9 days post-differentiation as compared to 3 days post-differentiation. These results suggest that ISIS Number 327901, the uniform 2′-MOE P═S oligomeric compound targeted to mature mir-143, interferes with the processing of the mir-143 pri-miRNA into the pre-miRNA by the Drosha RNase III enzyme. Alternatively, the compound interferes with the processing of the mir-143 pre-miRNA into the mature mir-143 miRNA by the Dicer enzyme. The decrease in levels of mature mir-143 miRNA in differentiating cells treated with ISIS Number 327901 (SEQ ID NO: 319) may also trigger a feedback mechanism that signals these cells to increase production of the mir-143 pri-miRNA molecule. Not mutually exclusive with the processing interference or the feedback mechanisms is the possibility that treatment with oligomeric compounds could stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-143 pri-miRNA or pre-miRNA molecules. Oligomeric compounds of the present invention are predicted to disrupt pri-miRNA and/or pre-miRNA structures, and sterically hinder Drosha and/or Dicer cleavage, respectively. Furthermore, oligomeric compounds which are capable of binding to the mature miRNA are also predicted to prevent the RISC-mediated binding of a miRNA to its target nucleic acid, either by cleavage or steric occlusion of the miRNA.
Example 20
Identification of RNA Transcripts Bound by miRNAs
The RACE-PCR method (Rapid Amplification of cDNA Ends) was used as a means of identifying candidate RNA transcripts bound and/or potentially regulated by miRNAs. RNA was prepared and isolated from preadipocytes, and, using the SMART RACE cDNA Amplification kit (BD Biosciences, Clontech, Palo Alto, Calif.) according to manufacturer's protocol, synthetic adaptor sequences were incorporated into both the 5′- and 3′-ends of the amplified cDNAs during first strand cDNA synthesis. 5′ RACE-PCR was then performed using the mature miRNA as the 3′-end primer along with the 5′ adapter primer from the kit to amplify the 5′-end of the candidate RNA transcript. 3′ RACE-PCR was performed using the antisense sequence of the miRNA as a primer along with the 3′ adapter primer from the kit to amplify the 3′-end of the candidate RNA transcript. In some embodiments, the primers 2-nucleotides shorter than the corresponding miRNA were used in order to identify targets with some mismatching nucleotides at the end of the miRNA (these primers are indicated by “3′-RACE-2nt” in Table 27 below).
For example, the antisense sequences of the mature mir-43, let-7g, mir-23b, mir-29c, mir-131, mir-143, mir-130b and mir-213 miRNAs were used as primers in 3′ RACE-PCR, and the mature mir-143 or mir-15a sequences were used in 5′ RACE-PCR. The RACE-PCR products employing the mir-143 miRNA, the mir-143 antisense sequence, the mir-131 antisense sequence or the mir-15a miRNA as primers were electrophoresed and gel purified, prominent bands were excised from the gel, and these products were subcloned using standard laboratory methods. The subcloned products from the RACE-PCR were then were sent to Retrogen, Inc. (San Diego, Calif.) for sequencing. Candidate RNA transcripts targeted by miRNAs were thereby identified.
Candidate RNA targets identified by RACE-PCR methods are shown in Table 27, where the miRNA-specific primer used to identify each transcript is indicated in the column entitled “primer”. (In some cases, the target was identified multiple times by more than one RACE-PCR method, and thus appears in the table more than once).







TABLE 27







Predicted RNA targets of mir-143















SEQ




GenBank

ID


Primer
Method
Accession
RNA transcript targeted by miRNA
NO














mir-143
5′RACE
NM_001753.2
caveolin 1, caveolae protein, 22 kDa
791


mir-143
5′RACE
NM_004652.1
ubiquitin specific protease 9, X-
792





linked (fat facets-like, Drosophila)



mir-143
5′RACE
NM_007126.2
valosin-containing protein
793


mir-143
5′RACE
NM_000031.1
aminolevulinate, delta-, dehydratase
794


mir-143
5′RACE
NM_007158.1
NRAS-related gene
795


mir-143
5′RACE
NM_015396.1
HSPC056 protein
796


mir-143
5′RACE
NM_001219.2
calumenin
797


mir-143
5′RACE
BC051889.1
RNA binding motif, single stranded
798





interacting protein 1



mir-143
5′RACE
BX647603.1
Homo sapiens mRNA; cDNA
799





DKFZp686L01105 (from clone






DKFZp686L01105)



mir-143
5′RACE
AB051447.1
KIAA1660 protein
800


mir-143
5′RACE
NM_007222.1
zinc-fingers and homeoboxes 1
801


mir-143
5′RACE
NM_001855.1
collagen, type XV, alpha 1
802


mir-143
3′RACE
NM_007222.1
zinc-fingers and homeoboxes 1
801


mir-143
3′RACE
NM_006732
FBJ murine osteosarcoma viral
803





oncogene homolog B



mir-143
3′RACE
NM_003718.2
cell division cycle 2-like 5
804





(cholinesterase-related cell






division controller)



mir-143
3′RACE
NM_005626.3
splicing factor, arginine/serine-
805





rich 4



mir-143
3′RACE
NM_002355.1
mannose-6-phosphate receptor (cation
806





dependent)



mir-143
3′RACE
NM_000100.1
cystatin B (stefin B)
807


mir-143
3′RACE
NM_015959.1
CGI-31 protein
808


mir-143
3′RACE
NM_006769.2
LIM domain only 4
809


mir-143
3′RACE
NM_003184.1
TAF2 RNA polymerase II, TATA box
810





binding protein (TBP)-associated






factor, 150 kDa



mir-143
3′RACE
NM_025107.1
myc target in myeloid cells 1
811


mir-143
3′RACE
NM_003113.1
nuclear antigen Sp100
812


mir-143
3′RACE
NM_002696.1
polymerase (RNA) II (DNA directed)
813





polypeptide G



mir-143
3′RACE
NM_004156.1
protein phosphatase 2 (formerly 2A),
814





catalytic subunit, beta isoform



mir-143
3′RACE
NM_031157
heterogeneous nuclear
815





ribonucleoprotein A1



mir-143
3′RACE
NM_004999.1
myosin VI
817


mir-143
3′RACE
NM_018036.1
chromosome 14 open reading frame 103
818


mir-143
3′RACE
NM_018312.2
chromosome 11 open reading frame 23
819


mir-143
3′RACE
NM_002950.1
ribophorin I
820


mir-143
3′RACE
NM_006708.1
glyoxalase I
821


mir-143
3′RACE
NM_014953.1
mitotic control protein dis3 homolog
822


mir-143
3′RACE
NM_004926.1
zinc finger protein 36, C3H type-
823





like 1



mir-143
3′RACE
NM_004530.1
matrix metalloproteinase 2
824





(gelatinase A, 72 kDa gelatinase,






72 kDa type IV collagenase)



mir-143
3′RACE
NM_015208.1
KIAA0874 protein
825


mir-143
3′RACE
NM_002582.1
poly (A)-specific ribonuclease
826





(deadenylation nuclease)



mir-143
3′RACE
NM_000297.2
polycystic kidney disease 2
827





(autosomal dominant)



mir-143
3′RACE
NM_001175
Rho GDP dissociation inhibitor (GDI)
828





beta



mir-143
3′RACE
XM_166529
glucocorticoid induced transcript 1,
837





GLCCI1



mir-143
3′RACE-
NM_001753.2
caveolin 1, caveolae protein, 22 kDa
791



2nt





mir-143
3′RACE-
NM_006732
FBJ murine osteosarcoma viral
803



2nt

oncogene homolog B



mir-143
3′RACE-
NM_000100.1
cystatin B (stefin B)
807



2nt





mir-143
3′RACE-
NM_015959.1
CGI-31 protein
808



2nt





mir-143
3′RACE-
NM_004156.1
protein phosphatase 2 (formerly 2A),
814



2nt

catalytic subunit, beta isoform



mir-143
3′RACE-
NM_031157
heterogeneous nuclear
815



2nt

ribonucleoprotein A1



mir-143
3′RACE-
NM_002582.1
poly(A)-specific ribonuclease
826



2nt

(deadenylation nuclease)



mir-143
3′RACE-
NM_000297.2
polycystic kidney disease 2
827



2nt

(autosomal dominant)



mir-143
3′RACE-
NM_006325.2
RAN, member RAS oncogene family
829



2nt





mir-143
3′RACE-
NM_004627.1
tryptophan rich basic protein
830



2nt





mir-143
3′RACE-
NM_012210.1
tripartite motif-containing 32
831



2nt





mir-143
3′RACE-
AJ131244.1
SEC24 related gene family, member A
832



2nt

(S. cerevisiae)



mir-143
3′RACE-
NM_031267.1
cell division cycle 2-like 5
833



2nt

(cholinesterase-related cell






division controller)



mir-143
3′RACE-
AL049367.1
guanine nucleotide binding protein
835



2nt

(G protein), gamma 12



mir-143
3′RACE-
NM_001344
defender against cell death 1
836



2nt





mir-131
3′RACE
AK001214.1
hypothetical protein FLJ10352
1735


mir-131
3′RACE
NM_001614
actin, gamma 1 (ACTG1), mRNA
1736


mir-131
3′RACE
NM_001948.1
dUTP pyrophosphatase (DUT), mRNA
1737


mir-131
3′RACE
NM_002387.1
mutated in colorectal cancers (MCC),
1738





mRNA



mir-131
3′RACE
NM_004109.1
ferredoxin 1 (FDX1), nuclear gene
1739





encoding mitochondrial protein, mRNA



mir-131
3′RACE
NM_004342.4
caldesmon 1 (CALD1), transcript
1740





variant 2, mRNA



mir-131
3′RACE
NM_005572.2
lamin A/C (LMNA), transcript variant
1741





2, mRNA



mir-131
3′RACE
NM_015640.1
PAI-1 mRNA-binding protein (PAI-
1742





RBP1), mRNA



mir-131
3′RACE
NM_017789.1
semaphorin 4C (SEMA4C), mRNA
1743


mir-131
3′RACE
NM_144697.1
hypothetical protein BC017397
1744





(LOC148523), mRNA



mir-131
3′RACE
NM_173710
NADH dehydrogenase 3 (MTND3), mRNA
1745


mir-15a
5′RACE
AF220018.1
Homo sapiens tripartite motif
1746





protein (TRIM2) mRNA



mir-15a
5′RACE
M98399.1
Human antigen CD36 mRNA
1747


mir-15a
5′RACE
Y00281.1
Human mRNA for ribophorin I
1748









Because these RNA transcripts in Table 27 were identified as being bound by one of the mir-143, mir-131, or mir-15a miRNAs, these miRNAs are predicted to serve a regulatory role in expression or activity of these transcripts identified by RACE-PCR. Additional candidate human RNA targets can be identified in the same manner.
Example 21
Effects of Oligomeric Compounds on Adipocyte Differentiation Hallmark Genes in Differentiated Adipocytes
The effect of the oligomeric compounds of the present invention targeting miRNAs on the expression of markers of cellular differentiation was examined in differentiated adipocytes.
The effects of the oligomeric compounds of the present invention on the hallmark genes known to be upregulated during adipocyte differentiation assayed in Example 13 were also assayed in differentiated adipoctyes. As previously described, the HSL, aP2, Glut4, and PPARγ genes play important rolls in the uptake of glucose and the metabolism and utilization of fats. Also as previously described, an increase in triglyceride content is another well-established marker for adipocyte differentiation. Human white preadipocytes (Zen-Bio Inc., Research Triangle Park, NC) were grown in preadipocyte media (ZenBio Inc.). After the cells reached confluence (approximately three days), they were exposed to differentiation media (Zen-Bio, Inc.) containing a PPAR-γ agonist, IBMX, dexamethasone, and insulin for three days. Cells were then fed Adipocyte Medium (Zen-Bio, Inc.), which was replaced at 2 to 3 day intervals. One day before transfection, 96-well plates were seeded with 3000 cells/well. Cells were then transfected on day nine post-differentiation, according to standard published procedures with 250 nM oligonucleotide in LIPOFECTINTM (Invitrogen Corporation, Carlsbad, Calif.) (Monia et al., J. Biol. Chem. 1993 268(19):14514-22). Oligomeric compounds were tested in triplicate on each 96-well plate, and the effect of TNF-α; known to inhibit adipocyte differentiation, was also measured in triplicate. Oligomeric compound treatments and transfectant-only negative controls may be measured up to six times per plate. On day twelve post-differentiation, cells were washed and lysed at room temperature, and the expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulation were measured in adipocytes transfected with the uniform 2′-MOE phosphorothioate (PS) previously described in Example 13 as well as the chimeric gapmer oligomeric compounds targeting the mir-143 miRNA and the mir-143 pri-miRNA described in Example 16. On day twelve post-differentiation, cells were lysed in a guanadinium-containing buffer and total RNA was harvested. The amount of total RNA in each sample was determined using a Ribogreen Assay (Molecular Probes, Eugene, Oreg.). Real-time PCR was performed on the total RNA using primer/probe sets for the adipocyte differentiation hallmark genes Glut4, HSL, aP2, and PPARγ. Triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed relative to control levels (control=treatment with ISIS-29848 (SEQ ID NO: 737)). The results of this experiment are shown in Table 28.







TABLE 28







Effects of oligomeric compounds targeting miRNAs on


expression of adipocyte differentiation markers













ISIS
SEQ ID




PPAR


Number
NO
TG
aP2
HSL
Glut4
gamma





327876
294
1.16
0.67
0.81
3.53
1.28


327878
296
1.08
0.13
0.19
0.17
0.85


327880
298
1.12
1.14
0.93
0.76
1.86


327888
306
1.13
0.73
0.84
0.56
1.69


327889
307
1.09
1.12
0.77
0.99
1.63


327890
308
1.13
0.35
0.42
0.37
1.05


327892
310
1.23
0.81
0.62
0.42
1.01


327901
319
1.12
1.28
1.47
2.20
1.34


327903
321
1.12
0.56
0.53
0.36
0.91


327905
323
1.18
0.85
0.65
0.58
1.31


327913
331
1.12
1.05
1.09
1.52
1.29


327919
337
1.15
1.20
0.83
1.82
1.80


327922
340
1.48
0.91
1.01
0.61
0.99


327925
343
1.33
0.78
1.20
0.74
1.30


327933
351
1.63
1.58
1.30
2.12
1.60


327934
352
1.43
1.50
1.97
1.52
1.54


327939
357
1.33
1.16
1.08
0.72
1.89


327941
359
1.33
0.90
1.17
0.90
1.66


327954
372
1.46
1.23
1.35
0.61
1.46


328382
491
1.33
0.92
0.53
0.75
0.97


338664
491
1.72
0.77
1.01
1.08
1.06


340927
319
1.61
0.71
0.64
0.96
1.21









From these data, it was observed that the compound targeting the mir-203 miRNA (ISIS Number 327878), exhibited a sustained reduction in the hallmark marker genes at the 12th day post differentiation. Treatment with this compound resulted in decreased expression of the aP2, HSL, Glut4 and PPARγ marker genes, indicating that this oligomeric compound may lead to reduced levels of mobilization of fatty acids from adipose tissue, and has the potential to ameliorate some of the symptoms of type 2 diabetes, obesity, hypertension, atherosclerosis, cardiovascular disease, insulin resistance, and certain cancers. Notably, the effect of treatment of differentiated adipocytes with this oligomeric compound targeting the mir-203 miRNA mirrors the effect of treating cells with the TNF-α positive control that inhibits adipocyte differentiation. This evidence suggests that the oligomeric compound targeting the mir-203 miRNA can act as a TNF-α mimetic compound, and potentially may be used in the suppression of cellular differentiation and the maintenance of cells in a quiescent state.
The oligomeric compound targeting the mir-203 miRNA was also tested in the insulin assay (see Example 18) and was observed to reduce expression of PEPCK-c, indicating that it may also be useful as an insulin mimetic and/or antidiabetic drug.
As an extension of these conclusions, one having ordinary skill in the art would appreciate that further modified oligomeric compounds could be designed to also target the mir-203 mature miRNA, or the pri-miRNA and pre-miRNA precursors. Such compounds are noted to be within the scope of the present invention.
Example 22
Effects of Oligomeric Compounds on Lymphocytic Leukemia Cells
Mir15-a-1 and mir-16-3 have been recently shown to reside in human chromosomal region (13q14) that is deleted in about 50% of chronic lymphocytic leukemia (CLL) patients. Mir-15 and 16 were found to be down-regulated in about 68% of CLL cases (Calin et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 15524-15529, which is incorporated herein by reference in its entirety). CLL B-cells develop chemotherapy resistance over time, possibly due to a defective apoptosis pathway.
Using the 5′RACE method (described in Example 20), the CD36 mRNA was identified as one target regulated by mir-15 and/or mir-16 miRNAs. CD36 is a scavenger receptor involved in fat uptake by macrophages and adipocytes. CD36 is reported to be upregulated in some CLL cell lines, and its expression may correlate with tumor invasiveness.
If the apoptosis pathway is defective and the deletion or down-regulation of mir-15 and/or mir-16 play a role in CLL chemo-resistance, then addition of mir-15 and/or mir-16 should be able to induce apoptosis in CLL and increase drug-induced apoptosis. RNA oligonucleotide molecules ISIS Number 338963 (TAGCAGCACATAATGGTTTGTG; SEQ ID NO: 269) representing mir-15a-1/mir-15a-2, ISIS Number 338961 (TAGCAGCACATCATGGTTTACA; SEQ ID NO: 246) representing mir-15b, and ISIS Number 338965 (TAGCAGCACGTAAATATTGGCG; SEQ ID NO: 196) representing mir-16-1/mir-16-2/mir-16-3 were synthesized and deprotected. Additionally, RNA oligonucleotides bearing imperfect complementarity to these miRNA mimics (mimicking the imperfect complementarity found in the pri-miRNA) were also synthesized and deprotected. These imperfect complements were ISIS Number 338964 (TGCAGGCCATATTGTGCTGCCT; SEQ ID NO: 840), which is partially complementary to ISIS Number 338963 and represents the imperfect complement of mir-15a-1/mir-15a-2; ISIS Number 338962 (TGCGAATCATTATTTGCTGCTC; SEQ ID NO: 841), which is partially complementary to ISIS Number 338961 and represents the imperfect complement of mir-15b; ISIS Number 338966 (CTCCAGTATTAACTGTGCTGCTG; SEQ ID NO: 842), which is partially complementary to ISIS Number 338965 and represents the imperfect complement of mir-16-1 and mir-16-2; and ISIS Number 338967 (CACCAATATTACTGTGCTGCTT; SEQ ID NO: 843), which is partially complementary to ISIS Number 338965 and represents the imperfect complement of mir-16-3. These RNA molecules were diluted in water, and the concentration determined by A260. Equimolar amounts of each of the miRNAs and their imperfect complementary RNA sequences were mixed together in the presence of Dharmacon 5× Universal buffer to form four “natural” double-stranded miRNA mimics ISIS Number 338965 (SEQ ID NO: 196) was used twice; once, it was hybridized to ISIS Number 338966, and once it was hybridized to ISIS Number 338967, to form two different “natural” double-stranded miRNA mimics, Mir-16-1/Mir-16-2 and Mir-16-3, with imperfect complementarity. The mixture of four “natural” miRNA mimics was incubated for 1-5 minutes at 90° C. (the time depends on the volume of the mixture) and then incubated at 37° C. for one hour. A260 readings were taken on the mixture for final concentration determination.
Heparinized peripheral blood from CLL patients was separated on a Ficoll density gradient to obtain greater than 95% pure CLL B-cells. These cells are tested for expression of the CD5/CD19/CD23 antigens. Positive expression of these three antigens indicates that the cells are CLL B-cells (Pederson et al., Blood, 2002, 100, 2965, which is incorporated herein by reference in its entirety). Additionally, cytogenetic analysis can be performed to ascertain that the cells have the 13q deletion. A mixture of all four “natural” miRNA mimics at 2 μM each was electroporated into the cells. The cells were cultured in the presence or absence of apoptosis-inducing agents fludarabine A, or Dexamethasone (which are known to employ the intrinsic mitochondrial apoptotic pathway) or the antitumor agent CDDO-Im (reported to function through an alternative extrinsic apoptotic pathway) for 24 hours. Following incubation, apoptosis was monitored by annexin/PI double staining as outlined in FIG. 1 of Pederson et al., Blood, 2002, 100, 2965. The double-stranded RNA oligomeric compounds representing mir-15 and mir-16 miRNA mimics were observed to play a role in the induction of spontaneous as well as drug-induced apoptosis. Thus, oligomeric compounds of the present invention may be useful in the treatment of CD36-related diseases and conditions such as chronic lymphocytic leukemia and other cancers.
Example 23
Effect of Oligomeric Compounds Targeting miRNAs In Vivo
As described herein, leptin-deficient (ob/ob) mice, leptin receptor-deficient (db/db) mice and diet-induced obesity (DIO) mice are used to model obesity and diabetes. In accordance with the present invention, oligomeric compounds targeting mir-143, mir-131 (also known as mir-9) and mir-203 were tested in the ob/ob and db/db models. The ob/ob mice were fed a high fat diet and were subcutaneously injected with the oligomeric compounds of the invention or a control compound at a dose of 25 mg/kg two times per week for 6 weeks. Saline-injected animals, leptin wildtype littermates (i.e. lean littermates) and ob/ob mice fed a standard rodent diet served as controls. The physiological effects resulting from inhibition of target RNA, such as the effects of target inhibition on glucose and insulin metabolism and the expression of genes that participate in lipid metabolism, cholesterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism, were assessed by methods disclosed herein. In brief, plasma levels of liver transaminases, cholesterol, triglycerides, free fatty acids and glucose were assessed weekly by tail bleed, with the tail bleed on week three taken under fasting conditions. After the treatment period, mice were sacrificed and liver, spleen, pancreas, muscle, kidney and heart, as well as brown adipose tissue (BAT) and white adipose tissue (WAT) tissues were collected. mRNA expression levels of the Glut4, aP2, HSL and PPARγ marker genes were evaluated. RNA isolation and target RNA expression level quantitation are performed as described.
Two oligomeric compounds targeting the mir-143 miRNA were compared for their effects on the physiological indications of obesity and diabetes. The oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319), 22-nucleotides in length, targets the mature mir-143, and is a uniform 2′-MOE oligonucleotide with phosphorothioate internucleoside linkages throughout. The oligomeric compound ISIS Number 340927 (SEQ ID NO: 319) is a 5-10-7 gapmer also designed to target the mature mir-143 miRNA. The effects of these oligomeric compounds targeting mir-143 on several physiological parameters and markers of obesity and/or diabetes were examined in vivo. Potential effects on food consumption were also monitored.
Plasma cholesterol levels were observed to slightly decrease over time in ob/ob mice treated with the gapmer oligomeric compound ISIS Number 340927 (SEQ ID NO: 319) targeted to mir-143. Similarly, plasma triglyceride and plasma glucose levels were generally slightly lower in ob/ob mice treated with this compound as compared to untreated mice, or mice treated with control compounds. mRNA expression levels of the Glut4, aP2 and HSL marker genes were slightly reduced by both oligomeric compounds ISIS Number 327901 and ISIS Number 340927 targeting mir-143. Thus, these oligomeric compounds targeting mir-143 may be useful compounds in the treatment of obesity or diabetes.
In addition, Northern blot analyses were performed to quantitate the expression of mature mir-143 in kidney samples of ob/ob mice treated with oligomeric compounds of the present invention. The mir-143 specific DNA oligonucleotide probe (SEQ ID NO: 319) described above was used to detect expression levels of the mir-143 miRNA in ob/ob mice treated (twice weekly at 25 mg/kg) with ISIS Numbers 327901, the uniform 2′-MOE oligomeric compound, or ISIS Number 340927, the 5-10-7 gapmer compound, both targeted to mir-143, versus saline treated animals or animals treated with ISIS 342672 (SEQ ID NO: 789), a uniform 2′-MOE scrambled negative control oligomeric compound. Expression levels were normalized against the U6 RNA and the expression levels of saline treated animals were set at 100%. Most notably, in kidney samples from ob/ob mice treated with ISIS Number 327901, the uniform 2′-MOE oligomeric compound targeted to mir-143 exhibited a nearly 40% decrease in in vivo expression levels of the mature mir-143 miRNA. In kidney samples from mice treated with the gapmer oligomeric compound targeting mir-143, ISIS Number 340927, a 23% reduction in in vivo expression levels of the mature mir-143 miRNA was observed.
Oligomeric compounds targeting the mir-131/mir-9 and the mir-203 miRNAs were also tested for their effects on the physiological indicators or markers of obesity and diabetes. The oligomeric compound, ISIS Number 327892 (SEQ ID NO: 310), targeted to mir-131/mir-9, 21-nucleotides in length, is a uniform 2′-MOE oligonucleotide with phosphorothioate internucleoside linkages throughout. The oligomeric compound ISIS Number 340926 (SEQ ID NO: 310) is a 5-10-6 gapmer oligomeric compound also designed to target the mir-131/mir-9 miRNA. The oligomeric compound ISIS Number 327878 (SEQ ID NO: 296) targeted to mir-203, 22-nucleotides in length, is a uniform 2′-MOE oligonucleotide with phosphorothioate internucleoside linkages throughout. The oligomeric compound ISIS Number 345349 (SEQ ID NO: 296) is a 5-10-7 gapmer oligomeric compound also designed to target the mir-203 miRNA. The effects of these oligomeric compounds were examined in vivo in the ob/ob model. Potential effects on food consumption were also monitored.
Fed plasma glucose levels in ob/ob mice treated with the oligomeric compounds ISIS Number 327892 (SEQ ID NO: 310) and ISIS Number 340926 (SEQ ID NO: 310) targeted to mir-131/mir-9, and ISIS Number 327878 (SEQ ID NO: 296) and ISIS Number 345349 (SEQ ID NO: 296) targeted to mir-203 were observed to be reduced beginning at approximately four weeks after the start of treatment and continuing to decrease on week five as compared to untreated mice, or mice treated with control compounds. Triglyceride levels were also observed to be reduced over time in mice treated with ISIS 340926 and 345349, the gapmer oligomeric compounds targeted to mir-131/mir-9 and mir-203, respectively. No signs of liver toxicity were indicated by weekly measurements of plasma transaminases upon treatment of ob/ob mice with any of the oligomeric compounds targeting mir-143, mir-203 or mir-131/mir9.
ob/ob mice in the fasted state on day 19 after treatment with the oligomeric compounds ISIS Number 327892 (SEQ ID NO: 310) and ISIS Number 340926 (SEQ ID NO: 310) targeted to mir-131/mir-9 also exhibited significant reductions in plasma glucose levels. Notably, the gapmer oligomeric compound ISIS Number 340926 (SEQ ID NO: 310) targeted to mir-131/mir-9 was even more potent than the corresponding uniform 2′-MOE oligonucleotide ISIS Number 327892 (SEQ ID NO: 310).
Furthermore, a decrease in food consumption was observed by the third week and this reduced level was maintained in the fourth week post-treatment of ob/ob mice with these oligomeric compounds. Therefore, the oligomeric compounds targeting the mir-131/mir-9 and mir-203 miRNAs have potential use as appetite suppressants, as well as in the treatment of obesity or diabetes.
The oligomeric compounds ISIS Number 327901 and ISIS Number 340927 both targeting mir-143, ISIS Number 327892 and ISIS Number 340926 both targeting mir-131/mir-9, and ISIS Number 327878 and ISIS Number 345349 both targeting mir-203 were also tested in db/db mice. Although treatment of db/db mice with the gapmer compounds targeting mir-143, mir-203 or mir-131/mir9 resulted in an approximately 2-fold increase in liver transaminases in db/db mice, the uniform 2′-MOE oligomeric compounds targeting mir-143, mir-203 or mir-131/mir-9 were not found to cause liver toxicity in db/db mice, as assessed by weekly measurements of plasma transaminase levels.
Additional oligomeric compounds targeting miRNAs were studied in ob/ob mice. Six week old ob/ob mice were treated (dose=25 mg/kg, twice weekly for four weeks) with uniform 2′-MOE and gapmer oligomeric compounds targeting mir-143, mir-23b, mir-221, let-7a, and mir-29b, and compared to saline treated animals or animals treated with ISIS 342672 (SEQ ID NO: 789), a uniform 2′-MOE scrambled negative control oligomeric compound bearing 13 base mismatches to mir-143. Expression levels were normalized against the U6 RNA and the expression levels of saline treated animals were set at 100%. Fed plasma samples were taken bi-weekly by tail bleed, and plasma levels of liver transaminases, cholesterol, triglycerides, free fatty acids and glucose were assessed, with the tail bleed on week three taken under fasting conditions. Ob/ob mice were treated with ISIS Numbers 327901 and 340927, the uniform 2′-MOE and gapmer oligomeric compounds, respectively, targeting mir-143 are described above. Additionally, ob/ob mice were also treated with the following compounds: ISIS Number 327889 (SEQ ID NO: 307), a phosphorothioate uniform 2′-MOE oligomeric compound, and ISIS Number 340925 (SEQ ID NO: 307), a 2′-MOE 5-10-8 gapmer oligomeric compound, each targeting mir-23b; ISIS Number 327919 (SEQ ID NO: 337), a uniform 2′-MOE oligomeric compound, and ISIS Number 345384 (SEQ ID NO: 337), a phosphorothioate 2′-MOE 5-10-8 gapmer oligomeric compound, each targeting mir-221; ISIS Number 327903 (SEQ ID NO: 321), a uniform 2′-MOE oligomeric compound, and ISIS Number 345370 (SEQ ID NO: 321), a phosphorothioate 2′-MOE 5-10-7 gapmer oligomeric compound, each targeting let-7a; and ISIS Number 327876 (SEQ ID NO: 294), a uniform 2′-MOE oligomeric compound, and ISIS Number 345347 (SEQ ID NO: 294), a phosphorothioate 2′-MOE 5-10-8 gapmer oligomeric compound, each targeted to mir-29b-1.
Ob/ob mice treated with the gapmer compounds ISIS 340925 and ISIS 345384, targeting mir-23b and mir-221, respectively, exhibited reductions in plasma glucose levels in the fed state at weeks two and four, as compared to untreated mice, or mice treated with control compounds. Furthermore, mice treated with ISIS 340925 exhibited a decrease in triglycerides in the fourth week. Ob/ob mice treated with ISIS 340925 did not exhibit an increase in plasma transaminases at weeks two or four. Thus, the oligomeric compounds ISIS Numbers 340925 and 345384 may be useful as agents for the treatment of obesity and/or diabetes.
In addition, Northern blot analyses were performed to quantitate the expression of mir-23b in kidney samples of ob/ob mice treated with oligomeric compounds of the present invention. To detect the mir-23b target, a target-specific DNA oligonucleotide probe with the sequence GTGGTAATCCCTGGCAATGTGAT (SEQ ID NO: 307) was synthesized by IDT (Coralville, Iowa). The oligo probes were 5′ end-labeled with T4 polynucleotide kinase with (γ-32P) ATP (Promega). The mir-23b specific DNA oligonucleotide probe was used to detect expression levels of the mir-23b miRNA in ob/ob mice treated (twice weekly at 25 mg/kg) with ISIS Numbers 327889, the uniform 2′-MOE oligomeric compound, or ISIS Number 340925, the 5-10-8 gapmer compound, both targeted to mir-23b, versus saline treated animals or animals treated with a control oligomeric compound, ISIS Number 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 844), a uniform 5-10-5 2′-MOE gapmer targeting an unrelated gene, PTEN. Expression levels were normalized against the U6 RNA and the expression levels of saline treated animals were set at 100%. Most notably, in kidney samples from ob/ob mice treated with ISIS Number 327889, the uniform 2′-MOE oligomeric compound targeted to mir-23b exhibited a nearly 64% decrease in in vivo expression levels of the mir-23b miRNA. In kidney samples from mice treated with the gapmer oligomeric compound targeting mir-23b, ISIS Number 340925, a 41% reduction in in vivo expression levels of the mir-23b miRNA was observed.
As described, supra, the C57BL/6 mouse strain is reported to be susceptible to hyperlipidemia-induced atherosclerotic plaque formation, and when these mice are fed a high-fat diet, they develop diet-induced obesity (DIO). Accordingly, the DIO mouse model is useful for the investigation of obesity and development of agents designed to treat these conditions. In one embodiment of the present invention, oligomeric compounds targeting miRNAs were tested in the DIO model. Normal C57/BL6 male mice were fed a high fat diet (40% fat, 41% carbohydrate, 18% protein) for 12 weeks before the study began. DIO mice were then randomized by weight and insulin values. Initial body fat composition was determined by Dual X-ray Absorptiometry (DEXA) Scan. DIO mice were then subcutaneously injected with oligomeric compounds of the invention at a dose of 25 mg/kg, twice weekly. DIO mice were treated with oligomeric compounds ISIS Numbers 327901 and 340927 targeting mir-143, ISIS Numbers 327892 and 340926 targeting mir-131/mir-9, ISIS Numbers 327878 and ISIS Number 345349 targeting mir-203, and ISIS Numbers 327889 and 340925, targeting mir-23b. As negative controls, “scrambled” oligomeric compounds were also designed: ISIS Number 342672 contains 13 mismatches with respect to the mature mir-143 miRNA; ISIS Number 353607 (ACTAGTTTTTCTTACGTCTGA; herein incorporated as SEQ ID NO: 845) is a phosphorothioate 5-10-6 2′-MOE gapmer oligomeric compound containing 12 mismatches with respect to mir-131/mir-9; ISIS Number 353608 (CTAGACATTAGCTTTGACATCC; herein incorporated as SEQ ID NO: 846) is a phosphorothioate 5-10-7 2′-MOE gapmer oligomeric compound containing 16 mismatches with respect to mir-203. DEXA scans were also performed at weeks 0, 3 and 5 after treatment with the oligomeric compounds to assess the fat mass to lean mass ratio. The effects of target inhibition on levels of plasma glucose and insulin, liver transaminases, cholesterol and triglycerides, were also assessed weekly by tail bleed, and after the treatment period, mice were sacrificed and liver and kidney heart, as well as white adipose tissue (WAT) tissues collected. The mRNA expression levels of the Glut4, aP2, HSL and PPARγ marker genes are also assessed. Treatment of DIO mice with the uniform 2′-MOE oligomeric compounds ISIS 327901 targeting mir-143, ISIS 327892 targeting mir-131/mir9, ISIS 327878 targeting mir-203, and ISIS 327889 targeting mir-23b did not appear to cause liver toxicity in these mice as assessed by weekly measurements of plasma transaminase levels. Similarly, the gapmer oligomeric compounds ISIS 340927 targeting mir-143, and ISIS 340926 targeting mir-131/mir-9, 340925 did not cause significant increases in liver toxicity, and the gapmer compound ISIS 340925 targeting mir-23b caused only an approximately 2-fold increase in the liver transaminase AST. Interestingly, the gapmer compounds ISIS Numbers 340927 targeting mir-143, 340926 targeting mir-131/mir-9, 345349 targeting mir-203, and 340925, targeting mir-23b were all effective at reducing insulin levels at the two and four week time points, as compared to saline-treated control mice. Furthermore, some improvement in body composition (a reduction in body weight and fat mass) was observed. These data from the DIO model suggest that oligomeric compounds targeting mir-143, mir-131/mir-9, mir-203 and mir-23b may be useful as agents for the treatment of obesity and/or diabetes.
Having the information disclosed herein, one of ordinary skill in the art would comprehend that of other classes of inhibitors targeting mir-143, mir-209, mir-131/mir-9 and mir-23b miRNAs, such as antibodies, small molecules, and inhibitory peptides, can be assessed for their effects on the physiological indicators of diseases in in vivo models, and these inhibitors can be developed for the treatment, amelioration or improvement of physiological conditions associated with a particular disease state or condition. Such inhibitors are envisioned as within the scope of the instant invention.
Example 24
Effects of Oligomeric Compounds on Cell Cycling
Cell Cycle Assay:
Cell cycle regulation is the basis for various cancer therapeutics. Cell cycle checkpoints are responsible for surveillance of proper completion of certain steps in cell division such as chromosome replication, spindle microtubule attachment and chromosome segregation, and it is believed that checkpoint functions are compromised in some cancerous cells. Furthermore, because the shift from quiescence to an actively growing state as well as the passage through mitotic checkpoints are essential transitions in cancer cells, most current chemotherapy agents target dividing cells. For example, by blocking the synthesis of new DNA required for cell division, an anticancer drug can block cells in S-phase of the cell cycle. These chemotherapy agents impact many healthy organs as well as tumors. In some cases, a cell cycle regulator will cause apoptosis in cancer cells, but allow normal cells to undergo growth arrest and therefore remain unaffected. Loss of tumor suppressors such as p53 sensitizes cells to certain anticancer drugs; however, cancer cells often escape apoptosis. Further disruption of cell cycle checkpoints in cancer cells can increase sensitivity to chemotherapy while allowing normal cells to take refuge in G1 and remain unaffected. A goal of these assays is to determine the effects of oligomeric compounds on the distribution of cells in various phases of the cell cycle.
In some embodiments, the effects of several oligomeric compounds of the present invention were examined in the normal human foreskin fibroblast BJ cell line, the mouse melanoma cell line B16-F10 (also known as B16 cells), as well as the breast carcinoma cell line, T47D. These cell lines can be obtained from the American Type Culture Collection (Manassas, Va.). BJ cells were routinely cultured in MEM high glucose with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate (all media and supplements from Invitrogen Life Technologies, Carlsbad, Calif.). B16-F10 cells were routinely cultured in DMEM high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). T47D cells were cultured in DMEM High glucose media (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum. Cells were routinely passaged by trypsinization and dilution when they reached 80 to 90% confluence. Cells were plated on collagen-coated 24-well plates (Falcon-Primaria #3047, BD Biosciences, Bedford, Mass.) at approximately 50,000 cells per well and allowed to attach to wells overnight.
As a negative control, a random-mer oligomeric compound, 20 nucleotides in length, ISIS 29848 (SEQ ID NO: 737) was used. In addition, a positive control, ISIS 183891 (CCGAGCTCTCTTATCAACAG; herein incorporated as SEQ ID NO: 847) was included; ISIS 183891 targets kinesin-like 1 (also known as Eg5) and inhibits cell cycle progression. Eg5 is known to induce apoptosis when inhibited. ISIS 29248 and ISIS 183891 are chimeric oligomeric compounds (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings” (a “5-10-5 gapmer”). The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the compound. All cytidine residues are 5-methylcytidines. ISIS 340348 (CTACCTGCACGAACAGCACTTT; herein incorporated as SEQ ID NO: 848) is a uniform 2′-MOE phosphorothioate oligomeric compound targeting the mir-93 miRNA, and ISIS 340365 (TACTTTATATAGAACACAAG; herein incorporated as SEQ ID NO: 849) is a 5-10-5 gapmer phosphorothioate oligomeric compound targeting the mir-92-2 miRNA.
Oligomeric compounds were mixed with LIPOFECTINTM (Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEMTM (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve a final concentration of 150 nM of oligomeric compound and 4.5 μg/ml LIPOFECTINTM. Before adding to cells, the oligomeric compound, LIPOFECTINTM and OPTI-MEMTM were mixed thoroughly and incubated for 0.5 hrs. The medium was removed from the plates and each well was washed in 250 μl of phosphate-buffered saline. The wash buffer in each well was replaced with 250 μL of the oligomeric compound/OPTI-MEMTM/LIPOFECTIN cocktail. Control cells received LIPOFECTINTM only. The plates were incubated for 4 hours at 37° C., after which the medium was removed. 100 μl of full growth medium was added to each well. After 72 hours, routine procedures were used to prepare cells for flow cytometry analysis and cells were fixed with ethanol and stained with propidium iodide to generate a cell cycle profile using a flow cytometer. The cell cycle profile was analyzed with the ModFit program (Verity Software House, Inc., Topsham Me.).
Fragmentation of nuclear DNA is a hallmark of apoptosis and produces an increase in cells with a hypodiploid DNA content. Cells with a hypodiploid DNA content are categorized as “subG1.” The cells in the G1, G2/M and S phases are considered to be cycling, and cells in the subG1 and aneuploid categories are considered to have left the cell cycle. An increase in cells in G1 phase is indicative of a cell cycle arrest prior to entry into S phase; an increase in cells in S phase is indicative of cell cycle arrest during DNA synthesis; and an increase in cells in the G2/M phase is indicative of cell cycle arrest just prior to or during mitosis. Data are are shown in Table 29 and expressed as percentage of cells in each phase of the cell cycle.







TABLE 29







Effects of oligomeric compounds targeting


miRNAs on cell cycling















SEQ
Pri-




aneu-


ISIS #
ID #
miRNA
SubG1
G1
S
G2/M
ploid

















UTC
N/A
N/A
8.1
59.6
27.5
12.9
7.3


ISIS-29848
737
N/A
9.6
57.8
26.5
15.6
12


n-mer









ISIS-183891
847
Kinesin-
20.8
33.1
39.2
27.6
11.5


Positive

like 1/Eg5







control









327878
296
mir-203
17.3
39.1
40.8
20
11.9


327888
306
mir-108-1
13.3
53.7
29.5
16.7
12.9


327889
307
mir-23b
8.2
53.1
32.5
14.4
10.5


327901
319
mir-143
12
34.7
44.9
20.3
13.6


327902
320
mir-192-1
10.6
50.7
33.9
15.3
13.4


327903
321
let-7a-3
11
53.7
30.9
15.4
13.4


327904
322
mir-181a
8.6
54.4
29.5
16.2
15.6


327905
323
mir-205
8.5
56.9
28.1
15
14.7


327906
324
mir-103-1
15.2
46.1
33
20.9
15.8


327907
325
mir-26a
17.8
49.5
32.8
17.6
17.8


327908
326
mir-33a
5.6
55.4
29.2
15.3
13.1


327909
327
mir-196-2
7.9
52.6
30.1
17.3
16.3


327910
328
mir-107
9.3
49.5
33
17.5
13.1


327911
329
mir-106
10.9
49.9
30.1
20
16.5


327914
332
mir-130a
8.5
55.8
28.9
15.3
16.2


327919
337
mir-221
10.8
54.3
30.3
15.4
16


327922
340
mir-19b-2
10
50.4
30.7
18.9
16.8


327928
346
mir-29a-1
6.6
56
27.9
16
15.9


327933
351
mir-145
10.2
49.6
31.3
19.1
15.9


327934
352
mir-213
6.6
54.4
28.2
17.4
17


327941
359
mir-181b
8.2
57.2
29.9
12.9
15.8


327951
369
mir-15a-1
4.3
60.9
24.8
14.3
16.7


328342
451
mir-203
4.8
62.3
24.9
12.8
15.2


328362
471
mir-108-1
9.1
51.2
33.6
15.1
12.9


328364
473
mir-23b
1.9
61.5
24.2
14.3
15.1


328382
491
mir-143
2.9
59.8
25.7
14.4
14.8


328388
497
let-7a-3
4.0
57.5
28
14.6
14.5


328394
503
mir-181a 
2.4
59.5
24.5
16
18.3


328396
505
mir-205
4.6
56.8
28.2
15
19.8


328419
528
mir-221
6.0
51.2
32.5
16.3
17.9


328423
532
mir-19b-2
4.9
52.9
32.4
14.8
15.3


328424
533
mir-19b-2
3.1
61.9
23.7
14.4
16.9


328436
545
mir-29a-1
3.5
59.2
26.9
13.9
17.4


328644
553
mir-145
7.2
58.4
27.6
14
17.5


328691
600
mir-145
7.7
60.5
24.4
15.1
16.6


328697
606
mir-181b
2.4
57.6
26.4
16
13.5


328773
682
mir-15a-2
2.7
56.4
26.9
16.7
11.7


340348
848
mir-93
14.1
53.9
31.8
14.3
12.3


340365
849
mir-92-2
4.3
55.2
29.4
15.4
18.3









From these data, it is evident that treatment with the oligomeric compounds targeting mir-143, ISIS Number 327901 (SEQ ID NO: 319); mir-203, ISIS Number 327878 (SEQ ID NO: 296); mir-103-1, ISIS Number 327906 (SEQ ID NO: 324); mir-106, ISIS Number 327911 (SEQ ID NO: 329); and mir-145, ISIS Number 327933 (SEQ ID NO: 351) resulted in an increased percentage of cells in the G2/M phase, indicating that these oligomeric compounds arrest or delay the cell cycle at or just prior to mitosis, potentially activating a mitotic checkpoint.
Treatment with the oligomeric compounds targeting mir-26a, ISIS Number 327907 (SEQ ID NO: 325); mir-205, ISIS Number 328396 (SEQ ID NO: 505); mir-181a, ISIS Number 328394 (SEQ ID NO: 503); and mir-92-2, ISIS Number 340365 (SEQ ID NO: 849) resulted in higher than average percentages of aneuploid cells, indicating that these oligomeric compounds interfere with proper chromosome segregation.
Treatment with the oligomeric compounds targeting mir-203, ISIS Number 327878 (SEQ ID NO: 296); mir-103-1, ISIS Number 327906 (SEQ ID NO: 324); mir-26a, ISIS Number 327907 (SEQ ID NO: 325); and mir-93, ISIS Number 340348 (SEQ ID NO: 848) resulted in an increased percentage of cells with hypodiploid DNA content (SubG1 phase) indicating that the oligomeric compound treatment may induce apoptotic events.
The effects of several oligomeric compounds of the present invention were also examined in the HeLa and A549 human carcinoma cell lines, both of which can be obtained from the American Type Culture Collection (Manassas, Va.).
In some embodiments, HeLa cells were plated on collagen-coated 24-well plates at 50,000-60,000 cells per well, and allowed to attach to wells overnight. In some embodiments, HeLa cells were synchronized by double thymidine block (cells were washed three times with PBS, then grown in 10% FBS containing 2 mM thymidine; then 19 hours later, cells were washed three times in PBS, 10% FBS for 9 hours; cells were then incubated in 10% FBS, 2 mM thymidine for 15 hours; then washed three times with PBS, 10% FBS and samples were taken every two hours over a 16 hour period). A portion of each time sample was fixed with ethanol and treated with propidium iodide and subjected to FACs analysis for determination of the percentage of cells in each phase of the cell cycle. Distinctive peaks were observed for G0-, S-, Early G2/M-, Late G2/M-, and G1-phases of the cell cycle at 0-, 4-, 6-, 8-, and 12-hours, respectively, indicating that the cells were synchronized. HeLa cells treated with 10 μM cisplatin or 100 ng/ml nocodazole were used as controls for G1-phase and late G2/M-phases, respectively. From the remaining portion of each of these time samples, total RNA was isolated and used to assess the expression of cell cycle marker mRNAs using the real-time RT-PCR methods and/or used to screen microarrays to assess the expression of miRNAs over the course of the cell cycle. It was observed that several miRNAs are expressed in a cell-cycle-dependent manner. Shown in Table 30 are the mRNA levels of the E2F1 transcription factor and topoisomerase 2A (Top2A), which vary over the course of the cell cycle and can be used for comparison to the experimental groups for the confirmation of cell cycle phase. Data are an average of three trials.







TABLE 30







Expression levels of cell cycle markers











treatment
E2F1 mRNA
Top2A mRNA














10 uM cisplatin
102
15



100 ng/ml nocodazole
23
176



0 hrs (G0-phase)
100
100



4 hrs (S-phase)
81
105



6 hrs (early G2/M-phase)
39
221



8 hrs (late G2/M-phase)
50
254



12 hrs (G1-phase)
61
124









In some embodiments, HeLa cells were also treated with oligomeric compounds targeting miRNAs. As described above, oligomeric compounds were mixed with LIPOFECTINTM in OPTI-MEMTM (Invitrogen Life Technologies, Carlsbad, Calif.) to a final concentration of 150 nM of oligomeric compound and 6 μg/ml LIPOFECTINTM. Before adding to cells, the oligomeric compound, LIPOFECTINTM and OPTI-MEMTM were mixed thoroughly and incubated for 0.5 hrs. The medium was removed from the plates. Each well was washed in 250 μl of PBS. The wash buffer in each well was replaced with 250 μL of the oligomeric compound/OPTI-MEMTM/LIPOFECTIN cocktail. Control cells received LIPOFECTINTM only. The plates were incubated for 4 hours at 37° C., after which the medium was removed. 1000 μl of full growth medium was added to each well. After 24 hours (Table 31) or 48 hours (Table 32), cells were prepared for flow cytometry analysis to generate a cell cycle profile. The cell cycle profile was analyzed with the ModFit program (Verity Software House, Inc., Topsham Me.).
The random-mer ISIS 29848 (SEQ ID NO: 737) was used as a negative control, and ISIS 183891 (SEQ ID NO: 847), targeting kinesin-like 1/Eg5, was included as a positive control. Results of these experiments are shown in Tables 31 and 32. Data are expressed as percentage of cells in each phase relative to the untreated control (UTC); values above 100 are considered to indicate a delay or arrest in that phase of the cell cycle. Table 31 shows the results from cells sampled 24 hours after oligomeric compound treatment, and Table 32 shows the results from cells sampled 48 hours after oligomeric compound treatment. In some cases, the same oligomeric compound was tested in repeated experiments.







TABLE 31







Effects of oligomeric compounds targeting 


miRNAs on cell cycling (24 hours)











ISIS 
SEQ
% cells in cell cycle phase




















G2/
aneu-


Pri-miRNA
#
ID #
subG1
G1
S
M
ploid

















UTC
N/A
N/A
100
100
100
100
100


n-mer
29848
737
120
116
81
108
76


Kinesin-
183891
847
251
21
109
231
95


like 1/Eg5









collagen,
338797
624
197
101
79
148
193


type I,









alpha 1/









hypothetical









miRNA-144









hypothetical 
338666
493
235
123
63
158
102


miRNA-039









hypothetical 
328111
413
62
127
75
99
50


miRNA-111









hypothetical 
338750
577
107
148
76
97
166


miRNA-111









hypothetical 
328115
417
177
90
87
147
59


miRNA-142









hypothetical 
328119
421
75
100
94
112
83


miRNA-154









hypothetical 
328724
633
155
91
90
135
197


miRNA-154









hypothetical 
328749
658
312
126
82
110
138


miRNA-179









hypothetical 
328780
689
124
96
87
136
149


miRNA-179









hypothetical
328136
438
330
125
81
88
51


miRNA-181









hypothetical 
338833
660
232
150
56
142
185


miRNA-181









let-7a-3
327903
321
118
92
104
106
98


let-7a-3
328388
375
120
110
83
115
85


mir-100-1
327957
497
197
91
88
145
66


mir-100-1
328707
616
188
36
93
195
166


mir-103-1
327906
324
228
153
47
107
65


mir-103-1
328397
506
134
93
86
142
91


mir-106
327911
329
158
130
62
122
104


mir-106
328403
512
284
70
85
197
53


mir-106
328403
512
189
86
75
179
82


mir-107
327910
328
174
154
42
118
73


mir-108-1
328362
471
114
101
87
126
66


mir-10a
327949
367
194
82
84
172
68


MiR-125a, 
341787
852
221
113
75
144
165


Mouse









mir-127, Mouse
341788
853
303
154
54
140
114


mir-130b
328687
596
231
80
98
131
149


mir-130b
338769
596
188
171
61
103
133


mir-131-2/mir-9
327892
310
153
86
111
103
80


mir-131-2/mir-9
328369
310
84
100
88
125
71


mir-131-2/mir-9
340926
478
286
98
91
121
83


mir-133b
338713
540
93
152
72
101
187


mir-141
338741
568
157
141
73
112
166


mir-143
327901
319
108
101
94
110
90


mir-143
328382
491
81
118
76
116
78


mir-143
328382
491
226
102
80
144
202


mir-143
340927
319
118
121
75
111
88


mir-143
340927
319
131
128
71
106
87


mir-145
327933
351
192
102
83
131
92


mir-145
327933
351
190
90
91
140
47


mir-145
328644
553
71
113
84
109
68


mir-145
345395
351
247
54
82
222
77


mir-149, Mouse
341785
854
125
152
92
53
158


mir-152
328727
636
245
133
81
105
161


mir-152
338809
636
106
159
82
69
210


mir-16-3
327877
295
154
107
66
159
62


mir-17/mir-91
327885
303
151
129
63
121
55


mir-181a-1
327904
322
114
99
102
99
89


mir-182
328744
653
229
31
108
167
111


mir-182
338826
653
145
148
79
90
138


mir-192-1
327902
320
178
57
106
176
66


mir-192-1
327902
320
175
44
121
163
98


mir-192-1
328383
492
314
55
82
222
92


mir-192-1
328383
492
289
63
97
183
98


mir-192-1
338665
340
173
85
76
175
193


mir-19b-2
327922
492
131
97
96
114
104


mir-19b-2
328424
533
60
110
85
112
74


mir-203
327878
296
124
96
94
122
73


mir-203
328342
451
192
33
95
238
67


mir-205
327905
323
144
99
88
129
50


mir-205
327905
323
149
94
95
121
98


mir-205
328396
505
97
94
87
139
88


mir-205
338678
505
162
122
75
131
202


mir-211
327946
364
225
90
84
156
43


mir-211
328674
583
564
125
93
84
69


mir-211
338756
583
137
147
75
99
166


mir-213/
327934
352
278
87
85
160
55


mir-181a-2









mir-213/
327934
352
204
118
66
137
77


mir-181a-2









mir-213/
328647
556
140
101
92
119
140


mir-181a-2









mir-216
327956
374
120
124
68
120
61


mir-216
328759
668
239
88
78
168
184


mir-22
327896
314
121
83
103
128
65


mir-22
328374
483
198
54
115
162
97


mir-220
327944
362
165
85
110
111
50


mir-221
327919
337
85
92
103
109
96


mir-221
328419
528
87
109
79
124
77


mir-23a
338836
663
153
185
53
105
150


mir-23b
327889
307
122
104
102
87
82


mir-23b
340925
307
151
103
89
117
73


mir-26a-1
327907
325
224
119
77
111
75


mir-26a-1
345373
325
196
66
94
176
68


mir-29b-1
327876
294
103
98
104
95
66


mir-29b-1
327876
294
149
93
92
131
75


mir-29b-1
328337
446
107
106
88
113
104


mir-29b-1
328337
446
99
108
88
109
64


mir-29b-2
328339
448
235
77
102
143
61


mir-29c
338690
517
149
124
78
123
194


mir-30a
328084
585
381
43
104
163
101


mir-30b
328676
585
139
99
86
134
169


mir-30b
338758
743
113
129
81
108
190


mir-30d
328421
530
288
47
105
200
70


mir-33a
327908
326
138
98
99
106
114


mir-92-1
327897
315
143
114
80
115
69


mir-92-1
327897
315
180
128
74
100
54


mir-92-2
340365
849
109
125
71
114
84


mir-95 
340350
855
218
183
54
104
94


(Mourelatos)









mir-96
338637
464
88
170
70
84
188
















TABLE 32







Effects of oligomeric compounds targeting


miRNAs on cell cycling (48 hours)











ISIS
SEQ
% cells in cell cycle phase




















G2/
aneu-


Pri-miRNA
#
ID #
subG1
G1
S
M
ploid

















UTC
N/A
N/A
100
100
100
100
100


n-mer
29848
737
86
87
121
117
109


Kinesin-
183891
847
173
19
124
331
72


like 1/Eg5









collagen, type I,
338797
624
813
66
124
168
175


alpha 1/









hypothetical









miRNA-144









hypothetical 
338666
493
1832
44
136
217
125


miRNA-039









hypothetical 
328111
413
371
84
126
119
90


miRNA-111









hypothetical 
338750
577
201
99
101
103
190


miRNA-111









hypothetical 
328115
417
195
92
114
107
86


miRNA-142









hypothetical
328119
421
767
75
145
124
81


miRNA-154









hypothetical 
328724
633
653
70
134
140
155


miRNA-154









hypothetical 
328749
658
962
37
129
246
65


miRNA-179









hypothetical 
328780
689
917
83
130
110
133


miRNA-179









hypothetical 
328136
438
140
83
133
113
85


miRNA-181









hypothetical 
338833
660
1091
44
106
258
154


miRNA-181









let-7a-3
327903
321
74
102
95
98
94


let-7a-3
328388
375
112
99
101
102
126


mir-100-1
327957
497
864
65
169
127
85


mir-100-1
328707
616
1486
46
134
213
155


mir-103-1
327906
324
57
100
98
103
83


mir-103-1
328397
506
74
97
101
109
96


mir-106
327911
329
65
99
96
109
101


mir-106
328403
512
863
61
177
131
85


mir-106
328403
512
108
82
148
106
80


mir-107
327910
328
53
99
91
111
92


mir-108-1
328362
471
87
96
104
108
97


mir-10a
327949
367
773
66
157
138
71


MiR-125a, 
341787
852
707
55
126
197
153


Mouse









mir-127, Mouse
341788
853
748
76
105
163
116


mir-130b
328687
596
1119
55
174
131
171


mir-130b
338769
596
482
76
116
149
194


mir-131-2/mir-9
327892
310
121
74
150
129
79


mir-131-2/mir-9
328369
310
72
99
95
109
109


mir-131-2/mir-9
340926
478
68
83
120
131
106


mir-133b
338713
540
426
95
104
109
194


mir-141
338741
568
185
100
101
99
170


mir-143
327901
319
93
98
104
103
104


mir-143
328382
491
71
102
92
103
109


mir-143
328382
491
350
83
122
120
133


mir-143
340927
319
95
91
107
121
113


mir-143
340927
319
83
91
107
122
108


mir-145
327933
351
91
76
135
138
86


mir-145
327933
351
438
80
133
123
75


mir-145
328644
553
52
101
101
98
82


mir-145
345395
351
213
51
192
157
87


mir-149, Mouse
341785
854
1148
82
126
116
166


mir-152
328727
636
846
68
152
124
140


mir-152
338809
636
345
86
110
129
157


mir-16-3
327877
295
755
59
152
168
80


mir-17/mir-91
327885
303
456
78
129
133
76


mir-181a-1
327904
322
116
87
126
114
80


mir-182
328744
653
1774
31
78
334
171


mir-182
338826
653
696
61
124
182
137


mir-192-1
327902
320
1176
39
171
208
81


mir-192-1
327902
320
202
44
166
205
87


mir-192-1
328383
492
303
53
217
124
90


mir-192-1
328383
492
940
54
178
150
90


mir-192-1
338665
340
1629
40
89
292
149


mir-19b-2
327922
492
81
96
105 
109
91


mir-19b-2
328424
533
89
103
91
101
111


mir-203
327878
296
50
89
119 
114
92


mir-203
328342
451
189
55
115 
225
107


mir-205
327905
323
719
48
194 
150
67


mir-205
327905
323
100
78
143 
122
99


mir-205
328396
505
88
89
114 
119
129


mir-205
338678
505
1158
81
78
188
179


mir-211
327946
364
431
72
150 
129
76


mir-211
328674
583
1663
69
160 
109
134


mir-211
338756
583
311
90
121 
100
169


mir-213/
327934
352
752
62
156 
152
92


mir-181a-2









mir-213/
327934
352
155
66
148 
155
117


mir-181a-2









mir-213/
328647
556
589
69
153 
118
136


mir-181a-2









mir-216
327956
374
184
91
106 
121
110


mir-216
328759
668
1744
50
31
343
148


mir-22
327896
314
886
55
140 
194
66


mir-22
328374
483
787
65
157 
143
71


mir-220
327944
362
490
75
129 
144
78


mir-221
327919
337
104
80
122 
139
104


mir-221
328419
528
83
99
96
107
112


mir-23a
338836
663
811
52
152
169
165


mir-23b
327889
307
133
78
137 
130
101


mir-23b
340925
307
89
87
130 
109
93


mir-26a-1
327907
325
116
92
111 
115
94


mir-26a-1
345373
325
116
75
132 
145
119


mir-29b-1
327876
294
41
87
120 
119
100


mir-29b-1
327876
294
251
76
141 
126
69


mir-29b-1
328337
446
66
92
105 
119
108


mir-29b-1
328337
446
662
73
143 
135
74


mir-29b-2
328339
448
678
73
153 
123
92


mir-29c
338690
517
413
91
110 
112
190


mir-30a
328084
585
1028
20
168 
241
57


mir-30b
328676
585
366
86
118 
118
172


mir-30b
338758
743
267
103
99
92
153


mir-30d
328421
530
1103
30
202 
198
64


mir-33a
327908
326
61
99
98
105
93


mir-92-1
327897
315
134
100
103 
95
84


mir-92-1
327897
315
125
94
114 
105
63


mir-92-2
340365
849
71
99
94
109
129


mir-95 
340350
855
1144
76
126 
134
125


(Mourelatos)









mir-96
338637
464
239
90
109 
117
210









Several oligomeric compounds were observed to result in an arrest or delay of the cell cycle, in some cases correlating with a cell-cycle-dependent expression profile as determined by miRNA microarray analysis.
For example, from these data, it was observed that treatment of HeLa cells with oligomeric compounds (MOE-gapmers and fully modified MOEs) targeting miRNAs caused an increase in the percentage of cells exhibiting a subG1-phase or aneuploid DNA content, indicating aberrant chromosome segregation. Treatment with oligomeric compounds ISIS Number 338797 (SEQ ID NO: 624) targeted to hypothetical miRNA-144, ISIS Number 338833 (SEQ ID NO: 660) targeted to hypothetical miRNA-181, and ISIS Number 328759 (SEQ ID NO: 668) targeted to mir-216, each appeared to cause an induce chromosome missegregation events at both the 24-hour and 48-hour timepoints. Thus, these compounds may be useful in triggering a checkpoint arrest in rapidly dividing cells, potentially useful in the treatment of hyperproliferative disorders such as cancer.
It was also observed that other oligomeric compounds (MOE-gapmers and fully modified MOEs) targeting miRNAs appeared to induce an arrest or delay in the G1-, S-, or G2/M-phases of the cell cycle. By miRNA microarray analysis, expression levels of the mir-205 miRNA were observed to increase in the S- and G1-phases of the cell cycle in HeLa cells. Treatment of HeLa cells with the oligomeric compound ISIS Number 327905 (SEQ ID NO: 323), targeting the mir-205 miRNA, was observed to arrest or delay the cell cycle in S-phase at the 48-hour time point, suggesting that the mir-205 miRNA may play a role in regulating DNA replication. It was also observed that treatment of HeLa cells with the oligomeric compound ISIS Number 338678 (SEQ ID NO: 505), targeted to the mir-205 pri-miRNA, resulted in an arrest or delay primarily in the G2/M-phase of the cell cycle, suggesting that this oligomeric compound may interfere with processing of the miRNA precursor into a mature miRNA, which appears to have an impact on mitosis.
Treatment of HeLa cells with oligomeric compounds ISIS Number 327892 (SEQ ID NO: 310), targeting mir-131/mir-9, and ISIS Number 327934 (SEQ ID NO: 352), targeting mir-213/mir-181a-2, was observed to arrest or delay the cell cycle in G2/M- and S-phases at the 48-hour time point, suggesting that the mir-131/mir-9 and mir-213/mir-181a-2 miRNAs are involved in regulating DNA replication and entry into mitosis.
Treatment of HeLa cells with oligomeric compound ISIS Number 345373 (SEQ ID NO: 325), targeting miR-26a-1, was observed to arrest or delay cells mainly in the G2/M-phase at 24 hrs after oligonucleotide-treatment, and at 48 hrs after oligonucleotide-treatment to arrest or delay cells mainly in S-phase of the cell cycle, suggesting that miR-26a-1 is involved in mitosis and that cells making it through a first round of cell division may harbor errors that cause them to arrest during a new round of DNA replication.
By miRNA microarray analysis, expression levels of the mir-145 miRNA were observed to increase in the G2/M-phase of the cell cycle in HeLa cells, and treatment of HeLa cells with the oligomeric compounds ISIS Number 327933 (SEQ ID NO: 351), a uniform 2′-MOE compound, and ISIS Number 345395 (SEQ ID NO: 351), a chimeric 2′-MOE gapmer compound, both targeting the mir-145 miRNA, were observed to arrest or delay the cell cycle in G2/M-phase at the 24-hour time point and at subG1-phase at the 48-hour time point, suggesting that the mir-145 miRNA plays a role in mitosis and that cells making it through a first round of cell division may harbor errors that cause them to arrest before a new round of DNA replication.
By miRNA microarray analysis, expression levels of the mir-192-1 miRNA were observed to increase in the G2/M-phase of the cell cycle in HeLa cells, and treatment of HeLa cells with the oligomeric compounds ISIS Number 327902 (SEQ ID NO: 320), a uniform 2′-MOE compound, and ISIS Number 328383 (SEQ ID NO: 492), a chimeric 2′-MOE gapmer compound, targeted against the mir-192-1 miRNA and the mir-192-1 precursor, respectively, were observed to arrest or delay the cell cycle in the G2/M-phase at 24-hours after oligonucleotide treatment, and at both the S- and G2/M-phases at the 48-hour time point, suggesting that the mir-192 miRNA is involved in mitosis, and that cells making it through a first round of cell division may harbor errors that cause them to arrest during a new round of DNA replication. A uniform 2′-MOE oligomeric compound ISIS Number 338665 targeting the mir-192-1 precursor was also observed to induce a G2/M-phase arrest at both time points.
Treatment of HeLa cells with the oligomeric compound ISIS Number 328744 (SEQ ID NO: 653), targeting the mir-182 miRNA, was observed to arrest or delay the cell cycle in G2/M-phase at 48-hours after oligonucleotide treatment, suggesting that the mir-182 miRNA plays a role in regulating mitosis.
Treatment of HeLa cells with the oligomeric compound ISIS Number 328421 (SEQ ID NO: 530), targeting miR-30d was also observed to arrest or delay cells mainly in the G2/M-phase at the 24-hour time point and at both the S- and G2/M-phases at the 48-hour time point after oligonucleotide treatment, suggesting that the mir-30d miRNA is involved in mitosis, and that a cell division error arising from the first round of division may allow cells to pass through mitosis and initiate a second round of division, but then a cell cycle checkpoint is set off before the cells are able to complete DNA synthesis.
Treatment of HeLa cells with the oligomeric compound ISIS Number 328403 (SEQ ID NO: 512), targeting mir-106 was also observed to arrest or delay cells in the G2/M-phase at the 24-hour time point and at both the S- and G2/M-phases at the 48-hour time point after oligonucleotide treatment, suggesting that the mir-106 miRNA is involved in mitosis, and that a cell division error arising from the first round of division may allow cells to pass through mitosis and initiate a second round of division, but then a cell cycle checkpoint is set off before the cells are able to complete DNA synthesis. Interestingly, the cell cycle regulatory transcription factor E2F1 mRNA is reported to be a target of the mir-106 miRNA (Lewis et al., Cell, 2003, 115, 787-798).
Treatment of HeLa cells with the oligomeric compound ISIS Number 328759 (SEQ ID NO: 668), targeting the mir-216 miRNA, was observed to arrest or delay the cell cycle in G2/M-phase at both 24- and 48-hours after oligonucleotide treatment, suggesting that the mir-216 miRNA plays a role in regulating mitosis.
Treatment of HeLa cells with the oligomeric compound ISIS Number 328342 (SEQ ID NO: 451), targeting the mir-203 miRNA, was observed to arrest or delay the cell cycle in G2/M-phase at both 24- and 48-hours after oligonucleotide treatment, suggesting that the mir-203 miRNA plays a role in regulating mitosis.
Treatment of HeLa cells with the oligomeric compound ISIS Number 328707 (SEQ ID NO: 616), targeting miR-100-1 was also observed to arrest or delay cells mainly in the G2/M-phase at both 24- and 48-hours after oligonucleotide treatment, suggesting that the miR-100-1 miRNA plays a role in regulating mitosis.
Dose Responsiveness:
In accordance with the present invention, certain oligomeric compounds targeting miRNAs were selected for dose response studies. Using the cell cycle assay described above, the cell cycle profiles of HeLa or A549 cells treated with varying concentrations of oligomeric compounds of the present invention were assessed.
HeLa cells were treated with 25-, 50-, 100- or 150 nM of the oligomeric compounds ISIS Numbers 327902 (SEQ ID NO: 320) and 328383 (SEQ ID NO: 492), both targeted against mir-192, and ISIS 327905 (SEQ ID NO: 323), targeting mir-205, and ISIS 328403 (SEQ ID NO: 512), targeting mir-106. Cells treated with increasing concentrations of oligomeric compounds were compared to untreated cells, to assess the dose-dependency of the observed delay or arrest. The random-mer ISIS 29848 was used as a negative control. Cells were prepared for flow cytometry 48-hours after oligonucleotide treatment, as described, supra. Oligomeric compounds targeted to miRNAs were tested in quadruplicate, and ISIS 29848 was tested in triplicate; data is presented as an average of the replicates. Results of these dose response studies are shown in Table 33, where data are expressed as percentage of cells in each phase.







TABLE 33







Dose response of oligomeric compounds targeting


miRNAs on cell cycling (48 hours)














Dose















oligomeric
% cells in cell cycle phase













ISIS #
compound
SubG1
G1
S
G2/M
Aneuploid





Untreated
 25 nM
1.3
56
24
20
12


control
 50 nM
1.4
56
24
20
14


(UTC)
100 nM
1.6
57
24
19
11



150 nM
1.6
57
23
20
15


 29848
 25 nM
2.0
55
25
20
12



 50 nM
1.5
56
25
19
12



100 nM
3.2
52
28
20
13



150 nM
4.2
48
31
21
15


327902
 25 nM
1.6
57
23
19
13



 50 nM
2.4
51
30
20
14



100 nM
3.1
43
30
27
11



150 nM
6.3
36
36
28
12


327905
 25 nM
1.7
57
24
18
12



 50 nM
2.1
50
30
20
12



100 nM
2.5
46
30
24
14



150 nM
4.5
38
38
24
12


328383
 25 nM
1.9
57
25
18
12



 50 nM
1.3
56
25
18
13



100 nM
9.3
36
34
30
10



150 nM
11.8 
29
36
34
11


328403
 25 nM
1.5
58
24
18
13



 50 nM
1.1
53
27
20
14



100 nM
3.5
48
29
23
11



150 nM
8.2
37
40
24
13









From these data, it was observed that 48-hours after treatment of HeLa cells with increasing doses of each of these four oligomeric compounds targeting miRNAs, a dose-responsive delay or arrest resulted, exhibited as an increasing percentage of cells in the S- and G2/M-phases of the cell cycle. Concomittent decreases in the percentage of cells in G1-phase of the cell cycle and increases in the percentage of hypodiploid (subG1) cells were also observed. Likewise, a dose-responsive G2/M delay or arrest was observed in A549 cells treated with 25-, 50-, 100-, or 150 nM of the oligomeric compounds ISIS 327902, ISIS 328383 and ISIS Number 328342.
In a further study, A549 cells were treated with 25-, 50-, 100- or 150 nM of the oligomeric compounds ISIS Numbers 338637 (SEQ ID NO: 464) targeted against mir-96, and ISIS 338769 (SEQ ID NO: 596) targeted against mir-130b, ISIS 338836 (SEQ ID NO: 663) targeted against mir-23a, and ISIS 340350 (SEQ ID NO: 855) targeted against mir-95 (Mourelatos). Cells treated with increasing concentrations of oligomeric compounds were compared to untreated cells, to assess the dose-responsiveness of the observed delay or arrest. The random-mer ISIS 29848 was used as a negative control. Cells were prepared for flow cytometry 24-hours after oligonucleotide treatment. Results of these dose response studies are shown in Table 34, where data are expressed as percentage of cells in each phase relative to the untreated control cells in that phase.







TABLE 34







Dose response of oligomeric compounds targeting


miRNAs on cell cycling (24 hours)














Dose















oligomeric
% cells in cell cycle phase













ISIS #
compound
SubG1
G1
S
G2/M
Aneuploid
















 29848
 25 nM
90
121
86
87
76



 50 nM
91
116
88
93
90



100 nM
272
125
74
112
116



150 nM
507
126
71
119
84


338637
 25 nM
89
100
99
101
99



 50 nM
86
110
89
107
120



100 nM
67
126
73
115
146



150 nM
216
123
66
144
135


338769
 25 nM
62
101
94
114
101



 50 nM
82
114
81
122
132



100 nM
130
124
75
113
157



150 nM
341
117
71
145
184


338836
 25 nM
76
97
103
97
99



 50 nM
232
113
89
98
111



100 nM
68
117
80
116
153



150 nM
178
117
69
149
114


340350
 25 nM
91
102
100
95
120



 50 nM
158
128
67
126
80



100 nM
267
125
60
155
107



150 nM
402
128
40
211
108









From these data, it was observed that 24-hours after treatment of A549 cells with increasing doses of the oligomeric compounds ISIS Numbers 338637 (SEQ ID NO: 464) targeted against mir-96, and ISIS 338769 (SEQ ID NO: 596) targeted against mir-130b, ISIS 338836 (SEQ ID NO: 663) targeted against mir-23a, and ISIS 340350 (SEQ ID NO:855) targeted against mir-95 (Mourelatos), a dose-responsive delay or arrest resulted, exhibited as an increasing percentage of cells in the G2/M-phases of the cell cycle. Concomittent decreases in the percentage of cells in S-phase of the cell cycle and increases in the percentage of hypodiploid (subG1) cells were also observed.
In further studies, additional cell lines were treated with oligomeric compounds targeted against miRNAs to assess the effects of each oligomeric compound on cell cycling. BJ, B16, T47D, and HeLa cells were cultured and transfected as described above. T47D cells are deficient in p53. T47Dp53 cells are T47D cells that have been transfected with and selected for maintenance of a plasmid that expresses a wildtype copy of the p53 gene (for example, pCMV-p53; Clontech, Palo Alto, Calif.), using standard laboratory procedures. BJ cells were treated with 200 nM of each oligomeric compound, and T47D, T47Dp53, HeLa, and B16 cells were treated with 150 nM of each oligomeric compound. The human foreskin fibroblast BJ cell line represents a non-cancer cell line, while HeLa, T47D, T47Dp53 cells and the mouse melanoma cell line B16-F10 represent cancerous cell lines. For comparison, oligomeric compounds ISIS 183891 (SEQ ID NO: 847) and ISIS 285717 (TCGGTTCTTTCCAAGGCTGA; herein incorporated as SEQ ID NO: 857), both targeting the kinesin-like 1/Eg5 mRNA, involved in cell cycling, were used as positive controls. The random-mer ISIS 29848 was used as a negative control. Additionally, the oligomeric compounds ISIS Number 25690 (ATCCCTTTCTTCCGCATGTG; herein incorporated as SEQ ID NO: 858) and ISIS Number 25691 (GCCAAGGCGTGACATGATAT; herein incorporated as SEQ ID NO: 859), targeted to nucleotides 3051-3070 and 3085-3104, respectively, of the mRNA encoding the Drosha RNase III (GenBank Accession NM—013235.2, incorporated herein as SEQ ID NO: 860) were also tested. ISIS Number 25690 and ISIS Number 25691 are 5-10-5 2′-MOE gapmer compounds, 20 nucleotides in length, with phosphorothioate internucleoside linkages throughout the oligomeric compound. All cytidine residues are 5-methylcytidines. Transfections were performed using the methods described herein. Cells were prepared for flow cytometry 48-hours after oligonucleotide treatment. Results of these studies are shown in Table 35, where data are expressed as percentage of cells in each phase relative to the untreated control cells in that phase.







TABLE 35







Effects of oligomeric compounds targeting


miRNAs on cell cycling (48 hours)













SEQ 

% cells in cell cycle phase















Cell
ISIS
ID 

Sub


G2/
aneu-


line
#
NO
target
G1
G1
S
M
ploid


















BJ
29848
737
N/A
187
100
99
100
105


B16
29848
737
N/A
143
98
98
110
99


HeLa
29848
737
N/A
403
83
113
106
155


T47D
29848
737
N/A
86
95
113
98
155


T47Dp53
29848
737
N/A
173
121
75
97
93


BJ
183891
847
kinesin-
422
58
173
287
158





like 1/eg5







B16
285717
857
kinesin- 
627
72
78
220
178





like 1/eg5







HeLa
183891
847
kinesin- 
1237
22
95
211
161





like 1/eg5







T47D
183891
847
kinesin- 
85
55
84
156
161





like 1/eg5







T47Dp53
183891
847
kinesin- 
351
71
53
189
84





like 1/eg5







HeLa
25690
858
Drosha, 
64
119
89
87
140





RNAse III







T47D
25690
858
Drosha, 
97
97
80
113
140





RNAse III







T47Dp53
25690
858
Drosha, 
193
97
108
114
144





RNAse III







BJ
25691
859
Drosha, 
183
94
116
125
209





RNAse III







B16
25691
859
Drosha, 
316
116
83
99
105





RNAse III







HeLa
25691
859
Drosha, 
881
53
141
113
203





RNAse III







T47D
25691
859
Drosha, 
125
94
104
104
203





RNAse III







T47Dp53
25691
859
Drosha, 
212
130
66
93
95





RNAse III







HeLa
338797
624
hypo-
144
104
89
115
125





thetical










miRNA-










144







HeLa
338666
493
hypo-
214
92
98
130
151





thetical










miRNA 










039







HeLa
338833
660
hypo-
255
87
100
136
136





thetical










miRNA










181







HeLa
328707
616
mir-100-1
125
103
87
122
140


BJ
328403
512
mir-106
81
102
95
92
114


B16
328403
512
mir-106
112
111
88
99
92


HeLa
328403
512
mir-106
89
125
89
80
175


T47D
328403
512
mir-106
49
104
112
89
175


T47Dp53
328403
512
mir-106
140
114
87
94
89


HeLa
341787
852
MiR-125a, 
324
88
96
145
177





Mouse







T47D
328687
596
mir-130b
142
101
92
115
169


T47Dp53
338769
596
mir-130b
116
103
87
123
87


B16
327933
351
mir-145
104
109
84
116
130


BJ
345395
351
mir-145
132
100
97
104
115


B16
345395
351
mir-145
147
106
87
115
150


HeLa
345395
351
mir-145
87
108
96
95
139


BJ
328744
653
mir-182
125
94
111
127
158


B16
328744
653
mir-182
153
108
87
110
115


HeLa
328744
653
mir-182
1057
53
110 
213
178


T47D
328744
653
mir-182
85
90
87
118
191


T47Dp53
328744
653
mir-182
90
130
59
101
100


BJ
327902
320
mir-192-1
91
99
88
108
82


B16
327902
320
mir-192-1
151
112
88
98
101


HeLa
327902
320
mir-192-1
94
108
96
93
162


T47D
327902
320
mir-192-1
102
75
120
116
162


T47Dp53
327902
320
mir-192-1
155
100
98
102
97


HeLa
338665
492
mir-192-1
322
92
92
142
138


HeLa
328342
451
mir-203
103
96
89
138
96


BJ
327905
323
mir-205
105
100
77
109
102


B16
327905
323
mir-205
142
107
89
106
94


HeLa
327905
323
mir-205
55
108
99
90
164


T47D
327905
323
mir-205
81
97
101 
103
164


T47Dp53
327905
323
mir-205
109
112
80
104
103


HeLa
338678
505
mir-205
129
103
94
105
132


BJ
328759
668
mir-216
164
91
117
141
160


B16
328759
668
mir-216
132
104
91
110
126


HeLa
328759
668
mir-216
797
40
82
203
223


T47D
328759
668
mir-216
123
86
87
122
223


T47Dp53
328759
668
mir-216
423
99
93
108
109


HeLa
327896
314
mir-22
95
103
94
106
144


HeLa
338836
660
mir-23a
303
97
96
121
114


HeLa
328084
743
mir-30a
286
89
92
153
125


HeLa
340350
855
mir-95
132
101
94
112
177





(Moure-










latos)














When treatment of cells with oligomeric compounds resulted in greater than 750% cells in subG1 phase, these oligomeric compounds were deemed to be “hits,” in that they appear to cause an increase in apoptosis, resulting in hypodiploid DNA contents. When treatment of cells with oligomeric compounds resulted in greater than 140% cells in G1-phase, these oligomeric compounds were deemed “hits,” as they appeared to cause an arrest or delay in G1-phase and/or blocked entry into S-phase of the cell cycle. When treatment of cells with oligomeric compounds resulted in greater than 140% cells in S-phase, these oligomeric compounds were deemed “hits,” as they appeared to cause an arrest or delay in DNA synthesis. When treatment of cells with oligomeric compounds resulted in greater than 140% cells in G2/M phase, these oligomeric compounds were deemed “hits,” as they appeared to cause an arrest or delay in the transition into mitosis, and/or in cell division, itself.
From these data, it was observed that 48-hours after treatment of the various cell lines with the oligomeric compounds, ISIS Number 183891 targeting the kinesin-like 1/Eg5 mRNA results in a delay or arrest in G2/M phase of the cell cycle for all cell lines. Treatment of HeLa cells with ISIS Number 25691, targeted against the Drosha RNase III mRNA, resulted in an increased percentage of cells in S-phase as well as a significant percentage of cells in the subG1 and aneuploid categories, indicating that this oligomeric compound may interfere with DNA replication and/or maintenance of the integrity of the proper complement of genetic material.
In HeLa cells, ISIS 341787 (SEQ ID NO: 852) targeted against mir-125a (mouse), resulted in an arrest or delay in G2/M as well as an increased percentage of cells in the subG1 and aneuploid categories, indicating that this oligomeric compound may interfere with cell division and equal chromosome segregation during mitosis.
In HeLa cells treated with ISIS 328744 (SEQ ID NO: 653) targeted against mir-182, an increase in the percentage of cells in the G2/M-phase of the cell cycle as well as in the subG1 category was observed, indicating that this oligomeric compound may interfere with cell division and equal chromosome segregation during mitosis. Notably, genetically normal cells (BJ and T47Dp53cells) were not affected by ISIS Number 328744, indicating that the oligomeric compound targeting miR-182 may selectively cause a cell cycle delay or arrest in cancer cells and not normal cells, and suggesting that this compound may be particularly useful as a therapeutic agent in the treatment of hyperproliferative disorders such as cancer.
In HeLa cells treated with ISIS 328759 (SEQ ID NO: 668) targeted against mir-216, a delay or arrest resulted in the G2/M-phase of the cell cycle was observed, as well as an increase in the percentage of cells in the subG1 and aneuploid categories, indicating that this oligomeric compound may interfere with cell division and equal chromosome segregation during mitosis.
Thus, it was observed that treatment of HeLa cells with oligomeric compounds targeting miRNAs is a effective means of identifying compounds that can block progression through various stages of the cell cycle. Notably, a transient increase in G1-phase was observed 24 hours after treatment of HeLa cells with oligomeric compounds targeting miRNAs; for example, oligomeric compounds ISIS Numbers 338769, 338836, 340350, and 338637 caused a transient increase in the percentage of cells delayed or arrested in G1-phase at the 24-hour time point, which, by the 48-hour time point, had shifted to a delay or arrest in S-phase. It was also noted that multiple oligomeric compounds targeting the same miRNA have the same effect on cell cycling. It was also observed that uniform 2′-MOE as well as 2′-MOE chimeric gapmer oligomeric compounds targeting the mature miRNA, as well as uniform 2′-MOE oligomeric compounds targeting the pri-miRNA often have the same effect.
Oligomeric compounds that delay, arrest or prevent cell cycle progression or induce apoptosis may be useful as therapeutic agents for the treatment of hyperproliferative disorders, such as cancer, cancer, as well as diseases associated with a hyperactivated immune response.
It is understood that BJ, B16, HeLa, A549, HMECs, T47D, T47Dp53, MCF7 or other cell lines can be treated with oligomeric compounds designed to mimic miRNAs in studies to examine their effects on progression through the cell cycle. Such oligomeric compounds are within the scope of the present invention.
Example 25
A Bioinformatic Approach to Identification of miRNA Targets
Several candidate RNA transcripts identified using the RACE-PCR methods described in Example 20 were the basis for a bioinformatic analysis of predicted targets bound to and/or potentially regulated by miRNAs. The complementarity between the miRNA used as a primer and the 3′-UTR of the RNA transcript identified by RACE-PCR was assessed using several methods. Transcripts identified by RACE-PCR were also analyzed using the FASTA sequence alignment program (accessible through the internet at, for example, www.ebi.ac.uk/fasta33) to find the best alignment between complementary sequences of the transcript and the miRNA used as a primer for RACE-PCR. When, using the default parameters, the FASTA alignment program resulted in the identification of the actual primer binding site (PBS) within the 3′-UTR of the RNA transcript as the sequence most complementary to the miRNA used as a primer in the RACE-PCR method, the candidate miRNA target transcript was specified by a plus sign (for example, see the “mir-143/PBS complementary?” column in Table 36 below). When the FASTA program failed to align the actual PBS with the sequence most complementary to the miRNA used in the RACE-PCR, the candidate miRNA target transcript was specified by a minus sign. When the FASTA program could be made to align with the sequence most complementary to the miRNA used in the RACE-PCR by decreasing the stringency of the FASTA program parameters, the candidate miRNA target transcript was specified by “±”.
A global alignment was also performed to assess whether the sequence of the PBS within the RNA transcript identified by RACE-PCR was conserved between human and mouse orthologs of the RNA target. For example, in Table 36, below, strong conservation of PBS in the human and murine orthologs (homology from 80-100%) was indicated by a plus sign; moderate conservation (homology between 70-80%) was indicated by “±”, and a minus sign indicates homology below 70%.
A variety of algorithms can be used to predict RNA secondary structures based on thermodynamic parameters and energy calculations. For example, secondary structure prediction can be performed using either M-fold or RNA Structure 2.52. M-fold can be accessed through the Internet at, for example, www.ibc.wustl.edu/-zuker/ma/form2.cgi or can be downloaded for local use on UNIX platforms. M-fold is also available as a part of GCG package. RNA Structure 2.52 is a windows adaptation of the M-fold algorithm and can be accessed through the Internet at, for example, 128.151.176.70/RNAstructure.html. The RNA Structure 2.52 program was used to analyze a series of 30-base fragments spanning the entire length of the human RNA transcript and their potential to hybridize with the miRNA used as a primer in the RACE-PCR, allowing the prediction of the lowest absolute free energy peak representing the most likely site of hybridization (including bulged regions) between the miRNA and the RNA target. If the free energy peak representing the hybridization between the miRNA and the PBS of the RNA transcript identified by RACE-PCR was among the top five peaks predicted by the RNA Structure 2.52 program, the transcript was given a plus sign, “+”. If the free energy peak representing the hybridization between the miRNA and the PBS was in the top five to ten peaks predicted by RNA Structure 2.52, the transcript was given a plus/minus sign, “±”, and if the peak representing the hybridization between the miRNA and the PBS was below the top ten peaks predicted by RNA Structure 2.52, the transcript was given a minus sign, “−”.
A list of the RNA transcript targets identified by RACE-PCR employing the mir-143 miRNA as a specific primer is shown in Table 36.







TABLE 36







Potential RNA targets of the mir-143 miRNA















mir-143/PBS



SEQ ID

RNA Structure
complementary


RNA transcript target
NO:
PBS conserved?
peak?
?





Matrix
819
+
−
+


metalloproteinase 2






Sec24
829
−
+/−
+


Tripartite motif-
828
+/−
+
+/−


containing 32






RAN
824
+/−
+
+


Cystatin B
802
−
+
+


Glucocorticoid
839
+
+/−
+


induced transcript 1






Protein phosphatase 2
809
+
+
+


Polycystic kidney
822
−
−
−


disease 2






Mannose-6-phosphate
801
+/−
−
+


receptor






Mitotic control
817
+
+
−


protein dis3 homolog






Chromosome 14 ORF 103
813
+
+/−
−


Rho GDP dissociation
823
−
−
−


inhibitor beta






Glyoxalase I
816
+
+
+


Zinc finger protein
818
+
+/−
+


36, C3H type-like 1






LIM domain only 4
804
+
+
+









Note that four genes (Sec24, cystatin B, polycystic kidney disease 2, and Rho GDP dissociation inhibitor beta) did not have murine orthologs to compare in a global analysis of the PBS. Because these RNA transcripts were identified as being bound by the mir-143 miRNA used as a primer in the RACE-PCR approach previously described, the mir-143 miRNA is predicted to serve a regulatory role in expression or activity of one or more or all of these RNA transcripts. Of particular note are three targets, protein phosphatase 2, glyoxalase I, and LIM domain only 4 (LMO4) mRNAs, for which all three analyses yielded a positive result. That all three parameters assessed yielded a positive result suggests that these mRNAs are probable targets of mir-143.
The well-studied C. elegans lin-4 miRNA interaction with its lin-28 mRNA target was used as the starting point for a bioinformatics approach to the identification of miRNA binding sites in target nucleic acids. Lin-4 has been experimentally determined to bind at a single site on the lin-28 mRNA. Herein, as a primary determinant of miRNA-target interactions, it was hypothesized that the bimolecular hybridization free energies (ΔG° 37) of the interaction of the miRNA with a true target site would be more negative than the ΔG°37 of other interactions of the miRNA with other binding sites. The nucleotide sequence of the lin-28 mRNA was assessed by computationally deriving 30-nucleotide windows, starting with the first nucleotide of the sequence and defining the first nucleotide in each window by shifting 1 nucleotide in the 3′ direction. Each window was assessed by hybridizing the 30-nucleotide sequence in the window with the lin-4 miRNA and disallowing unimolecular interactions, thereby spanning the entire length of the lin-28 mRNA, and the resulting ΔG°37 value was plotted against the start position of the window. It was observed that the bimolecular hybridization between the true lin-4 binding site and the lin-28 mRNA had the lowest ΔG° 37 value, supporting our hypothesis and our bioinformatic approach to the identification of miRNA binding sites in target nucleic acids.
The mitogen-activated protein kinase 7/extracellular signal-regulated kinase 5 (ERK5) (GenBank Accession NM—139032.1, incorporated herein as SEQ ID NO: 861) mRNA transcript was previously computationally predicted to be regulated by mir-143 miRNA binding in the 3′-UTR regions (Lewis et al., Cell, 2003, 115, 787-798). In order to identify mir-143 binding sites in the ERK5 mRNA, a bimolecular hybridization free energy assessment was performed by performing a hybridization walk to assess possible mir-143 binding sites along the entire length of the ERK5 mRNA. A strong negative ΔG°37 value (−20.1) was found at the previously predicted mir-143 binding site in the 3′-UTR, lending further support to our method. Surprisingly, two additional, and novel, mir-143 binding sites with more negative ΔG°37 values, as well as a third mir-143 binding site with a comparable ΔG°37 value were also identified. Using the ERK5 sequence (GenBank Accession NM—139032.1) as a reference, these binding sites encompass nucleotides 937-966 with a ΔG°37 value of (−22.8), nucleotides 2041-2070 with a ΔG°37 value of (−20.6) and nucleotides 2163-2192 with a ΔG° 37 value of (−19.3). See FIG. 1. Thus, three novel mir-143 binding sites (and, thus, a potential regulatory sites) were identified within the coding sequence of the ERK5 gene. Thus, this method of screening for miRNA binding sites by a bimolecular hybridization free energy assessment can be used to confirm previously predicted sites, and further allows the identification of novel miRNA target nucleic acid binding sites. It is believed that this method may more closely mimic the energetic mechanism by which a miRNA scans a target nucleic acid to find its interaction site. In subsequent experiments, the predicted mir-143 binding sites within the ERK5 coding sequence were also tested using the reporter system described below.
Example 26
Northern Analysis of miRNA Expression
As described in the adipocyte differentiation assay, the oligomeric compounds ISIS Number 327889 (SEQ ID NO: 307), targeted to mir-23b, and ISIS Number 327876 (SEQ ID NO: 294), targeted to mir-29b-1, were found to reduce the expression of several hallmark genes of adipocyte differentiation, indicating that mir-23b and mir-29b-1 may play a role in adipocyte differentiation, and that oligomeric compounds targeting these miRNAs may be useful as agents blocking cellular differentiation. Therefore, the expression of mir-23b and mir-29b was assessed by Northern blot of total RNA from multiple tissues. To detect the mir-23b and mir-29b-1 targets, target specific DNA oligonucleotide probes with the sequences GTGGTAATCCCTGGCAATGTGAT (SEQ ID NO: 307) and AACACTGATTTCAAA TGGTGCTA (SEQ ID NO: 294), respectively, were synthesized by IDT (Coralville, Iowa). The oligo probes were 5′ end-labeled with T4 polynucleotide kinase with (γ-32P) ATP (Promega). To normalize for variations in loading and transfer efficiency membranes are stripped and probed for U6 RNA. Total RNA from mouse and human tissues as well as total RNA from human adipocytes and HepG2 cells was probed in Northern blot analyses, using methods described in Example 14.
By Northern analyses, the mir-23b miRNA was found to be most highly expressed in human kidney tissue as well as in adipose tissue from ob/ob mice, and was also highly expressed in human liver, adipocytes, preadipocytes and HepG2 cells. Moderate expression of mir-23b was also noted in murine kidney tissue. The mir-29b-1 miRNA was found to be most highly expressed in human and mouse kidney, and was also expressed in human liver, adipocytes, preadipocytes, and HepG2 cells, as well as in murine adipose tissue and liver. Levels of both the mir-23b and mir-29b-1 miRNAs were also found to be upregulated in human differentiated adipocytes.
Similarly, target specific DNA oligonucleotide probes for mir-16, mir-15a, and let-7a were designed and used in Northern blot analyses to assess expression of these miRNAs in human and mouse tissues. The mir-16 and mir-15a miRNAs were each found to be most highly expressed in human spleen, heart, testes, and kidney, and expression was also observed in liver as well as HEK293 and T47D cells. Additionally, low levels of expression of the mir-16 miRNA were observed in NT2 cells. The let-7a miRNA was most highly expressed in human and murine kidney, and expression was also observed in human and murine liver. Additionally, low levels of let-7a expression were found in HepG2 cells.
To detect the mir-21 miRNA in Northern blot analyses, a target specific DNA oligonucleotide probe with the sequences TCAACATCAGTCTGATAAGCTA (SEQ ID NO: 335) was synthesized by IDT (Coralville, Iowa). The oligo probes were 5′ end-labeled with T4 polynucleotide kinase with (γ-32P) ATP (Promega). Twenty micrograms of total RNA from human Promyelocytic Leukemia HL-60 cells, A549, HeLa, HEK293, T47D, HepG2, T-24, MCF7, and Jurkat cells was was fractionated by electrophoresis through 15% acrylamide urea gels using a TBE buffer system (Invitrogen). RNA was transferred from the gel to HYBONDTM-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by electroblotting in an Xcell SureLockTM Minicell (Invitrogen). Membranes were fixed by UV cross-linking using a STRATALINKERTM UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using Rapid Hyb buffer solution (Amersham) using manufacturer's recommendations for oligonucleotide probes. To normalize for variations in loading and transfer efficiency membranes are stripped and probed for U6 RNA. High levels of expression of mir-21 were observed in A549 and HeLa cells; in fact, levels of mir-21 expression were noted to be among the highest of any of the miRNAs observed in HeLa cells.
Example 27
Reporter Systems for Assaying Activity of Oligomeric Compounds Targeting or Mimicking miRNAs
Reporter systems have been developed herein to assess the ability of miRNA mimics to provoke a gene silencing response and to assess whether antisense oligomeric compounds targeting miRNAs can inhibit gene silencing activity. The T-RExTM-HeLa cell line (Invitrogen Corp., Carlsbad, Calif.) was used for either stable or transient transfections with plasmids constitutively expressing miRNAs, pre-miRNAs, pri-miRNAs or mimics thereof, and, in some cases, antisense oligomeric compounds targeting the expressed miRNA were also transfected into the cells. It is understood that other mammalian cells lines can also be used in this reporter system. T-RExTM-HeLa cells were routinely cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.), supplemented with 10% fetal bovine serum (Invitrogen Corporation). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were harvested when they reached 90% confluence, and on the day before transfection with expression or reporter plasmids (described in detail below), the T-RExTM-HeLa cells were seeded onto 24-well plates at 50,000 cells/well. The following day, cells were transfected according to standard published procedures with various combinations of plasmids using 2 μg LipofectamineTM 2000 Reagent (Invitrogen) per μg of plasmid DNA. When transfecting oligomeric compounds, 1-3 μg of LipofectamineTM 2000 Reagent was used per 100 nM oligomeric compound.
Plasmids used are as follows: The pcDNA3.1©/NT-GFP (Invitrogen) plasmid, containing a CMV promoter controlling expression of a GFP reporter sequence at the N-terminus of the transcription start site was used as a control plasmid. The pcDNA3.1©/NT-GFP-mir-143 sensor plasmid contains (in addition to the elements above) three 22-nucleotide sites encoding the mir-143 miRNA binding site, downstream of the GFP coding sequence and upstream of the polyadenylation signal. The pCR3-pri-mir-143 plasmid (“pri-mir-143”) is a CMV promoter-driven constitutive expression plasmid which expresses the 110-nucleotide mir-143 pri-miRNA sequence (SEQ ID NO: 38), to act as a mir-143 pri-miRNA mimic. The pCR3-pri-mir control (“pri-mir-control”) is a CMV promotor-driven constitutive expression plasmid which is designed to express a similar 110-nucleotide pri-miRNA sequence (AGCAGCGCAGCGCCCTGTCTCCCAGCCAAGGTGGAACCTTCTGGGA AGCGGTCAGTTGGGAGTCCCTTCCCTGAAGGTTCCTCCTTGGAAGAGAGAAGTTGTTCTG CAGC; SEQ ID NO: 862) wherein the mature mir-143 sequence has been replaced with an unrelated sequence and the predicted complementary strand opposite it within the pri-miRNA structure is replaced with a nearly complementary sequence in order to preserve the stem loop as well as the bulge structure of the natural mir-143 pri-miRNA. Additionally, in order to test the effect of an oligomeric compound targeting a miRNA, the T-RExTM-HeLa cells were also transfected with the uniform 2′-MOE phosphorothioate (PS) antisense oligomeric compound ISIS Number 327901 (SEQ ID NO: 319), targeted to mir-143 previously described.
Twenty-four hours post-transfection, cells were trypsinized and GFP fluorescence was analyzed by flow cytometry. Results are shown in Table 37.







TABLE 37







Mean GFP fluorescence after transfection of T-REx TM-HeLa cells








Treatment
Mean












pri-mir


GFP mir-143
327901
fluores-


control
pri-mir-143
GFP control
sensor
oligo
cence















−
−
−
−
−
2.2


+
−
−
−
−
2.7


−
+
−
−
−
2.6


−
−
+
−
−
7.9


+
−
+
−
−
22.7


−
+
+
−
−
9.6


−
−
−
+
−
12.4


+
−
−
+
−
21.8


−
+
−
+
−
5.3


−
+
−
+
+
4.1


−
−
−
+
+
4.2


−
+
−
−
+
3.7









Plus signs, “+”, indicate the presence of the expression plasmid or oligomeric construct in transfectants; minus signs “−”, indicate the absence of same. Mean fluorescence is measured in arbitrary units.
In cells transfected with the sensor plasmid and expressing the mir-143 pri-miRNA mimic from the pCR3-pri-mir-143 plasmid, the mir-143 miRNA is expected to be processed endogenously, allowing it to bind as a mature miRNA to the RNA transcript encoding GFP and containing the mir-143 binding sites expressed from the reporter plasmid, resulting in cleavage of the reporter transcript and a decrease in fluorescence as compared to the control plasmid. From the data shown in Table 37, it was observed that expression of the pCR3-pri-mir-143 plasmid results in an inhibition of expression of GFP indicated by a decrease in fluorescence produced by the pcDNA3.1©/NT-GFP-mir-143 sensor plasmid, whereas expression of the pCR3-pri-mir control plasmid had no effect on GFP reporter expression. Neither the pCR3-pri-mir control nor the pCR3-pri-mir-143 plasmid had any inhibitory effect on GFP expression from the pcDNA3.1©/NT-GFP control plasmid. Thus, the mir-143 pri-miRNA mimic oligomeric compound silences the expression of RNA transcribed from a reporter plasmid containing mir-143 target sites.
In a further study, T-RExTM-HeLa cells transfected with the pcDNA3.1©/NT-GFP-mir-143 sensor plasmid were treated at various dosages with the following oligomeric compounds: 1) a double-stranded RNA oligomeric compound (“ds-mir-143”) composed of ISIS Number 342199 (TGAGATGAAGCACTGTAGCTCA; SEQ ID NO: 220) representing the mir-143 sense sequence, hybridized to its perfect complement ISIS Number 342200 (TGAGCTACAGTGCTTCATCTCA; SEQ ID NO: 319); 2) a negative control dsRNA (“ds-Control”), representing a 10-base mismatched sequence antisense to the unrelated PTP1B mRNA, composed of ISIS Number 342427 (CCTTCCCTGAAGGTTCCTCC; SEQ ID NO: 863) hybridized to its perfect complement ISIS Number 342430 (GGAGGAACCTTCAGGGAAGG; SEQ ID NO: 864); 3) the pCR3-pri-mir-143 expression plasmid (“pCR3-pri-mir-143”) which expresses the 110-nucleotide mir-143 pri-miRNA; 4) the pCR3-pri-mir control (“pri-mir-control”); 5) an in vitro transcribed RNA oligomeric compound (“hairpin mir-143”) representing the 110 bp fragment of the mir-143 pri-miRNA molecule (SEQ ID NO: 38) plus an additional two cytosine nucleobases from the T7 promoter at the 5′ end; and 6) an in vitro transcribed RNA oligomeric compound (“hairpin control”) (SEQ ID NO: 862) representing a similar hairpin structure except that the mature mir-143 sequence and its complementary sequence within the pri-miRNA hairpin structure were replaced with sequences unrelated to mir-143. The RNA hairpin oligomeric compounds were in vitro transcribed using the MAXIscript Kit (Ambion Inc., Austin, Tex.) according to the manufacturer's protocol, beginning with a DNA template amplified by PCR. GFP fluorescence of treated cells was assessed using the methods described above, and it was observed that the ds-mir-143 oligomeric compound mimic inhibited expression of GFP from the sensor plasmid in a dose dependent manner. In a further embodiment, pcDNA3.1©/NT-GFP-mir-143 sensor-expressing cells treated with 20 nM mir-143 dsRNA oligomeric compound were additionally treated with 4-, 20- or 100 nM uniform 2′-MOE oligomeric compound ISIS Number 327901 (SEQ ID NO: 319), or 4-, 20- or 100 nM uniform 2′-MOE scrambled mir-143 control ISIS Number 342673 (SEQ ID NO: 758) to assess the ability of compounds to inhibit the inhibitory effect of the mir-143 dsRNA mimic. At all three concentrations, the oligomeric compound ISIS Number 327901 targeting mir-143 blocked the inhibitory effect of the mir-143 dsRNA oligomeric compound, exhibited as a recovery of GFP fluorescence.
In one embodiment, an expression system based on the pGL3-Control (Promega Corp., Madison Wis.) vector containing a CMV promoter controlling expression of a luciferase reporter sequence was used in transient transfections of HeLa cells with plasmids expressing miRNA or pri-miRNA mimics To assess the ability of miRNA mimics to bind and regulate the expression of the luciferase reporter gene, two reporter plasmids were constructed: 1) a synthetic DNA fragment comprising two sites perfectly complementary to mir-143 were inserted into the pGL3-Control luciferase reporter vector, to create the pGL3-mir-143 sensor plasmid, and 2) a DNA fragment comprising the 3′-UTR of the LIM domain only 4 (LMO4) gene (predicted to be regulated by mir-143) was inserted into pGL3-Control to create pGL3-LMO4; this fragment was PCR-amplified using a primer beginning at position 1261 of the LMO4 sequence (GenBank Accession NM—006769.2, incorporated herein as SEQ ID: 809) and the downstream primer hybridizing to the poly-A tail. In each of these plasmids, the target site was placed downstream of the luciferase coding sequence and upstream of the polyadenylation signal in the 3′-UTR of the luciferase reporter vector. The unmodified pGL3-Control luciferase reporter vector was used as a control.
HeLa cells were routinely cultured and passaged as described, and on the day before transfection with expression or reporter plasmids, the HeLa cells were seeded onto 24-well plates 50,000 cells/well. Cells were transfected according to standard published procedures with various combinations of plasmids using 2 μg LipofectamineTM 2000 Reagent (Invitrogen) per μg of plasmid DNA, or, when transfecting oligomeric compounds, 1.25 μg of LipofectamineTM 2000 Reagent per 100 nM oligonucleotide or double-stranded RNA. The luciferase signal in each well was normalized to the Renilla luciferase (RL) activity produced from a co-transfected plasmid, pRL-CMV, which was transfected at 0.5 μg per well. Cells were treated at various dosages (4 nM, 20 nM, and 100 nM) with the following oligomeric compound mimics: 1) “ds-mir-143,” 2) “ds-Control,” 3) “pCR3-pri-mir-143,” or 4) “pri-mir-control,” as described supra. In accordance with methods described in Example 12, supra, a luciferase assay was performed 48-hours after transfection. Briefly, cells were lysed in passive lysis buffer (PLB; Promega), and 20 ul of the lysate was then assayed for RL activity using a Dual Luciferase Assay kit (Promega) according to the manufacturer's protocol. The results below are an average of three trials and are presented as percent pGL3-Control luciferase expression normalized to pRL-CMV expression (RL). The data are shown in Table 38.







TABLE 38







Luciferase assays showing effects of oligomeric


compounds mimicking mir-143









luciferase expression



(% lucif. only control)











pGL3-
pGL3-
pGL3-


treatment
Control
mir-143 sensor
LMO4













no luciferase (pRL)
0.3
0.3
0.4


luciferase (pRL) only
100.0
101.0
100.0


ds-mir-143 (4 nM)
101.5
14.5
151.6


ds-mir-143 (20 nM)
123.8
8.0
140.1


ds-mir-143 (100 nM)
131.8
7.1
128.4


ds-Control (4 nM)
133.6
144.5
172.4


ds-Control (20 nM)
126.1
169.8
151.6


ds-Control (100 nM)
123.0
151.3
151.5


pCR3-pri-mir-143 (0.25 ug)
75.6
58.6
101.9


pCR3-pri-mir-143
76.6
50.7
95.7


precursor (0.5 ug)





pCR3-pri-mir-143 (1 ug)
64.7
35.0
82.5


pri-mir control (0.25 ug)
90.3
78.3
114.8


pri-mir control (0.5 ug)
57.3
61.8
95.4


pri-mir control (1 ug)
67.9
64.9
74.8









From these data, it was observed that the mir-143 dsRNA oligomeric compound and the mir-143 pri-miRNA mimic expressed from the pCR3-pri-mir-143 expression plasmid both inhibited luciferase activity from the pGL3-mir-143 sensor plasmid in a dose-dependent manner.
In another embodiment, HeLa cells were transfected with 0.03 μg pGL3-mir-143 sensor plasmid and 0.01 μg pRL-CMV plasmid, and, in addition, (except those samples described below as “without mir-143 pri-miRNA,”) were also transfected with 0.01 μg of an expression plasmid designed to express a mir-143 pri-miRNA mimic comprising a larger 430-nt fragment of the mir-143 primary miRNA transcript, referred to as “pCR3-pri-mir-143 (430)” (AGGTTTGGTCCTGGGTGCTCAAATGGCAGGCCACAGACAGGAAACACAG TTGTGAGGAATTACAACAGCCTCCCGGCCAGAGCTGGAGAGGTGGAGCCCAGGTCCCCT CTAACACCCCTTCTCCTGGCCAGGTTGGAGTCCCGCCACAGGCCACCAGAGCGGAGCAG CGCAGCGCCCTGTCTCCCAGCCTGAGGTGCAGTGCTGCATCTCTGGTCAGTTGGGAGTCT GAGATGAAGCACTGTAGCTCAGGAAGAGAGAAGTTGTTCTGCAGCCATCAGCCTGGAAG TGGTAAGTGCTGGGGGGTTGTGGGGGGCCATAACAGGAAGGACAGAGTGTTTCCAGACT CCATACTATCAGCCACTTGTGATGCTGGGGAAGTTCCTCTACACAAGTTCCCCTGGTGCC ACGATCTGCTTCACGAGTCTGGGCA; SEQ ID NO: 871). It was observed that the mir-143 pri-miRNA mimic expressed by pCR3-pri-mir-143 (430) inhibits luciferase expression from the pGL3-mir-143 sensor plasmid. To further evaluate the ability of the mir-143 pri-miRNA mimic to inhibit luciferase activity from the sensor plasmid, and to assess the ability of oligomeric compounds to interfere with the inhibition of pGL3-mir-143 sensor luciferase expression by the mir-143 pri-miRNA mimic, pGL3-mir-143 sensor-expressing HeLa cells treated with pCR3-pri-mir-143 (430) were additionally treated with varying concentrations (0-, 6.7- or 20 nM) of the following oligomeric compounds: 1) ISIS Number 327901 (SEQ ID NO: 319), a uniform 2′-MOE oligomeric compound targeting mir-143; 2) ISIS Number 342673 (SEQ ID NO: 758), a uniform 2′-MOE scrambled control; or 3) ISIS Number 327924 (SEQ ID NO: 342) targeting an unrelated miRNA (mir-129-2). ISIS Numbers 342673 and 327924 were used as negative controls. HeLa cells transfected with the pRL-CMV and pGL3-mir-143 sensor plasmids, but not treated with the pCR3-pri-mir-143 (430) hairpin precursor served as a control. In this experiment, the luciferase assay was performed 24-hours after transfection. The data are presented in Table 39 as relative luciferase activity (normalized to RL expression levels). Where present, “N.D.” indicates “no data.”







TABLE 39







Effects of oligomeric compounds on mir-143 pri-miRNA


mimic-mediated inhibition of luciferase expression











Relative luciferase activity



SEQ ID
Dose of oligomeric compound











Treatment
NO
0 nM
6.7 nM
20 nM














327901
319
0.97
4.0
6.4


342673
758
0.97
1.3
1.5


negative control






327924
342
0.97
0.8
1.2


negative control






without pCR3-pri-mir-143(430)
N/A
13.8
N.D.
N.D.









From these data, it was observed that the oligomeric compound ISIS Number 327901 targeting mir-143 blocked the inhibitory effect of the mir-143 pri-miRNA mimic, exhibited as a 4- to 6.4-fold recovery of luciferase activity in HeLa cells expressing the pGL3-mir-143 sensor plasmid.
More than four-hundred target genes have been predicted to be regulated by miRNA binding to the 3′-UTR regions of the mRNA transcript (Lewis et al., Cell, 2003, 115, 787-798). For example, at least six genes have been reported to bear regulatory sequences in their 3′-UTRs which are predicted to be bound by the mir-143 miRNA; these include the inwardly rectifying potassium channel Kir2.2 (GenBank Accession AB074970, incorporated herein as SEQ ID NO: 872), synaptotagmin III (GenBank Accession BC028379, incorporated herein as SEQ ID NO: 873), mitogen-activated protein kinase 7/extracellular signal-regulated kinase 5 (ERK5) (GenBank Accession NM—139032.1, SEQ ID NO: 861), protein phosphatase 2 (formerly 2A), catalytic subunit, beta isoform (PPP2CB) (GenBank Accession NM—004156.1, SEQ ID NO: 814), glyoxalase I (GLO1) (GenBank Accession NM—006708.1, SEQ ID NO: 821), and LIM domain only 4 (LMO4) (GenBank Accession NM—006769.2, SEQ ID NO: 809). It should be noted that one third of miRNA targets predicted in the study by Lewis, et al. are expected to be false positives (Lewis et al., Cell, 2003, 115, 787-798).
Because the present inventors independently identified the PPP2CB and GLO1 genes as potential targets of mir-143 by the RACE-PCR methods as described in Example 20, these targets were selected for further study. In addition, and described in Example 25, a novel mir-143 binding site (and, thus, a potential regulatory site) was identified within the coding sequence of the ERK5 gene; this predicted mir-143 binding site within the ERK5 coding sequence was also tested in these reporter systems.
In some embodiments, an expression system based on the pGL3-Control (Promega Corp., Madison Wis.) reporter vector and comprising predicted miRNA binding sites was used in stable transfections of HeLa cells, selecting for cells that have integrated the reporter plasmid into their genome. Because pGL3-based reporter vectors have no selectable marker for antibiotic resistance, a neomycin-resistance (Genetecin) gene was cloned into the pCR2 plasmid (Invitrogen Life Technologies, Carlsbad, Calif.) to create the pCR2-neo plasmid, and pCR2-neo was co-transfected into HeLa cells with the pGL3-mir-143-sensor plasmid at a ratio of one pCR2-neo plasmid to ten pGL3-mir-143-sensor plasmids. Co-transfected cells were then selected for the presence of the Genetecin marker and assayed for luciferase activity; Genetecin-resistant cells are very likely to have also integrated the luciferase reporter into their genome.
Establishment of Stably-Transfected Cells:
One day prior to transfection, approximately 750,000 HeLa cells are seeded onto a 10 cm dish or T-75 flask and grown in complete medium overnight at 37° C. The next day, 10 μg of pGL3-mir-143-sensor plasmid and 1 μg pCR2-neo are mixed in 2 ml OPTI-MEMTM (Invitrogen Corporation, Carlsbad, Calif.). (Linearization of circular plasmids by digestion with restriction enzyme may increase the number of stable transfectants per μg transforming DNA, but is not an essential step). 10 μl LIPOFECTINTM reagent (Invitrogen Corporation, Carlsbad, Calif.) is mixed with 2 ml OPTI-MEMTM. The plasmid/OPTI-MEMTM and OPTI-MEMTM/LIPOFECTINTM mixtures are then mixed together, and an additional 11 ml OPTI-MEMTM is added, and the resulting 15 ml cocktail is added to the cells. Cells are incubated in the plasmid/OPTI-MEMTM/LIPOFECTINTM cocktail for approximately 4 hours at 37° C., after which the cocktail is removed and replaced with fresh complete medium. The following day, cells are trypsinized and transferred to a T-175 flask. Media containing the selection agent, 500 μg/ml G418 (Geneticin; GIBCO/Life Technologies, Gaithersburg, Md.), is added and cells are grown at 37° C. Cells are re-fed daily with fresh media containing the selection agent until the majority of the cells appear to have died off and isolated colonies of neomycin-resistant cells appear. In cases where subcloning is desired, selected neomycin-resistant cells are trypsinized and plated at a concentration of 0.5 cells/well in 96-well plates, maintaining the cells in 500 μg/ml G418 selection media.
In one embodiment, five stably-transfected, neomycin-resistant, luciferase-positive, pGL3-mir-143-sensor cell clones were isolated, subcloned and selected for further testing with oligomeric compounds of the present invention. Cells stably expressing the luciferase reporter and comprising one or more miRNA binding sites were then transfected with oligomeric compounds mimicking miRNAs, pre-miRNAs or pri-miRNAs in order to assess the ability of these miRNA mimics to bind and regulate the expression of the luciferase reporter.
An expression system based on the pGL3-Control (Promega Corp., Madison Wis.) reporter vector and comprising predicted miRNA binding sites was used in transient transfections of HeLa cells with plasmids expressing oligomeric compounds mimicking miRNAs, pre-miRNAs or pri-miRNAs in order to assess the ability of these miRNA mimics to bind and regulate the expression of the luciferase reporter. The effect of increasing the copy number of the miRNA-binding site in the target was also tested by including multiple binding sites in artificial reporter constructs. It is understood that the presence of multiple miRNA-binding sites in a target can include binding sites for different miRNAs.
The following reporter plasmids were constructed by cloning the specified fragment into the XbaI site of the pGL3-control plasmid, placing the potential miRNA-binding site in the 3′-UTR of the luciferase reporter: The reporter plasmid pGL3-bulge(x3) contains three contiguous copies of the sequence (TGAGCTACAGCTTCATCTCA; herein incorporated as SEQ ID NO: 874) which represents a sequence complementary to the mir-143 miRNA except that it is missing 2 nucleotides such that the mir-143 miRNA is presumed to adopt a bulged structure when it hybridizes to this target sequence. The pGL3-GLO1 reporter plasmid contains a DNA fragment comprising the 3′-UTR of the GLO1 sequence; this fragment was PCR-amplified using a primer beginning at nucleotide number 621 of the GLO1 sequence (GenBank Accession NM—006708.1, SEQ ID NO: 821) and the downstream primer hybridizing to the poly A tail. The pGL3-PP2A reporter plasmid contains a DNA fragment comprising the 3′-UTR of the PP2A gene; this fragment was PCR-amplified using a primer beginning at nucleotide number 921 of the PP2A sequence (GenBank Accession NM—004156.1) and the downstream primer hybridizing to the poly A tail. The reporter plasmid pGL3-ERK5-3′-UTR(x1) contains one copy of the sequence TATTCTGCAGGTTCATCTCAG (herein incorporated as SEQ ID NO: 875), found in the 3′-UTR of ERK5 and predicted by Lewis, et al. to be bound by the mir-143 miRNA, and the reporter plasmid pGL3-ERK5-3′UTR(x3) has three contiguous copies of this sequence. The reporter plasmid pGL3-ERK5-3′UTR(ext) contains one copy of the sequence CGGCTTGGATTATTCTGCAGGTTCATCTCAGACCCACCTTT (herein incorporated as SEQ ID NO: 876), which includes an additional ten nucleotides at either end of the mir-143 binding site in 3′-UTR of ERK5 predicted by Lewis, et al. (Lewis et al., Cell, 2003, 115, 787-798). The plasmids pGL3-ERK5-cds(x1), pGL3-ERK5-cds(x2), pGL3-ERK5-cds(x3), and pGL3-ERK5-cds(x5) contain one, two, three or five contiguous copies, respectively, of the novel mir-143 binding site (TTGAGCCCAGCGCTCGCATCTCA; herein incorporated as SEQ ID NO: 877) we identified within the coding sequence of ERK5. The unmodified pGL3-Control luciferase reporter vector was used as a negative control, and the pGL3-mir-143 sensor reporter plasmid was used as a positive control.
HeLa cells were routinely cultured and passaged as described. In some embodiments, HeLa cells were transfected with 0.05 μg of the relevant pGL3-sensor plasmid and 0.01 μg pRL-CMV plasmid. Additionally, in some embodiments, cells were treated at various dosages (11 nM, 33 nM, and 100 nM) with the following oligomeric compound mimics: 1) ds-mir-143, or 2) ds-Control as described. In accordance with methods described in Example 12, a luciferase assay was performed 24-hours after transfection. The results, shown in Tables 40 and 41, are an average of three trials. Data are presented as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.







TABLE 40







Effects of oligomeric compounds mimicking


mir-143 on luciferase expression









Reporter
ds-mir-143
ds-control













plasmid
11 nM
33 nM
100 nM
11 nM
33 nM
100 nM





pGL3-Control
90.7
94.2
72.5
113.4
79.6
87.0


pGL3-bulge(x3)
50.7
35.4
17.2
111.3
82.6
84.7


pGL3-ERK5-
81.9
84.7
62.2
103.2
79.6
77.6


3′UTR(x1)









From these data, it was observed that, while treatment of HeLa cells expressing the reporter plasmids with the ds-control did not appear to significantly affect luciferase expression, the mir-143 dsRNA mimic compound inhibited luciferase activity from the pGL3-bulge(x3) sensor plasmid in a dose-dependent manner.







TABLE 41







Effects of oligomeric compounds mimicking


mir-143 on luciferase expression









Reporter
ds-mir-143
ds-control













plasmid
11 nM
33 nM
100 nM
11 nM
33 nM
100 nM
















pGL3-Control
110.2
124.3
92.3
114.1
95.6
103.0


pGL3-mir-143 
15.0
15.0
11.1
114.5
108.9
97.1


sensor








pGL3-bulge(x3)
36.1
33.9
22.2
109.5
103.2
92.4


pGL3-ERK5-
92.2
108.1
81.9
106.2
99.6
90.1


3′UTR(x1)








pGL3-ERK5-
51.7
51.0
28.2
104.6
103.4
95.7


3′UTR(x3)








pGL3-ERK5-
101.3
115.4
77.4
100.6
102.1
96.2


cds(x1)








pGL3-ERK5-
92.7
113.8
63.6
111.3
99.2
90.4


cds(x2)








pGL3-ERK5-
73.5
77.9
49.4
105.2
96.6
79.9


cds(x3)








pGL3-ERK5-
49.4
44.5
23.9
103.0
113.4
89.9


cds(x5)








pGL3-ERK5-
89.0
106.7
81.4
96.8
108.9
89.4


3′UTR(ext)









From these data it was observed that treatment of HeLa cells expressing the pGL3-bulge(x3) reporter plasmid with the ds-mir-143 miRNA mimic oligomeric compound resulted in a dose-dependent inhibition of luciferase activity while the ds-control oligomeric compound had no effect as described previously. Treatment of HeLa cells expressing the pGL3-ERK5-3′UTR(x1) (containing one copy of the mir-143 binding site predicted by Lewis, et al.) with the ds-mir-143 mimic oligomeric compound did not inhibit luciferase activity, although increasing the number of potential mir-143 binding sites in the pGL3-ERK5-3′UTR(x3) reporter plasmid to three appeared to favor the binding of the ds-mir-143 mimic and inhibition of luciferase activity. Treatment of cells expressing the pGL3-ERK5-cds(x1) or pGL3-ERK5-cds(x2) reporter plasmids bearing a one or two copies, respectively, of the novel mir-143 binding site identified in the coding sequence of the ERK5 gene with 11- or 33 nM of the ds-mir-143 mimic oligomeric compound did not appear to inhibit luciferase activity, although treatment with 100 nM of the ds-mir-143 mimic did reduce luciferase expression. Treatment of cells expressing the pGL3-ERK5-cds(x3) or pGL3-ERK5-cds(x5) reporter plasmids, bearing three or five of copies, respectively, of the novel mir-143 binding site in the ERK5 coding sequence, with the ds-mir-143 mimic oligomeric compound resulted in a reduction in luciferase activity. The pGL3-ERK5-cds(x5) reporter plasmid exhibited a dose-responsiveness with increasing concentration of the mir-143 mimic oligomeric compound. Taken together, these results support the conclusion that multiple miRNAs and miRNA binding sites may cooperate to silence gene expression.
In order to assess the ability of miRNAs to bind predicted miRNA binding sites and regulate the expression of the luciferase reporter, in some embodiments, expression systems based on the pGL3-Control (Promega Corp., Madison Wis.) reporter vector and comprising either a mir-15a, mir-21, or a mir-23b miRNA binding site were developed and used in transient transfections of HeLa cells to determine whether the endogenous mir-15a, mir-21, or mir-23b miRNAs, respectively, could repress luciferase reporter gene expression.
The pGL3-mir-15a sensor plasmid was created by cloning the sequence (CACAAACCATTATGTGCTGCTA; SEQ ID NO: 369), complementary to the mir-15a miRNA, into the Xba site of the pGL3-Control plasmid, placing the potential miRNA-binding site in the 3′UTR of the luciferase reporter. This reporter plasmid was used to transfect HeLa cells and it was observed that the endogenous mir-15a miRNA was able to inhibit luciferase expression from the pGL3-mir-15a sensor plasmid. Thus, to further evaluate the ability of the mir-15a miRNA to bind this target site encoded by the pGL3-mir-15a sensor plasmid, and to assess the ability of oligomeric compounds to interfere with mir-15a-mediated silencing, pGL3-mir-15a sensor-expressing HeLa cells were treated with varying concentrations (3-, 10- or 30 nM) of the following oligomeric compounds: ISIS Number 327951 (SEQ ID NO: 369) is a uniform 2′-MOE compound targeting the mature mir-15a-1 miRNA. ISIS Numbers 356213 (SEQ ID NO: 878), 356215 (SEQ ID NO: 879), 356216 (SEQ ID NO: 880), 356218 (SEQ ID NO: 881), 356221 (SEQ ID NO: 882), 356227 (SEQ ID NO: 883) and 356229 (SEQ ID NO: 884) are phosphorothioate, uniform 2′-MOE oligomeric compounds designed and synthesized to target the entire length of the mir-15a pri-miRNA molecule (described in detail in Example 28, below). The uniform 2′-MOE phosphorothioate oligomeric compounds ISIS Number 327901 (SEQ ID NO: 319), targeting an unrelated miRNA (mir-143) and ISIS Number 342673 (AGACTAGCGGTATCTTTATCCC; herein incorporated as SEQ ID NO: 758), containing 15 mismatches with respect to the mature mir-143 miRNA, were used as negative controls. The data presented in Table 42 are the average of three trials and are presented as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.







TABLE 42







Effects of oligomeric compounds on mir-15a


miRNA-mediated inhibition of luciferase expression











Relative luciferase activity



SEQ ID
Dose of oligomeric compound











Treatment
NO
3 nM
10 nM
30 nM














327901
319
83.6
96.6
88.2


negative control






342673
758
104.5
82.6
85.7


negative control






327951
369
151.0
207.6
137.1


356213
878
101.2
80.5
109.9


356215
879
98.0
116.7
79.6


356216
880
102.8
84.7
113.2


356218
881
91.6
110.3
85.7


356221
882
106.8
74.0
81.2


356227
883
86.1
117.8
101.5


356229
884
109.7
100.3
97.5









From these data, it was observed that the oligomeric compound ISIS Number 327951 targeting the mature mir-15a miRNA blocked the inhibitory effect of mir-15a, exhibited as a recovery and increase in luciferase activity in HeLa cells expressing the pGL3-mir-15a sensor plasmid.
The pGL3-mir-23b sensor plasmid was created by cloning the sequence (GTGGTAATCCCTGGCAATGTGAT; SEQ ID NO: 307), representing a sequence complementary to the mir-23b miRNA, into the Xba site of the pGL3-Control plasmid, placing the potential miRNA-binding site in the 3′UTR of the luciferase reporter. This reporter plasmid was used to transfect HeLa cells and it was observed that the endogenous mir-23b miRNA was able to inhibit luciferase expression from the pGL3-mir-23b sensor plasmid. Thus, to further evaluate the ability of the mir-23b miRNA to bind this target site encoded by the pGL3-mir-23b sensor plasmid, and to assess the ability of oligomeric compounds to interfere with mir-23b-mediated silencing, pGL3-mir-23b sensor-expressing HeLa cells were treated with varying concentrations (1.3-, 5- or 20 nM) of the following oligomeric compounds: ISIS Number 327889 (SEQ ID NO: 307), a phosphorothioate uniform 2′-MOE oligomeric compound, and ISIS Number 340925 (SEQ ID NO: 307), a 2′-MOE 5-10-8 gapmer oligomeric compound, both targeting mir-23b. The uniform 2′-MOE phosphorothioate oligomeric compound ISIS Number 327924 (SEQ ID NO: 342) targeting an unrelated miRNA (mir-129-2) was used as a negative control. The data are the average of three trials, and are presented in Table 43 as relative luciferase activity (normalized to pRL-CMV luciferase plasmid only, not treated with oligomeric compound).







TABLE 43







Effects of oligomeric compounds on mir-23b miRNA-


mediated inhibition of luciferase expression











Fold change luciferase



SEQ ID
Dose of oligomeric compound











Treatment
NO
1.3 nM
5 nM
20 nM





327924
342
1.15
0.68
0.92


negative control






327889-uniform MOE
307
3.75
3.46
7.40


340925-gapmer
307
0.99
1.41
1.19









From these data, it was observed that, at all doses, ISIS Number 327889, the uniform 2′-MOE oligomeric compound targeting the mature mir-23b miRNA, de-repressed the expression of the luciferase reporter. Thus, ISIS 327889 reversed the silencing effect of the mir-23b miRNA, apparently by inhibiting the binding of mir-23b to its target site encoded by the pGL3-mir-23b sensor plasmid.
The pGL3-mir-21 sensor plasmid was created by cloning the sequence (TCAACATCAGTCTGATAAGCTA; SEQ ID NO: 335), representing a sequence complementary to the mir-21 miRNA, into the Xba site of the pGL3-Control plasmid, placing the potential miRNA-binding site in the 3′UTR of the luciferase reporter. This reporter plasmid was used to transfect HeLa cells and it was observed that the endogenous mir-21 miRNA was able to inhibit luciferase expression from the pGL3-mir-21 sensor plasmid. Thus, to further evaluate the ability of the mir-21 miRNA to bind this target site encoded by the pGL3-mir-21 sensor plasmid, and to assess the ability of oligomeric compounds to interfere with mir-21-mediated silencing, pGL3-mir-21 sensor-expressing HeLa cells were treated with varying concentrations (10 nM or 50 nM) of the following oligomeric compounds: ISIS Number 327917 (SEQ ID NO: 335), a phosphorothioate uniform 2′-MOE oligomeric compound; ISIS Number 338697 (TGCCATGAGATTCAACAGTC; herein incorporated as SEQ ID NO: 524), a uniform 2′-MOE oligomeric compound targeting the mir-21 pri-miRNA molecule; and ISIS Number 328415 (SEQ ID NO: 524), a 2′-MOE 5-10-5 gapmer oligomeric compound targeting the mir-21 pri-miRNA. The uniform 2′-MOE phosphorothioate oligomeric compound ISIS Number 327901 (SEQ ID NO: 319) targeting an unrelated miRNA (mir-143) was used as a negative control. The data are the average of three trials and are presented in Table 44 as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.







TABLE 44







Effects of oligomeric compounds on mir-21 miRNA-


mediated inhibition of luciferase expression












% UTC




SEQ ID
Dose of oligomeric compound













Treatment
NO
10 nM
50 nM















327901
319
74.2
83.1



negative control






327917
335
1037.6
847.5



338697
524
87.0
84.8



328415
524
66.0
104.4









From these data, it was observed that, at both doses, treatment of HeLa cells with ISIS Number 327917, the uniform 2′-MOE oligomeric compound targeting the mature mir-21 miRNA, de-repressed the expression of the luciferase reporter. Thus, ISIS 327917 reversed the silencing effect of the endogenous mir-21 miRNA, apparently by inhibiting the binding of mir-21 to its target site encoded by the pGL3-mir-21 sensor plasmid.
Therefore, oligomeric compounds targeting and/or mimicking the mir-143, mir-15a, mir-23b and mir-21 miRNAs and their corresponding pri-miRNA molecules have been demonstrated to bind to target RNA transcripts and silence reporter gene expression.
Example 28
Effects of Oligomeric Compounds on Expression of Pri-miRNAs 
As described above in Example 19, pri-miRNAs, often hundreds of nucleotides in length, are processed by a nuclear enzyme in the RNase III family known as Drosha, into approximately 70 nucleotide-long pre-miRNAs (also known as stem-loop structures, hairpins, pre-mirs or foldback miRNA precursors), and pre-miRNAs are subsequently exported from the nucleus to the cytoplasm, where they are processed by human Dicer into double-stranded miRNAs, which are subsequently processed by the Dicer RNase into mature miRNAs. It is believed that, in processing the pri-miRNA into the pre-miRNA, the Drosha enzyme cuts the pri-miRNA at the base of the mature miRNA, leaving a 2-nt 3′overhang (Lee, et al., Nature, 2003, 425, 415-419). The 3′ two-nucleotide overhang structure, a signature of RNaseIII cleavage, has been identified as a critical specificity determinant in targeting and maintaining small RNAs in the RNA interference pathway (Murchison, et al., Curr. Opin. Cell Biol., 2004, 16, 223-9).
The oligomeric compounds of the present invention are believed to disrupt pri-miRNA and/or pre-miRNA structures, and sterically hinder Drosha and/or Dicer cleavage, respectively. Additionally, oligomeric compounds capable of binding to the mature miRNA are believed to prevent the RISC-mediated binding of a miRNA to its mRNA target, either by cleavage or steric occlusion of the miRNA.
Using the real-time RT-PCR methods described in Example 19, the expression levels of the mir-15a pri-miRNA were compared in HepG2 cells treated with a nested series of chimeric gapmer oligomeric compounds, targeting and spanning the entire length of the mir-15a pri-miRNA; these compounds are shown in Table 45, below. Each gapmer is 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. Using the transfection methods described herein, HepG2 cells were treated with 100 nM of each of these gapmer oligomeric compounds. Total RNA was isolated from HepG2 cells by lysing cells in 1 mL TRIZOLTM (Invitrogen) using the manufacturer's recommended protocols. Real-time RT-PCR analysis was performed using a primer/probe set specific for the mir-15a pri-miRNA molecule to assess the effects of these compounds on expression of the mir-15a pri-miRNA molecule. ISIS 339317 (GTGTGTTTAAAAAAAATAAAACCTTGGA; SEQ ID NO.: 885) was used as the forward primer, ISIS 339318 (TGGCCTGCACCTTTTCAAA; SEQ ID NO.: 886) was used as the reverse primer, and ISIS 339319 (AAAGTAGCAGCACATAATGGTTTGTGG; SEQ ID NO.: 887) was used as the probe. Total RNA was quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target are normalized to 5.8S rRNA, and values are expressed relative to the untreated control Inhibition of expression of the mir15a pri-miRNA by these gapmer oligomeric compounds is expressed as a percentage of RNA levels in untreated control cells. Results of these experiments are described in Table 45 below:







TABLE 45







Effects of chimeric oligomeric


compounds on expression of the mir-15a pri-miRNA











SEQ

Expression of


ISIS
ID

mir-15a pri-miRNA


Number
NO
Sequence
(% UTC)













347964
878
TATAACATTGATGTAATATG
13.7


347965
888
GCTACTTTACTCCAAGGTTT
86.0


347966
879
TGCTACTTTACTCCAAGGTT
39.2


347967
880
GCACCTTTTCAAAATCCACA
152.3


347968
889
CCTGCACCTTTTCAAAATCC
8.4


347969
881
TGGCCTGCACCTTTTCAAAA
39.5


347970
890
ATATGGCCTGCACCTTTTCA
2.2


347971
891
ACAATATGGCCTGCACCTTT
92.8


347972
882
AGCACAATATGGCCTGCACC
98.6


347973
892
GGCAGCACAATATGGCCTGC
143.3


347974
893
TGAGGCAGCACAATATGGCC
98.1


347975
894
TTTTGAGGCAGCACAATATG
9.2


347976
895
TATTTTTGAGGCAGCACAAT
73.0


347977
896
TTGTATTTTTGAGGCAGCAC
111.3


347978
883
TCCTTGTATTTTTGAGGCAG
51.1


347979
897
AGATCCTTGTATTTTTGAGG
74.9


347980
884
AGATCAGATCCTTGTATTTT
3.6


347981
898
AGAAGATCAGATCCTTGTAT
N/D


347982
899
TTCAGAAGATCAGATCCTTG
82.2


347983
900
AAATATATTTTCTTCAGAAG
13.0









From these data, it was observed that oligomeric compounds ISIS Numbers 347964, 347966, 347968, 347970, 347975, 347980 and 347983 show significant inhibition of expression of the mir-15a pri-miRNA molecule. Thus, it is believed that the antisense oligomeric compounds ISIS Numbers 347964, 347966, 347968, 347970, 347975, 347980 and 347983 bind to the mir-15a pri-miRNA and/or pre-miRNA molecules and cause their degradation and cleavage.
From these data, it was observed that oligomeric compounds ISIS Numbers 347967, 347977 and 347973 stimulate an increase in expression levels of the mir-15a pri-miRNA. It is believed that the oligomeric compounds ISIS Numbers 347967, 347977 and 347973 bind to the mir-15a pri-miRNA and inhibit its processing into the mature mir-15a miRNA. It is believed that, in addition to the increase in the levels of the mir-15a pri-miRNA observed upon treatment of cells with the oligomeric compounds ISIS Numbers 347977, 347967 and 347973, a drop in expression levels of the fully processed mature mir-15a miRNA may also trigger a feedback mechanism signaling these cells to increase production of the mir-15a pri-miRNA.
The gapmer oligomeric compounds targeting the mir-15b and mir-15-a-1 mature miRNAs described above were also transfected into T47D cells according to standard procedures. In addition, uniform 2′-MOE and 2′-MOE gapmer oligomeric compounds targeting the mature mir-15a-1 and mir-15b miRNAs were also transfected into T47D cells, for analysis of their effects on mir-15a-1 and mir-15b pri-miRNA levels. The oligomeric compounds ISIS Number 327927 (SEQ ID NO: 345), a uniform 2′-MOE compound and ISIS Number 345391 (SEQ ID NO: 345), a 2′-MOE 5-10-7 gapmer compound, both target mir-15b. The oligomeric compounds ISIS Number 327951 (SEQ ID NO: 369), a uniform 2′-MOE compound, and ISIS Number 345411 (SEQ ID NO: 369), a 2′-MOE 5-10-7 gapmer compound, both target mir-15a-1. Oligomeric compounds ISIS Number 129686 (CGTTATTAACCTCCGTTGAA; SEQ ID NO: 901), and ISIS Number 129691 (ATGCATACTACGAAAGGCCG; SEQ ID NO:902), both universal scrambled controls, as well as ISIS Number 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 844) targeting an unrelated gene, PTEN, were used as negative controls. ISIS Numbers 129686, 129691, and 116847 are phosphorothiated 2′-MOE 5-10-5 gapmers, and all cytosines are 5-methylcytosines. T47D cells (seeded in 12-well plates) were treated with these oligomeric compounds, and RNA was isolated from the treated cells by lysing in 1 mL TRIZOLTM (Invitrogen) and total RNA was prepared using the manufacturer's recommended protocols. To assess the effects of these compounds on expression of the mir-15a or mir-15b pri-miRNA molecules, real-time RT-PCR analysis was performed using either the primer/probe set specific for the mir-15a pri-miRNA molecule described above, or a primer probe set specific for the mir-15b pri-miRNA molecule: ISIS 339320 (CCTACATTTTTGAGGCCTTAAAGTACTG; SEQ ID NO: 903) was used as the forward primer for the mir-15b pri-miRNA, ISIS 339321 (CAAATAATGATTCGCATCTTG ACTGT; SEQ ID NO: 904) was used as the reverse primer for the mir-15b pri-miRNA, and ISIS 339322 (AGCAGCACATCATGGTTTACATGC; SEQ ID NO: 905) was used as the probe. Total RNA was quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target were normalized to 5.8S rRNA, and values are expressed relative to the untreated control Inhibition of expression of the mir15a or mir-15b pri-miRNA molecules upon treatment with these oligomeric compounds is was assessed and expressed as a percentage of RNA levels in untreated control cells.
On multiple repeats of these experiments, it was observed that the uniform 2′-MOE oligomeric compounds ISIS Number 327927 (SEQ ID NO: 345) and ISIS Number 327951 (SEQ ID NO: 369), targeted to the mature mir-15b and mir-15a-1 miRNAs, respectively, each stimulate an approximately 2.5- to 3.5-fold increase in expression of the mir-15a pri-miRNA molecule and an approximately 1.5- to 2.5-fold increase in the expression of the mir-15b pri-miRNA molecule. Therefore, it is believed that ISIS Numbers 327927 and 327951 can bind to the mir-15a and/or mir-15b pri-miRNA or pre-miRNA molecules and interfere with their processing into the mature mir-15a or mir-15b miRNAs. It is also recognized that a decrease in levels of the mature, processed forms of the mir-15a or mir-15b miRNAs in T47D cells treated with ISIS Number 345411 (SEQ ID NO: 369), ISIS Number 327927 (SEQ ID NO: 345) or ISIS Number 327951 (SEQ ID NO: 369) may also trigger a feedback mechanism that signals these cells to increase production of the mir-15a and/or mir-15b pri-miRNA molecules.
In accordance with the present invention, a nested series of uniform 2′-MOE oligomeric compounds were designed and synthesized to target the entire length of the mir-15a pri-miRNA molecule. Each compound is 19 nucleotides in length, composed of 2′-methoxyethoxy (2′-MOE) nucleotides and phosphorothioate (P═S) internucleoside linkages throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds are shown in Table 46. The compounds can be analyzed for their effect on mature miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or the function of targets downstream of miRNAs.







TABLE 46







Uniform 2′-MOE


PS Compounds targeting the mir-15a pri-miRNA











ISIS
SEQ ID




Number
NO
Sequence







356213
878
TATAACATTGATGTAATATG



356214
879
GCTACTTTACTCCAAGGTTT



356215
880
TGCTACTTTACTCCAAGGTT



356216
881
GCACCTTTTCAAAATCCACA



356217
882
CCTGCACCTTTTCAAAATCC



356218
883
TGGCCTGCACCTTTTCAAAA



356219
884
ATATGGCCTGCACCTTTTCA



356220
888
ACAATATGGCCTGCACCTTT



356221
889
AGCACAATATGGCCTGCACC



356222
890
GGCAGCACAATATGGCCTGC



356223
891
TGAGGCAGCACAATATGGCC



356224
892
TTTTGAGGCAGCACAATATG



356225
893
TATTTTTGAGGCAGCACAAT



356226
894
TTGTATTTTTGAGGCAGCAC



356227
895
TCCTTGTATTTTTGAGGCAG



356228
896
AGATCCTTGTATTTTTGAGG



356229
897
AGATCAGATCCTTGTATTTT



356230
898
AGAAGATCAGATCCTTGTAT



356231
899
TTCAGAAGATCAGATCCTTG



356232
900
AAATATATTTTCTTCAGAAG










Using the real-time RT-PCR methods described, the expression levels of the mir-15a pri-miRNA were compared in T47D cells treated with the nested series of uniform 2′-MOE oligomeric compounds, targeting and spanning the entire length of the mir-15a pri-miRNA. The region encompassing the mir-15a primary transcript (the complement of nucleotides 31603159 to 31603468 of GenBank Accession number NT—024524.13; AAATAATTATG CATATTACATCAATGTTATAATGTTTAAACATAGATTTTTTTACATGCATTCTTTTTTTCCT GAAAGAAAATATTTTTTATATTCTTTAGGCGCGAATGTGTGTTTAAAAAAAATAAAACCT TGGAGTAAAGTAGCAGCACATAATGGTTTGTGGATTTTGAAAAGGTGCAGGCCATATTG TGCTGCCTCAAAAATACAAGGATCTGATCTTCTGAAGAAAATATATTTCTTTTTATTCATA GCTCTTATGATAGCAATGTCAGCAGTGCCTTAGCAGCACGTAAATATTGGCGTTAAG) is incorporated herein as SEQ ID NO: 906. ISIS Number 356215 (SEQ ID NO: 879) targets a region flanking and immediately 5′ to the predicted 5′ Drosha cleavage site in the mir-15a pri-miRNA. ISIS Number 356218 (SEQ ID NO: 881) targets a region in the loop of the mir-15a pri-miRNA. ISIS 356227 (SEQ ID NO: 883) targets a region flanking and immediately 3′ to the predicted 3′ Drosha cleavage site in the mir-15a pri-miRNA. Additionally, oligomeric compound ISIS 327951 (SEQ ID NO: 369), a uniform 2′-MOE compound targeting the mature mir-15a-1 miRNA, was tested for comparison. Oligomeric compounds ISIS 327901 (SEQ ID NO: 319) targeting the mature mir-143 miRNA; ISIS 129690, (TTAGAATACGTCGCGTTATG; SEQ ID NO: 907), a phosphorothioate 5-10-5 MOE gapmer used as a universal scrambled control; and ISIS 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 844), a uniform 5-10-5 2′-MOE gapmer targeting an unrelated gene, PTEN, were used as negative controls. Using the transfection methods previously described, T47D cells were treated with 100 nM of each of these oligomeric compounds. Total RNA was isolated by lysing cells in 1 mL TRIZOLTM (Invitrogen) using the manufacturer's recommended protocols. real-time RT-PCR analysis was performed using a primer/probe set specific for the mir-15a pri-miRNA molecule [forward primer=ISIS 339317 (SEQ ID NO.: 885), reverse primer=ISIS 339318 (SEQ ID NO.: 886), and probe=ISIS 339319 (SEQ ID NO.: 887)]. Total RNA was quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target were normalized to 5.8S rRNA, and values were expressed relative to the untreated control (UTC). Effects on expression of the mir-15a pri-miRNA molecule resulting from treatment of T47D cells with these uniform 2′-MOE oligomeric compounds is expressed as a percentage of RNA levels in untreated control cells. Results of these experiments are described in Table 47 below:







TABLE 47







Effects of uniform 2′-MOE


oligomeric compounds on mir-15a pri-miRNA expression











SEQ




ISIS #
ID NO:
target
% UTC













UTC
N/A
N/A
100


129690
XXX
N/A
121


scrambled control





327901
319
mir-143
132


116847
844
PTEN mRNA
132


327951
369
mature mir-15a-1
713


356213
878
>100 bp upstream of mature mir-15a
171


356215
879
flanking 5′ Drosha cleavage site
1005




of mir-15a-1 pri-miRNA



356216
880
mir-15a-1 pri-miRNA
503


356218
881
loop of mir-15a-1 pri-miRNA
392


356221
882
mir-15a-1 pri-miRNA
444


356224
894
mir-15a-1 pri-miRNA
592


356227
883
flanking 3′ Drosha cleavage site
879




of mir-15a-1 pri-miRNA



356229
884
mir-15a-1 pri-miRNA
818


356231
899
mir-15a-1 pri-miRNA
811


356232
900
mir-15a-1 pri-miRNA
631









From these data, it was observed that the uniform 2′-MOE oligomeric compounds ISIS Numbers 327927, 327951, 356215, 356216, 356218, 356221, 356224, 356227, 356229, 356231 and 356232 stimulate an increase in levels of the mir-15a pri-miRNA molecule as detected by real-time RT-PCR. Notably, oligomeric compounds ISIS Numbers 356215 and 356227 which target the regions immediately flanking the predicted 5′ and 3′ Drosha cleavage sites in the mir-15a pri-miRNA, respectively, were observed to stimulate the greatest increases in expression of the mir-15a pri-miRNA. It is believed that these oligomeric compounds bind to the mir-15a pri-miRNA and/or pre-miRNA molecules and interfere with their processing into the mature mir-15a miRNA, possibly by interfering with the activity of RNase III-like enzymes such as human Dicer and/or Drosha. The resultant decrease in levels of the processed mature mir-15a miRNA may trigger a feedback mechanism leading to an upregulation of production of the mir-15a pri-miRNA molecule. Not mutually exclusive with the processing interference and the feedback mechanisms is the possibility that treatment with oligomeric compounds could stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-15a pri-miRNA or pre-miRNA molecules. It is understood that such oligomeric compound-triggered mechanisms may be operating not only upon regulation of mir-15a production and processing, but may also be found to regulate the production and processing of other miRNAs.
The expression levels of mir-24-2, let-7i, and let-7d were assessed in HeLa or T-24 cells treated with various uniform 2′-MOE oligomeric compounds targeting mature miRNAs. For example, using the transfection methods previously described, HeLa cells were treated with 100 nM of the oligomeric compound ISIS Number 327945 (SEQ ID NO: 363) targeting the mir-24-2 mature miRNA. Total RNA was isolated and expression levels of the mir-24-2 pri-miRNA were analyzed by real-time quantitative RT-PCR using a primer/probe set specific for the mir-24-2 pri-miRNA molecule [forward primer=ISIS 359358 (CCCTGGGCTCTGCCT; herein incorporated as SEQ ID NO.: 908), reverse primer=ISIS 359359 (TGTACACAAACCAACTGTGTTTC; herein incorporated as SEQ ID NO.: 909), and probe=ISIS 359360 (CGTGCCTACTGAGC; herein incorporated as SEQ ID NO.: 910)]. An approximately 35-fold increase in expression levels of the mir-24-2 pri-miRNA molecule was observed in HeLa cells treated with the oligomeric compound ISIS 327945 as detected by real-time RT-PCR.
Using the transfection methods previously described, HeLa cells were treated with 100 nM of the oligomeric compound ISIS Number 327890 (SEQ ID NO: 308) targeting the let-7i mature miRNA. Total RNA was isolated and expression levels of the let-7i pri-miRNA were analyzed by real-time quantitative RT-PCR using a primer/probe set specific for the let-7i pri-miRNA molecule [forward primer=ISIS 341684 (TGAGGTAGTAGTTTGTGCTGTTGGT; herein incorporated as SEQ ID NO.: 777), reverse primer=ISIS 341685 (AGGCAGTAGCTTGCGCAGTTA; herein incorporated as SEQ ID NO.: 778), and probe=ISIS 341686 (TTGTGACATTGCCCGCTGTGGAG; herein incorporated as SEQ ID NO.: 779)]. An approximately 4-fold increase in expression levels of the let-7i pri-miRNA molecule was observed in HeLa cells treated with the oligomeric compound ISIS 327890 as detected by real-time RT-PCR.
Using the transfection methods previously described, supra, T-24 cells were treated with 100 nM of the oligomeric compound ISIS Number 327926 (SEQ ID NO: 344) targeting the let-7d mature miRNA. Total RNA was isolated and expression levels of the let-7d pri-miRNA were analyzed by real-time quantitative RT-PCR using a primer/probe set specific for the let-7d pri-miRNA molecule (forward primer=ISIS 341678 (CCTAGGAAGAGGTAG TAGGTTGCA; herein incorporated as SEQ ID NO.: 771), reverse primer=ISIS 341679 (CAGCAGGTCGTATAGTTACCTCCTT; herein incorporated as SEQ ID NO.: 772), and probe=ISIS 341680 (AGTTTTAGGGCAGGGATTTTGCCCA; herein incorporated as SEQ ID NO.: 773)). An approximately 1.7-fold increase in expression levels of the let-7d pri-miRNA molecule was observed in T-24 cells treated with the oligomeric compound ISIS 327926 as detected by real-time RT-PCR.
Thus, treatment with uniform 2′-MOE oligomeric compounds targeting mature miRNAs appears to result in an induction of expression of the corresponding pri-miRNA molecule.
In one embodiment, the expression of mir-21 (noted to be expressed at high levels in HeLa cells) was assessed in cells treated with oligomeric compounds. Using the transfection methods previously described, HeLa cells were treated with 100 nM of the uniform 2′-MOE oligomeric compound ISIS Number 327917 (SEQ ID NO: 335) targeting the mir-21 mature miRNA. Total RNA was isolated by lysing cells in 1 mL TRIZOLTM (Invitrogen) using the manufacturer's recommended protocols. By Northern blot analysis of total RNA from HeLa cells treated with ISIS 327917, expression levels of the mir-21 mature miRNA were observed to be reduced to 50% of those of untreated control cells. Furthermore, expression levels of the mir-21 pri-miRNA were found to increase in these HeLa cells treated with the oligomeric compound ISIS 327917. Real-time RT-PCR analysis was also performed on HeLa cells treated with ISIS 327917 using a primer/probe set specific for the mir-21 pri-miRNA molecule [forward primer=ISIS 339332 (GCTGTACCACCTTGTCGGGT; herein incorporated as SEQ ID NO.: 911), reverse primer=ISIS 339333 (TCGACTGGTGTTGCCATGA; herein incorporated as SEQ ID NO.: 912), and probe=ISIS 339334 (CTTATCAGACTGATGTTGACTGTTGAAT; herein incorporated as SEQ ID NO.: 913)]. Total RNA was quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for the target were normalized to 5.8S rRNA, and values were expressed relative to an untreated control (UTC). ISIS Number 327917 was observed to stimulate an approximately 2-fold increase in levels of the mir-21 pri-miRNA molecule as detected by real-time RT-PCR.
Thus, it is believed that, in addition to binding the mir-21 mature miRNA and interfering with the RISC-mediated binding of mir-21 to its mRNA target, the oligomeric compound, ISIS 327917, binds to the mir-21 pri-miRNA and/or pre-miRNA molecules and interferes with their processing into the mature mir-21 miRNA, inhibiting expression of the mature mir-21 miRNA in HeLa cells, possibly by interfering with the activity of RNase III-like enzymes such as human Dicer or Drosha. The resultant decrease in levels of mature mir-21 miRNA may trigger a feedback mechanism leading to an upregulation of production of the mir-21 pri-miRNA molecule. Treatment with this oligomeric compound could also stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-21 pri-miRNA or pre-miRNA molecules.
In accordance with the present invention, a nested series of uniform 2′-MOE oligomeric compounds were designed and synthesized to target the entire length of the mir-21 pri-miRNA molecule. The region encompassing the mir-21 primary transcript (nucleotides 16571584 to Ser. No. 16/571,864 of GenBank Accession number NT—010783.14; CTGGGTTTTTTTGGTTTGTTTTTGTTTTTGTTTTTTTATCAAATCCTGCCTGACTGTCTGCTT GTTTTGCCTACCATCGTGACATCTCCATGGCTGTACCACCTTGTCGGGTAGCTTATCAGAC TGATGTTGACTGTTGAATCTCATGGCAACACCAGTCGATGGGCTGTCTGACATTTTGGTA TCTTTCATCTGACCATCCATATCCAATGTTCTCATTTAAACATTACCCAGCATCATTGTTT ATAATCAGAAACTCTGGTCCTTCTGTCTGGTGGCAC) is incorporated herein as SEQ ID NO: 914. Each compound is 20 nucleotides in length, composed of 2′-methoxyethoxy (2′-MOE) nucleotides and phosphorothioate (P═S) internucleoside linkages throughout the compound. All cytidine residues are 5-methylcytidines. The compounds are shown in Table 48. The compounds can be analyzed for their effect on mature miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or the function of targets downstream of miRNAs.







TABLE 48







Uniform 2′-MOE PS Compounds targeting the mir-21 pri-miRNA











ISIS
SEQ ID




Number
NO
Sequence






358765
915
ACAAGCAGACAGTCAGGCAG



358766
916
GGTAGGCAAAACAAGCAGAC



358767
917
GGAGATGTCACGATGGTAGG



358768
918
AGGTGGTACAGCCATGGAGA



358769
919
GATAAGCTACCCGACAAGGT



358770
920
AGTCTGATAAGCTACCCGAC



358771
921
CAACAGTCAACATCAGTCTG



358772
922
GAGATTCAACAGTCAACATC



358773
923
CTGGTGTTGCCATGAGATTC



358774
924
CATCGACTGGTGTTGCCATG



358775
925
ACAGCCCATCGACTGGTGTT



358776
926
TGTCAGACAGCCCATCGACT



358777
927
CCAAAATGTCAGACAGCCCA



358778
928
GATACCAAAATGTCAGACAG



358779
929
GGTCAGATGAAAGATACCAA



358780
930
AACATTGGATATGGATGGTC



358781
931
TAATGTTTAAATGAGAACAT



358782
932
AACAATGATGCTGGGTAATG



358783
933
GAGTTTCTGATTATAAACAA



358784
934
CGACAAGGTGGTACAGCCAT



358785
935
GAAAGATACCAAAATGTCAG









Using the real-time RT-PCR methods, the expression levels of the mir-21 pri-miRNA were compared in HeLa cells treated with this nested series of uniform 2′-MOE oligomeric compounds, targeting and spanning the entire length of the mir-21 pri-miRNA. ISIS Number 358768 (SEQ ID NO: 918) targets a region flanking the predicted 5′ Drosha cleavage site in the mir-21 pri-miRNA. ISIS Number 358777 (SEQ ID NO: 927) targets a region spanning the 3′ Drosha cleavage site in the mir-21 pri-miRNA. ISIS 358779 (SEQ ID NO: 929) targets a region flanking the predicted 3′ Drosha cleavage site in the mir-21 pri-miRNA. Additionally, oligomeric compounds ISIS 327917 (SEQ ID NO: 335), a uniform 2′-MOE compound targeting the mature mir-21 miRNA, and ISIS 345382 (TCAACATCAGTCTGATAAGCTA; SEQ ID NO: 335), a 5-10-7 phosphorothioate 2′-MOE gapmer targeting mir-21, were tested for comparison. Oligomeric compound ISIS 327863 (ACGCTAGCCTAATAGCGAGG; herein incorporated as SEQ ID NO: 936), a phosphorothioate 5-10-5 2′-MOE gapmer, was used as scrambled control. Using the transfection methods previously described, HeLa cells were treated with 100 nM of each of these oligomeric compounds. Total RNA was isolated by lysing cells in 1 mL TRIZOLTM (Invitrogen) using the manufacturer's recommended protocols. real-time RT-PCR analysis was performed using the primer/probe set specific for the mir-21 pri-miRNA molecule [forward primer=ISIS 339332 (SEQ ID NO.: 911), reverse primer=ISIS 339333 (SEQ ID NO.: 912), and probe=ISIS 339334 (SEQ ID NO.: 913)]. Total RNA was quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.), expression levels observed for each target were normalized to 5.8S rRNA, and values were expressed relative to the untreated control (UTC). Effects on expression of the mir-21 pri-miRNA molecule resulting from treatment of HeLa cells with these uniform 2′-MOE oligomeric compounds is expressed as a percentage of RNA levels in untreated control cells. Results of these experiments are shown in Table 49 below:







TABLE 49







Effects of oligomeric compounds on mir-21 pri-miRNA expression











SEQ




ISIS #
ID NO:
target
% UTC





UTC
N/A
N/A
100


327863
936
N/A
107


gapmer control





327917
335
mature mir-21
249


uniform 2′-MOE





345382
335
mature mir-21
119


5-10-7 2′-MOE





gapmer





358765
915
mir-21 pri-miRNA
133


358766
916
mir-21 pri-miRNA
142


358767
917
mir-21 pri-miRNA
248


358768
918
flanking 5′ Drosha cleavage site
987




of mir-21 pri-miRNA



358769
919
mir-21 pri-miRNA
265


358770
920
mir-21 pri-miRNA
250


358771
921
mir-21 pri-miRNA
181


358772
922
mir-21 pri-miRNA
245


358773
923
mir-21 pri-miRNA
148


358774
924
mir-21 pri-miRNA
104


358775
925
mir-21 pri-miRNA
222


358776
926
mir-21 pri-miRNA
367


358777
927
spanning 3′ Drosha cleavage site
536




of mir-21 pri-miRNA



358778
928
mir-21 pri-miRNA
503


358779
929
flanking 3′ Drosha cleavage site
646




of mir-21 pri-miRNA



358780
930
mir-21 pri-miRNA
269


358781
931
mir-21 pri-miRNA
122


358782
932
mir-21 pri-miRNA
155


358783
933
mir-21 pri-miRNA
133


358784
934
mir-21 pri-miRNA
358


358785
935
mir-21 pri-miRNA
257









From these data, it was observed that the uniform 2′-MOE oligomeric compounds ISIS Numbers 327917, 358767, 358768, 358769, 358770, 358772, 358775, 358776, 358777, 358778, 358779, 358780, 358784 and 358785 stimulate an increase in levels of the mir-21 pri-miRNA molecule as detected by real-time RT-PCR. Notably, oligomeric compounds ISIS Numbers 358768 and 358779 which target the regions flanking the predicted 5′ and 3′ Drosha cleavage sites, respectively, and ISIS Number 358777, which targets a region spanning the 3′ Drosha cleavage site in the mir-21 pri-miRNA were observed to stimulate the greatest increases in expression of the mir-21 pri-miRNA. Furthermore, treatment of HeLa cells with increasing concentrations (25, 50, 100, and 200 nM) of ISIS Numbers 358768, 358779, and 327917 was observed to result in a dose-responsive induction of mir-21 pri-miRNA levels. Thus, it is believed that these oligomeric compounds bind to the mir-21 pri-miRNA and/or pre-miRNA molecules and interfere with their processing into the mature mir-21 miRNA, possibly by interfering with the activity of RNase III-like enzymes such as human Dicer and/or Drosha. The resultant decrease in levels of the processed mature mir-21 miRNA may trigger a feedback mechanism leading to an upregulation of production of the mir-21 pri-miRNA molecule. Not mutually exclusive with the processing interference and the feedback mechanisms is the possibility that treatment with oligomeric compounds could stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies the mir-21 pri-miRNA or pre-miRNA molecules. It is understood that such oligomeric compound-triggered mechanisms may be operating not only upon regulation of mir-21 production and processing, but may also be found to regulate the production and processing of other miRNAs or target nucleic acids.
In one embodiment, the oligomeric compounds ISIS Number 327917 (SEQ ID NO: 335), the phosphorothioate uniform 2′-MOE targeting mature mir-21; ISIS Number 358768 (SEQ ID NO: 918), the uniform 2′-MOE targeting the mir-21 pri-miRNA which stimulated the largest increase in pri-miRNA expression levels by real time quantitative RT-PCR; and ISIS Number 345382 (SEQ ID NO: 335), the 5-10-7 phosphorothioate 2′-MOE gapmer targeting mature mir-21 were selected for dose response studies in HeLa cells using the luciferase reporter system described in Example 27. ISIS Number 342683 (SEQ ID NO: 790), representing the scrambled nucleotide sequence of an unrelated PTP1B antisense oligonucleotide, was used as a negative control. HeLa cells expressing the pGL3-mir-21 sensor plasmid (described in Example 27) were treated with 1.9, 5.5, 16.7, and 50 nM of these oligomeric compounds, to assess the ability of oligomeric compounds to interfere with endogenous mir-21-mediated silencing of the pGL3-mir-21 sensor plasmid. The data are presented in Table 50 as percent untreated control (luciferase plasmid only, not treated with oligomeric compound) luciferase expression, normalized to pRL-CMV levels.







TABLE 50







Effects of oligomeric compounds on


mir-21 miRNA-mediated inhibition of luciferase expression











% UTC




Dose of oligomeric compound













Treatment
1.9 nM
5.5 nM
16.7 nM
50 nM

















342683
127
171
104
108



negative control







327917
522
1293
2470
4534



358768
103
163
146
118



345382
101
135
117
95










From these data, it was observed that, at all doses, treatment of HeLa cells with ISIS Number 327917, the uniform 2′-MOE oligomeric compound targeting the mature mir-21 miRNA, de-repressed the expression of the luciferase reporter, in a dose-dependent fashion. Thus, ISIS 327917 reversed the silencing effect of the endogenous mir-21 miRNA, possibly by inhibiting the binding of mir-21 to its target site encoded by the pGL3-mir-21 sensor plasmid.
Example 29
Diseases Associated with miRNA-Containing Loci
Using the public databases Online Mendelian Inheritance in Man (OMIM) (accessible through the Internet at, for example, ftp.ncbi.nih.gov/repository/OMIM/) and LocusLink (accessible at, for example, ftp.ncbi.nlm.nih.gov/refseq/LocusLink/), a bioinformatic analysis was performed which allowed the prediction of miRNAs associated with several human diseases. First, miRNAs encoded within genes having LocusLink identification numbers were identified, and these were compared to tables (for example, “mim2loc,” which connects LocusLink identification numbers with OMIM identification numbers, as well as “genemap,” “genemap.key,” “mim-title,” and “morbidmap” tables) for the construction of a new database called “db1.mdb” linking miRNAs to LocusLink and OMIM identification numbers and linking these to human diseases.
It was observed that, beginning with 95 pri-miRNAs, a subset of 49 had OMIM identification numbers, 48 of which were linked to OMIM names. Six of these miRNAs were associated with specific diseased patients (some in each category were duplicates). Thus, the majority of miRNAs with OMIM identification numbers are not directly linked to observed diseases, but are likely to be important in pathways (such as cholesterol homeostasis) associated with diseases. Tables 51 and 52 summarize information retrieved from these studies.







TABLE 51







miRNA genes associated with specific diseases









OMIM




ID:
locus containing miRNA
Disease association:





120150
collagen, type I, alpha 1/
Osteogenesis imperfecta,



hypothetical miRNA-144
type I, 166200


114131
calcitonin receptor containing
Osteoporosis,



hypothetical miRNA 30
postmenopausal




susceptibility, 166710


605317
forkhead box P2/
Speech-language disorder-



hypothetical miRNA 169
1, 602081


600700
LIM domain-containing preferred
Lipoma; Leukemia, myeloid



translocation partner in lipoma




containing miR-28



160710
myosin, heavy polypeptide 6,
Cardiomyopathy, familial



cardiac muscle, alpha
hypertrophic, 192600



(cardiomyopathy, hypertrophic 1)




containing miR-208



606157
hypothetical protein FLJ11729
Neurodegeneration,



containing mir-103-2
pantothenate kinase-




associated, 234200









The previous table shows miRNAs associated with an OMIM record that were also associated with diseased patients.
The following table, Table 52, describes diseases or disease-related phenotypes found to be associated with genetic loci associated with a miRNA.







TABLE 52







miRNAs associated with disease phenotypes









OMIM




ID:
Locus containing miRNA
Disease association:





114131
calcitonin receptor containing
Osteoporosis, 



hypothetical miRNA-30
postmenopausal,




susceptibility, 166710


120150
collagen, type I, alpha 1/
Osteogenesis imperfecta,



hypothetical miRNA-144
type I, 166200


138247
glutamate receptor, ionotropic,
cerebellar long-term



AMPA 2/hypothetical 
depression



miRNA-171



160710
myosin, heavy polypeptide 6,
Cardiomyopathy, familial



cardiac muscle, alpha
hypertrophic, 192600



(cardiomyopathy, hypertrophic 1)




containing miR-208



184756
sterol regulatory element-binding
Emery-Dreifuss muscular



protein-1/mir-33b
dystrophy, 310300; dilated




cardiomyopathy (CMD1A),




115200; familial partial




lipodystrophy (FPLD), 




151660


300093
gamma-aminobutyric acid
early-onset parkinsonism, or



(GABA) A receptor, epsilon
Waisman syndrome, 311510;




and MRX3 X-linked mental




retardation, 309541


305660
gamma-aminobutyric acid
manic depressive illness,



(GABA) A receptor, alpha 3
colorblindness, and G6PD



containing miR-105 (Mourelatos)




and miR-105-2



305915
glutamate receptor, ionotrophic,
complex bipolar disorder;



AMPA 3/hypothetical
drug addiction



miRNA-033



600150
potassium large conductance
cardiovascular disease



calcium-activated channel,




subfamily M, alpha member 1




containing hypothetical 




miRNA-172



600395
glypican 1 containing miR-149
angiogenesis


600481
Sterol regulatory element binding
LDL and cholesterol



transcription factor 2 containing
homeostasis



mir-33a



600592
Minichromosome maintenance
increased chromosomal loss,



deficient (S. cerevisiae) 7
DNA replication and



containing miR-93 (Mourelatos)
recombination



and miR-25 and miR-94



600700
LIM domain-containing preferred
Lipoma; Leukemia, myeloid



translocation partner in lipoma




containing miR-28



600758
Focal adhesion kinase, p125/
oncogenesis



mir-151



601009
tight junction protein 1 (zona
peptic ulcer disease and



occludens 1)/hypothetical 
gastric carcinoma



miRNA-183



601029
mesoderm specific transcript
intrauterine and postnatal



(mouse) homolog containing mir-
growth retardation



240* (Kosik)



601698
protein tyrosine phosphatase,
insulin-dependent diabetes



receptor type, N polypeptide 2
mellitus (IDDM)



containing mir-153-2



601773
protein tyrosine phosphatase,
insulin-dependent diabetes



receptor type, N containing mir-
mellitus (IDDM), 222100



153-1



603576
melastatin 1 containing mir-211
metastatic human melanoma


603634
ribosomal protein L5/
colorectal cancers



hypothetical miRNA 168-2



603745
slit (Drosophila) homolog 3
congenital diaphragmatic



containing mir-218-2
hernia


603746
slit (Drosophila) homolog 2
retinal ganglion cell axon



containing mir-218-1
guidance


603803
dachshund (Drosophila) homolog
cell proliferation during



containing hypothetical
mammalian retinogenesis and



miRNA-083
pituitary development


605317
forkhead box P2/hypothetical
autism & speech-language



miRNA 169
disorder-1, 602081


605547
follistatin-like 1 containing
systemic rheumatic diseases



mir-198



605575
SMC4 (structural maintenance of
cell proliferation



chromosomes 4, yeast)-like 1




containing mir-16-3 and mir-15b



605766
deleted in lymphocytic leukemia,
B-cell chronic lymphocytic



2 containing mir-16-1 and mir-
leukemia



15a-1



606157
hypothetical protein FLJ11729
Neurodegeneration,



containing mir-103-2
pantothenate kinase-




associated, 234200 (3);


606160
pantothenate kinase containing
pantothenate kinase-



mir-107
associated neurodegeneration


606161
hypothetical protein FLJ12899
pantothenate kinase-



containing mir-103-1
associated neurodegeneration









From these data, it was observed that several miRNAs are predicted to be associated with human disease states. For example, several studies of autistic disorder have demonstrated linkage to a similar region of 7q (the AUTS1 locus), leading to the proposal that a single genetic factor on 7q31 contributes to both autism and language disorders, and it has been reported that the FOXP2 gene, located on human 7q31, encoding a transcription factor containing a polyglutamine tract and a forkhead domain, is mutated in a severe monogenic form of speech and language impairment, segregating within a single large pedigree, and is also disrupted by a translocation. In one recent study, association and mutation screening analysis of the FOXP2 gene was performed to assess the impact of this gene on complex language impairments and autism, and it was concluded that coding-region variants in FOXP2 do not underlie the AUTS1 linkage and that the gene is unlikely to play a role in autism or more common forms of language impairment (Newbury, et al., Am. J. Hum. Genet. 2002, 70, 1318-27). However, hypothetical mir-169 is also encoded by this same genetic locus, and it is possible that mutations affecting the hypothetical mir-169 miRNA could underlie the AUTS1 linkage and play a role in language impairment. To this end, oligomeric compounds targeting or mimicking the mir-169 miRNA may prove useful in the study, diagnosis, treatment or amelioration of this disease.
Example 30
Effects of Oligomeric Compounds Targeting miRNAs on Insulin Signaling and Hallmark Gene Expression in HepG2 Cells
Additional oligomeric compounds were screened in the assays described in Example 18. As stated above, insulin inhibits the expression of IGFBP-1, PEPCK-c and follistatin mRNAs.
Protocols for treatment of HepG2 cells and transfection of oligomeric compounds are as described in Example 18. Also as described in Example 18, forty-four hours post-transfection, the cells in the transfected wells were treated with either no insulin (“basal” Experiment 3 (below), for identification of insulin-mimetic compounds) or with 1 nM insulin (“insulin treated” Experiment 4 (below), for identification of insulin sensitizers) for four hours. At the same time, in both plates, cells in some of the un-transfected control wells are treated with 100 nM insulin to determine maximal insulin response. At the end of the insulin or no-insulin treatment (forty-eight hours post-transfection), total RNA is isolated from both the basal and insulin treated (1 nM) 96-well plates, and the amount of total RNA from each sample is determined using a Ribogreen assay (Molecular Probes, Eugene, Oreg.). Real-time PCR is performed on all the total RNA samples using primer/probe sets for three insulin responsive genes: PEPCK-c, IGFBP-1 and follistatin. Expression levels for each gene are normalized to total RNA, and values±standard deviation are expressed relative to the transfectant only and negative control oligonucleotides. The compound ISIS Number 186515 (AGGTAGCTTTGATTATGTAA; SEQ ID NO: 939) is targeted to IGFBP-1 and is a phosphorothioate 5-10-5 MOE gapmer where all cytosines are 5-methylcytosines, as is used as a transfection control. The oligomeric compound ISIS Number 340341 (TAGCTTATCAGACTGATGTTGA; SEQ ID NO: 236) is a uniform 2′-MOE phosphorothioate compound targeted to mir-104 (Mourelatos), ISIS 340362 (GACTGTTGAATCTCATGGCA; SEQ ID NO: 937) is a 5-10-5 gapmer compound also targeted to mir-104 (Mourelatos), and ISIS Number 341813 (AGACACGTGCACTGTAGA; SEQ ID NO: 938) is a uniform 2′-MOE phosphorothioate compound targeted to mir-139. Results of these experiments are shown in Tables 53 and 54.







TABLE 53







Experiment 3: Effects of oligomeric compounds targeting


miRNAs on insulin-repressed gene expression in HepG2 cells













SEQ






ISIS
ID

IGFBP-1
PEPCK-c
Follistatin


NO:
NO
Pri-miRNA
(% UTC)
(% UTC)
(% UTC)















UTC
N/A
N/A
100
100
100


29848
737
N/A
104
100
90


n-mer







186515
939
IGFBP-1
193
70
67


328384
493
hypothetical
139
142
110




miRNA-039





328677
586
hypothetical
208
145
130




miRNA-120





328685
594
mir-219
157
219
100


328691
600
mir-145
105
108
93


328759
668
mir-216
356
98
266


328761
670
hypothetical
118
48
91




miRNA-138





328765
674
mir-215
88
93
87


328773
682
mir-15a-2
148
138
131


328779
688
hypothetical mir-177
135
123
109


340341
236
mir-104 (Mourelatos)
110
129
94


340362
937
mir-104 (Mourelatos)
157
168
123


341813
938
mir-139
137
121
100









Under “basal” conditions (without insulin), treatments of HepG2 cells with oligomeric compounds of the present invention resulting in decreased mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that the oligomeric compounds have an insulin mimetic effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin repression of mRNA expression of these genes.
From these data, it is evident that the oligomeric compound ISIS Number 328761 targeting hypothetical mir-138, for example, results in a 52% decrease in PEPCK-c mRNA, a marker widely considered to be insulin-responsive. Thus, this oligomeric compound may be useful as a pharmaceutical agent with insulin mimetic properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.







TABLE 54







Experiment 4: Effects of oligomeric compounds targeting


miRNAs on insulin-sensitization of gene expression in HepG2 cells













SEQ






ISIS
ID

IGFBP-1
PEPCK-c
Follistatin


NO:
NO
Pri-miRNA
(% UTC)
(% UTC)
(% UTC)















UTC
N/A
N/A
100
100
100


(1 nm







insulin)







29848
737
N/A
92
90
95


n-mer







186515
939
IGFBP-1
105
40
39


328384
493
hypothetical
102
114
121




miRNA-039





328677
586
hypothetical
159
117
118




miRNA-120





328685
594
mir-219
143
184
157


328691
600
mir-145
101
97
104


328759
668
mir-216
212
92
224


328761
670
hypothetical
93
55
98




miRNA-138





328765
674
mir-215
94
73
97


328773
682
mir-15a-2
136
93
148


328779
688
hypothetical mir-177
128
78
119


340341
236
mir-104 (Mourelatos)
113
115
120


340362
937
mir-104 (Mourelatos)
129
104
119


341813
938
mir-139
117
88
102









In HepG2 cells treated with 1 nM insulin, treatments with oligomeric compounds of the present invention resulting in a decrease in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds have an insulin sensitization effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin response of repression of mRNA expression of these genes.
From these data, it is evident that the oligomeric compounds, ISIS Number 328761 targeting hypothetical mir-138 and ISIS Number 328765 targeting mir-215, for example, were observed to result in a 45% and a 27% reduction, respectively, of PEPCK-c mRNA expression, widely considered to be a marker of insulin-responsiveness. Furthermore, mRNA levels of the IGFBP-1 and follistatin genes were also reduced. Thus, these oligomeric compounds may be useful as pharmaceutical agents with insulin-sensitizing properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.
Example 31
Adipocyte Assay of Oligomeric Compounds
The effect of several oligomeric compounds of the present invention targeting miRNA target nucleic acids on the expression of markers of cellular differentiation was examined in differentiating adipocytes.
As described in Example 13, some genes known to be upregulated during adipocyte differentiation include HSL, aP2, Glut4 and PPARγ. These genes play important roles in the uptake of glucose and the metabolism and utilization of fats. An increase in triglyceride content is another well-established marker for adipocyte differentiation.
For assaying adipocyte differentiation, expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulation were measured as previously described in adipocytes transfected with uniform 2′-MOE or chimeric gapmer phosphorothioate (PS) oligomeric compounds. Triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed as a percentage of untreated control (UTC) levels. Results are shown in Table 55.







TABLE 55







Effects of oligomeric compounds targeting miRNAs


on expression of adipocyte differentiation markers













ISIS
SEQ




PPAR


Number
ID NO
TG
HSL
AP2
Glut4
gamma
















UTC
N/A
100
100
100
100
100


ISIS-29848
737
89
84
89
96
100


n-mer








327877
295
109
82
77
119
85


327888
306
132
134
102
84
103


327904
322
56
42
65
40
54


327909
327
132
130
88
132
96


327927
345
125
120
114
120
108


327928
346
45
52
77
39
57


327933
351
127
132
82
127
100


327937
355
81
77
76
63
92


327951
369
76
100
91
81
84


327953
371
94
94
92
112
90


327956
374
80
90
102
69
91


327960
378
47
52
52
34
76


328093
395
59
89
97
73
99


328112
414
92
89
73
97
79


328114
416
110
134
123
116
106


328132
434
120
89
81
67
94


328340
449
76
130
85
112
110


328362
471
73
83
59
80
78


328400
509
60
40
34
18
67


328417
526
83
98
87
68
94


328434
543
91
96
85
83
79


328651
560
93
109
84
78
106


328677
586
34
68
61
44
89


328685
594
50
100
73
69
91


328691
600
130
156
166
144
105


328759
668
87
105
108
66
95









For these data, values for triglyceride accumulation above 100 are considered to indicate that the compound has the ability to stimulate triglyceride accumulation, whereas values at or below 100 indicate that the compound inhibits triglyceride accumulation. With respect to leptin secretion, values above 100 are considered to indicate that the compound has the ability to stimulate secretion of the leptin hormone, and values at or below 100 indicate that the compound has the ability to inhibit secretion of leptin. With respect to the four adipocyte differentiation hallmark genes, values above 100 are considered to indicate induction of cell differentiation, whereas values at or below 100 indicate that the compound inhibits differentiation.
Several compounds were found to have remarkable effects. For example, the oligomeric compounds ISIS Number 327904 (SEQ ID NO: 322), targeted to mir-181a-1, ISIS Number 327928 (SEQ ID NO: 346), targeted to mir-29a, ISIS Number 327960 (SEQ ID NO: 378), targeted to mir-215, ISIS Number 328400 (SEQ ID NO: 509), targeted to mir-196-2, and ISIS Number 328677 (SEQ ID NO: 586), targeted to hypothetical miRNA-120 were shown to reduce the expression levels of all five markers of adipocyte differentiation, indicating that these oligomeric compounds have the ability to block adipocyte differentiation. Therefore, these oligomeric compounds may be useful as therapeutic agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as in the maintenance of the pluripotent phenotype of stem or precursor cells.
The oligomeric compounds ISIS Number 328691 (SEQ ID NO: 600) targeted to mir-145, ISIS Number 328114 (SEQ ID NO: 416) targeted to hypothetical miRNA-138, and ISIS Number 327927 (SEQ ID NO: 345) targeted to mir-15b are examples of compounds which exhibit an increase in all five markers of adipocyte differentiation. Additionally, the oligomeric compound ISIS Number 327909 (SEQ ID NO: 327) targeted to mir-196-2 exhibited an increase in three of the five markers of adipocyte differentiation. Thus, these oligomeric compounds may be useful as pharmaceutical agents in the treatment of diseases in which the induction of adipocyte differentiation is desirable, such as anorexia, or for conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which regeneration of neural tissue would be beneficial. Furthermore, these oligomeric compounds may be useful in the treatment, attenuation or prevention of diseases in which it is desirable to induce cellular differentiation and/or quiescence, for example in the treatment of hyperproliferative disorders such as cancer.
Example 32
Effects of Oligomeric Compounds on Endothelial Tube Formation Assay
Angiogenesis is the growth of new blood vessels (veins and arteries) by endothelial cells. This process is important in the development of a number of human diseases, and is believed to be particularly important in regulating the growth of solid tumors. Without new vessel formation it is believed that tumors will not grow beyond a few millimeters in size. In addition to their use as anti-cancer agents, inhibitors of angiogenesis have potential for the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis (Carmeliet and Jain, Nature, 2000, 407, 249-257; Freedman and Isner, J. Mol. Cell. Cardiol., 2001, 33, 379-393; Jackson et al., Faseb J., 1997, 11, 457-465; Saaristo et al., Oncogene, 2000, 19, 6122-6129; Weber and De Bandt, Joint Bone Spine, 2000, 67, 366-383; Yoshida et al., Histol. Histopathol., 1999, 14, 1287-1294). Endothelial tube formation assay as a measure of angiogenesis:
Angiogenesis is stimulated by numerous factors that promote interaction of endothelial cells with each other and with extracellular matrix molecules, resulting in the formation of capillary tubes. This morphogenic process is necessary for the delivery of oxygen to nearby tissues and plays an essential role in embryonic development, wound healing, and tumor growth (Carmeliet and Jain, Nature, 2000, 407, 249-257). Moreover, this process can be reproduced in a tissue culture assay that evaluates the formation of tube-like structures by endothelial cells. There are several different variations of the assay that use different matrices, such as collagen I (Kanayasu et al., Lipids, 1991, 26, 271-276), Matrigel (Yamagishi et al., J. Biol. Chem., 1997, 272, 8723-8730) and fibrin (Bach et al., Exp. Cell Res., 1998, 238, 324-334), as growth substrates for the cells. In this assay, human umbilical vein endothelial cells (HuVECs) are plated on a matrix derived from the Engelbreth-Holm-Swarm mouse tumor, which is very similar to Matrigel (Kleinman et al., Biochemistry, 1986, 25, 312-318; Madri and Pratt, J. Histochem. Cytochem., 1986, 34, 85-91). Untreated HuVECs form tube-like structures when grown on this substrate. Loss of tube formation in vitro has been correlated with the inhibition of angiogenesis in vivo (Carmeliet and Jain, Nature, 2000, 407, 249-257; Zhang et al., Cancer Res., 2002, 62, 2034-2042), which supports the use of in vitro tube formation as an endpoint for angiogenesis.
In one embodiment, primary human umbilical vein endothelial cells (HuVECs) were used to measure the effects of oligomeric compounds targeted to miRNAs on tube formation activity. HuVECs were routinely cultured in EBM (Clonetics Corporation, Walkersville, Md.) supplemented with SingleQuots supplements (Clonetics Corporation, Walkersville, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence and were maintained for up to 15 passages. HuVECs are plated at 3000 cells/well in 96-well plates. One day later, cells are transfected with oligomeric compounds. The tube formation assay is performed using an in vitro Angiogenesis Assay Kit (Chemicon International, Temecula, Calif.).
A scrambled control compound, ISIS 29848 (NNNNNNNNNNNNNNNNNNNN; where N is A,T,C or G; herein incorporated as SEQ ID NO: 737) served as a negative control. ISIS 196103 (AGCCCATTGCTGGACATGCA; incorporated herein as SEQ ID NO: 940) targets integrin beta 3 and was used as a positive control to inhibit endothelial tube formation. ISIS 29248 and ISIS 196103 are chimeric 5-10-5 2′-MOE gapmer oligonucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotides. All cytidine residues are 5-methylcytidines. ISIS 342672 (SEQ ID NO: 789) contains 13 mismatches with respect to the mature mir-143 miRNA, and was also used as a negative control. ISIS 342672 is a uniform 2′-MOE phosphorothioate oligomeric compound 22 nucleotides in length. All cytidine residues are 5-methylcytidines.
Oligomeric compound was mixed with LIPOFECTINTM (Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEMTM (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve a final concentration of 75 nM of oligomeric compound and 2.25 μg/mL LIPOFECTINTM. Before adding to cells, the oligomeric compound, LIPOFECTINTM and OPTI-MEMTM were mixed thoroughly and incubated for 0.5 hrs. Untreated control cells received LIPOFECTINTM only. The medium was removed from the plates and the plates were tapped on sterile gauze. Each well was washed in 150 μl of phosphate-buffered saline. The wash buffer in each well was replaced with 100 μL of the oligomeric compound/OPTI-MEMTM/LIPOFECTINTM cocktail. Compounds targeted to miRNAs were tested in triplicate, and ISIS 29848 was tested in up to six replicates. The plates were incubated for 4 hours at 37° C., after which the medium was removed and the plate was tapped on sterile gauze. 100 μl of full growth medium was added to each well. Fifty hours after transfection, cells are transferred to 96-well plates coated with ECMa-trixTM (Chemicon Inter-national). Under these conditions, untreated HuVECs form tube-like structures. After an overnight incubation at 37° C., treated and untreated cells are inspected by light microscopy. Individual wells are assigned discrete scores from 1 to 5 depending on the extent of tube formation. A score of 1 refers to a well with no tube formation while a score of 5 is given to wells where all cells are forming an extensive tubular network. Results are expressed as a percentage of the level of the tube formation observed in cultures not treated with oligonucleotide, and are shown in Tables 56-59.







TABLE 56







Effect of compounds targeting


miRNAs on Tube Formation Activity in HuVECs











SEQ ID

% Activity Relative


ISIS NO:
NO:
Pri-miRNA
to UTC













UTC
N/A
N/A
100


196103
940
Integrin beta 3
35.7


positive control





342672
789
N/A
46.4


negative control





327873
291
mir-140
100.0


327875
293
mir-34
71.4


327876
294
mir-29b-1
50.0


327877
295
mir-16-3
78.6


327878
296
mir-203
57.1


327879
297
mir-7-1
71.4


327880
298
mir-10b
57.1


327881
299
mir-128a
50.0


327882
300
mir-153-1
107.1


327883
301
mir-27b
92.9


327884
302
mir-96
78.6


327885
303
mir-17as/mir-91
50.0


327886
304
mir-123/mir-126as
42.9


327887
305
mir-132
57.1


327888
306
mir-108-1
100.0


327889
307
mir-23b
50.0


327890
308
let-7i
92.9


327891
309
mir-212
50.0


327892
310
mir-131-2/mir-9
57.1


327893
311
let-7b
100.0


327894
312
mir-1d
100.0


327895
313
mir-122a
100.0


327896
314
mir-22
64.3


327898
316
mir-142
100.0









From these data, it was observed that ISIS Number 327886 targeted to mir-123/mir126as suppressed tube formation, indicating that this compound may be useful as an angiogenesis inhibitor and/or anti-tumor agent, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.







TABLE 57







Effect of compounds targeting


miRNAs on Tube Formation Activity in HuVECs













SEQ ID

% Activity



ISIS NO:
NO:
Pri-miRNA
Relative to UTC
















UTC
N/A
N/A
100



196103
940
Integrin beta 3
24.1



positive control






342672
789
N/A
58.6



negative control






327899
317
mir-183
34.5



327900
318
mir-214
55.2



327901
319
mir-143
48.3



327902
320
mir-192-1
41.4



327903
321
let-7a-3
103.5



327904
322
mir-181a
89.7



327905
323
mir-205
48.3



327906
324
mir-103-1
69.0



327907
325
mir-26a
62.1



327908
326
mir-33a 
103.5



327909
327
mir-196-2
96.6



327910
328
mir-107
55.2



327911
329
mir-106
75.9



327913
331
mir-29c
69.0



327914
332
mir-130a
82.8



327915
333
mir-218-1
69.0



327916
334
mir-124a-2
96.6



327917
335
mir-21
82.8



327918
336
mir-144
96.6



327919
337
mir-221
103.5



327920
338
mir-222
41.4



327921
339
mir-30d
96.6



327922
340
mir-19b-2
89.7



327923
341
mir-128b
48.3










From these data, it was observed that ISIS Number 327899 targeted to mir-183, ISIS Number 327902 targeted to mir-192-1, and ISIS Number 327920 targeted to mir-222 suppressed tube formation, indicating that these compounds may be useful as an angiogenesis inhibitors and/or anti-tumor agents, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.







TABLE 58







Effect of compounds targeting


miRNAs on Tube Formation Activity in HuVECs











SEQ ID

% Activity Relative


ISIS NO:
NO:
Pri-miRNA
to UTC













UTC
N/A
N/A
100


196103
940
Integrin beta 3
29.6


positive control





342672
789
N/A
55.6


negative control





327924
342
mir-129-2
88.9


327925
343
mir-133b
44.4


327926
344
let-7d
96.3


327927
345
mir-15b
59.3


327928
346
mir-29a-1
37.0


327929
347
mir-199b
51.9


327930
348
let-7e
88.9


327931
349
let-7c
103.7


327932
350
mir-204
51.9


327933
351
mir-145
59.3


327934
352
mir-213/mir-181a
51.9


327935
353
mir-20
74.1


327936
354
mir-133a-1
51.9


327937
355
mir-138-2
88.9


327938
356
mir-98
96.3


327939
357
mir-125b-1
66.7


327940
358
mir-199a-2
59.3


327941
359
mir-181b
74.1


327942
360
mir-141
74.1


327943
361
mir-18
81.5


327944
362
mir-220
37.0


327945
363
mir-24-2
59.3


327946
364
mir-211
51.9


327947
365
mir-101-3
81.5









From these data, it was observed that ISIS Number 327925 targeted to mir-133b, ISIS Number 327928 targeted to mir-29a-1, and ISIS Number 327944 targeted to mir-220 suppressed tube formation, indicating that these compounds may be useful as an angiogenesis inhibitors and/or anti-tumor agents, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.







TABLE 59







Effect of compounds targeting


miRNAs on Tube Formation Activity in HuVECs











SEQ ID

% Activity Relative


ISIS Number
NO:
Pri-miRNA
to UTC













UTC
N/A
N/A
100


196103
940
Integrin beta 3
26.7


positive control





342672
789
N/A
60.0


negative control





327874
292
mir-30a
46.7


327897
315
mir-92-1
40.0


327901
319
mir-143
100.0


327948
366
mir-30b
33.3


327949
367
mir-10a
66.7


327950
368
mir-19a
73.3


327951
369
mir-15a-1
73.3


327952
370
mir-137
53.3


327953
371
mir-219
53.3


327954
372
mir-148b
53.3


327955
373
mir-130b
46.7


327956
374
mir-216
46.7


327957
375
mir-100-1
66.7


327958
376
mir-187
40.0


327959
377
mir-210
40.0


327960
378
mir-215
53.3


327961
379
mir-223
53.3


327962
380
mir-30c
53.3


327963
381
mir-26b
93.3


327964
382
mir-152
86.7


327965
383
mir-135-1
100.0


327966
384
mir-217
40.0


327967
385
let-7g
93.3


327968
386
mir-33b
93.3









From these data, it was observed that ISIS Number 327948 targeted to mir-30b, ISIS Number 327958 targeted to mir-187, ISIS Number 327959 targeted to mir-210, and ISIS Number 327966 targeted to mir-217 suppressed tube formation, indicating that these compounds may be useful as an angiogenesis inhibitors and/or anti-tumor agents, with potential therapeutic applications in the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis, psoriasis, as well as cancer.
Example 33
Effect of Oligomeric Compounds on miRNA Target Protein Expression
Several mRNA transcripts have been predicted to be regulated by miRNAs (Lewis et al., Cell, 2003, 115, 787-798). For example, the mRNAs encoded by six genes, 1) inwardly rectifying potassium channel Kir2.2 (GenBank Accession AB074970, SEQ ID NO: 872); 2) synaptotagmin III (GenBank Accession BC028379, SEQ ID NO: 873); 3) mitogen-activated protein kinase 7/extracellular signal-regulated kinase 5 (ERK5) (GenBank Accession NM—139032.1, SEQ ID NO: 861); 4) protein phosphatase 2 (formerly 2A), catalytic subunit, beta isoform (PPP2CB) (GenBank Accession NM—004156.1, SEQ ID NO: 814); 5) glyoxalase I (GenBank Accession NM—006708.1, SEQ ID NO: 821); and 6) LIM domain only 4 (LMO4) (GenBank Accession NM—006769.2, SEQ ID NO: 865), are believed to have mir-143 binding sites within their 3′-UTRs. The latter three genes encode mRNAs that were identified as potential targets of mir-143 by the RACE-PCR experiments described, supra. Thus, the mir-143 miRNA is predicted to regulate some or all of these genes.
When miRNAs have effects on the expression of downstream genes or proteins encoded by these genes, it is advantageous to measure the protein levels of those gene products, and to do this, western blot (immunoblot) analysis is employed Immunoblot analysis is carried out using standard methods. Briefly, preadipocytes and differentiating adipocytes were cultured as described previously, and differentiating adipocytes are sampled at several timepoints after stimulation of differentiation. Cells were treated with 250 nM oligomeric compounds and harvested 16-20 h after oligomeric compound treatment. Cells were washed, lysed in RIPA buffer with protease inhibitor cocktail (Roche Diagnostics Corporation, Indianapolis, Ind.), suspended in Laemmli buffer (20 ul/well), boiled for 5 minutes and loaded onto either an 8% SDS-PAGE or a 4-20% gradient SDS-PAGE gel. Gels are run for approximately 1.5 hours at 150 V, and transferred to PVDF membrane for western blotting. Appropriate primary antibody directed to a target is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Because expression levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein remain constant, an antibody recognizing the GAPDH protein (Abcam, Cambridge, Mass.) can be used in a re-probing of the membrane to verify equal protein loading. It is also understood that antisense oligomeric compounds specifically targeting and known to inhibit the expression of the mRNA and protein endproducts of the gene of interest can be used as controls in these experiments. Bands are visualized and quantitated using a PHOSPHORIMAGERTM (Molecular Dynamics, Sunnyvale Calif.) or the ChemiDocTM system (Bio-Rad Laboratories, Inc., Hercules, Calif.). Thus, the effects of treatment of many cell types (including, but not limited to, preadipocytes, differentiating adipocytes, HeLa, T-24 and A549 cells) with oligomeric compounds of the present invention on the levels of gene expression products can be assessed. It is understood that one of ordinary skill in the art can use immunoblot analysis to examine the expression of any protein predicted to be the downstream expression product of a target of a miRNA Similarly, using methods described above, real-time RT-PCR methods can also be used to examine the mRNA expression levels of any of these predicted targets of the mir-143 miRNA. More specifically, immunoblot analysis and/or real-time RT-PCR methods can be used to examine the effects of treatment with oligomeric compounds on the protein or mRNA levels, respectively, produced by the Kir2.2, synaptotagmin III, ERK5, PPP2CB, glyoxalase I, and/or LMO4 genes in a variety of cell types.
In one embodiment of the invention, immunoblot analysis was used to assess the effects of the oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319) targeting mir-143, on expression levels of the PPP2CB protein in differentiating adipocytes. It was observed that, upon treatment with ISIS 327901, PPP2CB protein levels were higher in differentiating adipocytes both 7- and 10-days post-differentiation than in pre-adipocytes or in untreated differentiating adipocytes from the same timepoints. Thus, mir-143 appears to negatively regulate the expression of the PPP2CB gene, presumably by inhibiting translation of the PPP2CB mRNA into protein, and upon treatment with the oligomeric compound ISIS 327901, this inhibition of PPP2CB protein expression was relieved.
In one embodiment of the invention, immunoblot analysis was used to assess the effects of the oligomeric compound, ISIS Number 327901 (SEQ ID NO: 319) targeting mir-143, on expression levels of the ERK5 protein in differentiating adipocytes. It was observed that, upon treatment of cells with ISIS 327901, ERK5 protein levels were approximately 2-2.5-fold higher in differentiating adipocytes both 7- and 10-days post-differentiation than in pre-adipocytes or in untreated differentiating adipocytes from the same timepoints. Thus, mir-143 appears to negatively regulate the expression of the ERK5 gene presumably by inhibiting translation of the ERK5 mRNA into protein, either directly (by mir-143 binding an ERK5 cis-regulatory sequence) or indirectly (by mir-143 regulating another target gene that regulates ERK5); upon treatment with the oligomeric compound ISIS 327901, this mir-143-dependent inhibition of ERK5 expression was relieved. It is known that ERK5 promotes cell growth and proliferation in response to tyrosine kinase signaling. In light of the involvement of mir-143 in adipocyte differentiation disclosed in several examples in the present invention, as well as the role of mir-143 in regulating ERK5, it is predicted that ERK5 and mir-143 are together involved regulating the balance between cellular proliferation and differentiation.
It is understood that the oligomeric compounds of the present invention, including miRNA mimics, can also be tested for their effects on the expression of the protein endproducts of targets of miRNAs. For example, an oligomeric compound such as a mir-143 mimic can be used to treat differentiating adipocytes, and is predicted to result in a reduction of Kir2.2, synaptotagmin III, ERK5, PPP2CB, glyoxalase I, and/or LMO4 protein expression levels.
The phosphatase and tensin homolog (mutated in multiple advanced cancers 1) (PTEN) tumor suppressor mRNA (GenBank Accession NM—000314, incorporated herein as SEQ ID NO: 941) has been predicted to be a potential target of the mir-19a miRNA (Lewis et al., Cell, 2003, 115, 787-798). Oligomeric compounds that target or mimic the mir-19a miRNA or mir-19a pri-miRNA can be used to treat cells and, using the methods described above, the effects of these oligomeric compounds on the expression of the PTEN protein and mRNA levels can be assessed. It is predicted that the mir-19a miRNA, or an oligomeric compound acting as a mir-19a mimic, would inhibit expression of the PTEN tumor suppressor mRNA and protein, and that treatment with oligomeric compounds targeting mir-19a would reverse this inhibition. It is also understood that other antisense oligomeric compounds specifically targeting and known to inhibit the expression of the mRNA and protein endproducts of the gene interest can be used as controls in these experiments.
Example 34
Additional Oligomeric Compounds Targeting miRNAs
In accordance with the present invention, oligomeric compounds were designed and synthesized to target or mimic one or more miRNA genes or gene products. Pri-miRNAs, pre-miRNAs and mature miRNAs represent target nucleic acids to which the oligomeric compounds of the present invention were designed and synthesized. Oligomeric compounds of the present invention can also be designed and synthesized to mimic the pri-miRNA, pre-miRNA or mature miRNA structure while incorporating certain chemical modifications that alter one or more properties of the mimic, thus creating a construct with superior properties as compared to the endogenous precursor or mature miRNA.
In accordance with the present invention, oligomeric compounds were designed to target or mimic one or more human, mouse, rat, or Drosophila pri-miRNAs, pre-miRNAs or mature miRNAs.
A list of human pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 60. “Pri-miRNA name” indicates the gene name for each of the pri-miRNAs. Also given in table 60 are the name and sequence of the mature miRNA derived from the pri-miRNA. Mature miRNA sequences from pri-miRNA precursors have been proposed by several groups; consequently, for a given pri-miRNA sequence, several miRNAs may be disclosed and given unique names, and thus a given pri-miRNA sequence may occur repeatedly in the table. The sequences are written in the 5′ to 3′ direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.







TABLE 60







Human pri-miRNA sequences and the corresponding mature miRNAs















SEQ



SEQ ID


ID


Pri-miRNA name
NO
Mature miRNA name
Mature miRNA sequence
NO














mir-27b
17
mir-27b
TTCACAGTGGCTAAGTTCTG
202


mir-27b
17
miR-27* (Michael
TTCACAGTGGCTAAGTTCTGC
1059




et al)




mir-23b
23
mir-23b
ATCACATTGCCAGGGATTACCAC
208


glutamate
36
hypothetical
TGTTATAGTATTCCACCTACC
1060


receptor,

miRNA-033




ionotrophic, AMPA






3/hypothetical






miRNA-033






LOC 114614
74
hypothetical
TGCTAATCGTGATAGGGGTTT
1061


containing miR-

miRNA-071




155/hypothetical






miRNA-071






LOC 114614
74
mir-155 (RFAM)
TTAATGCTAATCGTGATAGGGG
1062


containing miR-






155/hypothetical






miRNA-071






collagen, type I,
147
hypothetical
AGACATGTTCAGCTTTGTGGA
1063


alpha 1/

miRNA-144




hypothetical






miRNA-144






sterol regulatory
168
mir-33b
GTGCATTGCTGTTGCATTG
286


element-binding






protein-1/mir-33b






tight junction
186
hypothetical
AGCCTGTGGAGCTGCGCTTAC
1064


protein 1 (zona

miRNA-183




occludens 1)/






hypothetical






miRNA-183






mir-140
4
mir-140
AGTGGTTTTACCCTATGGTAG
192


mir-140
4
miR-140-as
TACCACAGGGTAGAACCACGGA
1065


mir-140
4
mir-239* (Kosik)
TACCACAGGGTAGAACCACGGACA
1066


mir-34
6
mir-34
TGGCAGTGTCTTAGCTGGTTGT
194


mir-34
6
miR-172 (RFAM-M. mu.)
TGGCAGTGTCTTAGCTGGTTGTT
1067


mir-203
10
mir-203
GTGAAATGTTTAGGACCACTAG
197


mir-203
10
miR-203 (RFAM-M. mu.)
TGAAATGTTTAGGACCACTAG
1068


mir-203
10
miR-203 (Tuschl)
TGAAATGTTTAGGACCACTAGA
1069


mir-7_1/mir-7_1*
11
mir-7_1*_Ruvkun
CAACAAATCACAGTCTGCCATA
1070


mir-7_1/mir-7_1*
11
mir-7
TGGAAGACTAGTGATTTTGTT
198


mir-10b
12
miR-10b (Tuschl)
CCCTGTAGAACCGAATTTGTGT
1071


mir-10b
12
mir-10b
TACCCTGTAGAACCGAATTTGT
199


mir-10b
12
miR-10b (Michael
TACCCTGTAGAACCGAATTTGTG
1072




et al)




mir-128a
13
mir-128 (Kosik)
TCACAGTGAACCGGTCTCTTT
1073


mir-128a
13
mir-128a
TCACAGTGAACCGGTCTCTTTT
200


mir-153_1
14
mir-153
TTGCATAGTCACAAAAGTGA
201


mir-153_2
15
mir-153
TTGCATAGTCACAAAAGTGA
201


hypothetical miR-
16
hypothetical
TATCAAACATATTCCTACAGT
1074


13/miR-190

miRNA-013




hypothetical miR-
16
miR-190
TGATATGTTTGATATATTAGGT
1075


13/miR-190






mir-123/mir-126
20
mir-123/mir-126as
CATTATTACTTTTGGTACGCG
205


mir-123/mir-126
20
mir-126
TCGTACCGTGAGTAATAATGC
1076


mir-132
21
miR-132 (RFAM-
TAACAGTCTACAGCCATGGTCG
1077




Human)




mir-132
21
mir-132
TAACAGTCTACAGCCATGGTCGC
206


mir-108_1
22
mir-108
ATAAGGATTTTTAGGGGCATT
207


let-7i
24
let-7i
TGAGGTAGTAGTTTGTGCT
209


let-7i
24
let-7i_Ruvkun
TGAGGTAGTAGTTTGTGCTGTT
1078


mir-212
25
mir-212
TAACAGTCTCCAGTCACGGCC
210


hypothetical miRNA
26
hypothetical
TGGGCAAGAGGACTTTTTAAT
1079


023

miRNA-023




mir-131_2/mir-9
27
mir-131
TAAAGCTAGATAACCGAAAGT
211


mir-131_2/mir-9
27
mir-131_Ruvkun
TAAAGCTAGATAACCGAAAGTA
1080


mir-131_2/mir-9
27
miR-9
TCTTTGGTTATCTAGCTGTATGA
1081


let-7b
28
let-7b
TGAGGTAGTAGGTTGTGTGGTT
212


let-7b
28
let-7b_Ruvkun
TGAGGTAGTAGGTTGTGTGGTTT
1082


mir-1d_1
29
miR-1 (RFAM)
TGGAATGTAAAGAAGTATGTA
1083


mir-1d_1
29
mir-1d
TGGAATGTAAAGAAGTATGTAT
213


mir-122a
30
miR-122a,b
TGGAGTGTGACAATGGTGTTTG
1084




(Tuschl)




mir-122a
30
mir-122a
TGGAGTGTGACAATGGTGTTTGT
214


mir-22
31
mir-22
AAGCTGCCAGTTGAAGAACTGT
215


hypothetical miRNA
33
hypothetical
TGACATCACATATACGGCAGC
1085


30

miRNA-030




mir-142
34
mir-142
CATAAAGTAGAAAGCACTAC
217


mir-142
34
miR-142-as
TGTAGTGTTTCCTACTTTATGG
1086


mir-142
34
miR-142as
TGTAGTGTTTCCTACTTTATGGA
1087




(Michael et al)




mir-183
35
mir-183
TATGGCACTGGTAGAATTCACTG
218


mir-214
37
mir-214
ACAGCAGGCACAGACAGGCAG
219


mir-143
38
miR-143 (Michael
TGAGATGAAGCACTGTAGCTC
1088




et al)




mir-143
38
mir-143
TGAGATGAAGCACTGTAGCTCA
220


mir-192_1
39
miR-192 (Tuschl)
CTGACCTATGAATTGACA
1089


mir-192_1
39
mir-192
CTGACCTATGAATTGACAGCC
221


mir-192_1
39
miR-192 (Michael
TGACCTATGAATTGACAGCCAG
1090




et al)




hypothetical miRNA
42
hypothetical
TAAGACTTGCAGTGATGTTTA
1091


039

miRNA-039




hypothetical miRNA
43
hypothetical
TGTCAACAAAACTGCTTACAA
1092


040

miRNA-040




hypothetical miRNA
44
hypothetical
TACCAGTTGTTTTCTCTGTGA
1093


041

miRNA-041




let-7a_3
45
let-7a
TGAGGTAGTAGGTTGTATAGTT
222


hypothetical miRNA
46
hypothetical
TGACAGGAAATCTTTGAGAGG
1094


043

miRNA-043




hypothetical miRNA
47
hypothetical
TTCCACTCTGTTTATCTGACA
1095


044

miRNA-044




mir-181a_1
48
mir-178 (Kosik)
AACATTCAACGCTGTCGGTGAG
1096


mir-181a_1
48
mir-181a
AACATTCAACGCTGTCGGTGAGT
223


let-7a_1
49
let-7a
TGAGGTAGTAGGTTGTATAGTT
222


mir-205
50
mir-205
TCCTTCATTCCACCGGAGTCTG
224


mir-33a
53
mir-33a
GTGCATTGTAGTTGCATTG
227


mir-196_2
54
miR-196 (Tuschl)
TAGGTAGTTTCATGTTGTTGG
1097


mir-196_2
54
mir-196
TAGGTAGTTTCATGTTGTTGGG
228


let-7f_1
57
let-7f (Michael
TGAGGTAGTAGATTGTATAGT
1098




et al)




let-7f_1
57
let-7f
TGAGGTAGTAGATTGTATAGTT
231


hypothetical miRNA
58
hypothetical
TTGCATGCCCTATTGATTCTC
1099


055

miRNA-055




mir-29c
59
mir-29c
CTAGCACCATTTGAAATCGGTT
232


mir-29c
59
miR-29c (Tuschl)
TAGCACCATTTGAAATCGGTTA
1100


mir-130a
60
mir-130a
CAGTGCAATGTTAAAAGGGC
233


mir-130a
60
mir-130 (Kosik)
CAGTGCAATGTTAAAAGGGCAT
1101


hypothetical miRNA
61
hypothetical
TGTCAGATGCTTAATGTTCTT
1102


058

miRNA-058




mir-218_1
62
mir-218
TTGTGCTTGATCTAACCATGT
234


mir-218_1
62
mir-253* (Kosik)
TTGTGCTTGATCTAACCATGTG
1103


mir-124a_2
63
mir-124a (Kosik)
TAAGGCACGCGGTGAATGCCA
1104


mir-124a_2
63
mir-124a
TTAAGGCACGCGGTGAATGCCA
235


mir-124a_2
63
mir-124a_Ruvkun
TTAAGGCACGCGGTGAATGCCAA
1105


mir-144
66
mir-144
TACAGTATAGATGATGTACTAG
237


mir-221
67
mir-221 (RFAM-
AGCTACATTGTCTGCTGGGTTT
1106




mmu)




mir-221
67
mir-221
AGCTACATTGTCTGCTGGGTTTC
238


mir-222
68
mir-222 (RFAM-
AGCTACATCTGGCTACTGGGTCT
1107




mmu)




mir-222
68
mir-222
AGCTACATCTGGCTACTGGGTCTC
239


mir-30d
69
mir-30d
TGTAAACATCCCCGACTGGAAG
240


mir-30d
69
mir-30d_Ruvkun
TGTAAACATCCCCGACTGGAAGCT
1108


mir-128b
71
mir-128 (Kosik)
TCACAGTGAACCGGTCTCTTT
1073


mir-128b
71
mir-128b
TCACAGTGAACCGGTCTCTTTC
242


mir-219_2
72
mir-219
TGATTGTCCAAACGCAATTCT
271


hypothetical miRNA
73
hypothetical
TCACATTTGCCTGCAGAGATT
1109


070

miRNA-070




mir-129_2
76
mir-129as/mir-
AAGCCCTTACCCCAAAAAGCAT
1110




258* (Kosik)




mir-129_2
76
mir-129
CTTTTTGCGGTCTGGGCTTGC
243


mir-129_2
76
miR-129b (RFAM-
CTTTTTGCGGTCTGGGCTTGCT
1111




Human)




mir-133b
77
mir-133b
TTGGTCCCCTTCAACCAGCTA
244


hypothetical miRNA
78
hypothetical
TGGTTAAAATATTAATGGGGC
1112


075

miRNA-075




let-7d
79
let-7d
AGAGGTAGTAGGTTGCATAGT
245


let-7d
79
let-7d_Ruvkun
AGAGGTAGTAGGTTGCATAGTT
1113


let-7d
79
let-7d* (RFAM-M. mu.)
CTATACGACCTGCTGCCTTTCT
1114


mir-15b
80
miR-15b (Michael
TAGCAGCACATCATGGTTTAC
1115




et al)




mir-15b
80
mir-15b
TAGCAGCACATCATGGTTTACA
246


mir-29a
81
mir-29a
CTAGCACCATCTGAAATCGGTT
247


mir-29a
81
mir-29a_Ruvkun
TAGCACCATCTGAAATCGGTTA
1116


hypothetical miRNA
82
hypothetical
TGATATGTTTGATATTGGG
1117


079

miRNA-079




mir-199b
83
mir-199b (human)
CCCAGTGTTTAGACTATCTGTTC
248


mir-199b
83
miR-199-as
TACAGTAGTCTGCACATTGGTT
1118


mir-129_1
84
mir-129
CTTTTTGCGGTCTGGGCTTGC
243


mir-129_1
84
miR-129b (RFAM-
CTTTTTGCGGTCTGGGCTTGCT
1111




Human)




let-7e
85
let-7e
TGAGGTAGGAGGTTGTATAGT
249


hypothetical miRNA
86
hypothetical
TTACATGGGGAAGCTATCATA
1119


083

miRNA-083




let-7c_1
87
let-7c
TGAGGTAGTAGGTTGTATGGTT
250


let-7c_1
87
let-7c_Ruvkun
TGAGGTAGTAGGTTGTATGGTTT
1120


mir-204
88
mir-204
TTCCCTTTGTCATCCTATGCCT
251


mir-204
88
miR-204 (Tuschl)
TTCCCTTTGTCATCCTATGCCTG
1121


mir-145
89
miR-145 (Michael
GTCCAGTTTTCCCAGGAATCC
1122




et al)




mir-145
89
mir-145
GTCCAGTTTTCCCAGGAATCCCTT
252


mir-124a_1
90
mir-124a (Kosik)
TAAGGCACGCGGTGAATGCCA
1104


mir-124a_1
90
mir-124a
TTAAGGCACGCGGTGAATGCCA
235


mir-124a_1
90
mir-124a_Ruvkun
TTAAGGCACGCGGTGAATGCCAA
1105


DiGeorge syndrome
91
hypothetical
TGTGATTTCCAATAATTGAGG
1123


critical region

miRNA-088




gene 8/






hypothetical






miRNA-088






mir-213/mir-
92
mir-178 (Kosik)
AACATTCAACGCTGTCGGTGAG
1096


181a_2






mir-213/mir-
92
mir-181a
AACATTCAACGCTGTCGGTGAGT
223


181a_2






mir-213/mir-
92
mir-213
ACCATCGACCGTTGATTGTACC
253


181a_2






hypothetical miRNA
93
hypothetical
TAGGCCAAATGGCGCATCAAT
1124


090

miRNA-090




mir-20
94
miR-20* (human)
ACTGCATTATGAGCACTTAAA
1125


mir-20
94
miR-20 (RFAM-
TAAAGTGCTTATAGTGCAGGTA
1126




Human)




mir-20
94
mir-20
TAAAGTGCTTATAGTGCAGGTAG
254


mir-133a_1
95
mir-133a
TTGGTCCCCTTCAACCAGCTGT
255


mir-138_2
96
mir-138
AGCTGGTGTTGTGAATC
256


mir-138_2
96
mir-138_Ruvkun
AGCTGGTGTTGTGAATCAGGCCG
1127


mir-196_1
98
miR-196 (Tuschl)
TAGGTAGTTTCATGTTGTTGG
1097


mir-196_1
98
mir-196
TAGGTAGTTTCATGTTGTTGGG
228


mir-125b_1
99
mir-125b
TCCCTGAGACCCTAACTTGTGA
258


mir-199a_2
100
miR-199-s
CCCAGTGTTCAGACTACCTGTT
1128


mir-199a_2
100
mir-199a
CCCAGTGTTCAGACTACCTGTTC
259


mir-199a_2
100
miR-199-as
TACAGTAGTCTGCACATTGGTT
1118


hypothetical miRNA
102
hypothetical
AGGCAGATAGAGAAGTCACAG
1272


099

miRNA-099




mir-181b_1
103
mir-181b
AACATTCATTGCTGTCGGTGGGTT
260


hypothetical miRNA
104
hypothetical
TGACAGTCAATTAACAAGTTT
1130


101

miRNA-101




mir-141
105
mir-141
AACACTGTCTGGTAAAGATGG
261


mir-131_1/mir-9
106
mir-131
TAAAGCTAGATAACCGAAAGT
211


mir-131_1/mir-9
106
mir-131_Ruvkun
TAAAGCTAGATAACCGAAAGTA
1080


mir-131_1/mir-9
106
miR-9
TCTTTGGTTATCTAGCTGTATGA
1081


mir-133a_2
107
mir-133a
TTGGTCCCCTTCAACCAGCTGT
255


hypothetical miRNA
108
miR-202 (human)
AGAGGTATAGGGCATGGGAAAA
1131


105






hypothetical miRNA
108
hypothetical
TTCCTATGCATATACTTCTTT
1132


105

miRNA-105




hypothetical miRNA
110
hypothetical
TGACAGTTTATTGGCTTTATC
1133


107

miRNA-107




mir-1d_2
111
miR-1 (RFAM)
TGGAATGTAAAGAAGTATGTA
1083


mir-1d_2
111
mir-1d
TGGAATGTAAAGAAGTATGTAT
213


mir-1d_2
111
miR-1d (Tuschl)
TGGAATGTAAAGAAGTATGTATT
1134


mir-220
113
mir-220
CCACACCGTATCTGACACTTT
263


hypothetical miRNA
114
hypothetical
TTCCTCCTCCTCCGACTCGGA
1135


111

miRNA-111




mir-7_3
115
mir-7
TGGAAGACTAGTGATTTTGTT
198


mir-218_2
116
mir-218
TTGTGCTTGATCTAACCATGT
234


mir-218_2
116
mir-253* (Kosik)
TTGTGCTTGATCTAACCATGTG
1103


mir-211
120
mir-211 (human)
TTCCCTTTGTCATCCTTCGCCT
1136


mir-30b
122
mir-30b
TGTAAACATCCTACACTCAGC
266


mir-30b
122
mir-30b_Ruvkun
TGTAAACATCCTACACTCAGCT
1137


hypothetical miRNA
123
hypothetical
TTACAGCAATCCAGTAATGAT
1138


120

miRNA-120




mir-10a
125
mir-10a (Tuschl)
TACCCTGTAGATCCGAATTTGT
1139


mir-10a
125
mir-10a
TACCCTGTAGATCCGAATTTGTG
267


let-7f_2
127
let-7f (Michael
TGAGGTAGTAGATTGTATAGT
1098




et al)




let-7f_2
127
let-7f
TGAGGTAGTAGATTGTATAGTT
231


mir-108_2
129
mir-108
ATAAGGATTTTTAGGGGCATT
207


mir-137
130
mir-137
TATTGCTTAAGAATACGCGTAG
270


mir-148b
132
mir-148b
TCAGTGCATCACAGAACTTTGT
272


mir-130b
133
mir-130b
CAGTGCAATGATGAAAGGGC
273


mir-130b
133
mir-266* (Kosik)
CAGTGCAATGATGAAAGGGCAT
1140


let-7a_4
135
let-7a
TGAGGTAGTAGGTTGTATAGTT
222


mir-216
136
mir-216
TAATCTCAGCTGGCAACTGTG
274


hypothetical miRNA
140
hypothetical
TAAACTGGCTGATAATTTTTG
1141


137

miRNA-137




hypothetical miRNA
141
hypothetical
TGCAAGTATGAAAATGAGATT
1142


138

miRNA-138




mir-124a_3
143
mir-124a (Kosik)
TAAGGCACGCGGTGAATGCCA
1104


mir-124a_3
143
mir-124a
TTAAGGCACGCGGTGAATGCCA
235


mir-124a_3
143
mir-124a_Ruvkun
TTAAGGCACGCGGTGAATGCCAA
1105


mir-7_2
144
mir-7
TGGAAGACTAGTGATTTTGTT
198


hypothetical miRNA
145
hypothetical
TGACGCTGCTCCCCACCTTCT
1143


142

miRNA-142




hypothetical miRNA
146
hypothetical
TGCAATTTGCTTGCAATTTTG
1144


143

miRNA-143




mir-210
148
mir-210
CTGTGCGTGTGACAGCGGCTG
277


mir-215
149
mir-215
ATGACCTATGAATTGACAGAC
278


mir-223
150
mir-223
TGTCAGTTTGTCAAATACCCC
279


mir-131_3/mir-9
151
mir-131
TAAAGCTAGATAACCGAAAGT
211


mir-131_3/mir-9
151
mir-131_Ruvkun
TAAAGCTAGATAACCGAAAGTA
1080


mir-131_3/mir-9
151
miR-9
TCTTTGGTTATCTAGCTGTATGA
1081


mir-199a_1
152
miR-199-s
CCCAGTGTTCAGACTACCTGTT
1128


mir-199a_1
152
mir-199a
CCCAGTGTTCAGACTACCTGTTC
259


mir-199a_1
152
miR-199-as
TACAGTAGTCTGCACATTGGTT
1118


mir-30c_1
153
mir-30c
TGTAAACATCCTACACTCTCAGC
280


mir-30c_1
153
mir-30c_Ruvkun
TGTAAACATCCTACACTCTCAGCT
1129


hypothetical miRNA
156
hypothetical
TGCAAGCAGATGCTGATAATA
1145


153

miRNA-153




hypothetical miRNA
157
hypothetical
TTAAAGTGGATGTGTGTTATT
1146


154

miRNA-154




mir-26b
158
miR-26b (RFAM-
TTCAAGTAATTCAGGATAGGT
1147




Human)




mir-26b
158
mir-26b
TTCAAGTAATTCAGGATAGGTT
281


hypothetical miRNA
159
hypothetical
TGCTTTCCCTCCTTCCTTCTT
1148


156

miRNA-156




mir-152
160
mir-152
TCAGTGCATGACAGAACTTGG
282


mir-135_1
161
miR-135 (RFAM-
TATGGCTTTTTATTCCTATGTGA
1149




Human)




mir-135_1
161
mir-135
TATGGCTTTTTATTCCTATGTGAT
283


non-coding RNA in
162
miR-135 (RFAM-
TATGGCTTTTTATTCCTATGTGA
1149


rhabdomyosarcoma/

Human)




mir-135_2






non-coding RNA in
162
mir-135
TATGGCTTTTTATTCCTATGTGAT
283


rhabdomyosarcoma/






mir-135_2






mir-217
163
mir-217 (human)
TACTGCATCAGGAACTGATTGGAT
284


hypothetical miRNA
164
hypothetical
TGGCCATAAACTTGTAGTCAT
1150


161

miRNA-161




mir-15a
165
mir-15_Ruvkun
TAGCAGCACATAATGGTTTGT
1151


mir-15a
165
mir-15a
TAGCAGCACATAATGGTTTGTG
269


let-7g
166
let-7g
TGAGGTAGTAGTTTGTACAGT
285


let-7g
166
let-7gL_Ruvkun
TGAGGTAGTAGTTTGTACAGTT
1152


hypothetical miRNA
167
hypothetical
TGCAAGGATTTTTATGTTTTG
1153


164

miRNA-164




hypothetical miRNA
169
hypothetical
TTCCAGTTGCAGCACCTGTAA
1154


166

miRNA-166




hypothetical miRNA
171
hypothetical
AGCCAGGTGCCTTCACCTGCT
1155


168_1/similar to

miRNA-168




ribosomal protein






L5






forkhead box
172
hypothetical
TGGCAGCTCTGGCATTTCATA
1156


P2/hypothetical

miRNA-169




miRNA-169






hypothetical miRNA
173
hypothetical
TGATCTTGCTCTAACACTTGG
1157


170

miRNA-170




glutamate
174
hypothetical
TGACAAGTATGTTTTATCGTT
1158


receptor,

miRNA-171




ionotropic, AMPA 2/






hypothetical






miRNA-171






hypothetical miRNA
175
hypothetical
TCCAACTGCAAGAAGTTACT
1159


172

miRNA-172




hypothetical miRNA
176
hypothetical
TAGTACGAGAAGAAGGAGGCT
1160


173

miRNA-173




mir-182
177
miR-182* (RFAM-
TGGTTCTAGACTTGCCAACTA
1161




Human)




mir-182
177
mir-182
TTTGGCAATGGTAGAACTCACA
287


hypothetical miRNA
178
hypothetical
TCTCCTTCAACCACCTGAGGT
1162


175

miRNA-175




hypothetical miRNA
179
hypothetical
TAGGAGTTTGATATGACATAT
1163


176

miRNA-176




hypothetical
180
hypothetical
AGACAAACATGCTACTCTCAC
1164


miRNA-177_1

miRNA-177




hypothetical miRNA
181
hypothetical
TAGCCTATCTCCGAACCTTCA
1165


178

miRNA-178




hypothetical miRNA
182
hypothetical
TGAAAGGCACTTTGTCCAATT
1166


179

miRNA-179




hypothetical miRNA
184
hypothetical
TCACCTGCTCTGGAAGTAGTT
1167


181

miRNA-181




mir-148a
185
mir-148a
TCAGTGCACTACAGAACTTTGT
288


hypothetical miRNA
188
hypothetical
TGATGGCCAGCTGAGCAGCTC
1168


185

miRNA-185




hypothetical
189
hypothetical
AGACAAACATGCTACTCTCAC
1164


miRNA-177_2/

miRNA-177




hypothetical miRNA






186






mir-181c
190
mir-181c
AACATTCAACCTGTCGGTGAGT
290


hypothetical miRNA
191
hypothetical
TGGTGAGGGGAATGAAAAGTA
1169


188

miRNA-188




mir-100_1
945
mir-100
AACCCGTAGATCCGAACTTGTG
275


mir-101_1
946
mir-101
TACAGTACTGTGATAACTGA
265


mir-101_1
946
miR-101 (RFAM-
TACAGTACTGTGATAACTGAAG
1170




Human)




mir-101_3
947
mir-101
TACAGTACTGTGATAACTGA
265


mir-101_3
947
miR-101 (RFAM-
TACAGTACTGTGATAACTGAAG
1170




Human)




mir-29b_2
948
miR-29b (RFAM-
TAGCACCATTTGAAATCAGT
1172




Human)




mir-29b_2
948
miR-29b (RFAM-M. mu.)
TAGCACCATTTGAAATCAGTGT
1173


mir-29b_2
948
mir-29b
TAGCACCATTTGAAATCAGTGTT
195


mir-29b_1
949
miR-29b (RFAM-
TAGCACCATTTGAAATCAGT
1172




Human)




mir-29b_1
949
miR-29b (RFAM-M. mu.)
TAGCACCATTTGAAATCAGTGT
1173


mir-29b_1
949
mir-29b
TAGCACCATTTGAAATCAGTGTT
195


mir-103_1
950
mir-103
AGCAGCATTGTACAGGGCTATGA
225


mir-106
951
mir-106 (human)
AAAAGTGCTTACAGTGCAGGTAGC
230


mir-107
952
mir-107
AGCAGCATTGTACAGGGCTATCA
229


mir-16_1
953
mir-16
TAGCAGCACGTAAATATTGGCG
196


mir-16_1
953
mir-16_Ruvkun
TAGCAGCACGTAAATATTGGCGT
1176


mir-16_3
954
mir-16
TAGCAGCACGTAAATATTGGCG
196


mir-16_3
954
mir-16_Ruvkun
TAGCAGCACGTAAATATTGGCGT
1176


mir-18
955
mir-18
TAAGGTGCATCTAGTGCAGATA
262


mir-18
955
mir-18_Ruvkun
TAAGGTGCATCTAGTGCAGATAG
1177


mir-19a
956
mir-19a
TGTGCAAATCTATGCAAAACTGA
268


mir-19b_1
957
mir-19b* (Michael
AGTTTTGCAGGTTTGCATCCAGC
1179




et al)




mir-19b_1
957
mir-19b
TGTGCAAATCCATGCAAAACTGA
241


mir-19b_2
958
mir-19b
TGTGCAAATCCATGCAAAACTGA
241


mir-21
959
mir-21
TAGCTTATCAGACTGATGTTGA
236


mir-23a
960
mir-23a
ATCACATTGCCAGGGATTTCC
289


mir-24_2
961
mir-24
TGGCTCAGTTCAGCAGGAACAG
264


mir-17/mir-91
962
mir-17 (human,
ACTGCAGTGAAGGCACTTGT
1180




rat)




mir-17/mir-91
962
mir-91_Ruvkun
CAAAGTGCTTACAGTGCAGGTAG
1181


mir-17/mir-91
962
mir-17as/mir-91
CAAAGTGCTTACAGTGCAGGTAGT
204


mir-92_1
963
miR-92 (RFAM-M. mu.)
TATTGCACTTGTCCCGGCCTG
1182


mir-92_1
963
mir-92
TATTGCACTTGTCCCGGCCTGT
216


mir-96
964
mir-96
TTTGGCACTAGCACATTTTTGC
203


mir-96
964
miR-96 (RFAM-M. mu.)
TTTGGCACTAGCACATTTTTGCT
1183


mir-30a
965
mir-30a
CTTTCAGTCGGATGTTTGCAGC
193


mir-30a
965
miR-30a-s
TGTAAACATCCTCGACTGGAAGC
1184


mir-98
966
mir-98
TGAGGTAGTAAGTTGTATTGTT
257


mir-104
967
miR-104
TCAACATCAGTCTGATAAGCTA
335


(Mourelatos)

(Mourelatos)




mir-105
968
miR-105
TCAAATGCTCAGACTCCTGT
1185


(Mourelatos)

(Mourelatos)




mir-27
969
miR-27
TTCACAGTGGCTAAGTTCC
1186


(Mourelatos)

(Mourelatos)




mir-27
969
miR-27a (RFAM-M. mu.)
TTCACAGTGGCTAAGTTCCGC
1187


(Mourelatos)






mir-27
969
miR-27a (RFAM-
TTCACAGTGGCTAAGTTCCGCC
1188


(Mourelatos)

Human)




mir-92_2
970
miR-92 (RFAM-M. mu.)
TATTGCACTTGTCCCGGCCTG
1182


mir-92_2
970
mir-92
TATTGCACTTGTCCCGGCCTGT
216


mir-93
971
miR-93
AAAGTGCTGTTCGTGCAGGTAG
1189


(Mourelatos)

(Mourelatos)




mir-93
971
miR-93 (Tuschl)
CAAAGTGCTGTTCGTGC
1190


(Mourelatos)






mir-93
971
miR-93 (RFAM-M. mu.)
CAAAGTGCTGTTCGTGCAGGTAG
1191


(Mourelatos)






mir-95
972
miR-95
TTCAACGGGTATTTATTGAGCA
1192


(Mourelatos)

(Mourelatos)




mir-99
973
miR-99
AACCCGTAGATCCGATCTTGTG
1193


(Mourelatos)

(Mourelatos)




mir-99
973
miR-99a (Tuschl)
ACCCGTAGATCCGATCTTGT
1194


(Mourelatos)






mir-25
974
miR-25 (Tuschl)
CATTGCACTTGTCTCGGTCTGA
1195


mir-28
975
miR-28 (Tuschl)
AAGGAGCTCACAGTCTATTGAG
1196


mir-31
976
miR-31 (RFAM-M. mu.)
AGGCAAGATGCTGGCATAGCTG
1197


mir-31
976
miR-31 (Tuschl)
GGCAAGATGCTGGCATAGCTG
1198


mir-32
977
miR-32 (Tuschl)
TATTGCACATTACTAAGTTGC
1199


mir-149
978
miR-149
TCTGGCTCCGTGTCTTCACTCC
1200


mir-30c_2
979
mir-30c
TGTAAACATCCTACACTCTCAGC
280


mir-30c_2
979
mir-30c_Ruvkun
TGTAAACATCCTACACTCTCAGCT
1129


mir-99b
980
miR-99b
CACCCGTAGAACCGACCTTGCG
1201


MiR-125a
981
miR-125a
TCCCTGAGACCCTTTAACCTGTG
1202


MiR-125b_2
982
mir-125b
TCCCTGAGACCCTAACTTGTGA
258


mir-26a_2
983
miR-26a (Michael
TTCAAGTAATCCAGGATAGGC
1203




et al)




mir-26a_2
983
mir-26a
TTCAAGTAATCCAGGATAGGCT
226


mir-127
984
mir-127_Ruvkun
TCGGATCCGTCTGAGCTTGG
1204


mir-127
984
miR-127
TCGGATCCGTCTGAGCTTGGCT
1205


mir-136
985
miR-136
ACTCCATTTGTTTTGATGATGGA
1206


mir-154
986
miR-154
TAGGTTATCCGTGTTGCCTTCG
1207


mir-26a_1
987
miR-26a (Michael
TTCAAGTAATCCAGGATAGGC
1203




et al)




mir-26a_1
987
mir-26a
TTCAAGTAATCCAGGATAGGCT
226


mir_186
988
miR-186
CAAAGAATTCTCCTTTTGGGCTT
1208


mir_198
989
mir-198
GGTCCAGAGGGGAGATAGG
1209


mir_191
990
mir-191
CAACGGAATCCCAAAAGCAGCT
1210


mir_191
990
mir-191_Ruvkun
CAACGGAATCCCAAAAGCAGCTGT
1211


mir_206
991
mir-206
TGGAATGTAAGGAAGTGTGTGG
1212


mir-94/mir-106b
992
miR-94
AAAGTGCTGACAGTGCAGAT
1213


mir-94/mir-106b
992
miR-106b (RFAM-M. mu.)
TAAAGTGCTGACAGTGCAGAT
1214


mir_184
993
miR-184
TGGACGGAGAACTGATAAGGGT
1215


mir_195
994
miR-195
TAGCAGCACAGAAATATTGGC
1216


mir_193
995
miR-193
AACTGGCCTACAAAGTCCCAG
1217


mir_185
996
miR-185
TGGAGAGAAAGGCAGTTC
1218


mir_188
997
miR-188
CATCCCTTGCATGGTGGAGGGT
1219


mir_197
998
miR-197a
TTCACCACCTTCTCCACCCAGC
1220


mir_194_1
999
miR-194
TGTAACAGCAACTCCATGTGGA
1221


mir_208
1000
miR-208
ATAAGACGAGCAAAAAGCTTGT
1222


mir_194_2
1001
miR-194
TGTAACAGCAACTCCATGTGGA
1221


mir_139
1002
miR-139
TCTACAGTGCACGTGTCT
1223


mir-200b
1003
miR-200a (RFAM-
CTCTAATACTGCCTGGTAATGATG
1224




Human)




mir-200b
1003
miR-200b (Michael
TAATACTGCCTGGTAATGATGA
1225




et al)




mir-200b
1003
miR-200b
TAATACTGCCTGGTAATGATGAC
1226


mir-200a
1004
miR-200a
TAACACTGTCTGGTAACGATG
1227


mir-200a
1004
miR-200a (RFAM-M. mu.)
TAACACTGTCTGGTAACGATGT
1228


mir-240* (Kosik)
1005
mir-240* (Kosik)
TCAAGAGCAATAACGAAAAATGT
1229


mir-232* (Kosik)
1006
mir-232* (Kosik)
CTGGCCCTCTCTGCCCTTCCGT
1230


mir-227*
1007
mir-226* (Kosik)
ACTGCCCCAGGTGCTGCTGG
1231


(Kosik)/mir-226*






(Kosik)






mir-227*
1007
mir-324-3p_Ruvkun
CCACTGCCCCAGGTGCTGCTGG
1232


(Kosik)/mir-226*






(Kosik)






mir-227*
1007
mir-227* (Kosik)
CGCATCCCCTAGGGCATTGGTGT
1233


(Kosik)/mir-226*






(Kosik)






mir-244* (Kosik)
1008
mir-244* (Kosik)
TCCAGCATCAGTGATTTTGTTGA
1234


mir-224* (Kosik)
1009
mir-224* (Kosik)
GCACATTACACGGTCGACCTCT
1235


mir-248* (Kosik)
1010
mir-248* (Kosik)
TCTCACACAGAAATCGCACCCGTC
1236


ribosomal protein
1011
hypothetical
AGCCAGGTGCCTTCACCTGCT
1155


L5/hypothetical

miRNA-168




miRNA 168_2






hypothetical
1012
hypothetical
AGACAAACATGCTACTCTCAC
1164


miRNA-177_3

miRNA-177




mir-138_3
1013
mir-138
AGCTGGTGTTGTGAATC
256


mir-138_3
1013
mir-138_Ruvkun
AGCTGGTGTTGTGAATCAGGCCG
1127


mir-138_4
1014
mir-138
AGCTGGTGTTGTGAATC
256


mir-181b_2
1015
mir-181b
AACATTCATTGCTGTCGGTGGGTT
260


mir-219_1
1016
mir-219
TGATTGTCCAAACGCAATTCT
271


mir-105_2
1017
miR-105
TCAAATGCTCAGACTCCTGT
1185




(Mourelatos)




hypothetical miRNA
1018
hypothetical
TTACAGCAATCCAGTAATGAT
1138


120_2

miRNA-120




cezanne 2/
1019
hypothetical
TCCTGTCAGACTTTGTTCGGT
1237


hypothetical

miRNA-180




miRNA-180






mir-103_2
1020
mir-103
AGCAGCATTGTACAGGGCTATGA
225


mir-147 (Sanger)
1021
miR-147 (RFAM-
GTGTGTGGAAATGCTTCTGC
1238




Human)




mir-224 (Sanger)
1022
miR-224 (RFAM-
CAAGTCACTAGTGGTTCCGTTTA
1239




Human)




mir-134 (Sanger)
1023
miR-134 (RFAM-
TGTGACTGGTTGACCAGAGGG
1240




Human)




mir-146 (Sanger)
1024
miR-146 (RFAM-
TGAGAACTGAATTCCATGGGTT
1241




Human)




mir-150 (Sanger)
1025
miR-150 (RFAM-
TCTCCCAACCCTTGTACCAGTG
1242




Human)




mir-30e (RFAM/mmu)
1026
miR-30e (RFAM-M. mu.)
TGTAAACATCCTTGACTGGA
1243


mir-30e (RFAM/mmu)
1026
miR-97 (Michael
TGTAAACATCCTTGACTGGAAG
1244




et al)




mir-296 (RFAM/mmu)
1027
miR-296 (RFAM-M. mu.)
AGGGCCCCCCCTCAATCCTGT
1245


mir-299 (RFAM/mmu)
1028
miR-299 (RFAM-M. mu.)
TGGTTTACCGTCCCACATACAT
1246


mir-301 (RFAM/mmu)
1029
miR-301 (RFAM-M. mu.)
CAGTGCAATAGTATTGTCAAAGC
1247


mir-301 (RFAM/mmu)
1029
mir-301_Ruvkun
CAGTGCAATAGTATTGTCAAAGCAT
1248


mir-302 (RFAM/mmu)
1030
miR-302 (RFAM-M. mu.)
TAAGTGCTTCCATGTTTTGGTGA
1249


mir-34a (RFAM/mmu)
1031
mir-34c (RFAM)
AGGCAGTGTAGTTAGCTGATTG
1250


mir-34a (RFAM/mmu)
1031
miR-34a (RFAM-M. mu.)
AGGCAGTGTAGTTAGCTGATTGC
1251


mir_320
1032
miR-320
AAAAGCTGGGTTGAGAGGGCGAA
1252


mir-321_1
1033
miR-321-1
TAAGCCAGGGATTGTGGGTTC
1253


mir-135b (Ruvkun)
1034
mir-135b (Ruvkun)
TATGGCTTTTCATTCCTATGTG
1254


mir-151* (Ruvkun)
1035
mir-151 (human)
ACTAGACTGAAGCTCCTTGAGG
1255


mir-151* (Ruvkun)
1035
mir-151* (Ruvkun)
TCGAGGAGCTCACAGTCTAGTA
1256


mir-340 (Ruvkun)
1036
mir-340 (Ruvkun)
TCCGTCTCAGTTACTTTATAGCC
1257


mir-331 (Ruvkun)
1037
mir-331 (Ruvkun)
GCCCCTGGGCCTATCCTAGAA
1258


mir_200c (RFAM)
1038
mir-200c (RFAM)
AATACTGCCGGGTAATGATGGA
1259


mir_34b (RFAM)
1039
mir-34b (RFAM)
AGGCAGTGTCATTAGCTGATTG
1260


mir_339_1 (RFAM)
1040
mir-339 (RFAM)
TCCCTGTCCTCCAGGAGCTCA
1261


mir_339_2 (RFAM)
1041
mir-339 (RFAM)
TCCCTGTCCTCCAGGAGCTCA
1261


mir-325 (Ruvkun)
1042
mir-325 (human)
CCTAGTAGGTGTCCAGTAAGTGT
1262


mir-326 (Ruvkun)
1043
miR-326 (Ruvkun)
CCTCTGGGCCCTTCCTCCAG
1263


mir-326 (Ruvkun)
1044
mir-326 (human)
CCTCTGGGCCCTTCCTCCAGC
1264


mir-329-1 (Ruvkun)
1045
mir-329 (human)
AACACACCTGGTTAACCTCTTT
1265


mir-329-2 (Ruvkun)
1046
mir-329 (human)
AACACACCTGGTTAACCTCTTT
1265


mir-330 (Ruvkun)
1047
mir-330 (human)
GCAAAGCACACGGCCTGCAGAGA
1266


mir-337 (Ruvkun)
1048
mir-337 (human)
TCCAGCTCCTATATGATGCCTTT
1267


mir-345 (Ruvkun)
1049
mir-345 (human)
TGCTGACTCCTAGTCCAGGGC
1268


mir-346 (Ruvkun)
1050
mir-346 (human)
TGTCTGCCCGCATGCCTGCCTCT
1269


mir-187
1051
miR-187 (RFAM-
TCGTGTCTTGTGTTGCAGCCG
1270




Human)




mir-187
1051
mir-187
TCGTGTCTTGTGTTGCAGCCGG
276


miR-24-1
1052
miR-189 (RFAM-
GTGCCTACTGAGCTGATATCAGT
1271




Human)




miR-24-1
1052
mir-24
TGGCTCAGTTCAGCAGGAACAG
264


mir-215
1053
mir-215
ATGACCTATGAATTGACAGAC
278









A list of mouse pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 61. “Pri-miRNA name” indicates the gene name for each of the pri-miRNAs. Also given in table 61 are the name and sequence of the mature miRNA derived from the pri-miRNA. Mature miRNA sequences from pri-miRNA precursors have been proposed by several groups; consequently, for a given pri-miRNA sequence, several miRNAs may be disclosed and given unique names, and thus a given pri-miRNA sequence may occur repeatedly in the table. The sequences are written in the 5′ to 3′ direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.







TABLE 61







Mouse pri-miRNA sequences and the corresponding mature miRNAs












SEQ


SEQ



ID


ID


Pri-miRNA name
NO
Mature miRNA name
Mature miRNA sequence
NO














mir-26b
1273 
miR-99
TTCAAGTAATTCAGGATAGGT
1147




(Mourelatos)




mir-26b
1273
miR-199-as
TTCAAGTAATTCAGGATAGGTT
281


mir-30a
1274
miR-199-as
CTTTCAGTCGGATGTTTGCAGC
193


mir-30a
1274
miR-26b (RFAM-
TGTAAACATCCTCGACTGGAAGC 
1184




Human)




mir-30c_1
1275
miR-32 (Tuschl)
TGTAAACATCCTACACTCTCAGC
280


mir-30c_1
1275
let-7c_Ruvkun
TGTAAACATCCTACACTCTCAGCT
1129


mir-128a
1276
mir-214
TCACAGTGAACCGGTCTCTTT
1073


mir-128a
1276 
miR-29b (RFAM-
TCACAGTGAACCGGTCTCTTTT
200




Human)




mir-29b_1
1277
mir-196
TAGCACCATTTGAAATCAGT
1172


mir-29b_1
1277
hypothetical
TAGCACCATTTGAAATCAGTGT
1173




miRNA-079




mir-29b_1
1277
mir-30c
TAGCACCATTTGAAATCAGTGTT
195


mir-29c
1278
mir-131_Ruvkun
CTAGCACCATTTGAAATCGGTT
232


mir-29c
1278
hypothetical
TAGCACCATTTGAAATCGGTTA
1100




miRNA-033




mir-123/mir-126
1279
mir-326 (rodent) 
CATTATTACTTTTGGTACGCG
205


mir-123/mir-126
1279
mir-126
TCGTACCGTGAGTAATAATGC
1076


mir-130a
1280
mir-23a
CAGTGCAATGTTAAAAGGGC
233


mir-130a
1280
hypothetical
CAGTGCAATGTTAAAAGGGCAT
1101




miRNA-040




mir-1d_1
1281
mir-132
TGGAATGTAAAGAAGTATGTA
1083


mir-1d_1
1281
mir-124a (Kosik)
TGGAATGTAAAGAAGTATGTAT
213


mir-1d_1
1281 
miR-200b
TGGAATGTAAAGAAGTATGTATT
1134


mir-124a_3
1282
mir-100
TAAGGCACGCGGTGAATGCCA
1104


mir-124a_3
1282
mir-212
TTAAGGCACGCGGTGAATGCCA
235


mir-124a_3
1282 
let-7a
TTAAGGCACGCGGTGAATGCCAA 
1105


mir-133a_2
1283 
miR-189 (RFAM-
TTGGTCCCCTTCAACCAGCTGT
255




Human)




mir-124a_2
1284
mir-181c
TAAGGCACGCGGTGAATGCCA
1104


mir-124a_2
1284
mir-108
TTAAGGCACGCGGTGAATGCCA
235


mir-124a_2
1284
mir-239* (Kosik)
TTAAGGCACGCGGTGAATGCCAA
1105


mir-131_1/mir-9
1285
mir-325 (rodent) 
TAAAGCTAGATAACCGAAAGT
211


mir-131_1/mir-9
1285
mir-19b
TAAAGCTAGATAACCGAAAGTA
1080


mir-131_1/mir-9
1285
mir-124a_Ruvkun
TCTTTGGTTATCTAGCTGTATGA
1081


mir-15b
1286
mir-152
TAGCAGCACATCATGGTTTAC
1115


mir-15b
1286
hypothetical
TAGCAGCACATCATGGTTTACA
246




miRNA-111




mir-16_3
1287
miR-104
TAGCAGCACGTAAATATTGGCG
196




(Mourelatos)




mir-16_3
1287
mir-128a
TAGCAGCACGTAAATATTGGCGT
1176


mir-137
1288
mir-30b
TATTGCTTAAGAATACGCGTAG
270


mir-101_1
1289
mir-18
TACAGTACTGTGATAACTGA
265


mir-101_1
1289
mir-128b
TACAGTACTGTGATAACTGAAG
1170


mir-29a
1291
miR-27a (RFAM- M.
CTAGCACCATCTGAAATCGGTT
247




mu.)




mir-29a
1291
mir-153
TAGCACCATCTGAAATCGGTTA 
1116


mir-29b_2
1292
mir-138_Ruvkun
TAGCACCATTTGAAATCAGT
1172


mir-29b_2
1292
hypothetical
TAGCACCATTTGAAATCAGTGT
1173




miRNA-075




mir-29b_2
1292
miR-30a-s
TAGCACCATTTGAAATCAGTGTT 
195


mir-148a
1293
miR-1d (Tuschl)
TCAGTGCACTACAGAACTTTGT
288


mir-141
1294
mir-16_Ruvkun
AACACTGTCTGGTAAAGATGG
261


mir-131_3/mir-9
1295
mir-124a (Kosik) 
TAAAGCTAGATAACCGAAAGT
211


mir-131_3/mir-9
1295
mir-7b (rodent)
TAAAGCTAGATAACCGAAAGTA
1080


mir-131_3/mir-9
1295
mir-19a
TCTTTGGTTATCTAGCTGTATGA
1081


mir-23a
1296
miR-1 (RFAM)
ATCACATTGCCAGGGATTTCC
289


mir-24_2
1297
mir-124a_Ruvkun
TGGCTCAGTTCAGCAGGAACAG
264


mir-140
1298
miR-199b (mouse) 
AGTGGTTTTACCCTATGGTAG
192


mir-140
1298
mir-205
TACCACAGGGTAGAACCACGGA 
1065


mir-140
1298
mir-26b
TACCACAGGGTAGAACCACGGACA
1066


let-7a_4
1299
mir-16_Ruvkun
TGAGGTAGTAGGTTGTATAGTT
222


mir-125b_1
1300
mir-131_Ruvkun
TCCCTGAGACCCTAACTTGTGA
258


mir-26a_1
1301
mir-29b
TTCAAGTAATCCAGGATAGGC
1203


mir-26a_1
1301
hypothetical
TTCAAGTAATCCAGGATAGGCT
226




miRNA-154




let-7i
1302
hypothetical
TGAGGTAGTAGTTTGTGCT
209




miRNA-179




let-7i
1302
miR-1d (Tuschl)
TGAGGTAGTAGTTTGTGCTGTT 
1078


mir-21
1303
mir-125b
TAGCTTATCAGACTGATGTTGA
236


mir-22
1304
mir-131
AAGCTGCCAGTTGAAGAACTGT
215


mir-142
1305
mir-131_Ruvkun
CATAAAGTAGAAAGCACTAC
217


mir-142
1305
hypothetical
TGTAGTGTTTCCTACTTTATGG
1086




miRNA-105




mir-142
1305
mir-218
TGTAGTGTTTCCTACTTTATGGA
1087


mir-144
1306
mir-26a
TACAGTATAGATGATGTACTAG
237


mir-152
1307
miR-99a (Tuschl) 
TCAGTGCATGACAGAACTTGG
282


mir-153_2
1308
mir-29c
TTGCATAGTCACAAAAGTGA
201


let-7a_1
1309
mir-16
TGAGGTAGTAGGTTGTATAGTT
222


let-7d
1310
mir-144
AGAGGTAGTAGGTTGCATAGT
245


let-7d
1310
hypothetical
AGAGGTAGTAGGTTGCATAGTT
1113




miRNA-171




let-7d
1310
miR-204 (Tuschl) 
CTATACGACCTGCTGCCTTTCT
1114


let-7f_1
1311
miR-9
TGAGGTAGTAGATTGTATAGT
1098


let-7f_1
1311
hypothetical
TGAGGTAGTAGATTGTATAGTT
231




miRNA-138




mir-23b
1312
mir-1d
ATCACATTGCCAGGGATTACCAC 
208


miR-24-1
1313
mir-124a (Kosik) 
GTGCCTACTGAGCTGATATCAGT
1271


miR-24-1
1313
hypothetical
TGGCTCAGTTCAGCAGGAACAG
264




miRNA-070




mir-27b
1314
miR-29c (Tuschl) 
TTCACAGTGGCTAAGTTCTG
202


mir-27b
1314
mir-135
TTCACAGTGGCTAAGTTCTGC
1059


mir-131_2/mir-9
1315
mir-107
TAAAGCTAGATAACCGAAAGT
211


mir-131_2/mir-9
1315
miR-224 (RFAM-
TAAAGCTAGATAACCGAAAGTA
1080




mouse)




mir-1312/mir-9
1315
mir-124a
TCTTTGGTTATCTAGCTGTATGA 
1081


mir-15a
1316
miR-20 (RFAM-
TAGCAGCACATAATGGTTTGT
1151




Human)




mir-15a
1316
miR-92 (RFAM-
TAGCAGCACATAATGGTTTGTG
269




M.mu.)




mir-16_1
1317
mir-98
TAGCAGCACGTAAATATTGGCG
196


mir-16_1
1317
mir-30c_Ruvkun
TAGCAGCACGTAAATATTGGCGT 
1176


mir-124a_1
1318
miR-132 (RFAM-
TAAGGCACGCGGTGAATGCCA
1104




Human)




mir-124a_1
1318
miR-140-as
TTAAGGCACGCGGTGAATGCCA
235


mir-124a_1
1318
hypothetical
TTAAGGCACGCGGTGAATGCCAA 
1105




miRNA-181




mir-18
1319
mir-124a
TAAGGTGCATCTAGTGCAGATA
262


mir-18
1319
miR-27
TAAGGTGCATCTAGTGCAGATAG 
1177




(Mourelatos)




mir-20
1320
mir-23b
TAAAGTGCTTATAGTGCAGGTA
1126


mir-20
1320
mir-199a
TAAAGTGCTTATAGTGCAGGTAG
254


mir-30b
1321
miR-31 (Tuschl)
TGTAAACATCCTACACTCAGC
266


mir-30b
1321
mir-18_Ruvkun
TGTAAACATCCTACACTCAGCT
1137


mir-30d
1322
miR-186
TGTAAACATCCCCGACTGGAAG
240


mir-30d
1322
let-7i_Ruvkun
TGTAAACATCCCCGACTGGAAGCT
1108


let-7b
1323
mir-135
TGAGGTAGTAGGTTGTGTGGTT
212


let-7b
1323
mir-133a
TGAGGTAGTAGGTTGTGTGGTTT 
1082


let7c_2
1324
let-7d* (RFAM- M.
TGAGGTAGTAGGTTGTATGGTT
250




mu.)




let7c_2
1324
hypothetical
TGAGGTAGTAGGTTGTATGGTTT 
1120




miRNA-170




let-7c_1
1325
let-7d
TGAGGTAGTAGGTTGTATGGTT
250


let-7c_1
1325
miR-135(RFAM-
TGAGGTAGTAGGTTGTATGGTTT
1120




Human)




mir-99
1326
miR-203(Tuschl)
AACCCGTAGATCCGATCTTGTG
1193


(Mourelatos)






mir-99
1326
mir-34
ACCCGTAGATCCGATCTTGT
1194


(Mourelatos)






LOC 114614
1327
mir-187
TTAATGCTAATTGTGATAGGGG
1459


containing miR-






155/hypothetical






miRNA-071






let-7e
1328
let-7a
TGAGGTAGGAGGTTGTATAGT
249


mir-1d_2
1329
miR-10b (Michael 
TGGAATGTAAAGAAGTATGTA
1083




et al)




mir-1d_2
1329
miR-139
TGGAATGTAAAGAAGTATGTAT
213


mir-1d_2
1329
mir-124a
TGGAATGTAAAGAAGTATGTATT 
1134


mir-133a_1
1330
mir-24
TTGGTCCCCTTCAACCAGCTGT
255


mir-143
1331
miR-15b (Michael 
TGAGATGAAGCACTGTAGCTC
1088




et al)




mir-143
1331
mir-253* (Kosik) 
TGAGATGAAGCACTGTAGCTCA
220


mir-145
1332
mir-148b
GTCCAGTTTTCCCAGGAATCC
1122


mir-145
1332
let-7f
GTCCAGTTTTCCCAGGAATCCCTT
252


mir-122a
1333
miR-172 (RFAM-M. 
TGGAGTGTGACAATGGTGTTTG
1084




mu.)




mir-122a
1333
mir-124a_Ruvkun
TGGAGTGTGACAATGGTGTTTGT 
214


mir-19b_2
1334
mir-22
TGTGCAAATCCATGCAAAACTGA 
241


let-7f_2
1335
hypothetical
TGAGGTAGTAGATTGTATAGT
1098




miRNA-137




let-7f_2
1335
mir-131
TGAGGTAGTAGATTGTATAGTT
231


mir-26a_2
1336
mir-29a_Ruvkun
TTCAAGTAATCCAGGATAGGC
1203


mir-26a_2
1336
hypothetical
TTCAAGTAATCCAGGATAGGCT
226




miRNA-153




mir-127
1337
mir-103
TCGGATCCGTCTGAGCTTGG
1204


mir-127
1337
mir-17as/mir-91
TCGGATCCGTCTGAGCTTGGCT
1205


mir-136
1338
mir-91_Ruvkun
ACTCCATTTGTTTTGATGATGGA 
1206


mir-154
1339
mir-17-3p (mouse)
TAGGTTATCCGTGTTGCCTTCG
1207


mir-149
1340
let-7gL_Ruvkun
TCTGGCTCCGTGTCTTCACTCC
1200


mir-30c_2
1341
miR-31 (RFAM-
TGTAAACATCCTACACTCTCAGC 
280




M.mu.)




mir-30c_2
1341
let-7c
TGTAAACATCCTACACTCTCAGCT
1129


mir-99b
1342
mir-101b (rodent)
CACCCGTAGAACCGACCTTGCG
1201


MiR-125a
1343
mir-106 (mouse)
TCCCTGAGACCCTTTAACCTGTG 
1202


MiR-125b_2
1344
miR-9
TCCCTGAGACCCTAACTTGTGA
258


mir-221
1345
miR-200a(RFAM-
AGCTACATTGTCTGCTGGGTTT
1106




Human)




mir-221
1345
miR-26a (Michael 
AGCTACATTGTCTGCTGGGTTTC 
238




et al)




mir-203
1346
mir-10b
GTGAAATGTTTAGGACCACTAG
197


mir-203
1346
mir-128 (Kosik)
TGAAATGTTTAGGACCACTAG
1068


mir-203
1346
mir-204
TGAAATGTTTAGGACCACTAGA
1069


let-7g
1347
hypothetical
TGAGGTAGTAGTTTGTACAGT
285




miRNA-176




let-7g
1347
mir-1d
TGAGGTAGTAGTTTGTACAGTT
1152


mir-101_3
1348
miR-200a
TACAGTACTGTGATAGCTGAAG
1460


mir-106
1349
miR-200a (RFAM-
CAAAGTGCTAACAGTGCAGGTA
1461




M. mu.)




mir-17/mir-91
1350
mir-123/mir-126as
ACTGCAGTGAGGGCACTTGT
1462


mir-17/mir-91
1350
mir-227* (Kosik) 
CAAAGTGCTTACAGTGCAGGTAG 
1181


mir-17/mir-91
1350
miR-195
CAAAGTGCTTACAGTGCAGGTAGT
204


mir-199b
1351
mir-226* (Kosik) 
CCCAGTGTTTAGACTACCTGTTC
1463


mir-199b
1351
mir-217 (rodent) 
TACAGTAGTCTGCACATTGGTT
1118


hypothetical
1352
mir-324-3p_Ruvkun
AGAGGTATAGCGCATGGGAAGA
1464


miRNA 105






hypothetical
1352
miR-127
TTCCTATGCATATACTTCTTT
1132


miRNA 105






mir-211
1353
mir-244* (Kosik) 
TTCCCTTTGTCATCCTTTGCCT
1465


mir-217
1354
mir-224* (Kosik) 
TACTGCATCAGGAACTGACTGGAT
1466


mir-224 (Sanger)
1355
mir-248* (Kosik)
TAAGTCACTAGTGGTTCCGTTTA
1467


mir-7_3
1356
mir-138
TGGAAGACTTGTGATTTTGTT
1468


mir-325 (Ruvkun)
1357
mir-138_Ruvkun
CCTAGTAGGTGCTCAGTAAGTGT
1469


mir-326 (Ruvkun)
1358
mir-181b
CCTCTGGGCCCTTCCTCCAG
1263


mir-326 (Ruvkun)
1358
miR-298
CCTCTGGGCCCTTCCTCCAGT
1470


mir-329-1
1359
mir-103
AACACACCCAGCTAACCTTTTT
1471


(Ruvkun)






mir-330 (Ruvkun)
1360
miR-134 (RFAM-
GCAAAGCACAGGGCCTGCAGAGA 
1472




Human)




mir-337 (Ruvkun)
1361
miR-146 (RFAM-
TTCAGCTCCTATATGATGCCTTT 
1473




Human)




mir-345 (Ruvkun)
1362
miR-30e (RFAM-
TGCTGACCCCTAGTCCAGTGC
1474




M.mu.)




mir-346 (Ruvkun)
1363
miR-97 (Michael
TGTCTGCCCGAGTGCCTGCCTCT 
1475




et al)




mir-151* (Ruvkun) 
1364
miR-193
ACTAGACTGAGGCTCCTTGAGG
1476


mir-151* (Ruvkun) 
1364
mir-340 (Ruvkun)
CTAGACTGAGGCTCCTTGAGG
1477


mir-151* (Ruvkun) 
1364
miR-299 (RFAM-
TCGAGGAGCTCACAGTCTAGTA
1256


M.mu.)






mir_34b (RFAM)
1365
mir-331 (Ruvkun) 
TAGGCAGTGTAATTAGCTGATTG 
1478


glutamate
1366
miR-143 (Michael
TGTTATAGTATTCCACCTACC
1060


receptor,

et al)




ionotrophic, AMPA






3/hypothetical






miRNA-033






mir-34
1367
mir-138
TGGCAGTGTCTTAGCTGGTTGT
194


mir-34
1367
mir-30a
TGGCAGTGTCTTAGCTGGTTGTT 
1067


mir-7_1/mir-7_1*
1368
mir-191_Ruvkun
CAACAAATCACAGTCTGCCATA
1070


mir-7_1/mir-7_1*
1368
mir-29b
TGGAAGACTAGTGATTTTGTT
198


mir-10b
1369
mir-210
CCCTGTAGAACCGAATTTGTGT
1071


mir-10b
1369
miR-29b (RFAM-
TACCCTGTAGAACCGAATTTGT
199




M.mu.)




mir-10b
1369
mir-34b (mouse)
TACCCTGTAGAACCGAATTTGTG 
1072


mir-132
1370
mir-130a
TAACAGTCTACAGCCATGGTCG
1077


mir-132
1370
miR-196 (Tuschl)
TAACAGTCTACAGCCATGGTCGC 
206


mir-108_1
1371
mir-130 (Kosik)
ATAAGGATTTTTAGGGGCATT
207


mir-212
1372
miR-1 (RFAM)
TAACAGTCTCCAGTCACGGCC
210


hypothetical
26
mir-143
TGGGCAAGAGGACTTTTTAAT
1079


miRNA 023






mir-214
37
mir-15b
ACAGCAGGCACAGACAGGCAG
219


hypothetical
43
mir-145
TGTCAACAAAACTGCTTACAA
1092


miRNA 040






hypothetical
1373
miR-145 (Michael
TGACAGGAAATCTTTGAGAGG
1094


miRNA 043

et al)




mir-205
1374
mir-101
TCCTTCATTCCACCGGAGTCTG
224


mir-33a
1375
miR-29b (RFAM-
GTGCATTGTAGTTGCATTG
227


M.mu.)






mir-196_2
1376
mir-7-1*_Ruvkun
TAGGTAGTTTCATGTTGTTGG
1097


mir-196_2
1376
mir-148a
TAGGTAGTTTCATGTTGTTGGG
228


hypothetical
1377
mir-122a
TTGCATGCCCTATTGATTCTC
1099


miRNA 055






hypothetical
1378
miR-122a, b
TGTCAGATGCTTAATGTTCTT
1102


miRNA 058

(Tuschl)




mir-218_1
1379
mir-140
TTGTGCTTGATCTAACCATGT
234


mir-218_1
1379
mir-196
TTGTGCTTGATCTAACCATGTG
1103


mir-222
1380
miR-200b (Michael
AGCTACATCTGGCTACTGGGTCT 
1107




et al)




mir-222
1380
let-7i
AGCTACATCTGGCTACTGGGTCTC
239


mir-128b
1381
mir-142
TCACAGTGAACCGGTCTCTTT
1073


mir-128b
1381
hypothetical
TCACAGTGAACCGGTCTCTTTC
242




miRNA-023




mir-219_2
1382
mir-30b_Ruvkun
TGATTGTCCAAACGCAATTCT
271


hypothetical
1383
mir-19b
TCACATTTGCCTGCAGAGATT
1109


miRNA 070






mir-129_2
1384
miR-196 (Tuschl) 
AAGCCCTTACCCCAAAAAGCAT
1110


mir-129_2
1384
mir-128 (Kosik)
CTTTTTGCGGTCTGGGCTTGC
243


mir-129_2
1384
miR-142-as
CTTTTTGCGGTCTGGGCTTGCT
1111


mir-133b
1385
miR-142as
TTGGTCCCCTTCAACCAGCTA
244




(Michael et al)




hypothetical
78 
let-7f
TGGTTAAAATATTAATGGGGC
1112


miRNA 075






hypothetical
1386
let-7f (Michael
TGATATGTTTGATATTGGG
1117


miRNA 079

et al)




mir-204
1387
let-7d_Ruvkun
TTCCCTTTGTCATCCTATGCCT
251


mir-204
1387
miR-10b (Tuschl) 
TTCCCTTTGTCATCCTATGCCTG
1121


mir-213/mir-
1388
mir-137
AACATTCAACGCTGTCGGTGAG
1096


181a_2






mir-213/mir-
1388
hypothetical
AACATTCAACGCTGTCGGTGAGT 
223


181a_2

miRNA-043




mir-213/mir-
1388
let-7f (Michael
ACCATCGACCGTTGATTGTACC
253


181a_2

et al)




hypothetical
1389
mir-26a
TAGGCCAAATGGCGCATCAAT
1124


miRNA 090






mir-138_2
1390
mir-92
AGCTGGTGTTGTGAATC
256


mir-138_2
1390
miR-27* (Michael 
AGCTGGTGTTGTGAATCAGGCCG 
1127




et al)




mir-196_1
1391
miR-29b (RFAM-
TAGGTAGTTTCATGTTGTTGG
1097




Human)




mir-1961
1391
mir-7
TAGGTAGTTTCATGTTGTTGGG
228


mir-199a_2
1392
miR-202 (mouse)
CCCAGTGTTCAGACTACCTGTT
1128


mir-199a_2
1392
mir-15a
CCCAGTGTTCAGACTACCTGTTC 
259


mir-199a_2
1392
mir-211 (rodent) 
TACAGTAGTCTGCACATTGGTT
1118


mir-181b_1
1393
mir-16
AACATTCATTGCTGTCGGTGGGTT
260


hypothetical
1394
miR-26a (Michael 
TGACAGTCAATTAACAAGTTT
1130


miRNA 101

et al)




hypothetical
1395
mir-127_Ruvkun
TTCCTCCTCCTCCGACTCGGA
1135


miRNA 111






mir-218_2
1396
mir-33a
TTGTGCTTGATCTAACCATGT
234


mir-218_2
1396
mir-24
TTGTGCTTGATCTAACCATGTG
1103


mir-148b
1397
mir-30d
TCAGTGCATCACAGAACTTTGT
272


mir-216
1398
mir-30d_Ruvkun
TAATCTCAGCTGGCAACTGTG
274


hypothetical
1399
miR-136
TAAACTGGCTGATAATTTTTG
1141


miRNA 137






hypothetical
1400
miR-154
TGCAAGTATGAAAATGAGATT
1142


miRNA 138






mir-210
1401
let-7c
CTGTGCGTGTGACAGCGGCTG
277


mir-223
1402
let-7c_Ruvkun
TGTCAGTTTGTCAAATACCCC
279


hypothetical
1403
miR-149
TGCAAGCAGATGCTGATAATA
1145


miRNA 153






hypothetical
1404
mir-30c
TTAAAGTGGATGTGTGTTATT
1146


miRNA 154






mir-135_1
1405
hypothetical
TATGGCTTTTTATTCCTATGTGA 
1149


miRNA-101






mir-135_1
1405
let-7e
TATGGCTTTTTATTCCTATGTGAT
283


non-coding RNA in 
1406
mir-181b
TATGGCTTTTTATTCCTATGTGA 
1149


rhabdomyosarcoma/






mir-135_2






non-coding RNA in 
1406
miR-155/
TATGGCTTTTTATTCCTATGTGAT
283


rhabdomyosarcoma/

hypothetical




mir-135_2

miRNA-071




hypothetical
1407
mir-30c_Ruvkun
TGATCTTGCTCTAACACTTGG
1157


miRNA 170






glutamate
174 
miR-99b
TGACAAGTATGTTTTATCGTT
1158


receptor,






ionotropic, AMPA






2/hypothetical






miRNA-171






hypothetical
179 
miR-125a
TAGGAGTTTGATATGACATAT
1163


miRNA 176






hypothetical
1408
mir-125b
TGAAAGGCACTTTGTCCAATT
1166


miRNA 179






hypothetical
1409
mir-221
TCACCTGCTCTGGAAGTAGTT
1167


miRNA 181






mir-181c
1410
mir-133a
AACATTCAACCTGTCGGTGAGT
290


mir-100_1
1411
let-7b
AACCCGTAGATCCGAACTTGTG
275


mir-103_1
950
mir-29a
AGCAGCATTGTACAGGGCTATGA
225


mir-107
1412
mir-141
AGCAGCATTGTACAGGGCTATCA 
229


mir-19a
1413
mir-20
TGTGCAAATCTATGCAAAACTGA
268


mir-19b_1
1414
mir-21
AGTTTTGCAGGTTTGCATCCAGC 
1179


mir-19b_1
1414
mir-223
TGTGCAAATCCATGCAAAACTGA 
241


mir-92_1
1415
hypothetical
TATTGCACTTGTCCCGGCCTG
1182




miRNA-090




mir-92_1
1415
miR-9
TATTGCACTTGTCCCGGCCTGT
216


mir-98
1416
mir-131
TGAGGTAGTAAGTTGTATTGTT
257


mir-104
1417
mir-221 (RFAM-
TCAACATCAGTCTGATAAGCTA
335


(Mourelatos)

mmu)




mir-27
1418
mir-213
TTCACAGTGGCTAAGTTCC
1186


(Mourelatos)






mir-27
1418
mir-222 (RFAM-
TTCACAGTGGCTAAGTTCCGC
1187


(Mourelatos)

mmu)




mir-27
1418
mir-203
TTCACAGTGGCTAAGTTCCGCC
1188


(Mourelatos)






mir-31
1419
mir-178 (Kosik)
AGGCAAGATGCTGGCATAGCTG
1197


mir-31
1419
miR-203 (RFAM-
GGCAAGATGCTGGCATAGCTG
1198




M.mu.)




mir-32
1420
let-7g
TATTGCACATTACTAAGTTGC
1199


mir_186
1421
miR-326 (Ruvkun) 
CAAAGAATTCTCCTTTTGGGCTT
1208


mir_191
1422
mir-329 (mouse)
CAACGGAATCCCAAAAGCAGCT
1210


mir_191
1422
miR-27a (RFAM-
CAACGGAATCCCAAAAGCAGCTGT
1211




Human)




mir_195
1423
mir-330 (rodent) 
TAGCAGCACAGAAATATTGGC
1216


mir_193
1424
mir-337 (rodent) 
AACTGGCCTACAAAGTCCCAG
1217


mir_188
1425
mir-345 (rodent)
CATCCCTTGCATGGTGGAGGGT
1219


mir_208
1426
mir-346 (mouse)
ATAAGACGAGCAAAAAGCTTGT
1222


mir_139
1427
mir-151* (Ruvkun)
TCTACAGTGCACGTGTCT
1223


mir-200b
1428
mir-151 (rodent)
CTCTAATACTGCCTGGTAATGATG
1224


mir-200b
1428
mir-216
TAATACTGCCTGGTAATGATGA
1225


mir-200b
1428
mir-219
TAATACTGCCTGGTAATGATGAC 
1226


mir-200a
1429
mir-181a
TAACACTGTCTGGTAACGATG
1227


mir-200a
1429
mir-151L (rodent)
TAACACTGTCTGGTAACGATGT
1228


mir-227*
1430
mir-191
ACTGCCCCAGGTGCTGCTGG
1231


(Kosik)/mir-226*






(Kosik)






mir-227*
1430
hypothetical
CCACTGCCCCAGGTGCTGCTGG
1232


(Kosik)/mir-226*

miRNA-058




(Kosik)






mir-227*
1430
hypothetical
CGCATCCCCTAGGGCATTGGTGT 
1233


(Kosik)/mir-226*

miRNA-055




(Kosik)






mir-244* (Kosik)
1431
mir-218
TCCAGCATCAGTGATTTTGTTGA 
1234


mir-224* (Kosik)
1432
mir-253* (Kosik) 
GCACATTACACGGTCGACCTCT
1235


mir-248* (Kosik)
1433
mir-222
TCTCACACAGAAATCGCACCCGTC
1236


mir-138_3
1434
mir-19b* (Michael
AGCTGGTGTTGTGAATC
256




et al)




mir-138_3
1434
mir-27b
AGCTGGTGTTGTGAATCAGGCCG 
1127


mir-181b_2
1435
mir-15_Ruvkun
AACATTCATTGCTGTCGGTGGGTT
260


mir-103_2
1436
miR-101 (RFAM-
AGCAGCATTGTACAGGGCTATGA 
225




Human)




mir-134 (Sanger)
1437
mir-129
TGTGACTGGTTGACCAGAGGG
1240


mir-146 (Sanger)
1438
mir-129as/mir-
TGAGAACTGAATTCCATGGGTT
1241




258* (Kosik)




mir-30e
1439
miR-129b (RFAM-
TGTAAACATCCTTGACTGGA
1243


(RFAM/mmu)

Human)




mir-30e
1439
miR-135 (RFAM-
TGTAAACATCCTTGACTGGAAG
1244


(RFAM/mmu)

Human)




mir-299
1440
mir-133b
TGGTTTACCGTCCCACATACAT
1246


(RFAM/mmu)






mir-340 (Ruvkun)
1441
miR-188
TCCGTCTCAGTTACTTTATAGCC 
1257


mir-331 (Ruvkun)
1442
miR-208
GCCCCTGGGCCTATCCTAGAA
1258


mir-187
1443
miR-199-s
TCGTGTCTTGTGTTGCAGCCG
1270


mir-187
1443
let-7b_Ruvkun
TCGTGTCTTGTGTTGCAGCCGG
276


miR-201
1444
miR-187 (RFAM-
TACTCAGTAAGGCATTGTTCT
1479




Human)




miR-207
1445
miR-201
GCTTCTCCTGGCTCTCCTCCCTC
1480


miR-291
1446
miR-291
AAAGTGCTTCCACTTTGTGTGCC 
1481


miR-291
1446
miR-207
CATCAAAGTGGAGGCCCTCTCT
1482


miR-292
1447
miR-291
AAGTGCCGCCAGGTTTTGAGTGT 
1483


miR-292
1447
miR-292
ACTCAAACTGGGGGCTCTTTTG
1484


miR-293
1448
miR-292
AGTGCCGCAGAGTTTGTAGTGT
1485


miR-294
1449
miR-293
AAAGTGCTTCCCTTTTGTGTGT
1486


miR-295
1450
miR-294
AAAGTGCTACTACTTTTGAGTCT 
1487


miR-300
1451
miR-295
TATGCAAGGGCAAGCTCTCTTC
1488


miR-322
1452
miR-300
AAACATGAAGCGCTGCAACA
1489


miR-344
1453
miR-322
TGATCTAGCCAAAGCCTGACTGT
1490


miR-350
1454
miR-344
TTCACAAAGCCCATACACTTTCAC
1491


miR-290
1455
miR-350
CTCAAACTATGGGGGCACTTTTT 
1492


miR-351
1456
miR-290
TCCCTGAGGAGCCCTTTGAGCCTG
1493


miR-341
1457
miR-351
TCGATCGGTCGGTCGGTCAGT
1494


miR-298
1458
miR-341
GGCAGAGGAGGGCTGTTCTTCC
1495









A list of rat pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 62. “Pri-miRNA name” indicates the gene name for each of the pri-miRNAs. Also given in table 62 are the name and sequence of the mature miRNA derived from the pri-miRNA. Mature miRNA sequences from pri-miRNA precursors have been proposed by several groups; consequently, for a given pri-miRNA sequence, several miRNAs may be disclosed and given unique names, and thus a given pri-miRNA sequence may occur repeatedly in the table. The sequences are written in the 5′ to 3′ direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.







TABLE 62







Rat pri-miRNA sequences and the corresponding mature miRNAs












SEQ


SEQ



ID


ID


Pri-miRNA name
NO
Mature miRNA name
Mature miRNA sequence
NO














mir-20
1496
miR-20* (mouse)
ACTGCATTACGAGCACTTACA
1608


mir-20
1496
miR-20 (RFAM-
TAAAGTGCTTATAGTGCAGGTA
1126




Human)




mir-20
1496
mir-20
TAAAGTGCTTATAGTGCAGGTAG
254


mir-151* (Ruvkun)
1497
mir-151L (rodent)
ACTAGACTGAGGCTCCTTGAGG
1476


mir-151* (Ruvkun) 
1497
mir-151 (rodent) 
CTAGACTGAGGCTCCTTGAGG
1477


mir-151* (Ruvkun) 
1497
mir-151* (Ruvkun)
TCGAGGAGCTCACAGTCTAGTA
1256


mir-346 (Ruvkun)
1498
miR-346 (rat)
TGTCTGCCTGAGTGCCTGCCTCT
1609


mir-143
1499
miR-143 (Michael 
TGAGATGAAGCACTGTAGCTC
1088




et al)




mir-143
1499
mir-143
TGAGATGAAGCACTGTAGCTCA
220


mir-203
1500
mir-203
GTGAAATGTTTAGGACCACTAG
197


mir-203
1500
miR-203(RFAM-M. 
TGAAATGTTTAGGACCACTAG
1068




mu.)




mir-203
1500
miR-203(Tuschl)
TGAAATGTTTAGGACCACTAGA
1069


mir-26b
1501
miR-26b (RFAM-
TTCAAGTAATTCAGGATAGGT
1147




Human)




mir-26b
1501
mir-26b
TTCAAGTAATTCAGGATAGGTT
281


mir-128a
1276
mir-128 (Kosik)
TCACAGTGAACCGGTCTCTTT
1073


mir-128a
1276
mir-128a
TCACAGTGAACCGGTCTCTTTT
200


mir-29b_1
1277
miR-29b (RFAM-
TAGCACCATTTGAAATCAGT
1172




Human)




mir-29b_1
1277
miR-29b (RFAM-M. 
TAGCACCATTTGAAATCAGTGT
1173




mu.)




mir-29b_1
1502
mir-29b
TAGCACCATTTGAAATCAGTGTT
195


mir-29c
1278
mir-29c
CTAGCACCATTTGAAATCGGTT
232


mir-29c
1278
miR-29c (Tuschl)
TAGCACCATTTGAAATCGGTTA
1100


mir-123/mir-126
1503
mir-123/mir-126as
CATTATTACTTTTGGTACGCG
205


mir-123/mir-126
1503
mir-126
TCGTACCGTGAGTAATAATGC
1076


mir-130a
1504
mir-130a
CAGTGCAATGTTAAAAGGGC
233


mir-130a
1504
mir-130 (Kosik)
CAGTGCAATGTTAAAAGGGCAT
1101


mir-124a_3
1282
mir-124a (Kosik) 
TAAGGCACGCGGTGAATGCCA
1104


mir-124a_3
1282
mir-124a
TTAAGGCACGCGGTGAATGCCA
235


mir-124a_3
1282
mir-124a_Ruvkun
TTAAGGCACGCGGTGAATGCCAA
1105


mir-15b
1286
miR-15b (Michael 
TAGCAGCACATCATGGTTTAC
1115




et al)




mir-15b
1286
mir-15b
TAGCAGCACATCATGGTTTACA
246


mir-163
1505
mir-16
TAGCAGCACGTAAATATTGGCG
196


mir-163
1505
mir-16_Ruvkun
TAGCAGCACGTAAATATTGGCGT
1176


mir-137
1288
mir-137
TATTGCTTAAGAATACGCGTAG
270


mir-101_1
1289
mir-101
TACAGTACTGTGATAACTGA
265


mir-101_1
1289
miR-101 (RFAM-
TACAGTACTGTGATAACTGAAG
1170




Human)




mir-29a
1291
mir-29a
CTAGCACCATCTGAAATCGGTT
247


mir-29a
1291
mir-29a_Ruvkun
TAGCACCATCTGAAATCGGTTA
1116


mir-29b_2
1292
miR-29b (RFAM-
TAGCACCATTTGAAATCAGT
1172




Human)




mir-29b_2
1292
miR-29b (RFAM-M. 
TAGCACCATTTGAAATCAGTGT
1173




mu.)




mir-29b_2
1292
mir-29b
TAGCACCATTTGAAATCAGTGTT
195


mir-131_3/mir-9
1506
mir-131
TAAAGCTAGATAACCGAAAGT
211


mir-131_3/mir-9
1506
mir-131_Ruvkun
TAAAGCTAGATAACCGAAAGTA
1080


mir-131_3/mir-9
1506
miR-9
TCTTTGGTTATCTAGCTGTATGA
1081


mir-23a
1507
mir-23a
ATCACATTGCCAGGGATTTCC
289


mir-140
1508
mir-140
AGTGGTTTTACCCTATGGTAG
192


mir-140
1508
miR-140-as
TACCACAGGGTAGAACCACGGA
1065


mir-140
1508
mir-239* (Kosik) 
TACCACAGGGTAGAACCACGGACA
1066


mir-125b_1
1509
mir-125b
TCCCTGAGACCCTAACTTGTGA
258


mir-26a_1
1510
miR-26a (Michael 
TTCAAGTAATCCAGGATAGGC
1203




et al)




mir-26a_1
1510
mir-26a
TTCAAGTAATCCAGGATAGGCT
226


let-7i
1302
let-7i
TGAGGTAGTAGTTTGTGCT
209


let-7i
1302
let-7i_Ruvkun
TGAGGTAGTAGTTTGTGCTGTT
1078


mir-21
1511
mir-21
TAGCTTATCAGACTGATGTTGA
236


mir-22
1512
mir-22
AAGCTGCCAGTTGAAGAACTGT
215


mir-142
1513
mir-142
CATAAAGTAGAAAGCACTAC
217


mir-142
1513
miR-142-as
TGTAGTGTTTCCTACTTTATGG
1086


mir-142
1513
miR-142as
TGTAGTGTTTCCTACTTTATGGA
1087




(Michael et al)




mir-144
1514
mir-144
TACAGTATAGATGATGTACTAG
237


mir-152
1515
mir-152
TCAGTGCATGACAGAACTTGG
282


mir-153_2
1516
mir-153
TTGCATAGTCACAAAAGTGA
201


let-7a_1
1517
let-7a
TGAGGTAGTAGGTTGTATAGTT
222


let-7d
1518
let-7d
AGAGGTAGTAGGTTGCATAGT
245


let-7d
1518
let-7d_Ruvkun
AGAGGTAGTAGGTTGCATAGTT
1113


let-7d
1518
let-7d* (RFAM-M. 
CTATACGACCTGCTGCCTTTCT
1114




mu.)




let-7f_1
1519
let-7f (Michael
TGAGGTAGTAGATTGTATAGT
1098




et al)




let-7f_1
1519
let-7f
TGAGGTAGTAGATTGTATAGTT
231


miR-24-1
1313
miR-189 (RFAM-
GTGCCTACTGAGCTGATATCAGT
1271




Human)




miR-24-1
1313
mir-24
TGGCTCAGTTCAGCAGGAACAG
264


mir-124a_1
1318
mir-124a (Kosik) 
TAAGGCACGCGGTGAATGCCA
1104


mir-124a_1
1318
mir-124a
TTAAGGCACGCGGTGAATGCCA
235


mir-124a_1
1318
mir-124a_Ruvkun
TTAAGGCACGCGGTGAATGCCAA
1105


mir-18
1319
mir-18
TAAGGTGCATCTAGTGCAGATA
262


mir-18
1319
mir-18_Ruvkun
TAAGGTGCATCTAGTGCAGATAG
1177


mir-30b
1520
mir-30b
TGTAAACATCCTACACTCAGC
266


mir-30b
1520
mir-30b_Ruvkun
TGTAAACATCCTACACTCAGCT
1137


mir-30d
1521
mir-30d
TGTAAACATCCCCGACTGGAAG
240


mir-30d
1521
mir-30d_Ruvkun
TGTAAACATCCCCGACTGGAAGCT
1108


let-7b
1522
let-7b
TGAGGTAGTAGGTTGTGTGGTT
212


let-7b
1522
let-7b_Ruvkun
TGAGGTAGTAGGTTGTGTGGTTT
1082


let-7e
1328
let-7e
TGAGGTAGGAGGTTGTATAGT
249


mir-133a_1
1330
mir-133a
TTGGTCCCCTTCAACCAGCTGT
255


mir-145
1332
miR-145 (Michael 
GTCCAGTTTTCCCAGGAATCC
1122




et al)




mir-145
1332
mir-145
GTCCAGTTTTCCCAGGAATCCCTT
252


mir-122a
1523
miR-122a, b
TGGAGTGTGACAATGGTGTTTG
1084




(Tuschl)




mir-122a
1523
mir-122a
TGGAGTGTGACAATGGTGTTTGT
214


let-7f_2
1335
let-7f (Michael
TGAGGTAGTAGATTGTATAGT
1098




et al)




let-7f_2
1335
let-7f
TGAGGTAGTAGATTGTATAGTT
231


mir-127
1337
mir-127_Ruvkun
TCGGATCCGTCTGAGCTTGG
1204


mir-127
1337
miR-127
TCGGATCCGTCTGAGCTTGGCT
1205


mir-136
1338
miR-136
ACTCCATTTGTTTTGATGATGGA
1206


mir-154
1339
miR-154
TAGGTTATCCGTGTTGCCTTCG
1207


mir-30c_2
1341
mir-30c
TGTAAACATCCTACACTCTCAGC
280


mir-30c_2
1341
mir-30c_Ruvkun
TGTAAACATCCTACACTCTCAGCT
1129


mir-99b
1342
miR-99b
CACCCGTAGAACCGACCTTGCG
1201


MiR-125a
1524
miR-125a
TCCCTGAGACCCTTTAACCTGTG
1202


mir-221
1525
mir-221 (RFAM-
AGCTACATTGTCTGCTGGGTTT
1106




mmu)




mir-221
1525
mir-221
AGCTACATTGTCTGCTGGGTTTC
238


mir-101_3
1526
mir-101b (rodent)
TACAGTACTGTGATAGCTGAAG
1460


mir-17/mir-91
1527
mir-17 (human,
ACTGCAGTGAAGGCACTTGT
1180




rat)




mir-17/mir-91
1527
mir-91_Ruvkun
CAAAGTGCTTACAGTGCAGGTAG
1181


mir-17/mir-91
1527
mir-17as/mir-91
CAAAGTGCTTACAGTGCAGGTAGT
204


hypothetical
1528
hypothetical
TTCCTATGCATATACTTCTTT
1132


miRNA 105

miRNA-105




mir-211
1529
mir-211 (rodent) 
TTCCCTTTGTCATCCTTTGCCT
1465


mir-217
1530 
mir-217 (rodent)
TACTGCATCAGGAACTGACTGGAT
1466


mir-7_3
1531
mir-7b (rodent)
TGGAAGACTTGTGATTTTGTT
1468


mir-325 (Ruvkun)
1357
mir-325 (rodent) 
CCTAGTAGGTGCTCAGTAAGTGT
1469


mir-326 (Ruvkun)
1532
miR-326 (Ruvkun) 
CCTCTGGGCCCTTCCTCCAG
1263


mir-326 (Ruvkun)
1532
mir-326 (rodent) 
CCTCTGGGCCCTTCCTCCAGT
1470


mir-330 (Ruvkun)
1533
mir-330 (rodent) 
GCAAAGCACAGGGCCTGCAGAGA
1472


mir-337 (Ruvkun)
1361
mir-337 (rodent)
TTCAGCTCCTATATGATGCCTTT
1473


mir-345 (Ruvkun)
1362
mir-345 (rodent)
TGCTGACCCCTAGTCCAGTGC
1474


mir_34b (RFAM)
1365
mir-34b (mouse)
TAGGCAGTGTAATTAGCTGATTG
1478


mir-34
1534
mir-34
TGGCAGTGTCTTAGCTGGTTGT
194


mir-34
1534
miR-172 (RFAM-M. 
TGGCAGTGTCTTAGCTGGTTGTT
1067




mu.)




mir-7_1/mir-7_1*
1535
mir-7_1*_Ruvkun
CAACAAATCACAGTCTGCCATA
1070


mir-7_1/mir-7_1*
1535
mir-7
TGGAAGACTAGTGATTTTGTT
198


mir-10b
1536
miR-10b (Tuschl)
CCCTGTAGAACCGAATTTGTGT
1071


mir-10b
1536
mir-10b
TACCCTGTAGAACCGAATTTGT
199


mir-10b
1536
miR-10b (Michael 
TACCCTGTAGAACCGAATTTGTG
1072




et al)




mir-132
1370
miR-132 (RFAM-
TAACAGTCTACAGCCATGGTCG
1077




Human)




mir-132
1370
mir-132
TAACAGTCTACAGCCATGGTCGC
206


mir-212
1537
mir-212
TAACAGTCTCCAGTCACGGCC
210


mir-108_1
1538
mir-108
ATAAGGATTTTTAGGGGCATT
207


hypothetical
26
hypothetical
TGGGCAAGAGGACTTTTTAAT
1079


miRNA 023

miRNA-023




mir-214
1539
mir-214
ACAGCAGGCACAGACAGGCAG
219


hypothetical
43
hypothetical
TGTCAACAAAACTGCTTACAA
1092


miRNA 040

miRNA-040




hypothetical
1540
hypothetical
TGACAGGAAATCTTTGAGAGG
1094


miRNA 043

miRNA-043




mir-205
1541
mir-205
TCCTTCATTCCACCGGAGTCTG
224


mir-33a
1542
mir-33a
GTGCATTGTAGTTGCATTG
227


mir-196_2
1543
miR-196 (Tuschl)
TAGGTAGTTTCATGTTGTTGG
1097


mir-196_2
1543
mir-196
TAGGTAGTTTCATGTTGTTGGG
228


mir-218_1
1544
mir-218
TTGTGCTTGATCTAACCATGT
234


mir-218_1
1544
mir-253* (Kosik) 
TTGTGCTTGATCTAACCATGTG
1103


mir-222
1545
mir-222 (RFAM-
AGCTACATCTGGCTACTGGGTCT
1107




mmu)




mir-222
1545
mir-222
AGCTACATCTGGCTACTGGGTCTC
239


mir-128b
1381
mir-128 (Kosik)
TCACAGTGAACCGGTCTCTTT
1073


mir-128b
1381
mir-128b
TCACAGTGAACCGGTCTCTTTC
242


mir-219_2
1546
mir-219
TGATTGTCCAAACGCAATTCT
271


hypothetical
1547
hypothetical
TCACATTTGCCTGCAGAGATT
1109


miRNA 070

miRNA-070




mir-129_2
1548
mir-129as/mir-
AAGCCCTTACCCCAAAAAGCAT
1110




258* (Kosik)




mir-129_2
1548
mir-129
CTTTTTGCGGTCTGGGCTTGC
243


mir-129_2
1548
miR-129b (RFAM-
CTTTTTGCGGTCTGGGCTTGCT
1111




Human)




mir-133b
1385
mir-133b
TTGGTCCCCTTCAACCAGCTA
244


hypothetical
78
hypothetical
TGGTTAAAATATTAATGGGGC
1112


miRNA 075

miRNA-075




mir-204
1549
mir-204
TTCCCTTTGTCATCCTATGCCT
251


mir-204
1549
miR-204 (Tuschl) 
TTCCCTTTGTCATCCTATGCCTG 
1121


mir-213/mir-
1550
mir-178 (Kosik)
AACATTCAACGCTGTCGGTGAG
1096


181a_2






mir-213/mir-
1550
mir-181a
AACATTCAACGCTGTCGGTGAGT 
223


181a_2






mir-213/mir-
1550
mir-213
ACCATCGACCGTTGATTGTACC
253


181a_2






hypothetical
1551
hypothetical
TAGGCCAAATGGCGCATCAAT
1124


miRNA 090

miRNA-090




mir-138_2
1552
mir-138
AGCTGGTGTTGTGAATC
256


mir-138_2
1552
mir-138_Ruvkun
AGCTGGTGTTGTGAATCAGGCCG 
1127


mir-199a_2
1553
miR-199-s
CCCAGTGTTCAGACTACCTGTT
1128


mir-199a_2
1553
mir-199a
CCCAGTGTTCAGACTACCTGTTC 
259


mir-199a_2
1553
miR-199-as
TACAGTAGTCTGCACATTGGTT
1118


hypothetical
1554
hypothetical
TGACAGTCAATTAACAAGTTT
1130


miRNA 101

miRNA-101




mir-148b
1397
mir-148b
TCAGTGCATCACAGAACTTTGT
272


mir-216
1555
mir-216
TAATCTCAGCTGGCAACTGTG
274


hypothetical
1399
hypothetical
TAAACTGGCTGATAATTTTTG
1141


miRNA 137

miRNA-137




hypothetical
1556
hypothetical
TGCAAGTATGAAAATGAGATT
1142


miRNA 138

miRNA-138




mir-210
1557
mir-210
CTGTGCGTGTGACAGCGGCTG
277


mir-223
1558
mir-223
TGTCAGTTTGTCAAATACCCC
279


hypothetical
1404
hypothetical
TTAAAGTGGATGTGTGTTATT
1146


miRNA 154

miRNA-154




non-coding RNA in
13
miR-135 (RFAM-
TATGGCTTTTTATTCCTATGTGA 
1149


rhabdomyosarcoma/

Human)




mir-1352






non-coding RNA in
13
mir-135
TATGGCTTTTTATTCCTATGTGAT
283


rhabdomyosarcoma/






mir-135_2






hypothetical
1559
hypothetical
TGATCTTGCTCTAACACTTGG
1157


miRNA 170

miRNA-170




glutamate
174
hypothetical
TGACAAGTATGTTTTATCGTT
1158


receptor,

miRNA-171




ionotropic, AMPA






2/hypothetical






miRNA-171






hypothetical
179
hypothetical
TAGGAGTTTGATATGACATAT
1163


miRNA 176

miRNA-176




hypothetical
1560
hypothetical
TGAAAGGCACTTTGTCCAATT
1166


miRNA 179

miRNA-179




hypothetical
1409
hypothetical
TCACCTGCTCTGGAAGTAGTT
1167


miRNA 181

miRNA-181




mir-181c
1410
mir-181c
AACATTCAACCTGTCGGTGAGT
290


mir-100_1
1561
mir-100
AACCCGTAGATCCGAACTTGTG
275


mir-103_1
950
mir-103
AGCAGCATTGTACAGGGCTATGA 
225


mir-107
1562
mir-107
AGCAGCATTGTACAGGGCTATCA 
229


mir-19a
1563
mir-19a
TGTGCAAATCTATGCAAAACTGA
268


mir-19b_1
1414
mir-19b* (Michael
AGTTTTGCAGGTTTGCATCCAGC
1179




et al)




mir-19b_1
1414
mir-19b
TGTGCAAATCCATGCAAAACTGA
241


mir-92_1
1564
miR-92 (RFAM-M.
TATTGCACTTGTCCCGGCCTG
1182




mu.)




mir-92_1
1564
mir-92
TATTGCACTTGTCCCGGCCTGT
216


mir-98
1565
mir-98
TGAGGTAGTAAGTTGTATTGTT
257


mir-104
1566
miR-104
TCAACATCAGTCTGATAAGCTA
335


(Mourelatos)

(Mourelatos)




mir-27
1567
miR-27
TTCACAGTGGCTAAGTTCC
1186


(Mourelatos)

(Mourelatos)




mir-27
1567
miR-27a (RFAM-M.
TTCACAGTGGCTAAGTTCCGC
1187


(Mourelatos)

mu.)




mir-27
1567
miR-27a (RFAM-
TTCACAGTGGCTAAGTTCCGCC
1188


(Mourelatos)

Human)




mir-31
1568
miR-31 (RFAM-M.
AGGCAAGATGCTGGCATAGCTG
1197




mu.)




mir-31
1568
miR-31 (Tuschl)
GGCAAGATGCTGGCATAGCTG
1198


mir-32
1569
miR-32 (Tuschl)
TATTGCACATTACTAAGTTGC
1199


mir 186
1570
miR-186
CAAAGAATTCTCCTTTTGGGCTT
1208


mir 191
1571
mir-191
CAACGGAATCCCAAAAGCAGCT
1210


mir 191
1422
mir-191Ruvkun
CAACGGAATCCCAAAAGCAGCTGT
1211


mir 195
1572
miR-195
TAGCAGCACAGAAATATTGGC
1216


mir 193
1573
miR-193
AACTGGCCTACAAAGTCCCAG
1217


mir 208
1574
miR-208
ATAAGACGAGCAAAAAGCTTGT
1222


mir 139
1427
miR-139
TCTACAGTGCACGTGTCT
1223


mir-200b
1428
miR-200a (RFAM-
CTCTAATACTGCCTGGTAATGATG
1224




Human)




mir-200b
1428
miR-200b (Michael
TAATACTGCCTGGTAATGATGA
1225




et al)




mir-200b
1428
miR-200b
TAATACTGCCTGGTAATGATGAC
1226


mir-200a
1429
miR-200a
TAACACTGTCTGGTAACGATG
1227


mir-200a
1429
miR-200a (RFAM-M.
TAACACTGTCTGGTAACGATGT
1228




mu.)




mir-227*
1430
mir-226* (Kosik) 
ACTGCCCCAGGTGCTGCTGG
1231


(Kosik)/mir-226*






(Kosik)






mir-227*
1430
mir-324-3p_Ruvkun
CCACTGCCCCAGGTGCTGCTGG
1232


(Kosik)/mir-226*






(Kosik)






mir-227*
1430
mir-227* (Kosik) 
CGCATCCCCTAGGGCATTGGTGT
1233


(Kosik)/mir-226*






(Kosik)






mir-244* (Kosik)
1431
mir-244* (Kosik)
TCCAGCATCAGTGATTTTGTTGA
1234


mir-224* (Kosik)
1432
mir-224* (Kosik)
GCACATTACACGGTCGACCTCT
1235


mir-248* (Kosik)
1433
mir-248* (Kosik) 
TCTCACACAGAAATCGCACCCGTC
1236


mir-138_3
1575
mir-138
AGCTGGTGTTGTGAATC
256


mir-138_3
1575
mir-138_Ruvkun
AGCTGGTGTTGTGAATCAGGCCG
1127


mir-181b_2
1576
mir-181b
AACATTCATTGCTGTCGGTGGGTT
260


mir-134 (Sanger)
1289
miR-134 (RFAM-
TGTGACTGGTTGACCAGAGGG
1240




Human)




mir-146 (Sanger)
1577
miR-146 (RFAM-
TGAGAACTGAATTCCATGGGTT
1241




Human)




mir-30e
1578
miR-30e (RFAM-M. 
TGTAAACATCCTTGACTGGA
1243


(RFAM/mmu)

mu.)




mir-30e
1578
miR-97 (Michael
TGTAAACATCCTTGACTGGAAG
1244


(RFAM/mmu)

et al)




mir-299
1440
miR-299 (RFAM-M.
TGGTTTACCGTCCCACATACAT
1246


(RFAM/mmu)

mu.)




mir-34a
1579
mir-34c (RFAM)
AGGCAGTGTAGTTAGCTGATTG
1250


(RFAM/mmu)






mir-34a
1579
miR-34a (RFAM-M. 
AGGCAGTGTAGTTAGCTGATTGC
1251


(RFAM/mmu)

mu.)




mir-135b (Ruvkun) 
1580
mir-135b (Ruvkun)
TATGGCTTTTCATTCCTATGTG
1254


mir-331 (Ruvkun)
1442
mir-331 (Ruvkun) 
GCCCCTGGGCCTATCCTAGAA
1258


mir-187
1443
miR-187 (RFAM-
TCGTGTCTTGTGTTGCAGCCG
1270




Human)




mir-187
1443
mir-187
TCGTGTCTTGTGTTGCAGCCGG
276


collagen, type I, 
1581
hypothetical
AGACATGTTCAGCTTTGTGGA
1063


alpha 1/

miRNA-144




hypothetical






miRNA-144






DiGeorge syndrome 
1582
hypothetical
TGTGATTTCCAATAATTGAGG
1123


critical region

miRNA-088




gene 8/






hypothetical






miRNA-088






hypothetical miR- 
1583
miR-190
TGATATGTTTGATATATTAGGT
1075


13/miR-190






hypothetical
1584
hypothetical
TAAGACTTGCAGTGATGTTTA
1091


miRNA 039

miRNA-039




hypothetical
1585
hypothetical
TACCAGTTGTTTTCTCTGTGA
1093


miRNA 041

miRNA-041




hypothetical
47
hypothetical
TTCCACTCTGTTTATCTGACA
1095


miRNA 044

miRNA-044




hypothetical
86
hypothetical
TTACATGGGGAAGCTATCATA
1119


miRNA 083

miRNA-083




hypothetical
1586
hypothetical
TGACAGTTTATTGGCTTTATC
1133


miRNA 107

miRNA-107




mir-10a
1587
mir-10a (Tuschl) 
TACCCTGTAGATCCGAATTTGT
1139


mir-10a
1587
mir-10a
TACCCTGTAGATCCGAATTTGTG
267


mir-130b
1588
mir-130b
CAGTGCAATGATGAAAGGGC
273


mir-130b
1588
mir-266* (Kosik) 
CAGTGCAATGATGAAAGGGCAT
1140


hypothetical
1589
hypothetical
AGACAAACATGCTACTCTCAC
1164


miRNA-177_1

miRNA-177




mir_185
1590
miR-185
TGGAGAGAAAGGCAGTTC
1218


mir_194_2
1591
miR-194
TGTAACAGCAACTCCATGTGGA
1221


mir-150 (Sanger)
1592
miR-150 (RFAM-
TCTCCCAACCCTTGTACCAGTG
1242




Human)




mir-301
1593
miR-301 (RFAM-M. 
CAGTGCAATAGTATTGTCAAAGC
1247


(RFAM/mmu)

mu.)




mir-301
1593
mir-301_Ruvkun
CAGTGCAATAGTATTGTCAAAGCAT
1248


(RFAM/mmu)






mir_320
1594
miR-320
AAAAGCTGGGTTGAGAGGGCGAA
1252


mir_200c (RFAM)
1595
mir-200c (RFAM) 
AATACTGCCGGGTAATGATGGA
1259


miR-322
1596
miR-322
AAACATGAAGCGCTGCAACA
1489


miR-341
1457
miR-341
TCGATCGGTCGGTCGGTCAGT
1494


miR-344
1597
miR-344
TGATCTAGCCAAAGCCTGACCGT 
1610


miR-350
1598
miR-350
TTCACAAAGCCCATACACTTTCAC
1491


miR-351
1599
miR-351
TCCCTGAGGAGCCCTTTGAGCCTG
1493


miR-290
1600
miR-290
CTCAAACTATGGGGGCACTTTTT 
1492


miR-291
1601
miR-291
AAAGTGCTTCCACTTTGTGTGCC 
1481


miR-291
1601
miR-291
CATCAAAGTGGAGGCCCTCTCT
1482


miR-292
1602
miR-292
AAGTGCCGCCAGGTTTTGAGTGT 
1483


miR-292
1602
miR-292
ACTCAAACTGGGGGCTCTTTTG
1484


miR-298
1603
miR-298
GGCAGAGGAGGGCTGTTCTTCC
1495


miR-300
1604
miR-300
TATGCAAGGGCAAGCTCTCTTC
1488


miR-333
1605
miR-333
GTGGTGTGCTAGTTACTTTT
1611


miR-336
1606
miR-336
TCACCCTTCCATATCTAGTCT
1612


miR-349
1607
miR-349
CAGCCCTGCTGTCTTAACCTCT
1613









A list of Drosophila pri-miRNAs and the mature miRNAs predicted to derive from them is shown in Table 63. “Pri-miRNA name” indicates the gene name for each of the pri-miRNAs, and “pri-miRNA sequence” indicates the sequence of the predicted primary miRNA transcript. Also given in table 63 are the name and sequence of the mature miRNA derived from the pri-miRNA. The sequences are written in the 5′ to 3′ direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.







TABLE 63







Drosophila pri-miRNA sequences and the corresponding mature miRNAs












Pri-

SEQ


SEQ


miRNA

ID
Mature miRNA

ID


name
Pri-miRNA sequence
NO
name
Mature miRNA sequence
NO





mir-14
GGAGCGAGACGGGGACTCACT
1614
miR-14
TCAGTCTTTTTCTCTCTCCTA
1616



GTGCTTATTAAATAGTCAGTC







TTTTTCTCTCTCCTATACAAA







TTGCGGGC






mir-
AATGATTTGACTACGAAACCG
1615
mir-Bantam
GTGAGATCATTTTGAAAGCTG
1617


bantam
GTTTTCGATTTGGTTTGACTG







TTTTTCATACAAGTGAGATCA







TTTTGAAAGCTGATTTTGTCA







ATGAATA









Oligomeric compounds targeting or mimicking pri-miRNAs, pre-miRNAs, or miRNAs were given internal numerical identifiers (ISIS Numbers) and are shown in Tables 64, 65, and 66 respectively. The sequences are written in the 5′ to 3′ direction and are represented in the DNA form. It is understood that a person having ordinary skill in the art would be able to convert the sequence of the targets to their RNA form by simply replacing the thymidine (T) with uracil (U) in the sequence.
Table 64 describes a series of oligomeric compounds designed and synthesized to target different regions of pri-miRNAs. These oligomeric compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets. In Table 64, “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. All compounds listed in Table 64 have phosphorothioate internucleoside linkages. In some embodiments, chimeric oligonucleotides (“gapmers”) are composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five nucleotide “wings,” wherein the wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. These chimeric compounds are indicated in the “Chemistry” column as “5-10-5 MOE gapmer.” In some embodiments, oligomeric compound consist of 2′-MOE ribonucleotides throughout, and these are indicated by “uniform MOE.”







TABLE 64







Phosphorothioate oligomeric compounds targeting pri-miRNAs












SEQ






ID





ISIS #
NO
sequence
chemistry
Pri-miRNA














338615
442
AGAACAGCATGACGTAACCT 
uniform MOE
mir-140, Human


338616
443
GCCCATCTGTGGCTTCACAG 
uniform MOE
mir-30a, Human


338617
444
GAAGTCCGAGGCAGTAGGCA 
uniform MOE
mir-30a, Human


338618
445
CTTCCTTACTATTGCTCACA 
uniform MOE
mir-34, Human


338619
446
GCTAGATACAAAGATGGAAA 
uniform MOE
mir-29b-1, Human


338620
447
CTAGACAATCACTATTTAAA 
uniform MOE
mir-29b-2, Human


338621
448
GCAGCGCAGCTGGTCTCCCC 
uniform MOE
mir-29b-2, Human


338622
449
TAATATATATTTCACTACGC 
uniform MOE
mir-16-3, Human


338623
450
TGCTGTATCCCTGTCACACT 
uniform MOE
mir-16-3, Human


338624
451
CAATTGCGCTACAGAACTGT 
uniform MOE
mir-203, Human


338625
452
TCGATTTAGTTATCTAAAAA 
uniform MOE
mir-7-1, Human


338626
453
CTGTAGAGGCATGGCCTGTG 
uniform MOE
mir-7-1, Human


338627
454
TGACTATACGGATACCACAC 
uniform MOE
mir-10b, Human


338628
455
GGAACAAGGCCAATTATTGC 
uniform MOE
mir-128a, Human


338629
456
AGAAATGTAAACCTCTCAGA 
uniform MOE
mir-128a, Human


338630
457
AGCTGTGAGGGAGAGAGAGA 
uniform MOE
mir-153-1, Human


338631
458
CTGGAGTGAGAATACTAGCT 
uniform MOE
mir-153-1, Human


338632
459
ACTGGGCTCATATTACTAGC 
uniform MOE
mir-153-2, Human


338633
460
TTGGATTAAATAACAACCTA 
uniform MOE
hypothetical miR-






13/miR-190, Human


338634
461
CCCGGAGACAGGGCAAGACA 
uniform MOE
hypothetical miR-






13/miR-190, Human


338635
462
AAAGCGGAAACCAATCACTG 
uniform MOE
chromosome 9 ORF3






containing mir-23b,






mir-24-2 and mir-27b,






Human


338636
463
GTCCCCATCTCACCTTCTCT 
uniform MOE
chromosome 9 ORF3






containing mir-23b,






mir-24-2 and mir-27b,






Human


338637
464
TCAGAGCGGAGAGACACAAG
uniform MOE
mir-96, Human


338638
465
TAGATGCACATATCACTACC
uniform MOE
miR-17/mir-91, Human


338639
466
CTTGGCTTCCCGAGGCAGCT
uniform MOE
miR-17/mir-91, Human


338640
467
AGTTTGAAGTGTCACAGCGC
uniform MOE
mir-123/mir-126, Human


338641
468
GCGTTTTCGATGCGGTGCCG
uniform MOE
mir-123/mir-126, Human


338642
469
GAGACGCGGGGGCGGGGCGC
uniform MOE
mir-132, Human


338643
470
TACCTCCAGTTCCCACAGTA
uniform MOE
mir-132, Human


338644
471
TGTGTTTTCTGACTCAGTCA
uniform MOE
mir-108-1, Human


338645
472
AGAGCACCTGAGAGCAGCGC
uniform MOE
chromosome 9 ORF3






containing mir-23b,






mir-24-2 and mir-27b,






Human


338646
473 
TCTTAAGTCACAAATCAGCA
uniform MOE
chromosome 9 ORF3






containing mir-23b,






mir-24-2 and mir-27b,






Human


338647
474
TCTCCACAGCGGGCAATGTC
uniform MOE
let-7i, Human


338648
475
GGCGCGCTGTCCGGGCGGGG
uniform MOE
mir-212, Human


338649
476
ACTGAGGGCGGCCCGGGCAG
uniform MOE
mir-212, Human


338650
477
GTCCTCTTGCCCAAGCAACA
uniform MOE
hypothetical miRNA-023,






Human


338651
478
GAAGACCAATACACTCATAC
uniform MOE
mir-131-2/miR-9, Human


338652
479
CCGAGGGGCAACATCACTGC
uniform MOE
let-7b, Human


338653
480 
TCCATAGCTTAGCAGGTCCA
uniform MOE
mir-1d-1, Human


338654
481
TTTGATAGTTTAGACACAAA
uniform MOE
mir-122a, Human


338655
482
GGGAAGGATTGCCTAGCAGT
uniform MOE
mir-122a, Human


338656
483 
AGCTTTAGCTGGGTCAGGAC
uniform MOE
mir-22, Human


338657
484
TACCATACAGAAACACAGCA
uniform MOE
mir-92-1, Human


338658
485
TCACAATCCCCACCAAACTC
uniform MOE
mir-92-1, Human


338659
486
TCACTCCTAAAGGTTCAAGT
uniform MOE
hypothetical miRNA-30,






Human


338660
487
CACCCTCCAGTGCTGTTAGT
uniform MOE
mir-142, Human


338661
488 
CTGACTGAGACTGTTCACAG
uniform MOE
mir-183, Human


338662
489 
CCTTTAGGGGTTGCCACACC
uniform MOE
glutamate receptor,






ionotrophic, AMPA 3/






hypothetical miRNA-033,






Human


338663
490
ACAGGTGAGCGGATGTTCTG
uniform MOE
mir-214, Human


338665
492
AGAGGGGAGACGAGAGCACT
uniform MOE
mir-192-1, Human


338666
493
TCACGTGGAGAGGAGTTAAA
uniform MOE
hypothetical miRNA-039,






Human


338667
494
AGTGCTAATACTTCTTTCAT
uniform MOE
hypothetical miRNA-040,






Human


338668
495 
ACCTGTGTAACAGCCGTGTA
uniform MOE
hypothetical miRNA-041,






Human


338669
496 
TTATCGGAACTTCACAGAGA
uniform MOE
hypothetical miRNA-041,






Human


338670
497
TCCCATAGCAGGGCAGAGCC
uniform MOE
let-7a-3, Human


338671
498
GGCACTTCATTGCTGCTGCC
uniform MOE
hypothetical miRNA-043,






Human


338672
499
GGAGCCTTGCGCTCAGCATT
uniform MOE
hypothetical miRNA-043,






Human


338673
500 
ATGGTAATTTCATTTCAGGC
uniform MOE
hypothetical miRNA-044,






Human


338674
501 
GATTGCACATCCACACTGTC
uniform MOE
hypothetical miRNA-044,






Human


338675
502 
GCTGGCCTGATAGCCCTTCT
uniform MOE
mir-181a, Human


338676
503 
GTTTTTTCAAATCCCAAACT
uniform MOE
mir-181a, Human


338677
504 
CCCAGTGGTGGGTGTGACCC
uniform MOE
let-7a-1, Human


338678
505
CTGGTTGGGTATGAGACAGA 
uniform MOE
mir-205, Human


338679
506
TTGATCCATATGCAACAAGG 
uniform MOE
mir-103-1, Human


338680
507
GCCATTGGGACCTGCACAGC 
uniform MOE
miR-26a-1, Human


338681
508
ATGGGTACCACCAGAACATG 
uniform MOE
mir-33a, Human


338682
509
AGTTCAAAACTCAATCCCAA 
uniform MOE
mir-196-2, Human


338683
510
GCCCTCGACGAAAACCGACT 
uniform MOE
mir-196-2, Human


338684
511
TTGAACTCCATGCCACAAGG 
uniform MOE
mir-107, Human


338685
512
AGGCCTATTCCTGTAGCAAA 
uniform MOE
mir-106, Human


338686
513
GTAGATCTCAAAAAGCTACC 
uniform MOE
mir-106, Human


338687
514
CTGAACAGGGTAAAATCACT 
uniform MOE
let-7f-1, Human


338688
515
AGCAAGTCTACTCCTCAGGG 
uniform MOE
let-7f-1, Human


338689
516
AATGGAGCCAAGGTGCTGCC 
uniform MOE
hypothetical miRNA-055,






Human


338690
517
TAGACAAAAACAGACTCTGA 
uniform MOE
mir-29c, Human


338691
518
GCTAGTGACAGGTGCAGACA 
uniform MOE
mir-130a, Human


338692
519
GGGCCTATCCAAAGTGACAG 
uniform MOE
hypothetical miRNA-058,






Human


338693
520
TACCTCTGCAGTATTCTACA 
uniform MOE
hypothetical miRNA-058,






Human


338694
521
TTTACTCATACCTCGCAACC 
uniform MOE
mir-218-1, Human


338695
522
AATTGTATGACATTAAATCA 
uniform MOE
mir-124a-2, Human


338696
523
CTTCAAGTGCAGCCGTAGGC 
uniform MOE
mir-124a-2, Human


338697
524
TGCCATGAGATTCAACAGTC 
uniform MOE
mir-21, Human


338698
525
ACATTGCTATCATAAGAGCT 
uniform MOE
mir-16-1, Human


338699
526
TAATTTTAGAATCTTAACGC 
uniform MOE
mir-16-1, Human


338700
527
AGTGTCTCATCGCAAACTTA 
uniform MOE
mir-144, Human


338701
528
TGTTGCCTAACGAACACAGA 
uniform MOE
mir-221, Human


338702
529
GCTGATTACGAAAGACAGGA 
uniform MOE
mir-222, Human


338703
530
GCTTAGCTGTGTCTTACAGC 
uniform MOE
mir-30d, Human


338704
531
GAGGATGTCTGTGAATAGCC 
uniform MOE
mir-30d, Human


338705
532
CCACATATACATATATACGC 
uniform MOE
mir-19b-2, Human


338706
533
AGGAAGCACACATTATCACA 
uniform MOE
mir-19b-2, Human


338707
534
GACCTGCTACTCACTCTCGT 
uniform MOE
mir-128b, Human


338708
535
GGTTGGCCGCAGACTCGTAC 
uniform MOE
hypothetical miRNA






069/mir-219-2, Human


338709
536
GATGTCACTGAGGAAATCAC 
uniform MOE
hypothetical miRNA-070,






Human


338710
537
TCAGTTGGAGGCAAAAACCC 
uniform MOE
LOC 114614/






hypothetical miRNA-071,






Human


338711
538
GGTAGTGCAGCGCAGCTGGT 
uniform MOE
mir-29b-2, Human


338712
539
CCGGCTATTGAGTTATGTAC 
uniform MOE
mir-129-2, Human


338713
540
ACCTCTCAGGAAGACGGACT 
uniform MOE
mir-133b, Human


338714
541
GAGCATGCAACACTCTGTGC 
uniform MOE
hypothetical miRNA-075,






Human


338715
542
CCTCCTTGTGGGCAAAATCC 
uniform MOE
let-7d, Human


338716
543
CGCATCTTGACTGTAGCATG 
uniform MOE
mir-15b, Human


338717
544
TCTAAGGGGTCACAGAAGGT 
uniform MOE
mir-29a-1, Human


338718
545
GAAAATTATATTGACTCTGA 
uniform MOE
mir-29a-1, Human


338719
546
GGTTCCTAATTAAACAACCC 
uniform MOE
hypothetical miRNA-079,






Human


338720
547
CCGAGGGTCTAACCCAGCCC 
uniform MOE
mir-199b, Human


338721
548
GACTACTGTTGAGAGGAACA 
uniform MOE
mir-129-1, Human


338722
549
TCTCCTTGGGTGTCCTCCTC 
uniform MOE
let-7e, Human


338723
550
TGCTGACTGCTCGCCCTTGC 
uniform MOE
hypothetical miRNA-083,






Human


338724
551
ACTCCCAGGGTGTAACTCTA 
uniform MOE
let7c-1, Human


338725
552
CATGAAGAAAGACTGTAGCC 
uniform MOE
mir-204, Human


338726
553
GACAAGGTGGGAGCGAGTGG 
uniform MOE
mir-145, Human


338727
554
TGCTCAGCCAGCCCCATTCT 
uniform MOE
mir-124a-1, Human


338728
555 
GCTTTTAGAACCACTGCCTC
uniform MOE
DiGeorge syndrome






critical region gene 8/






hypothetical miRNA-088,






Human


338729
556
GGAGTAGATGATGGTTAGCC
uniform MOE
mir-213/ mir-181a,






Human


338730
557 
ACTGATTCAAGAGCTTTGTA
uniform MOE
hypothetical miRNA-090,






Human


338731
558
GTAGATAACTAAACACTACC
uniform MOE
mir-20, Human


338732
559
AATCCATTGAAGAGGCGATT
uniform MOE
mir-133a-1, Human


338733
560
GGTAAGAGGATGCGCTGCTC
uniform MOE
mir-138-2, Human


338734
561
GGCCTAATATCCCTACCCCA
uniform MOE
mir-98, Human


338735
562
GTGTTCAGAAACCCAGGCCC
uniform MOE
mir-196-1, Human


338736
563
TCCAGGATGCAAAAGCACGA
uniform MOE
mir-125b-1, Human


338737
564
TACAACGGCATTGTCCTGAA
uniform MOE
mir-199a-2, Human


338738
565
TTTCAGGCTCACCTCCCCAG
uniform MOE
hypothetical miRNA-099,






Human


338739
566
AAAAATAATCTCTGCACAGG
uniform MOE
mir-181b, Human


338740
567
AGAATGAGTTGACATACCAA
uniform MOE
hypothetical miRNA-101,






Human


338741
568
GCTTCACAATTAGACCATCC
uniform MOE
mir-141, Human


338742
569
AGACTCCACACCACTCATAC
uniform MOE
mir-131-1/miR-9, Human


338743
570
ATCCATTGGACAGTCGATTT
uniform MOE
mir-133a-2, Human


338744
571
GGCGGGCGGCTCTGAGGCGG
uniform MOE
hypothetical miRNA-105,






Human


338745
572
CTCTTTAGGCCAGATCCTCA
uniform MOE
hypothetical miRNA-105,






Human


338746
573
TAATGGTATGTGTGGTGATA
uniform MOE
hypothetical miRNA-107,






Human


338747
574 
ATTACTAAGTTGTTAGCTGT
uniform MOE
miR-ld-2, Human


338748
575 
GATGCTAATCTACTTCACTA
uniform MOE
mir-18, Human


338749
576 
TCAGCATGGTGCCCTCGCCC
uniform MOE
mir-220, Human


338750
577 
TCCGCGGGGGCGGGGAGGCT
uniform MOE
hypothetical miRNA-111,






Human


338751
578
AGACCACAGCCACTCTAATC
uniform MOE
mir-7-3, Human


338752
579
TCCGTTTCCATCGTTCCACC
uniform MOE
mir-218-2, Human


338753
580
GCCAGTGTACACAAACCAAC
uniform MOE
mir-24-2, Human


338754
581
AAGGCTTTTTGCTCAAGGGC
uniform MOE
chromosome 9 ORF3






containing mir-23b,






mir-24-2 and mir-27b,






Human


338755
582
TTGACCTGAATGCTACAAGG
uniform MOE
mir-103-2, Human


338756
583
TGCCCTGCTCAGAGCCCTAG
uniform MOE
mir-211, Human


338757
584
TCAATGTGATGGCACCACCA
uniform MOE
mir-101-3, Human


338758
585
ACCTCCCAGCCAATCCATGT
uniform MOE
mir-30b, Human


338759
586
TCCTGGATGATATCTACCTC
uniform MOE
hypothetical miRNA-120,






Human


338760
587
TCTCCCTTGATGTAATTCTA
uniform MOE
let-7a-4, Human


338761
588
AGAGCGGAGTGTTTATGTCA
uniform MOE
mir-10a, Human


338762
589 
TCATTCATTTGAAGGAAATA
uniform MOE
mir-19a, Human


338763
590
TCCAAGATGGGGTATGACCC
uniform MOE
let-7f-2, Human


338764
591
TTTTTAAACACACATTCGCG
uniform MOE
mir-15a-1, Human


338765
592
AGATGTGTTTCCATTCCACT
uniform MOE
mir-108-2, Human


338766
593
CCCCCTGCCGCTGGTACTCT
uniform MOE
mir-137, Human


338767
594
CGGCCGGAGCCATAGACTCG
uniform MOE
mir-219-1, Human


338768
595
CTTTCAGAGAGCCACAGCCT
uniform MOE
mir-148b, Human


338769
596
GCTTCCCAGCGGCCTATAGT
uniform MOE
mir-130b, Human


338770
597
CAGCAGAATATCACACAGCT
uniform MOE
mir-19b-1, Human


338771
598
TACAATTTGGGAGTCCTGAA
uniform MOE
mir-199b, Human


338772
599
GCCTCCTTCATATATTCTCA
uniform MOE
mir-204, Human


338773
600
CCCCATCTTAGCATCTAAGG
uniform MOE
mir-145, Human


338774
601
TTGTATGGACATTTAAATCA
uniform MOE
mir-124a-1, Human


338775
602
TTTGATTTTAATTCCAAACT
uniform MOE
mir-213/ mir-181a,






Human


338776
603
CAAACGGTAAGATTTGCAGA
uniform MOE
hypothetical miRNA-090,






Human


338777
604
GGATTTAAACGGTAAACATC
uniform MOE
mir-125b-1, Human


338778
605
CTCTAGCTCCCTCACCAGTG
uniform MOE
hypothetical miRNA-099,






Human


338779
606
GCTTGTCCACACAGTTCAAC
uniform MOE
mir-181b, Human


338780
607
GCATTGTATGTTCATATGGG
uniform MOE
miR-ld-2, Human


338781
608
TGTCGTAGTACATCAGAACA
uniform MOE
mir-7-3, Human


338782
609
AGCCAGTGTGTAAAATGAGA
uniform MOE
chromosome 9 ORF3






containing mir-23b,






mir-24-2 and mir-27b,






Human


338783
610
TTCAGATATACAGCATCGGT
uniform MOE
mir-101-3, Human


338784
611
TGACCACAAAATTCCTTACA
uniform MOE
mir-10a, Human


338785
612
ACAACTACATTCTTCTTGTA
uniform MOE
mir-19a, Human


338786
613
TGCACCTTTTCAAAATCCAC
uniform MOE
mir-15a-1, Human


338787
614
AACGTAATCCGTATTATCCA
uniform MOE
mir-137, Human


338788
615
CGTGAGGGCTAGGAAATTGC
uniform MOE
mir-216, Human


338789
616
GCAACAGGCCTCAATATCTT
uniform MOE
mir-100-1, Human


338790
617
ACGAGGGGTCAGAGCAGCGC
uniform MOE
mir-187, Human


338791
618
GGCAGACGAAAGGCTGACAG
uniform MOE
hypothetical miRNA-137,






Human


338792
619
CTGCACCATGTTCGGCTCCC
uniform MOE
hypothetical miRNA-138,






Human


338793
620
GGGGCCCTCAGGGCTGGGGC
uniform MOE
mir-124a-3, Human


338794
621
CCGGTCCACTCTGTATCCAG
uniform MOE
mir-7-2, Human


338795
622
GCTGGGAAAGAGAGGGCAGA
uniform MOE
hypothetical miRNA-142,






Human


338796
623
TCAGATTGCCAACATTGTGA
uniform MOE
hypothetical miRNA-143,






Human


338797
624
CTGGGGAGGGGGTTAGCGTC
uniform MOE
collagen, type I, alpha






1/ hypothetical miRNA-






144, Human


338798
625
TGGGTCTGGGGCAGCGCAGT
uniform MOE
mir-210, Human


338799
626
TTGAAGTAGCACAGTCATAC
uniform MOE
mir-215, Human


338800
627
TCTACCACATGGAGTGTCCA
uniform MOE
mir-223, Human


338801
628
AGTGCCGCTGCCGCGCCGTG
uniform MOE
mir-131-3/miR-9, Human


338802
629
ACACATTGAGAGCCTCCTGA
uniform MOE
mir-199a-1, Human


338803
630
GTCGCTCAGTGCTCTCTAGG
uniform MOE
mir-30c-1, Human


338804
631
AGGCTCCTCTGATGGAAGGT
uniform MOE
mir-101-1, Human


338805
632
GCTGTGACTTCTGATATTAT
uniform MOE
hypothetical miRNA-153,






Human


338806
633
GACATCATGTGATTTGCTCA
uniform MOE
hypothetical miRNA-154,






Human


338807
634
CACCCCAAGGCTGCAGGGCA
uniform MOE
mir-26b, Human


338808
635
TGTCAAGCCTGGTACCACCA
uniform MOE
hypothetical miRNA-156,






Human


338809
636
CTGCTCCAGAGCCCGAGTCG
uniform MOE
mir-152, Human


338810
637
ACCCTCCGCTGGCTGTCCCC
uniform MOE
mir-135-1, Human


338811
638
TAGAGTGAATTTATCTTGGT
uniform MOE
non-coding RNA in






rhabdomyosarcoma/ mir-






135-2, Human


338812
639
TGGTGACTGATTCTTATCCA
uniform MOE
mir-217, Human


338813
640
CAATATGATTGGATAGAGGA
uniform MOE
hypothetical miRNA-161,






Human


338814
641
TTTAAACACACATTCGCGCC
uniform MOE
mir-15a-1, Human


338815
642
ACCGGGTGGTATCATAGACC 
uniform MOE
let-7g, Human


338816
643
TGCATACCTGTTCAGTTGGA 
uniform MOE
hypothetical miRNA-164,






Human


338817
644
GCCCGCCTCTCTCGGCCCCC 
uniform MOE
sterol regulatory






element-binding






protein-1/ mir-33b,






Human


338818
645
TCGCCCCCTCCCAGGCCTCT 
uniform MOE
hypothetical miRNA-166,






Human


338819
646
ACAACTGTAGAGTATGGTCA 
uniform MOE
mir-16-1, Human


338820
647
GCTGACCATCAGTACTTTCC 
uniform MOE
hypothetical miRNA 168-






1/similar to ribosomal






protein L5, Human


338821
648
TTATAGAACAGCCTCCAGTG 
uniform MOE
forkhead box






P2/hypothetical miRNA-






169, Human


338822
649
TTCAGGCACTAGCAGTGGGT 
uniform MOE
hypothetical miRNA-170,






Human


338823
650
AGTACTGCGAGGTTAACCGC 
uniform MOE
glutamate receptor,






ionotropic, AMPA 2 /






hypothetical miRNA-171,






Human


338824
651
GGACCTTTAAGATGCAAAGT 
uniform MOE
hypothetical miRNA-172,






Human


338825
652
TTCATATTATCCACCCAGGT 
uniform MOE
hypothetical miRNA-173,






Human


338826
653
CGGATCCTGTTACCTCACCA 
uniform MOE
mir-182, Human


338827
654
TGGTGCCTGCCACATCTTTG 
uniform MOE
hypothetical miRNA-175,






Human


338828
655
TGGGAGGCTGAATCAAGGAC 
uniform MOE
hypothetical miRNA-176,






Human


338829
656
TGACAACCAGGAAGCTTGTG 
uniform MOE
hypothetical miRNA-177-






1, Human


338830
657
GCCAGGCAGCGAGCTTTTGA 
uniform MOE
hypothetical miRNA-178,






Human


338831
658
CAGCCTGCCACCGCCGCTTT 
uniform MOE
hypothetical miRNA-179,






Human


338832
659
CTGCCCCCGTGGACCGAACA 
uniform MOE
cezanne 2/ hypothetical






miRNA-180, Human


338833
660
TCGTGCACCTGAGGAGTCTG 
uniform MOE
hypothetical miRNA-181,






Human


338834
661
CAAACGTGCTGTCTTCCTCC 
uniform MOE
mir-148a, Human


338835
662
AAGGACTCAGCAGTGTTTCA 
uniform MOE
tight junction protein






1 (zona occludens 1)/






hypothetical miRNA-183,






Human


338836
663
TCCTCGGTGGCAGAGCTCAG 
uniform MOE
mir-23a, Human


338837
664
AGACAATGAGTACACAGTTC 
uniform MOE
hypothetical miRNA-185,






Human


338838
665
CTGCAAGCACTGGTTCCCAT 
uniform MOE
hypothetical miRNA-177-






2/ hypothetical miRNA






186, Human


338839
666
TTGCCTGAGCTGCCCAAACT 
uniform MOE
mir-181c, Human


338840
667
TCCATCACACTGTCCTATGA 
uniform MOE
hypothetical miRNA-188,






Human


338841
668
GAGGGATTGTATGAACATCT 
uniform MOE
mir-216, Human


338842
669
GCTTGTGCGGACTAATACCA 
uniform MOE
mir-100-1, Human


338843
670
GCAGGCTAAAAGAAATAAGC 
uniform MOE
hypothetical miRNA-138,






Human


338844
671
ATTGTATAGACATTAAATCA 
uniform MOE
mir-124a-3, Human


338845
672
GTTGAGCGCAGTAAGACAAC 
uniform MOE
mir-7-2, Human


338846
673
AGATGTTTCTGGCCTGCGAG 
uniform MOE
hypothetical miRNA-142,






Human


338847
674
GACAAACTCAGCTATATTGT 
uniform MOE
mir-215, Human


338848
675
ACGGCTCTGTGGCACTCATA 
uniform MOE
mir-131-3/miR-9, Human


338849
676
GCTTTCTTACTTTCCACAGC 
uniform MOE
mir-30c-1, Human


338850
677
TACCTTTAGAATAGACAGCA 
uniform MOE
mir-101-1, Human


338851
678
AGGCTGGACAGCACACAACC 
uniform MOE
mir-26b, Human


338852
679
AGCAGGAGCCTTATCTCTCC 
uniform MOE
hypothetical miRNA-156,






Human


338853
680
ATGAGTGAGCAGTAGAATCA 
uniform MOE
mir-135-1, Human


338854
681
TGAGACTTTATTACTATCAC 
uniform MOE
non-coding RNA in






rhabdomyosarcoma/ mir-






135-2, Human


338855
682
TACTTTACTCCAAGGTTTTA 
uniform MOE
mir-15a-1, Human


338856
683
GCACCCGCCTCACACACGTG 
uniform MOE
sterol regulatory






element-binding






protein-1/ mir-33b,






Human


338857
684
TTCCCGACCTGCCTTTACCT 
uniform MOE
hypothetical miRNA-166,






Human


338858
685
TCCTGTAATTATAGGCTAGC 
uniform MOE
forkhead box






P2/hypothetical miRNA-






169, Human


338859
686
GGATCATATCAATAATACCA 
uniform MOE
hypothetical miRNA-172,






Human


338860
687
TGCTGAGACACACAATATGT 
uniform MOE
hypothetical miRNA-176,






Human


338861
688
TGTTTGTCTCCAAGAAACGT 
uniform MOE
hypothetical miRNA-177-






1, Human


338862
689
TGTCATGGACAGGATGAATA 
uniform MOE
hypothetical miRNA-179,






Human


338863
690
TCTATCATACTCAGAGTCGG 
uniform MOE
mir-148a, Human


338864
691
TTGTGACAGGAAGCAAATCC 
uniform MOE
mir-23a, Human


338865
692
CATCAGAGTCACCAACCCCA 
uniform MOE
hypothetical miRNA-185,






Human


338866
693
CAAGAGATGTCTCGTTTTGC 
uniform MOE
hypothetical miRNA-177-






2/ hypothetical miRNA






186, Human


340342
937
GACTGTTGAATCTCATGGCA 
uniform MOE
miR-104 (Mourelatos),






Human


340344
1656
GCATGAGCAGCCACCACAGG 
uniform MOE
miR-105 (Mourelatos),






Human


340346
1626
ACGACTTGGTGTGGACCCTG 
uniform MOE
miR-27 (Mourelatos),






Human


340347
849
TACTTTATATAGAACACAAG 
uniform MOE
mir-92-2/ miR-92






(Mourelatos), Human


340349
1632
AGGTTGGGTAATCACACTAC 
uniform MOE
miR-93 (Mourelatos),






Human


340351
1621
AATGTAACGCATTTCAATTC 
uniform MOE
miR-95 (Mourelatos),






Human


340353
1694
TGTGCGGTCCACTTCACCAC 
uniform MOE
miR-99 (Mourelatos),






Human


340355
1671
GTCCAGCAATTGCCCAAGTC 
uniform MOE
miR-25, Human


340357
1662
GGAAAGTCAGAAAGGTAACT 
uniform MOE
miR-28, Human


340359
1635
CAGGTTCCCAGTTCAACAGC 
uniform MOE
miR-31, Human


340361
1636
CATTGAGGCCGTGACAACAT 
uniform MOE
miR-32, Human


340363
1656
GCATGAGCAGCCACCACAGG 
5-10-5 MOE
miR-105 (Mourelatos),





gapmer
Human


340364
1626
ACGACTTGGTGTGGACCCTG 
5-10-5 MOE
miR-27 (Mourelatos),





gapmer
Human


340366
1632 
AGGTTGGGTAATCACACTAC
5-10-5 MOE
miR-93 (Mourelatos),





gapmer
Human


340367
1621 
AATGTAACGCATTTCAATTC
5-10-5 MOE
miR-95 (Mourelatos),





gapmer
Human


340368
1694 
TGTGCGGTCCACTTCACCAC
5-10-5 MOE
miR-99 (Mourelatos),





gapmer
Human


340369
1671 
GTCCAGCAATTGCCCAAGTC
5-10-5 MOE
miR-25, Human





gapmer



340370
1662 
GGAAAGTCAGAAAGGTAACT
5-10-5 MOE
miR-28, Human





gapmer



340371
1635 
CAGGTTCCCAGTTCAACAGC
5-10-5 MOE
miR-31, Human





gapmer



340372
1636 
CATTGAGGCCGTGACAACAT
5-10-5 MOE
miR-32, Human





gapmer



341817
1630
AGCCACCTTGAGCTCACAGC
uniform MOE
miR-30c-2, Human


341818
1695 
TGTGTGCGGCGAAGGCCCCG
uniform MOE
miR-99b, Human


341819
1657
GCCAGGCTCCCAAGAACCTC
uniform MOE
MiR-125a, Human


341820
1653 
GATGTTACTAAAATACCTCA
uniform MOE
MiR-125b-2, Human


341822
1679 
TCCGATGATCTTTCTGAATC
uniform MOE
miR-127, Human


341825
1646
CTTAAAATAAAACCAGAAAG
uniform MOE
miR-186, Human


341826
1618
AAAATCACAGGAACCTATCT
uniform MOE
miR-198, Human


341827
1688
TGGAATGCTCTGGAGACAAC
uniform MOE
miR-191, Human


341828
1677
TCCATAGCAAAGTAATCCAT
uniform MOE
miR-206, Human


341829
1668
GGTAGCACGGAGAGGACCAC
uniform MOE
miR-94, Human


341830
1624
ACACTTACAGTCACAAAGCT
uniform MOE
miR-184, Human


341831
1654
GCAGACTCGCTTCCCTGTGC
uniform MOE
miR-195, Human


341832
1684 
TGATCCGACACCCTCATCTC
uniform MOE
miR-193, Human


341833
1641 
CCTGGGGAGGGGACCATCAG
uniform MOE
miR-185, Human


341834
1676 
TCAGAAAGCTCACCCTCCAC
uniform MOE
miR-188, Human


341835
1648
GAGCTCTTACCTCCCACTGC
uniform MOE
miR-197, Human


341836
1686 
TGGAAATTGGTACACAGTCC
uniform MOE
miR-194-1, Human


341837
1642
CGTGAGCATCAGGTATAACC
uniform MOE
miR-208, Human


341838
1687
TGGAACCAGTGGGCACTTCC
uniform MOE
miR-194-2, Human


341839
1638
CCAGCCTCCGAGCCACACTG
uniform MOE
miR-139, Human


341840
1628
AGACCTGACTCCATCCAATG
uniform MOE
miR-200b, Human


341841
1629
AGAGTCAAGCTGGGAAATCC
uniform MOE
miR-200a, Human


341843
1630
AGCCACCTTGAGCTCACAGC
5-10-5 MOE
miR-30c-2, Human





gapmer



341844
1695 
TGTGTGCGGCGAAGGCCCCG
5-10-5 MOE
miR-99b, Human





gapmer



341845
1657 
GCCAGGCTCCCAAGAACCTC
5-10-5 MOE
MiR-125a, Human





gapmer



341846
1653
GATGTTACTAAAATACCTCA
5-10-5 MOE
MiR-125b-2, Human





gapmer



341848
1679
TCCGATGATCTTTCTGAATC
5-10-5 MOE
miR-127, Human





gapmer



341851
1646
CTTAAAATAAAACCAGAAAG
5-10-5 MOE
miR-186, Human





gapmer



341852
1618 
AAAATCACAGGAACCTATCT
5-10-5 MOE
miR-198, Human





gapmer



341853
1688
TGGAATGCTCTGGAGACAAC
5-10-5 MOE
miR-191, Human





gapmer



341854
1677 
TCCATAGCAAAGTAATCCAT
5-10-5 MOE
miR-206, Human





gapmer



341855
1668
GGTAGCACGGAGAGGACCAC
5-10-5 MOE
miR-94, Human





gapmer



341856
1624
ACACTTACAGTCACAAAGCT
5-10-5 MOE
miR-184, Human





gapmer



341857
1654
GCAGACTCGCTTCCCTGTGC
5-10-5 MOE
miR-195, Human





gapmer



341858
1684
TGATCCGACACCCTCATCTC
5-10-5 MOE
miR-193, Human





gapmer



341859
1641
CCTGGGGAGGGGACCATCAG 
5-10-5 MOE
miR-185, Human





gapmer



341860
1676
TCAGAAAGCTCACCCTCCAC 
5-10-5 MOE
miR-188, Human





gapmer



341861
1648
GAGCTCTTACCTCCCACTGC
5-10-5 MOE
miR-197, Human





gapmer



341862
1686
TGGAAATTGGTACACAGTCC
5-10-5 MOE
miR-194-1, Human





gapmer



341863
1642
CGTGAGCATCAGGTATAACC 
5-10-5 MOE
miR-208, Human





gapmer



341864
1687
TGGAACCAGTGGGCACTTCC 
5-10-5 MOE
miR-194-2, Human





gapmer



341865
1638
CCAGCCTCCGAGCCACACTG
5-10-5 MOE
miR-139, Human





gapmer



341866
1628
AGACCTGACTCCATCCAATG
5-10-5 MOE
miR-200b, Human





gapmer



341867
1629
AGAGTCAAGCTGGGAAATCC 
5-10-5 MOE
miR-200a, Human





gapmer



344731
1619
AACGGTTTATGACAAACATT 
uniform MOE
mir-240* (Kosik), Human


344732
1665
GGGCTGTATGCACTTTCTCC 
uniform MOE
mir-232* (Kosik), Human


344733
1667
GGGTCTCCAGCTTTACACCA 
uniform MOE
mir-227* (Kosik)/mir-






226* (Kosik), Human


344734
1649
GAGTCGCCTGAGTCATCACT 
uniform MOE
mir-244* (Kosik), Human


344735
1658
GCCATAAATAAAGCGAACGC 
uniform MOE
mir-224* (Kosik), Human


344736
1678
TCCATTAACCATGTCCCTCA 
uniform MOE
mir-248* (Kosik), Human


344737
1619
AACGGTTTATGACAAACATT 
5-10-5 MOE
mir-240* (Kosik), Human





gapmer



344738
1665
GGGCTGTATGCACTTTCTCC 
5-10-5 MOE
mir-232* (Kosik), Human





gapmer



344739
1667
GGGTCTCCAGCTTTACACCA
5-10-5 MOE
mir-227* (Kosik)/mir-





gapmer
226* (Kosik), Human


344740
1649
GAGTCGCCTGAGTCATCACT 
5-10-5 MOE
mir-244* (Kosik), Human





gapmer



344741
1658
GCCATAAATAAAGCGAACGC 
5-10-5 MOE
mir-224* (Kosik), Human





gapmer



344742
1678
TCCATTAACCATGTCCCTCA 
5-10-5 MOE
mir-248* (Kosik), Human





gapmer



346787
1689
TGGCTTCCATAGTCTGGTGT 
uniform MOE
miR-147 (Sanger), Human


346788
1623
ACAATGCACAATCATCTACT 
uniform MOE
miR-224 (Sanger), Human


346789
1669
GGTGAACACAGTGCATGCCC 
uniform MOE
miR-134 (Sanger), Human


346790
1682
TCTGACACTGACACAACCCA 
uniform MOE
miR-146 (Sanger), Human


346791
1631
AGGGTCTGAGCCCAGCACTG 
uniform MOE
miR-150 (Sanger), Human


346792
1637
CCAAGAGACGTTTCATTTTG 
uniform MOE
hypothetical miRNA-177-






3, Human


346793
1683
TCTGATTGGCAACGGCCTGA 
uniform MOE
mir-138-3, Human


346794
1627
ACTGTCCATCTTAGTTCAGA 
uniform MOE
mir-138-4, Human


346795
1634
AGTTGATTCAGACTCAAACC 
uniform MOE
mir-181b-2, Human


346796
1655
GCATAAGCAGCCACCACAGG 
uniform MOE
miR-105-2, Human


346797
1691
TGTATGATATCTACCTCAGG 
uniform MOE
hypothetical miRNA-120-






2, Human


346798
1689
TGGCTTCCATAGTCTGGTGT
5-10-5 MOE
miR-147 (Sanger), Human





gapmer



346799
1623
ACAATGCACAATCATCTACT
5-10-5 MOE
miR-224 (Sanger), Human





gapmer



346800
1669
GGTGAACACAGTGCATGCCC
5-10-5 MOE
miR-134 (Sanger), Human





gapmer



346801
1682
TCTGACACTGACACAACCCA 
5-10-5 MOE
miR-146 (Sanger), Human





gapmer



346802
1631
AGGGTCTGAGCCCAGCACTG
5-10-5 MOE
miR-150 (Sanger), Human





gapmer



346803
1637
CCAAGAGACGTTTCATTTTG
5-10-5 MOE
hypothetical miRNA-177-





gapmer
3, Human


346804
1683
TCTGATTGGCAACGGCCTGA
5-10-5 MOE
mir-138-3, Human





gapmer



346805
1627
ACTGTCCATCTTAGTTCAGA
5-10-5 MOE
mir-138-4, Human





gapmer



346806
1634
AGTTGATTCAGACTCAAACC
5-10-5 MOE
mir-181b-2, Human





gapmer



346807
1655
GCATAAGCAGCCACCACAGG
5-10-5 MOE
miR-105-2, Human





gapmer



346808
1691
TGTATGATATCTACCTCAGG
5-10-5 MOE
hypothetical miRNA-120-





gapmer
2, Human


348225
1620
AAGAGAAGGCGGAGGGGAGC
5-10-5 MOE
miR-320, Human





gapmer



348226
1643
CTCGAACCCACAATCCCTGG
5-10-5 MOE
miR-321-1, Human





gapmer



354006
1650
GAGTTTGGGACAGCAATCAC
5-10-5 MOE
mir-135b (Ruvkun),





gapmer
Human


354007
1633
AGTAGGGGATGAGACATACT
5-10-5 MOE
mir-151* (Ruvkun),





gapmer
Human


354008
1639
CCCACAAACGACATATGACA
5-10-5 MOE
mir-340 (Ruvkun), Human





gapmer



354009
1664
GGCCTGGTTTGATCTGGGAT
5-10-5 MOE
mir-331 (Ruvkun), Human





gapmer



354010
1647
GAGACTCCCAACCGCACCCA
5-10-5 MOE
miR-200c (RFAM-Human)





gapmer



354011
1700
TTGTAACCACCACAGTACAA
5-10-5 MOE
miR-34b (RFAM-Human)





gapmer



354012
1663
GGAGGACAGGGAGAGCGGCC
5-10-5 MOE
mir-339-1 (RFAM-Human)





gapmer



354013
1675
TCACAGGCAGGCACACGTGA
5-10-5 MOE
mir-339-1 (RFAM-Human)





gapmer



354014
1698
TTCAGAGCTACAGCATCGGT
5-10-5 MOE
mir-101-3, Mouse





gapmer



354015
1670
GTAGAACTCAAAAAGCTACC
5-10-5 MOE
mir-106, Mouse





gapmer



354016
1673
TAGATGCACACATCACTACC
5-10-5 MOE
miR-17/mir-91, Mouse





gapmer



354017
1690
TGTACAATTTGGGAGTCCTG
5-10-5 MOE
mir-199b, Human





gapmer



354018
1644
CTCTTTAGACCAGATCCACA
5-10-5 MOE
hypothetical miRNA-105,





gapmer
Mouse


354019
1640
CCTCACTCAGAGGCCTAGGC
5-10-5 MOE
mir-211, Mouse





gapmer



354020
1666
GGGGATTAAGTCTTATCCAG
5-10-5 MOE
mir-217, Mouse





gapmer



354021
1622
ACAATGCACAAACCATCTAC
5-10-5 MOE
miR-224 (Sanger), Mouse





gapmer



354022
1693
TGTCATATCATATCAGAACA
5-10-5 MOE
mir-7-3, Mouse





gapmer



354023
1672
TAGATGACGACACACTACCT
5-10-5 MOE
mir-20, Rat





gapmer



354024
1692
TGTCACAAACACTTACTGGA
5-10-5 MOE
mir-325 (Ruvkun), Human





gapmer



354025
1625
ACGAATTATGTCACAAACAC
5-10-5 MOE
mir-325 (Ruvkun), Mouse





gapmer



354026
1651
GATCTGAGCACCACCCGCCT
5-10-5 MOE
mir-326 (Ruvkun), Human





gapmer



354027
1652
GATCTGAGCATAACCCGCCT
5-10-5 MOE
mir-326 (Ruvkun), Mouse





gapmer



354028
1697
TGTTTCGTCCTCATTAAAGA
5-10-5 MOE
mir-329-1 (Ruvkun),





gapmer
Human


354029
1699
TTCTCATCAAAGAAACAGAG
5-10-5 MOE
mir-329-1 (Ruvkun),





gapmer
Mouse


354030
1696
TGTTTCGTCCTCAATAAAGA
5-10-5 MOE
mir-329-2 (Ruvkun),





gapmer
Human


354031
1681
TCGGTTGATCTTGCAGAGCC
5-10-5 MOE
mir-330 (Ruvkun), Human





gapmer



354032
1685
TGCTCGTTGGATCTTGAAGA
5-10-5 MOE
mir-330 (Ruvkun), Mouse





gapmer



354033
1661
GCTGGATAACTGTGCATCAA
5-10-5 MOE
mir-337 (Ruvkun), Human





gapmer



354034
1645
CTGAATGGCTGTGCAATCAA
5-10-5 MOE
mir-337 (Ruvkun), Mouse





gapmer



354035
1659
GCCCACCAGCCATCACGAGC
5-10-5 MOE
mir-345 (Ruvkun), Human





gapmer



354036
1660
GCCCAGTAGCCACCACAAGC
5-10-5 MOE
mir-345 (Ruvkun), Mouse





gapmer



354037
1680
TCCTTCAGAGCAACAGAGAG
5-10-5 MOE
mir-346 (Ruvkun), Human





gapmer



354038
1674
TAGTAGGGAGGAGACATACT
5-10-5 MOE
mir-151* (Ruvkun),





gapmer
Mouse


354039
1701
TTGTCAGCACCGCACTACAA
5-10-5 MOE
miR-34b (RFAM-Mouse)





gapmer









In accordance with the present invention, a further series of oligomeric compounds were designed and synthesized to target different regions of miRNAs. These oligomeric compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels by quantitative real-time PCR, or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets. The compounds are shown in Table 65, where “pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. Oligomeric compounds having phosphorothioate internucleoside linkages are indicated by “PS” in the “Chemistry” column of Table 65, whereas compounds having phosphodiester internucleoside linkages are indicated by “PO.” In some embodiments, chimeric oligonucleotides (“gapmers”) are composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by nucleotide “wings” two to ten nucleotides in length. The wings are composed of 2′-methoxyethoxy (2′-MOE) ribonucleotides. In some embodiments, chimeric oligonucleotides are of the “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Chimeric oligonucleotides of this type are also known in the art and are indicated in Table 65 as “hemimers.” For example, “PO/6MOE-10deoxy hemimer,” describes a chimeric oligomeric compound consisting of six 2′-MOE ribonucleotides at the 5′-terminus, followed by ten deoxyribonucleotides on the 3′-terminal end, with a phosphodiester backbone throughout the hemimer







TABLE 65







Oligomeric compounds targeting miRNAs












SEQ






ID





ISIS #
NO
sequence
Chemistry
Pri-miRNA














340343
1780
ACAGGAGTCTGAGCATTTGA
PS/MOE
miR-105






(Mourelatos)


340345
1882
GGAACTTAGCCACTGTGAA
PS/MOE
miR-27






(Mourelatos)


340350
855
TGCTCAATAAATACCCGTTGAA
PS/MOE
miR-95






(Mourelatos)


340352
1821
CACAAGATCGGATCTACGGGTT
PS/MOE
miR-99






(Mourelatos)


340354
1903
TCAGACCGAGACAAGTGCAATG
PS/MOE
miR-25 (Tuschl)


340356
1853
CTCAATAGACTGTGAGCTCCTT
PS/MOE
miR-28 (Tuschl)


340358
1825
CAGCTATGCCAGCATCTTGCC
PS/MOE
miR-31 (Tuschl)


340360
1865
GCAACTTAGTAATGTGCAATA
PS/MOE
miR-32 (Tuschl)


340924
298
ACAAATTCGGTTCTACAGGGTA
PS/MOE 5-10-7
mir-10b





gapmer



340925
307
GTGGTAATCCCTGGCAATGTGAT
PS/MOE 5-10-8
mir-23b





gapmer



340928
322
ACTCACCGACAGCGTTGAATGTT
PS/MOE 5-10-8
mir-181a





gapmer



340929
331
AACCGATTTCAAATGGTGCTAG
PS/MOE 5-10-7
mir-29c





gapmer



340930
342
GCAAGCCCAGACCGCAAAAAG
PS/MOE 5-10-6
mir-129





gapmer



340931
346
AACCGATTTCAGATGGTGCTAG
PS/MOE 5-10-7
mir-29a





gapmer



340932
349
AACCATACAACCTACTACCTCA
PS/MOE 5-10-7
let-7c





gapmer



340933
352
GGTACAATCAACGGTCGATGGT
PS/MOE 5-10-7
mir-213





gapmer



340934
356
AACAATACAACTTACTACCTCA
PS/MOE 5-10-7
mir-98





gapmer



340935
373
GCCCTTTCATCATTGCACTG
PS/MOE 5-10-5
mir-130b





gapmer



340936
385
ACTGTACAAACTACTACCTCA
PS/MOE 5-10-6
let-7g





gapmer



341785
854
GGAGTGAAGACACGGAGCCAGA
PS/MOE
miR-149


341786
1845
CGCAAGGTCGGTTCTACGGGTG
PS/MOE
miR-99b


341787
852
CACAGGTTAAAGGGTCTCAGGGA
PS/MOE
miR-125a


341788
853
AGCCAAGCTCAGACGGATCCGA
PS/MOE
miR-127


341789
1909
TCCATCATCAAAACAAATGGAGT
PS/MOE
miR-136


341790
1843
CGAAGGCAACACGGATAACCTA
PS/MOE
miR-154


341791
1880
GCTTCCAGTCGAGGATGTTTACA
PS/MOE
miR-30a-s


341792
1911
TCCGTGGTTCTACCCTGTGGTA
PS/MOE
miR-140-as


341793
1836
CCATAAAGTAGGAAACACTACA
PS/MOE
miR-142-as


341794
1761
AACAGGTAGTCTGAACACTGGG
PS/MOE
miR-199-s


341795
1762
AACCAATGTGCAGACTACTGTA
PS/MOE
miR-199-as


341796
1904
TCATACAGCTAGATAACCAAAGA
PS/MOE
miR-9


341797
1773
ACAAGTGCCTTCACTGCAGT
PS/MOE
miR-17


341798
1871
GCATTATTACTCACGGTACGA
PS/MOE
miR-126a


341799
1787
ACCTAATATATCAAACATATCA
PS/MOE
miR-190


341800
1766
AAGCCCAAAAGGAGAATTCTTTG
PS/MOE
miR-186


341801
1839
CCTATCTCCCCTCTGGACC
PS/MOE
miR-198a


341802
1806
AGCTGCTTTTGGGATTCCGTTG
PS/MOE
miR-191c


341803
760
CCACACACTTCCTTACATTCCA
PS/MOE
miR-206d


341804
761
ATCTGCACTGTCAGCACTTT
PS/MOE
miR-94


341805
762
ACCCTTATCAGTTCTCCGTCCA
PS/MOE
miR-184


341806
763
GCCAATATTTCTGTGCTGCTA
PS/MOE
miR-195


341807
764
CTGGGACTTTGTAGGCCAGTT
PS/MOE
miR-193


341808
1861
GAACTGCCTTTCTCTCCA
PS/MOE
miR-185


341809
1786
ACCCTCCACCATGCAAGGGATG
PS/MOE
miR-188


341810
1879
GCTGGGTGGAGAAGGTGGTGAA
PS/MOE
miR-197a


341811
1906
TCCACATGGAGTTGCTGTTACA
PS/MOE
miR-194


341812
1771
ACAAGCTTTTTGCTCGTCTTAT
PS/MOE
miR-208


341814
1887
GTCATCATTACCAGGCAGTATTA
PS/MOE
miR-200b


341815
1831
CATCGTTACCAGACAGTGTTA
PS/MOE
miR-200a


342946
1897
TAGGAGAGAGAAAAAGACTGA
PS/MOE
miR-14


342947
1827
CAGCTTTCAAAATGATCTCAC
PS/MOE
miR-Bantam


343875
321
AACTATACAACCTACTACCTCA
PO/MOE
let-7a


344267
1769
ACAAATTCGGATCTACAGGGTA
PS/MOE
miR-10 (Tuschl)


344268
1774
ACACAAATTCGGTTCTACAGGG
PS/MOE
miR-10b






(Tuschl)


344269
1890
TAACCGATTTCAAATGGTGCTA
PS/MOE
miR-29c






(Tuschl)


344270
1867
GCACGAACAGCACTTTG
PS/MOE
miR-93 (Tuschl)


344271
1770
ACAAGATCGGATCTACGGGT
PS/MOE
miR-99a






(Tuschl)


344272
1816
CAAACACCATTGTCACACTCCA
PS/MOE
miR-122a,b






(Tuschl)


344273
1920
TGTCAATTCATAGGTCAG
PS/MOE
miR-192






(Tuschl)


344274
1832
CCAACAACATGAAACTACCTA
PS/MOE
miR-196






(Tuschl)


344275
1912
TCTAGTGGTCCTAAACATTTCA
PS/MOE
miR-203






(Tuschl)


344276
1828
CAGGCATAGGATGACAAAGGGAA
PS/MOE
miR-204






(Tuschl)


344277
1767
AATACATACTTCTTTACATTCCA
PS/MOE
miR-1d (Tuschl)


344278
1769
ACAAATTCGGATCTACAGGGTA
PS/MOE 5-10-7
miR-10 (Tuschl)





gapmer



344279
1774
ACACAAATTCGGTTCTACAGGG
PS/MOE 5-10-7
miR-10b





gapmer
(Tuschl)


344280
1890
TAACCGATTTCAAATGGTGCTA
PS/MOE 5-10-7
miR-29c





gapmer
(Tuschl)


344281
1867
GCACGAACAGCACTTTG
PS/MOE 5-10-2
miR-93 (Tuschl)





gapmer



344282
1770
ACAAGATCGGATCTACGGGT
PS/MOE 5-10-5
miR-99a





gapmer
(Tuschl)


344283
1816
CAAACACCATTGTCACACTCCA
PS/MOE 5-10-7
miR-122a,b





gapmer
(Tuschl)


344284
1920
TGTCAATTCATAGGTCAG
PS/MOE 5-10-3
miR-192





gapmer
(Tuschl)


344285
1832
CCAACAACATGAAACTACCTA
PS/MOE 5-10-6
miR-196





gapmer
(Tuschl)


344286
1912
TCTAGTGGTCCTAAACATTTCA
PS/MOE 5-10-7
miR-203





gapmer
(Tuschl)


344287
1828
CAGGCATAGGATGACAAAGGGAA
PS/MOE 5-10-8
miR-204





gapmer
(Tuschl)


344288
1767
AATACATACTTCTTTACATTCCA
PS/MOE 5-10-8
miR-1d (Tuschl)





gapmer



344336
1918
TGGCATTCACCGCGTGCCTTA
PS/MOE
mir-124a






(Kosik)


344337
1754
AAAGAGACCGGTTCACTGTGA
PS/MOE
mir-128 (Kosik)


344338
1812
ATGCCCTTTTAACATTGCACTG
PS/MOE
mir-130 (Kosik)


344339
1854
CTCACCGACAGCGTTGAATGTT
PS/MOE
mir-178 (Kosik)


344340
1921
TGTCCGTGGTTCTACCCTGTGGTA
PS/MOE
mir-239*






(Kosik)


344341
1823
CACATGGTTAGATCAAGCACAA
PS/MOE
mir-253*






(Kosik)


344342
1814
ATGCTTTTTGGGGTAAGGGCTT
PS/MOE
mir-129as/mir-






258* (Kosik)


344343
1811
ATGCCCTTTCATCATTGCACTG
PS/MOE
mir-266*






(Kosik)


344344
1918
TGGCATTCACCGCGTGCCTTA
PS/MOE 5-10-6
mir-124a





gapmer
(Kosik)


344345
1754
AAAGAGACCGGTTCACTGTGA
PS/MOE 5-10-6
mir-128 (Kosik)





gapmer



344346
1812
ATGCCCTTTTAACATTGCACTG
PS/MOE 5-10-7
mir-130 (Kosik)





gapmer



344347
1854
CTCACCGACAGCGTTGAATGTT
PS/MOE 5-10-7
mir-178 (Kosik)





gapmer



344348
1921
TGTCCGTGGTTCTACCCTGTGGTA
PS/MOE 5-10-9
mir-239*





gapmer
(Kosik)


344349
1823
CACATGGTTAGATCAAGCACAA
PS/MOE 5-10-7
mir-253*





gapmer
(Kosik)


344350
1814
ATGCTTTTTGGGGTAAGGGCTT
PS/MOE 5-10-7
mir-129as/mir-





gapmer
258* (Kosik)


344351
1811
ATGCCCTTTCATCATTGCACTG
PS/MOE 5-10-7
mir-266*





gapmer
(Kosik)


344611
1785
ACATTTTTCGTTATTGCTCTTGA
PS/MOE
mir-240*






(Kosik)


344612
1790
ACGGAAGGGCAGAGAGGGCCAG
PS/MOE
mir-232*






(Kosik)


344613
1775
ACACCAATGCCCTAGGGGATGCG
PS/MOE
mir-227*






(Kosik)


344614
1834
CCAGCAGCACCTGGGGCAGT
PS/MOE
mir-226*






(Kosik)


344615
1900
TCAACAAAATCACTGATGCTGGA
PS/MOE
mir-244*






(Kosik)


344616
1800
AGAGGTCGACCGTGTAATGTGC
PS/MOE
mir-224*






(Kosik)


344617
1862
GACGGGTGCGATTTCTGTGTGAGA
PS/MOE
mir-248*






(Kosik)


344618
1785
ACATTTTTCGTTATTGCTCTTGA
PS/MOE 5-10-8
mir-240*





gapmer
(Kosik)


344619
1790
ACGGAAGGGCAGAGAGGGCCAG
PS/MOE 5-10-7
mir-232*





gapmer
(Kosik)


344620
1775
ACACCAATGCCCTAGGGGATGCG
PS/MOE 5-10-8
mir-227*





gapmer
(Kosik)


344621
1834
CCAGCAGCACCTGGGGCAGT
PS/MOE 5-10-5
mir-226*





gapmer
(Kosik)


344622
1900
TCAACAAAATCACTGATGCTGGA
PS/MOE 5-10-8
mir-244*





gapmer
(Kosik)


344623
1800
AGAGGTCGACCGTGTAATGTGC
PS/MOE 5-10-7
mir-224*





gapmer
(Kosik)


344624
1862
GACGGGTGCGATTTCTGTGTGAGA
PS/MOE 5-10-9
mir-248*





gapmer
(Kosik)


345344
291
CTACCATAGGGTAAAACCACT
PS/MOE 5-10-6
mir-140





gapmer



345345
292
GCTGCAAACATCCGACTGAAAG
PS/MOE 5-10-7
mir-30a





gapmer



345346
293
ACAACCAGCTAAGACACTGCCA
PS/MOE 5-10-7
mir-34





gapmer



345347
294
AACACTGATTTCAAATGGTGCTA
PS/MOE 5-10-8
mir-29b





gapmer



345348
295
CGCCAATATTTACGTGCTGCTA
PS/MOE 5-10-7
mir-16





gapmer



345350
297
AACAAAATCACTAGTCTTCCA
PS/MOE 5-10-6
mir-7





gapmer



345351
299
AAAAGAGACCGGTTCACTGTGA
PS/MOE 5-10-7
mir-128a





gapmer



345352
300
TCACTTTTGTGACTATGCAA
PS/MOE 5-10-5
mir-153





gapmer



345353
301
CAGAACTTAGCCACTGTGAA
PS/MOE 5-10-5
mir-27b





gapmer



345354
302
GCAAAAATGTGCTAGTGCCAAA
PS/MOE 5-10-7
mir-96





gapmer



345355
303
ACTACCTGCACTGTAAGCACTTTG
PS/MOE 5-10-9
mir-17as/mir-91





gapmer



345356
304
CGCGTACCAAAAGTAATAATG
PS/MOE 5-10-6
mir-123/mir-





gapmer
126as


345357
305
GCGACCATGGCTGTAGACTGTTA
PS/MOE 5-10-8
mir-132





gapmer



345358
306
AATGCCCCTAAAAATCCTTAT
PS/MOE 5-10-6
mir-108





gapmer



345359
308
AGCACAAACTACTACCTCA
PS/MOE 5-10-4
let-7i





gapmer



345360
309
GGCCGTGACTGGAGACTGTTA
PS/MOE 5-10-6
mir-212





gapmer



345361
311
AACCACACAACCTACTACCTCA
PS/MOE 5-10-7
let-7b





gapmer



345362
312
ATACATACTTCTTTACATTCCA
PS/MOE 5-10-7
mir-1d





gapmer



345363
313
ACAAACACCATTGTCACACTCCA
PS/MOE 5-10-8
mir-122a





gapmer



345364
314
ACAGTTCTTCAACTGGCAGCTT
PS/MOE 5-10-7
mir-22





gapmer



345365
315
ACAGGCCGGGACAAGTGCAATA
PS/MOE 5-10-7
mir-92





gapmer



345366
316
GTAGTGCTTTCTACTTTATG
PS/MOE 5-10-5
mir-142





gapmer



345367
317
CAGTGAATTCTACCAGTGCCATA
PS/MOE 5-10-8
mir-183





gapmer



345368
318
CTGCCTGTCTGTGCCTGCTGT
PS/MOE 5-10-6
mir-214





gapmer



345369
320
GGCTGTCAATTCATAGGTCAG
PS/MOE 5-10-6
mir-192





gapmer



345370
321
AACTATACAACCTACTACCTCA
PS/MOE 5-10-7
let-7a





gapmer



345371
323
CAGACTCCGGTGGAATGAAGGA
PS/MOE 5-10-7
mir-205





gapmer



345372
324
TCATAGCCCTGTACAATGCTGCT
PS/MOE 5-10-8
mir-103





gapmer



345373
325
AGCCTATCCTGGATTACTTGAA
PS/MOE 5-10-7
mir-26a





gapmer



345374
326
CAATGCAACTACAATGCAC
PS/MOE 5-10-4
mir-33a





gapmer



345375
327
CCCAACAACATGAAACTACCTA
PS/MOE 5-10-7
mir-196





gapmer



345376
328
TGATAGCCCTGTACAATGCTGCT
PS/MOE 5-10-8
mir-107





gapmer



345377
329
GCTACCTGCACTGTAAGCACTTTT
PS/MOE 5-10-9
mir-106





gapmer



345378
330
AACTATACAATCTACTACCTCA
PS/MOE 5-10-7
let-7f





gapmer



345379
332
GCCCTTTTAACATTGCACTG
PS/MOE 5-10-5
mir-130a





gapmer



345380
333
ACATGGTTAGATCAAGCACAA
PS/MOE 5-10-6
mir-218





gapmer



345381
334
TGGCATTCACCGCGTGCCTTAA
PS/MOE 5-10-7
mir-124a





gapmer



345382
335
TCAACATCAGTCTGATAAGCTA
PS/MOE 5-10-7
mir-21





gapmer



345383
336
CTAGTACATCATCTATACTGTA
PS/MOE 5-10-7
mir-144





gapmer



345384
337
GAAACCCAGCAGACAATGTAGCT
PS/MOE 5-10-8
mir-221





gapmer



345385
338
GAGACCCAGTAGCCAGATGTAGCT
PS/MOE 5-10-9
mir-222





gapmer



345386
339
CTTCCAGTCGGGGATGTTTACA
PS/MOE 5-10-7
mir-30d





gapmer



345387
340
TCAGTTTTGCATGGATTTGCACA
PS/MOE 5-10-8
mir-19b





gapmer



345388
341
GAAAGAGACCGGTTCACTGTGA
PS/MOE 5-10-7
mir-128b





gapmer



345389
343
TAGCTGGTTGAAGGGGACCAA
PS/MOE 5-10-6
mir-133b





gapmer



345390
344
ACTATGCAACCTACTACCTCT
PS/MOE 5-10-6
let-7d





gapmer



345391
345
TGTAAACCATGATGTGCTGCTA
PS/MOE 5-10-7
mir-15b





gapmer



345392
347
GAACAGATAGTCTAAACACTGGG
PS/MOE 5-10-8
mir-199b





gapmer



345393
348
ACTATACAACCTCCTACCTCA
PS/MOE 5-10-6
let-7e





gapmer



345394
350
AGGCATAGGATGACAAAGGGAA
PS/MOE 5-10-7
mir-204





gapmer



345395
351
AAGGGATTCCTGGGAAAACTGGAC
PS/MOE 5-10-9
mir-145





gapmer



345396
353
CTACCTGCACTATAAGCACTTTA
PS/MOE 5-10-8
mir-20





gapmer



345397
354
ACAGCTGGTTGAAGGGGACCAA
PS/MOE 5-10-7
mir-133a





gapmer



345398
355
GATTCACAACACCAGCT
PS/MOE 5-10-2
mir-138





gapmer



345399
357
TCACAAGTTAGGGTCTCAGGGA
PS/MOE 5-10-7
mir-125b





gapmer



345400
358
GAACAGGTAGTCTGAACACTGGG
PS/MOE 5-10-8
mir-199a





gapmer



345401
359
AACCCACCGACAGCAATGAATGTT
PS/MOE 5-10-9
mir-181b





gapmer



345402
360
CCATCTTTACCAGACAGTGTT
PS/MOE 5-10-6
mir-141





gapmer



345403
361
TATCTGCACTAGATGCACCTTA
PS/MOE 5-10-7
mir-18





gapmer



345404
362
AAAGTGTCAGATACGGTGTGG
PS/MOE 5-10-6
mir-220





gapmer



345405
363
CTGTTCCTGCTGAACTGAGCCA
PS/MOE 5-10-7
mir-24





gapmer



345406
364
AGGCGAAGGATGACAAAGGGAA
PS/MOE 5-10-7
mir-211





gapmer



345407
365
TCAGTTATCACAGTACTGTA
PS/MOE 5-10-5
mir-101





gapmer



345408
366
GCTGAGTGTAGGATGTTTACA
PS/MOE 5-10-6
mir-30b





gapmer



345409
367
CACAAATTCGGATCTACAGGGTA
PS/MOE 5-10-8
mir-10a





gapmer



345410
368
TCAGTTTTGCATAGATTTGCACA
PS/MOE 5-10-8
mir-19a





gapmer



345411
369
CACAAACCATTATGTGCTGCTA
PS/MOE 5-10-7
mir-15a





gapmer



345412
370
CTACGCGTATTCTTAAGCAATA
PS/MOE 5-10-7
mir-137





gapmer



345413
371
AGAATTGCGTTTGGACAATCA
PS/MOE 5-10-6
mir-219





gapmer



345414
372
ACAAAGTTCTGTGATGCACTGA
PS/MOE 5-10-7
mir-148b





gapmer



345415
374
CACAGTTGCCAGCTGAGATTA
PS/MOE 5-10-6
mir-216





gapmer



345416
375
CACAAGTTCGGATCTACGGGTT
PS/MOE 5-10-7
mir-100





gapmer



345417
376
CCGGCTGCAACACAAGACACGA
PS/MOE 5-10-7
mir-187





gapmer



345418
377
CAGCCGCTGTCACACGCACAG
PS/MOE 5-10-6
mir-210





gapmer



345419
378
GTCTGTCAATTCATAGGTCAT
PS/MOE 5-10-6
mir-215





gapmer



345420
379
GGGGTATTTGACAAACTGACA
PS/MOE 5-10-6
mir-223





gapmer



345421
380
GCTGAGAGTGTAGGATGTTTACA
PS/MOE 5-10-8
mir-30c





gapmer



345422
381
AACCTATCCTGAATTACTTGAA
PS/MOE 5-10-7
mir-26b





gapmer



345423
382
CCAAGTTCTGTCATGCACTGA
PS/MOE 5-10-6
mir-152





gapmer



345424
383
ATCACATAGGAATAAAAAGCCATA
PS/MOE 5-10-9
mir-135





gapmer



345425
384
ATCCAATCAGTTCCTGATGCAGTA
PS/MOE 5-10-9
mir-217





gapmer



345426
386
CAATGCAACAGCAATGCAC
PS/MOE 5-10-4
mir-33b





gapmer



345427
387
TGTGAGTTCTACCATTGCCAAA
PS/MOE 5-10-7
mir-182





gapmer



345428
388
ACAAAGTTCTGTAGTGCACTGA
PS/MOE 5-10-7
mir-148a





gapmer



345429
389
GGAAATCCCTGGCAATGTGAT
PS/MOE 5-10-6
mir-23a





gapmer



345430
390
ACTCACCGACAGGTTGAATGTT
PS/MOE 5-10-7
mir-181c





gapmer



345431
391
ACTGTAGGAATATGTTTGATA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-013


345432
392
ATTAAAAAGTCCTCTTGCCCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-023


345433
393
GCTGCCGTATATGTGATGTCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-030


345434
394
GGTAGGTGGAATACTATAACA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-033


345435
395
TAAACATCACTGCAAGTCTTA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-039


345436
396
TTGTAAGCAGTTTTGTTGACA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-040


345437
397
TCACAGAGAAAACAACTGGTA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-041


345438
398
CCTCTCAAAGATTTCCTGTCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-043


345439
399
TGTCAGATAAACAGAGTGGAA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-044


345440
400
GAGAATCAATAGGGCATGCAA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-055


345441
401
AAGAACATTAAGCATCTGACA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-058


345442
402
AATCTCTGCAGGCAAATGTGA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-070


345443
403
AAACCCCTATCACGATTAGCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-071


345444
404
GCCCCATTAATATTTTAACCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-075


345445
405
CCCAATATCAAACATATCA
PS/MOE 5-10-4
hypothetical





gapmer
miRNA-079


345446
406
TATGATAGCTTCCCCATGTAA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-083


345447
407
CCTCAATTATTGGAAATCACA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-088


345448
408
ATTGATGCGCCATTTGGCCTA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-090


345449
409
CTGTGACTTCTCTATCTGCCT
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-099


345450
410
AAACTTGTTAATTGACTGTCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-101


345451
411
AAAGAAGTATATGCATAGGAA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-105


345452
412
GATAAAGCCAATAAACTGTCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-107


345453
413
TCCGAGTCGGAGGAGGAGGAA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-111


345454
414
ATCATTACTGGATTGCTGTAA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-120


345455
415
CAAAAATTATCAGCCAGTTTA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-137


345456
416
AATCTCATTTTCATACTTGCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-138


345457
417
AGAAGGTGGGGAGCAGCGTCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-142


345458
418
CAAAATTGCAAGCAAATTGCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-143


345459
419
TCCACAAAGCTGAACATGTCT
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-144


345460
420
TATTATCAGCATCTGCTTGCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-153


345461
421
AATAACACACATCCACTTTAA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-154


345462
422
AAGAAGGAAGGAGGGAAAGCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-156


345463
423
ATGACTACAAGTTTATGGCCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-161


345464
424
CAAAACATAAAAATCCTTGCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-164


345465
425
TTACAGGTGCTGCAACTGGAA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-166


345466
426
AGCAGGTGAAGGCACCTGGCT
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-168


345467
427
TATGAAATGCCAGAGCTGCCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-169


345468
428
CCAAGTGTTAGAGCAAGATCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-170


345469
429
AACGATAAAACATACTTGTCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-171


345470
430
AGTAACTTCTTGCAGTTGGA
PS/MOE 5-10-5
hypothetical





gapmer
miRNA-172


345471
431
AGCCTCCTTCTTCTCGTACTA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-173


345472
432
ACCTCAGGTGGTTGAAGGAGA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-175


345473
433
ATATGTCATATCAAACTCCTA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-176


345474
434
GTGAGAGTAGCATGTTTGTCT
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-177


345475
435
TGAAGGTTCGGAGATAGGCTA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-178


345476
436
AATTGGACAAAGTGCCTTTCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-179


345477
437
ACCGAACAAAGTCTGACAGGA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-180


345478
438
AACTACTTCCAGAGCAGGTGA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-181


345479
439
GTAAGCGCAGCTCCACAGGCT
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-183


345480
440
GAGCTGCTCAGCTGGCCATCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-185


345481
441
TACTTTTCATTCCCCTCACCA
PS/MOE 5-10-6
hypothetical





gapmer
miRNA-188


345482
236
TAGCTTATCAGACTGATGTTGA
PS/MOE 5-10-7
miR-104





gapmer
(Mourelatos)


345483
1780
ACAGGAGTCTGAGCATTTGA
PS/MOE 5-10-5
miR-105





gapmer
(Mourelatos)


345484
1882
GGAACTTAGCCACTGTGAA
PS/MOE 5-10-4
miR-27





gapmer
(Mourelatos)


345485
848
CTACCTGCACGAACAGCACTTT
PS/MOE 5-10-7
miR-93





gapmer
(Mourelatos)


345486
855
TGCTCAATAAATACCCGTTGAA
PS/MOE 5-10-7
miR-95





gapmer
(Mourelatos)


345487
1821
CACAAGATCGGATCTACGGGTT
PS/MOE 5-10-7
miR-99





gapmer
(Mourelatos)


345488
1903
TCAGACCGAGACAAGTGCAATG
PS/MOE 5-10-7
miR-25 (Tuschl)





gapmer



345489
1853
CTCAATAGACTGTGAGCTCCTT
PS/MOE 5-10-7
miR-28 (Tuschl)





gapmer



345490
1825
CAGCTATGCCAGCATCTTGCC
PS/MOE 5-10-6
miR-31 (Tuschl)





gapmer



345491
1865
GCAACTTAGTAATGTGCAATA
PS/MOE 5-10-6
miR-32 (Tuschl)





gapmer



345492
1897
TAGGAGAGAGAAAAAGACTGA
PS/MOE 5-10-6
miR-14





gapmer



345493
854
GGAGTGAAGACACGGAGCCAGA
PS/MOE 5-10-7
miR-149





gapmer



345494
1845
CGCAAGGTCGGTTCTACGGGTG
PS/MOE 5-10-7
miR-99b





gapmer



345495
852
CACAGGTTAAAGGGTCTCAGGGA
PS/MOE 5-10-8
miR-125a





gapmer



345496
853
AGCCAAGCTCAGACGGATCCGA
PS/MOE 5-10-7
miR-127





gapmer



345497
1909
TCCATCATCAAAACAAATGGAGT
PS/MOE 5-10-8
miR-136





gapmer



345498
1843
CGAAGGCAACACGGATAACCTA
PS/MOE 5-10-7
miR-154





gapmer



345499
1880
GCTTCCAGTCGAGGATGTTTACA
PS/MOE 5-10-8
miR-30a-s





gapmer



345500
1911
TCCGTGGTTCTACCCTGTGGTA
PS/MOE 5-10-7
miR-140-as





gapmer



345501
1836
CCATAAAGTAGGAAACACTACA
PS/MOE 5-10-7
miR-142-as





gapmer



345502
1761
AACAGGTAGTCTGAACACTGGG
PS/MOE 5-10-7
miR-199-s





gapmer



345503
1762
AACCAATGTGCAGACTACTGTA
PS/MOE 5-10-7
miR-199-as





gapmer



345504
1904
TCATACAGCTAGATAACCAAAGA
PS/MOE 5-10-8
miR-9





gapmer



345505
1773
ACAAGTGCCTTCACTGCAGT
PS/MOE 5-10-5
miR-17





gapmer



345506
1871
GCATTATTACTCACGGTACGA
PS/MOE 5-10-6
miR-126a





gapmer



345507
1787
ACCTAATATATCAAACATATCA
PS/MOE 5-10-7
miR-190





gapmer



345508
1766
AAGCCCAAAAGGAGAATTCTTTG
PS/MOE 5-10-8
miR-186





gapmer



345509
1839
CCTATCTCCCCTCTGGACC
PS/MOE 5-10-4
miR-198a





gapmer



345510
1806
AGCTGCTTTTGGGATTCCGTTG
PS/MOE 5-10-7
miR-191c





gapmer



345511
760
CCACACACTTCCTTACATTCCA
PS/MOE 5-10-7
miR-206d





gapmer



345512
761
ATCTGCACTGTCAGCACTTT
PS/MOE 5-10-5
miR-94





gapmer



345513
762
ACCCTTATCAGTTCTCCGTCCA
PS/MOE 5-10-7
miR-184





gapmer



345514
763
GCCAATATTTCTGTGCTGCTA
PS/MOE 5-10-6
miR-195





gapmer



345515
764
CTGGGACTTTGTAGGCCAGTT
PS/MOE 5-10-6
miR-193





gapmer



345516
1861
GAACTGCCTTTCTCTCCA
PS/MOE 5-10-3
miR-185





gapmer



345517
1786
ACCCTCCACCATGCAAGGGATG
PS/MOE 5-10-7
miR-188





gapmer



345518
1879
GCTGGGTGGAGAAGGTGGTGAA
PS/MOE 5-10-7
miR-197a





gapmer



345519
1906
TCCACATGGAGTTGCTGTTACA
PS/MOE 5-10-7
miR-194





gapmer



345520
1771
ACAAGCTTTTTGCTCGTCTTAT
PS/MOE 5-10-7
miR-208





gapmer



345521
938
AGACACGTGCACTGTAGA
PS/MOE 5-10-3
miR-139





gapmer



345522
1887
GTCATCATTACCAGGCAGTATTA
PS/MOE 5-10-8
miR-200b





gapmer



345523
1831
CATCGTTACCAGACAGTGTTA
PS/MOE 5-10-6
miR-200a





gapmer



345524
1827
CAGCTTTCAAAATGATCTCAC
PS/MOE 5-10-6
miR-Bantam





gapmer



345922
1783
ACAGTGCTTCATCTCA
PO/6MOE-10deoxy
mir-143





hemimer



345923
1848
CTACAGTGCTTCATCTC
PO/6MOE-11deoxy
mir-143





hemimer



345924
1876
GCTACAGTGCTTCATCT
PO/6MOE-11deoxy
mir-143





hemimer



345925
1875
GCTACAGTGCTTCATC
PO/6MOE-10deoxy
mir-143





hemimer



345926
1803
AGCTACAGTGCTTCAT
PO/6MOE-10deoxy
mir-143





hemimer



345927
1863
GAGCTACAGTGCTTCA
PO/6MOE-10deoxy
mir-143





hemimer



345928
1916
TGAGCTACAGTGCTTC
PO/6MOE-10deoxy
mir-143





hemimer



346685
1884
GGCGGAACTTAGCCACTGTGAA
PS/MOE
miR-27a (RFAM-






Human)


346686
1857
CTTCAGTTATCACAGTACTGTA
PS/MOE
miR-101 (RFAM-






Human)


346687
1802
AGCAAGCCCAGACCGCAAAAAG
PS/MOE
miR-129b (RFAM-






Human)


346688
1898
TAGTTGGCAAGTCTAGAACCA
PS/MOE
miR-182* (RFAM-






Human)


346689
1830
CATCATTACCAGGCAGTATTAGAG
PS/MOE
miR-200a (RFAM-






Human)


346690
1792
ACTGATATCAGCTCAGTAGGCAC
PS/MOE
miR-189 (RFAM-






Human)


346691
1870
GCAGAAGCATTTCCACACAC
PS/MOE
miR-147 (RFAM-






Human)


346692
1889
TAAACGGAACCACTAGTGACTTG
PS/MOE
miR-224 (RFAM-






Human)


346693
1838
CCCTCTGGTCAACCAGTCACA
PS/MOE
miR-134 (RFAM-






Human)


346694
1763
AACCCATGGAATTCAGTTCTCA
PS/MOE
miR-146 (RFAM-






Human)


346695
1824
CACTGGTACAAGGGTTGGGAGA
PS/MOE
miR-150 (RFAM-






Human)


346696
1893
TACCTGCACTATAAGCACTTTA
PS/MOE
mir-20


346697
1788
ACCTATCCTGAATTACTTGAA
PS/MOE
mir-26b


346698
1793
ACTGATTTCAAATGGTGCTA
PS/MOE
mir-29b


346699
1847
CGGCTGCAACACAAGACACGA
PS/MOE
miR-187 (RFAM-






Human)


346700
1844
CGACCATGGCTGTAGACTGTTA
PS/MOE
miR-132 (RFAM-






Human)


346701
1901
TCACATAGGAATAAAAAGCCATA
PS/MOE
miR-135 (RFAM-






Human)


346702
1893
TACCTGCACTATAAGCACTTTA
PS/MOE 5-10-7
mir-20





gapmer



346703
1788
ACCTATCCTGAATTACTTGAA
PS/MOE 5-10-6
mir-26b





gapmer



346704
1884
GGCGGAACTTAGCCACTGTGAA
PS/MOE 5-10-7
miR-27a (RFAM-





gapmer
Human)


346705
1857
CTTCAGTTATCACAGTACTGTA
PS/MOE 5-10-7
miR-101 (RFAM-





gapmer
Human)


346706
1793
ACTGATTTCAAATGGTGCTA
PS/MOE 5-10-5
mir-29b





gapmer



346707
1847
CGGCTGCAACACAAGACACGA
PS/MOE 5-10-6
miR-187 (RFAM-





gapmer
Human)


346708
1844
CGACCATGGCTGTAGACTGTTA
PS/MOE 5-10-7
miR-132 (RFAM-





gapmer
Human)


346709
1901
TCACATAGGAATAAAAAGCCATA
PS/MOE 5-10-8
miR-135 (RFAM-





gapmer
Human)


346710
1802
AGCAAGCCCAGACCGCAAAAAG
PS/MOE 5-10-7
miR-129b (RFAM-





gapmer
Human)


346711
1898
TAGTTGGCAAGTCTAGAACCA
PS/MOE 5-10-6
miR-182* (RFAM-





gapmer
Human)


346712
1830
CATCATTACCAGGCAGTATTAGAG
PS/MOE 5-10-9
miR-200a (RFAM-





gapmer
Human)


346713
1792
ACTGATATCAGCTCAGTAGGCAC
PS/MOE 5-10-8
miR-189 (RFAM-





gapmer
Human)


346714
1870
GCAGAAGCATTTCCACACAC
PS/MOE 5-10-5
miR-147 (RFAM-





gapmer
Human)


346715
1889
TAAACGGAACCACTAGTGACTTG
PS/MOE 5-10-8
miR-224 (RFAM-





gapmer
Human)


346716
1838
CCCTCTGGTCAACCAGTCACA
PS/MOE 5-10-6
miR-134 (RFAM-





gapmer
Human)


346717
1763
AACCCATGGAATTCAGTTCTCA
PS/MOE 5-10-7
miR-146 (RFAM-





gapmer
Human)


346718
1824
CACTGGTACAAGGGTTGGGAGA
PS/MOE 5-10-7
miR-150 (RFAM-





gapmer
Human)


346905
1907
TCCAGTCAAGGATGTTTACA
PS/MOE
miR-30e (RFAM-






M. musculus)


346906
1781
ACAGGATTGAGGGGGGGCCCT
PS/MOE
miR-296 (RFAM-






M. musculus)


346907
1815
ATGTATGTGGGACGGTAAACCA
PS/MOE
miR-299 (RFAM-






M. musculus)


346908
1881
GCTTTGACAATACTATTGCACTG
PS/MOE
miR-301 (RFAM-






M. musculus)


346909
1902
TCACCAAAACATGGAAGCACTTA
PS/MOE
miR-302 (RFAM-






M. musculus)


346910
1866
GCAATCAGCTAACTACACTGCCT
PS/MOE
miR-34a (RFAM-






M. musculus)


346911
1776
ACACTGATTTCAAATGGTGCTA
PS/MOE
miR-29b (RFAM-






M. musculus)


346912
1851
CTAGTGGTCCTAAACATTTCA
PS/MOE
miR-203 (RFAM-






M. musculus)


346913
1795
AGAAAGGCAGCAGGTCGTATAG
PS/MOE
let-7d* (RFAM-






M. musculus)


346914
1810
ATCTGCACTGTCAGCACTTTA
PS/MOE
miR-106b (RFAM-






M. musculus)


346915
1784
ACATCGTTACCAGACAGTGTTA
PS/MOE
miR-200a (RFAM-






M. musculus)


346916
1874
GCGGAACTTAGCCACTGTGAA
PS/MOE
miR-27a (RFAM-






M. musculus)


346917
1826
CAGCTATGCCAGCATCTTGCCT
PS/MOE
miR-31 (RFAM-M. musculus)


346918
1829
CAGGCCGGGACAAGTGCAATA
PS/MOE
miR-92 (RFAM-M. musculus)


346919
1849
CTACCTGCACGAACAGCACTTTG
PS/MOE
miR-93 (RFAM-M. musculus)


346920
1801
AGCAAAAATGTGCTAGTGCCAAA
PS/MOE
miR-96 (RFAM-M. musculus)


346921
1759
AACAACCAGCTAAGACACTGCCA
PS/MOE
miR-172 (RFAM-






M. musculus)


346922
1907
TCCAGTCAAGGATGTTTACA
PS/MOE 5-10-5
miR-30e (RFAM-





gapmer
M. musculus)


346923
1781
ACAGGATTGAGGGGGGGCCCT
PS/MOE 5-10-6
miR-296 (RFAM-





gapmer
M. musculus)


346924
1815
ATGTATGTGGGACGGTAAACCA
PS/MOE 5-10-7
miR-299 (RFAM-





gapmer
M. musculus)


346925
1881
GCTTTGACAATACTATTGCACTG
PS/MOE 5-10-8
miR-301 (RFAM-





gapmer
M. musculus)


346926
1902
TCACCAAAACATGGAAGCACTTA
PS/MOE 5-10-8
miR-302 (RFAM-





gapmer
M. musculus)


346927
1866
GCAATCAGCTAACTACACTGCCT
PS/MOE 5-10-8
miR-34a (RFAM-





gapmer
M. musculus)


346928
1776
ACACTGATTTCAAATGGTGCTA
PS/MOE 5-10-7
miR-29b (RFAM-





gapmer
M. musculus)


346929
1851
CTAGTGGTCCTAAACATTTCA
PS/MOE 5-10-6
miR-203 (RFAM-





gapmer
M. musculus)


346930
1795
AGAAAGGCAGCAGGTCGTATAG
PS/MOE 5-10-7
let-7d* (RFAM-





gapmer
M. musculus)


346931
1810
ATCTGCACTGTCAGCACTTTA
PS/MOE 5-10-6
miR-106b (RFAM-





gapmer
M. musculus)


346932
1784
ACATCGTTACCAGACAGTGTTA
PS/MOE 5-10-7
miR-200a (RFAM-





gapmer
M. musculus)


346933
1874
GCGGAACTTAGCCACTGTGAA
PS/MOE 5-10-6
miR-27a (RFAM-





gapmer
M. musculus)


346934
1826
CAGCTATGCCAGCATCTTGCCT
PS/MOE 5-10-7
miR-31 (RFAM-M. musculus)





gapmer



346935
1829
CAGGCCGGGACAAGTGCAATA
PS/MOE 5-10-6
miR-92 (RFAM-M. musculus)





gapmer



346936
1849
CTACCTGCACGAACAGCACTTTG
PS/MOE 5-10-8
miR-93 (RFAM-M. musculus)





gapmer



346937
1801
AGCAAAAATGTGCTAGTGCCAAA
PS/MOE 5-10-8
miR-96 (RFAM-M. musculus)





gapmer



346938
1759
AACAACCAGCTAAGACACTGCCA
PS/MOE 5-10-8
miR-172 (RFAM-





gapmer
M. musculus)


347385
1782
ACAGTGCTTCATCTC
PO/6MOE-9deoxy
mir-143





hemimer



347386
1848
CTACAGTGCTTCATCTC
PO/6MOE-11deoxy
mir-143





hemimer



347387
1876
GCTACAGTGCTTCATCT
PO/6MOE-11deoxy
mir-143





hemimer



347388
1875
GCTACAGTGCTTCATC
PO/6MOE-10deoxy
mir-143





hemimer



347389
1803
AGCTACAGTGCTTCAT
PO/6MOE-10deoxy
mir-143





hemimer



347390
1863
GAGCTACAGTGCTTCA
PO/6MOE-10deoxy
mir-143





hemimer



347391
1916
TGAGCTACAGTGCTTC
PO/6MOE-10deoxy
mir-143





hemimer



347452
1783
ACAGTGCTTCATCTCA
PO/6MOE-10deoxy
mir-143





hemimer



347453
1783
ACAGTGCTTCATCTCA
PO/6MOE-10deoxy
mir-143





hemimer



348116
1922
TTCGCCCTCTCAACCCAGCTTTT
PS/MOE
miR-320


348117
1860
GAACCCACAATCCCTGGCTTA
PS/MOE
miR-321-1


348118
1886
GTAAACCATGATGTGCTGCTA
PS/MOE
miR-15b






(Michael et al)


348119
1908
TCCATAAAGTAGGAAACACTACA
PS/MOE
miR-142as






(Michael et al)


348120
1864
GAGCTACAGTGCTTCATCTCA
PS/MOE
miR-143






(Michael et al)


348121
1883
GGATTCCTGGGAAAACTGGAC
PS/MOE
miR-145






(Michael et al)


348122
1905
TCATCATTACCAGGCAGTATTA
PS/MOE
miR-200b






(Michael et al)


348123
1791
ACTATACAATCTACTACCTCA
PS/MOE
let-7f (Michael






et al)


348124
1820
CACAAATTCGGTTCTACAGGGTA
PS/MOE
miR-10b






(Michael et al)


348125
1878
GCTGGATGCAAACCTGCAAAACT
PS/MOE
miR-19b






(Michael et al)


348126
1873
GCCTATCCTGGATTACTTGAA
PS/MOE
miR-26a






(Michael et al)


348127
1869
GCAGAACTTAGCCACTGTGAA
PS/MOE
miR-27*






(Michael et al)


348128
1858
CTTCCAGTCAAGGATGTTTACA
PS/MOE
miR-97 (Michael






et al)


348129
1855
CTGGCTGTCAATTCATAGGTCA
PS/MOE
miR-192






(Michael et al)


348130
1922
TTCGCCCTCTCAACCCAGCTTTT
PS/MOE 5-10-8
miR-320





gapmer



348131
1860
GAACCCACAATCCCTGGCTTA
PS/MOE 5-10-6
miR-321-1





gapmer



348132
1886
GTAAACCATGATGTGCTGCTA
PS/MOE 5-10-6
miR-15b





gapmer
(Michael et al)


348133
1908
TCCATAAAGTAGGAAACACTACA
PS/MOE 5-10-8
miR-142as





gapmer
(Michael et al)


348134
1864
GAGCTACAGTGCTTCATCTCA
PS/MOE 5-10-6
miR-143





gapmer
(Michael et al)


348135
1883
GGATTCCTGGGAAAACTGGAC
PS/MOE 5-10-6
miR-145





gapmer
(Michael et al)


348136
1905
TCATCATTACCAGGCAGTATTA
PS/MOE 5-10-7
miR-200b





gapmer
(Michael et al)


348137
1791
ACTATACAATCTACTACCTCA
PS/MOE 5-10-6
let-7f (Michael





gapmer
et al)


348138
1820
CACAAATTCGGTTCTACAGGGTA
PS/MOE 5-10-8
miR-10b





gapmer
(Michael et al)


348139
1878
GCTGGATGCAAACCTGCAAAACT
PS/MOE 5-10-8
miR-19b





gapmer
(Michael et al)


348140
1873
GCCTATCCTGGATTACTTGAA
PS/MOE 5-10-6
miR-26a





gapmer
(Michael et al)


348141
1869
GCAGAACTTAGCCACTGTGAA
PS/MOE 5-10-6
miR-27*





gapmer
(Michael et al)


348142
1858
CTTCCAGTCAAGGATGTTTACA
PS/MOE 5-10-7
miR-97 (Michael





gapmer
et al)


348143
1855
CTGGCTGTCAATTCATAGGTCA
PS/MOE 5-10-7
miR-192





gapmer
(Michael et al)


354040
1751
AAACCACACAACCTACTACCTCA
PS/MOE
let-7b-Ruvkun


354041
1752
AAACCATACAACCTACTACCTCA
PS/MOE
let-7c-Ruvkun


354042
1764
AACTATGCAACCTACTACCTCT
PS/MOE
let-7d-Ruvkun


354043
1765
AACTGTACAAACTACTACCTCA
PS/MOE
let-7gL-Ruvkun


354044
1760
AACAGCACAAACTACTACCTCA
PS/MOE
let-7i-Ruvkun


354045
1924
TTGGCATTCACCGCGTGCCTTAA
PS/MOE
mir-124a-Ruvkun


354046
1833
CCAAGCTCAGACGGATCCGA
PS/MOE
mir-127-Ruvkun


354047
1896
TACTTTCGGTTATCTAGCTTTA
PS/MOE
mir-131-Ruvkun


354048
1846
CGGCCTGATTCACAACACCAGCT
PS/MOE
mir-138-Ruvkun


354049
1768
ACAAACCATTATGTGCTGCTA
PS/MOE
mir-15-Ruvkun


354050
1789
ACGCCAATATTTACGTGCTGCTA
PS/MOE
mir-16-Ruvkun


354051
1852
CTATCTGCACTAGATGCACCTTA
PS/MOE
mir-18-Ruvkun


354052
1779
ACAGCTGCTTTTGGGATTCCGTTG
PS/MOE
mir-191-Ruvkun


354053
1891
TAACCGATTTCAGATGGTGCTA
PS/MOE
mir-29a-Ruvkun


354054
1813
ATGCTTTGACAATACTATTGCACTG
PS/MOE
mir-301-Ruvkun


354055
1805
AGCTGAGTGTAGGATGTTTACA
PS/MOE
mir-30b-Ruvkun


354056
1804
AGCTGAGAGTGTAGGATGTTTACA
PS/MOE
mir-30c-Ruvkun


354057
1807
AGCTTCCAGTCGGGGATGTTTACA
PS/MOE
mir-30d-Ruvkun


354058
1835
CCAGCAGCACCTGGGGCAGTGG
PS/MOE
mir-324-3p-






Ruvkun


354059
1899
TATGGCAGACTGTGATTTGTTG
PS/MOE
mir-7-1*-Ruvkun


354060
1850
CTACCTGCACTGTAAGCACTTTG
PS/MOE
mir-91-Ruvkun


354061
1822
CACATAGGAATGAAAAGCCATA
PS/MOE
mir-135b






(Ruvkun)


354062
1895
TACTAGACTGTGAGCTCCTCGA
PS/MOE
mir-151*






(Ruvkun)


354063
1885
GGCTATAAAGTAACTGAGACGGA
PS/MOE
mir-340






(Ruvkun)


354064
1923
TTCTAGGATAGGCCCAGGGGC
PS/MOE
mir-331






(Ruvkun)


354065
1892
TACATACTTCTTTACATTCCA
PS/MOE
miR-1 (RFAM)


354066
1817
CAATCAGCTAACTACACTGCCT
PS/MOE
miR-34c (RFAM)


354067
1837
CCCCTATCACGATTAGCATTAA
PS/MOE
miR-155 (RFAM)


354068
1910
TCCATCATTACCCGGCAGTATT
PS/MOE
miR-200c (RFAM)


354069
1818
CAATCAGCTAATGACACTGCCT
PS/MOE
miR-34b (RFAM)


354070
1753
AAACCCAGCAGACAATGTAGCT
PS/MOE
mir-221 (RFAM-






M. musculus)


354071
1796
AGACCCAGTAGCCAGATGTAGCT
PS/MOE
mir-222 (RFAM-






M. musculus)


354072
1917
TGAGCTCCTGGAGGACAGGGA
PS/MOE
mir-339-1






(RFAM)


354073
1925
TTTAAGTGCTCATAATGCAGT
PS/MOE
miR-20* (human)


354074
1926
TTTTCCCATGCCCTATACCTCT
PS/MOE
miR-202 (human)


354075
1856
CTTCAGCTATCACAGTACTGTA
PS/MOE
miR-101b


354076
1894
TACCTGCACTGTTAGCACTTTG
PS/MOE
miR-106a


354077
1772
ACAAGTGCCCTCACTGCAGT
PS/MOE
miR-17-3p


354078
1859
GAACAGGTAGTCTAAACACTGGG
PS/MOE
miR-199b






(mouse)


354079
1915
TCTTCCCATGCGCTATACCTCT
PS/MOE
miR-202 (mouse)


354080
1808
AGGCAAAGGATGACAAAGGGAA
PS/MOE
miR-211 (mouse)


354081
1809
ATCCAGTCAGTTCCTGATGCAGTA
PS/MOE
miR-217 (mouse)


354082
1888
TAAACGGAACCACTAGTGACTTA
PS/MOE
miR-224 (RFAM






mouse)


354083
1758
AACAAAATCACAAGTCTTCCA
PS/MOE
miR-7b


354084
1919
TGTAAGTGCTCGTAATGCAGT
PS/MOE
miR-20* (mouse)


354085
1778
ACACTTACTGGACACCTACTAGG
PS/MOE
mir-325 (human)


354086
1777
ACACTTACTGAGCACCTACTAGG
PS/MOE
mir-325 (mouse)


354087
1877
GCTGGAGGAAGGGCCCAGAGG
PS/MOE
mir-326 (human)


354088
1794
ACTGGAGGAAGGGCCCAGAGG
PS/MOE
mir-326 (mouse)


354089
1755
AAAGAGGTTAACCAGGTGTGTT
PS/MOE
mir-329-1






(human)


354090
1750
AAAAAGGTTAGCTGGGTGTGTT
PS/MOE
mir-329-1






(mouse)


354091
1914
TCTCTGCAGGCCGTGTGCTTTGC
PS/MOE
mir-330 (human)


354092
1913
TCTCTGCAGGCCCTGTGCTTTGC
PS/MOE
mir-330 (mouse)


354093
1757
AAAGGCATCATATAGGAGCTGGA
PS/MOE
mir-337 (human)


354094
1756
AAAGGCATCATATAGGAGCTGAA
PS/MOE
mir-337 (mouse)


354095
1872
GCCCTGGACTAGGAGTCAGCA
PS/MOE
mir-345 (human)


354096
1868
GCACTGGACTAGGGGTCAGCA
PS/MOE
mir-345 (mouse)


354097
1799
AGAGGCAGGCATGCGGGCAGACA
PS/MOE
mir-346 (human)


354098
1798
AGAGGCAGGCACTCGGGCAGACA
PS/MOE
mir-346 (mouse)


354099
1840
CCTCAAGGAGCCTCAGTCTAG
PS/MOE
miR-151 (mouse)


354100
1841
CCTCAAGGAGCCTCAGTCTAGT
PS/MOE
miR-151 (rat)


354101
1797
AGAGGCAGGCACTCAGGCAGACA
PS/MOE
miR-346 (rat)


354102
1819
CAATCAGCTAATTACACTGCCTA
PS/MOE
miR-34b (mouse)


354103
1842
CCTCAAGGAGCTTCAGTCTAGT
PS/MOE
miR-151 (hum)


354104
1751
AAACCACACAACCTACTACCTCA
PS/MOE 5-10-8
let-7b-Ruvkun





gapmer



354105
1752
AAACCATACAACCTACTACCTCA
PS/MOE 5-10-8
let-7c-Ruvkun





gapmer



354106
1764
AACTATGCAACCTACTACCTCT
PS/MOE 5-10-7
let-7d-Ruvkun





gapmer



354107
1765
AACTGTACAAACTACTACCTCA
PS/MOE 5-10-7
let-7gL-Ruvkun





gapmer



354108
1760
AACAGCACAAACTACTACCTCA
PS/MOE 5-10-7
let-7i-Ruvkun





gapmer



354109
1924
TTGGCATTCACCGCGTGCCTTAA
PS/MOE 5-10-8
mir-124a-Ruvkun





gapmer



354110
1833
CCAAGCTCAGACGGATCCGA
PS/MOE 5-10-5
mir-127-Ruvkun





gapmer



354111
1896
TACTTTCGGTTATCTAGCTTTA
PS/MOE 5-10-7
mir-131-Ruvkun





gapmer



354112
1846
CGGCCTGATTCACAACACCAGCT
PS/MOE 5-10-8
mir-138-Ruvkun





gapmer



354113
1768
ACAAACCATTATGTGCTGCTA
PS/MOE 5-10-6
mir-15-Ruvkun





gapmer



354114
1789
ACGCCAATATTTACGTGCTGCTA
PS/MOE 5-10-8
mir-16-Ruvkun





gapmer



354115
1852
CTATCTGCACTAGATGCACCTTA
PS/MOE 5-10-8
mir-18-Ruvkun





gapmer



354116
1779
ACAGCTGCTTTTGGGATTCCGTTG
PS/MOE 5-10-9
mir-191-Ruvkun





gapmer



354117
1891
TAACCGATTTCAGATGGTGCTA
PS/MOE 5-10-7
mir-29a-Ruvkun





gapmer



354118
1813
ATGCTTTGACAATACTATTGCACTG
PS/MOE 5-10-10
mir-301-Ruvkun





gapmer



354119
1805
AGCTGAGTGTAGGATGTTTACA
PS/MOE 5-10-7
mir-30b-Ruvkun





gapmer



354120
1804
AGCTGAGAGTGTAGGATGTTTACA
PS/MOE 5-10-9
mir-30c-Ruvkun





gapmer



354121
1807
AGCTTCCAGTCGGGGATGTTTACA
PS/MOE 5-10-9
mir-30d-Ruvkun





gapmer



354122
1835
CCAGCAGCACCTGGGGCAGTGG
PS/MOE 5-10-7
mir-324-3p-





gapmer
Ruvkun


354123
1899
TATGGCAGACTGTGATTTGTTG
PS/MOE 5-10-7
mir-7-1*-Ruvkun





gapmer



354124
1850
CTACCTGCACTGTAAGCACTTTG
PS/MOE 5-10-8
mir-91-Ruvkun





gapmer



354125
1822
CACATAGGAATGAAAAGCCATA
PS/MOE 5-10-7
mir-135b





gapmer
(Ruvkun)


354126
1895
TACTAGACTGTGAGCTCCTCGA
PS/MOE 5-10-7
mir-151*





gapmer
(Ruvkun)


354127
1885
GGCTATAAAGTAACTGAGACGGA
PS/MOE 5-10-8
mir-340





gapmer
(Ruvkun)


354128
1923
TTCTAGGATAGGCCCAGGGGC
PS/MOE 5-10-6
mir-331





gapmer
(Ruvkun)


354129
1892
TACATACTTCTTTACATTCCA
PS/MOE 5-10-6
miR-1 (RFAM)





gapmer



354130
1817
CAATCAGCTAACTACACTGCCT
PS/MOE 5-10-7
miR-34c (RFAM)





gapmer



354131
1837
CCCCTATCACGATTAGCATTAA
PS/MOE 5-10-7
miR-155 (RFAM)





gapmer



354132
1910
TCCATCATTACCCGGCAGTATT
PS/MOE 5-10-7
miR-200c (RFAM)





gapmer



354133
1818
CAATCAGCTAATGACACTGCCT
PS/MOE 5-10-7
miR-34b (RFAM)





gapmer



354134
1753
AAACCCAGCAGACAATGTAGCT
PS/MOE 5-10-7
mir-221 (RFAM-





gapmer
M. musculus)


354135
1796
AGACCCAGTAGCCAGATGTAGCT
PS/MOE 5-10-8
mir-222 (RFAM-





gapmer
M. musculus)


354136
1917
TGAGCTCCTGGAGGACAGGGA
PS/MOE 5-10-6
mir-339-1





gapmer
(RFAM)


354137
1925
TTTAAGTGCTCATAATGCAGT
PS/MOE 5-10-6
miR-20* (human)





gapmer



354138
1926
TTTTCCCATGCCCTATACCTCT
PS/MOE 5-10-7
miR-202 (human)





gapmer



354139
1856
CTTCAGCTATCACAGTACTGTA
PS/MOE 5-10-7
miR-101b





gapmer



354140
1894
TACCTGCACTGTTAGCACTTTG
PS/MOE 5-10-7
miR-106a





gapmer



354141
1772
ACAAGTGCCCTCACTGCAGT
PS/MOE 5-10-5
miR-17-3p





gapmer



354142
1859
GAACAGGTAGTCTAAACACTGGG
PS/MOE 5-10-8
miR-199b





gapmer
(mouse)


354143
1915
TCTTCCCATGCGCTATACCTCT
PS/MOE 5-10-7
miR-202 (mouse)





gapmer



354144
1808
AGGCAAAGGATGACAAAGGGAA
PS/MOE 5-10-7
miR-211 (mouse)





gapmer



354145
1809
ATCCAGTCAGTTCCTGATGCAGTA
PS/MOE 5-10-9
miR-217 (mouse)





gapmer



354146
1888
TAAACGGAACCACTAGTGACTTA
PS/MOE 5-10-8
miR-224 (RFAM





gapmer
mouse)


354147
1758
AACAAAATCACAAGTCTTCCA
PS/MOE 5-10-6
miR-7b





gapmer



354148
1919
TGTAAGTGCTCGTAATGCAGT
PS/MOE 5-10-6
miR-20* (mouse)





gapmer



354149
1778
ACACTTACTGGACACCTACTAGG
PS/MOE 5-10-8
mir-325 (human)





gapmer



354150
1777
ACACTTACTGAGCACCTACTAGG
PS/MOE 5-10-8
mir-325 (mouse)





gapmer



354151
1877
GCTGGAGGAAGGGCCCAGAGG
PS/MOE 5-10-6
mir-326 (human)





gapmer



354152
1794
ACTGGAGGAAGGGCCCAGAGG
PS/MOE 5-10-6
mir-326 (mouse)





gapmer



354153
1755
AAAGAGGTTAACCAGGTGTGTT
PS/MOE 5-10-7
mir-329-1





gapmer
(human)


354154
1750
AAAAAGGTTAGCTGGGTGTGTT
PS/MOE 5-10-7
mir-329-1





gapmer
(mouse)


354155
1914
TCTCTGCAGGCCGTGTGCTTTGC
PS/MOE 5-10-8
mir-330 (human)





gapmer



354156
1913
TCTCTGCAGGCCCTGTGCTTTGC
PS/MOE 5-10-8
mir-330 (mouse)





gapmer



354157
1757
AAAGGCATCATATAGGAGCTGGA
PS/MOE 5-10-8
mir-337 (human)





gapmer



354158
1756
AAAGGCATCATATAGGAGCTGAA
PS/MOE 5-10-8
mir-337 (mouse)





gapmer



354159
1872
GCCCTGGACTAGGAGTCAGCA
PS/MOE 5-10-6
mir-345 (human)





gapmer



354160
1868
GCACTGGACTAGGGGTCAGCA
PS/MOE 5-10-6
mir-345 (mouse)





gapmer



354161
1799
AGAGGCAGGCATGCGGGCAGACA
PS/MOE 5-10-8
mir-346 (human)





gapmer



354162
1798
AGAGGCAGGCACTCGGGCAGACA
PS/MOE 5-10-8
mir-346 (mouse)





gapmer



354163
1840
CCTCAAGGAGCCTCAGTCTAG
PS/MOE 5-10-6
miR-151 (mouse)





gapmer



354164
1841
CCTCAAGGAGCCTCAGTCTAGT
PS/MOE 5-10-7
miR-151 (rat)





gapmer



354165
1797
AGAGGCAGGCACTCAGGCAGACA
PS/MOE 5-10-8
miR-346 (rat)





gapmer



354166
1819
CAATCAGCTAATTACACTGCCTA
PS/MOE 5-10-8
miR-34b (mouse)





gapmer



354167
1842
CCTCAAGGAGCTTCAGTCTAGT
PS/MOE 5-10-7
miR-151 (human)





gapmer









In accordance with the present invention, oligomeric compounds were designed to mimic one or more miRNAs, pre-miRNAs or pri-miRNAs. The oligomeric compounds of the present invention can also be designed to mimic a pri-miRNA, a pre-miRNA or a single- or double-stranded miRNA while incorporating certain chemical modifications that alter one or more properties of the mimic, thus creating a construct with superior properties over the endogenous pri-miRNA, pre-miRNA or miRNA. Oligomeric compounds representing synthesized miRNAs or chemically modified miRNA mimics were given internal numerical identifiers (ISIS Numbers) and are shown in Table 66. These oligomeric compounds can be analyzed for their effect on miRNA, pre-miRNA or pri-miRNA levels or for their effect on downstream target RNA transcripts by quantitative real-time PCR or they can be used in other assays to investigate the role of miRNAs or miRNA downstream targets. In Table 66, “pri-miRNA” indicates the particular pri-miRNA from which the mature miRNA is normally processed when it occurs in the cellular environment. All compounds listed in Table 66 are ribonucleotides. The miRNA mimics consist of phosphorothioate internucleoside linkages, indicated by “PS” in the “Chemistry” column of Table 66, whereas synthesized miRNA oligomeric compounds with phosphodiester internucleoside linkages are indicated by “PO.”







TABLE 66







miRNAs and miRNA mimics












SEQ






ID

Linkage



ISIS #
NO
sequence
chemistry
Pri-miRNA














343092
437
ACCGAACAAAGTCTGACAGGA
PO
hypothetical miRNA-180


343098
1780
ACAGGAGTCTGAGCATTTGA
PO
miR-105 (Mourelatos)


343099
1882
GGAACTTAGCCACTGTGAA
PO
miR-27 (Mourelatos)


343101
855
TGCTCAATAAATACCCGTTGAA
PO
miR-95 (Mourelatos)


343102
1821
CACAAGATCGGATCTACGGGTT
PO
miR-99 (Mourelatos)


343103
1903
TCAGACCGAGACAAGTGCAATG
PO
miR-25 (Tuschl)


343104
1853
CTCAATAGACTGTGAGCTCCTT
PO
miR-28 (Tuschl)


343105
1825
CAGCTATGCCAGCATCTTGCC
PO
miR-31 (Tuschl)


343106
1865
GCAACTTAGTAATGTGCAATA
PO
miR-32 (Tuschl)


343107
854
GGAGTGAAGACACGGAGCCAGA
PO
miR-149


343108
1845
CGCAAGGTCGGTTCTACGGGTG
PO
miR-99b


343109
852
CACAGGTTAAAGGGTCTCAGGGA
PO
miR-125a


343110
853
AGCCAAGCTCAGACGGATCCGA
PO
miR-127


343111
1909
TCCATCATCAAAACAAATGGAGT
PO
miR-136


343112
1843
CGAAGGCAACACGGATAACCTA
PO
miR-154


343113
1880
GCTTCCAGTCGAGGATGTTTACA
PO
miR-30a-s


343114
1911
TCCGTGGTTCTACCCTGTGGTA
PO
miR-140-as


343115
1836
CCATAAAGTAGGAAACACTACA
PO
miR-142-as


343117
1762
AACCAATGTGCAGACTACTGTA
PO
miR-199-as


343118
1904
TCATACAGCTAGATAACCAAAGA
PO
miR-9


343119
1773
ACAAGTGCCTTCACTGCAGT
PO
miR-17


343120
1871
GCATTATTACTCACGGTACGA
PO
miR-126a


343121
1787
ACCTAATATATCAAACATATCA
PO
miR-190


343122
1766
AAGCCCAAAAGGAGAATTCTTTG
PO
miR-186


343123
1839
CCTATCTCCCCTCTGGACC
PO
miR-198a


343124
1806
AGCTGCTTTTGGGATTCCGTTG
PO
miR-191c


343125
760
CCACACACTTCCTTACATTCCA
PO
miR-206d


343126
761
ATCTGCACTGTCAGCACTTT
PO
miR-94


343127
762
ACCCTTATCAGTTCTCCGTCCA
PO
miR-184


343128
763
GCCAATATTTCTGTGCTGCTA
PO
miR-195


343129
764
CTGGGACTTTGTAGGCCAGTT
PO
miR-193


343130
1861
GAACTGCCTTTCTCTCCA
PO
miR-185


343131
1786
ACCCTCCACCATGCAAGGGATG
PO
miR-188


343132
1879
GCTGGGTGGAGAAGGTGGTGAA
PO
miR-197a


343133
1906
TCCACATGGAGTTGCTGTTACA
PO
miR-194


343134
1771
ACAAGCTTTTTGCTCGTCTTAT
PO
miR-208


343135
938
AGACACGTGCACTGTAGA
PO
miR-139


343136
1887
GTCATCATTACCAGGCAGTATTA
PO
miR-200b


343137
1831
CATCGTTACCAGACAGTGTTA
PO
miR-200a


343138
291
CTACCATAGGGTAAAACCACT
PS
mir-140


343139
292
GCTGCAAACATCCGACTGAAAG
PS
mir-30a


343140
293
ACAACCAGCTAAGACACTGCCA
PS
mir-34


343141
294
AACACTGATTTCAAATGGTGCTA
PS
mir-29b


343142
295
CGCCAATATTTACGTGCTGCTA
PS
mir-16


343143
296
CTAGTGGTCCTAAACATTTCAC
PS
mir-203


343144
297
AACAAAATCACTAGTCTTCCA
PS
mir-7


343145
298
ACAAATTCGGTTCTACAGGGTA
PS
mir-10b


343146
299
AAAAGAGACCGGTTCACTGTGA
PS
mir-128a


343147
300
TCACTTTTGTGACTATGCAA
PS
mir-153


343148
301
CAGAACTTAGCCACTGTGAA
PS
mir-27b


343149
302
GCAAAAATGTGCTAGTGCCAAA
PS
mir-96


343150
303
ACTACCTGCACTGTAAGCACTTTG
PS
mir-17as/mir-91


343151
304
CGCGTACCAAAAGTAATAATG
PS
mir-123/mir-126as


343152
305
GCGACCATGGCTGTAGACTGTTA
PS
mir-132


343153
306
AATGCCCCTAAAAATCCTTAT
PS
mir-108


343154
307
GTGGTAATCCCTGGCAATGTGAT
PS
mir-23b


343155
308
AGCACAAACTACTACCTCA
PS
let-7i


343156
309
GGCCGTGACTGGAGACTGTTA
PS
mir-212


343157
310
ACTTTCGGTTATCTAGCTTTA
PS
mir-131


343158
311
AACCACACAACCTACTACCTCA
PS
let-7b


343159
312
ATACATACTTCTTTACATTCCA
PS
mir-1d


343160
313
ACAAACACCATTGTCACACTCCA
PS
mir-122a


343161
314
ACAGTTCTTCAACTGGCAGCTT
PS
mir-22


343162
315
ACAGGCCGGGACAAGTGCAATA
PS
mir-92


343163
316
GTAGTGCTTTCTACTTTATG
PS
mir-142


343164
317
CAGTGAATTCTACCAGTGCCATA
PS
mir-183


343165
318
CTGCCTGTCTGTGCCTGCTGT
PS
mir-214


343166
319
TGAGCTACAGTGCTTCATCTCA
PS
mir-143


343167
320
GGCTGTCAATTCATAGGTCAG
PS
mir-192


343168
321
AACTATACAACCTACTACCTCA
PS
let-7a


343169
322
ACTCACCGACAGCGTTGAATGTT
PS
mir-181a


343170
323
CAGACTCCGGTGGAATGAAGGA
PS
mir-205


343171
324
TCATAGCCCTGTACAATGCTGCT
PS
mir-103


343172
325
AGCCTATCCTGGATTACTTGAA
PS
mir-26a


343173
326
CAATGCAACTACAATGCAC
PS
mir-33a


343174
327
CCCAACAACATGAAACTACCTA
PS
mir-196


343175
328
TGATAGCCCTGTACAATGCTGCT
PS
mir-107


343176
329
GCTACCTGCACTGTAAGCACTTTT
PS
mir-106


343177
330
AACTATACAATCTACTACCTCA
PS
let-7f


343178
331
AACCGATTTCAAATGGTGCTAG
PS
mir-29c


343179
332
GCCCTTTTAACATTGCACTG
PS
mir-130a


343180
333
ACATGGTTAGATCAAGCACAA
PS
mir-218


343181
334
TGGCATTCACCGCGTGCCTTAA
PS
mir-124a


343182
335
TCAACATCAGTCTGATAAGCTA
PS
mir-21


343183
336
CTAGTACATCATCTATACTGTA
PS
mir-144


343184
337
GAAACCCAGCAGACAATGTAGCT
PS
mir-221


343185
338
GAGACCCAGTAGCCAGATGTAGCT
PS
mir-222


343186
339
CTTCCAGTCGGGGATGTTTACA
PS
mir-30d


343187
340
TCAGTTTTGCATGGATTTGCACA
PS
mir-19b


343188
341
GAAAGAGACCGGTTCACTGTGA
PS
mir-128b


343189
342
GCAAGCCCAGACCGCAAAAAG
PS
mir-129


343190
343
TAGCTGGTTGAAGGGGACCAA
PS
mir-133b


343191
344
ACTATGCAACCTACTACCTCT
PS
let-7d


343192
345
TGTAAACCATGATGTGCTGCTA
PS
mir-15b


343193
346
AACCGATTTCAGATGGTGCTAG
PS
mir-29a


343194
347
GAACAGATAGTCTAAACACTGGG
PS
mir-199b


343195
348
ACTATACAACCTCCTACCTCA
PS
let-7e


343196
349
AACCATACAACCTACTACCTCA
PS
let-7c


343197
350
AGGCATAGGATGACAAAGGGAA
PS
mir-204


343198
351
AAGGGATTCCTGGGAAAACTGGAC
PS
mir-145


343199
352
GGTACAATCAACGGTCGATGGT
PS
mir-213


343200
353
CTACCTGCACTATAAGCACTTTA
PS
mir-20


343201
354
ACAGCTGGTTGAAGGGGACCAA
PS
mir-133a


343202
355
GATTCACAACACCAGCT
PS
mir-138


343203
356
AACAATACAACTTACTACCTCA
PS
mir-98


343204
357
TCACAAGTTAGGGTCTCAGGGA
PS
mir-125b


343205
358
GAACAGGTAGTCTGAACACTGGG
PS
mir-199a


343206
359
AACCCACCGACAGCAATGAATGTT
PS
mir-181b


343207
360
CCATCTTTACCAGACAGTGTT
PS
mir-141


343208
361
TATCTGCACTAGATGCACCTTA
PS
mir-18


343209
362
AAAGTGTCAGATACGGTGTGG
PS
mir-220


343210
363
CTGTTCCTGCTGAACTGAGCCA
PS
mir-24


343211
364
AGGCGAAGGATGACAAAGGGAA
PS
mir-211


343212
365
TCAGTTATCACAGTACTGTA
PS
mir-101


343213
366
GCTGAGTGTAGGATGTTTACA
PS
mir-30b


343214
367
CACAAATTCGGATCTACAGGGTA
PS
mir-10a


343215
368
TCAGTTTTGCATAGATTTGCACA
PS
mir-19a


343216
369
CACAAACCATTATGTGCTGCTA
PS
mir-15a


343217
370
CTACGCGTATTCTTAAGCAATA
PS
mir-137


343218
371
AGAATTGCGTTTGGACAATCA
PS
mir-219


343219
372
ACAAAGTTCTGTGATGCACTGA
PS
mir-148b


343220
373
GCCCTTTCATCATTGCACTG
PS
mir-130b


343221
374
CACAGTTGCCAGCTGAGATTA
PS
mir-216


343222
375
CACAAGTTCGGATCTACGGGTT
PS
mir-100


343223
376
CCGGCTGCAACACAAGACACGA
PS
mir-187


343224
377
CAGCCGCTGTCACACGCACAG
PS
mir-210


343225
378
GTCTGTCAATTCATAGGTCAT
PS
mir-215


343226
379
GGGGTATTTGACAAACTGACA
PS
mir-223


343227
380
GCTGAGAGTGTAGGATGTTTACA
PS
mir-30c


343228
381
AACCTATCCTGAATTACTTGAA
PS
mir-26b


343229
382
CCAAGTTCTGTCATGCACTGA
PS
mir-152


343230
383
ATCACATAGGAATAAAAAGCCATA
PS
mir-135


343231
384
ATCCAATCAGTTCCTGATGCAGTA
PS
mir-217


343232
385
ACTGTACAAACTACTACCTCA
PS
let-7g


343233
386
CAATGCAACAGCAATGCAC
PS
mir-33b


343234
387
TGTGAGTTCTACCATTGCCAAA
PS
mir-182


343235
388
ACAAAGTTCTGTAGTGCACTGA
PS
mir-148a


343236
389
GGAAATCCCTGGCAATGTGAT
PS
mir-23a


343237
390
ACTCACCGACAGGTTGAATGTT
PS
mir-181c


343238
391
ACTGTAGGAATATGTTTGATA
PS
hypothetical miRNA-013


343239
392
ATTAAAAAGTCCTCTTGCCCA
PS
hypothetical miRNA-023


343240
393
GCTGCCGTATATGTGATGTCA
PS
hypothetical miRNA-030


343241
394
GGTAGGTGGAATACTATAACA
PS
hypothetical miRNA-033


343242
395
TAAACATCACTGCAAGTCTTA
PS
hypothetical miRNA-039


343243
396
TTGTAAGCAGTTTTGTTGACA
PS
hypothetical miRNA-040


343244
397
TCACAGAGAAAACAACTGGTA
PS
hypothetical miRNA-041


343245
398
CCTCTCAAAGATTTCCTGTCA
PS
hypothetical miRNA-043


343246
399
TGTCAGATAAACAGAGTGGAA
PS
hypothetical miRNA-044


343247
400
GAGAATCAATAGGGCATGCAA
PS
hypothetical miRNA-055


343248
401
AAGAACATTAAGCATCTGACA
PS
hypothetical miRNA-058


343249
402
AATCTCTGCAGGCAAATGTGA
PS
hypothetical miRNA-070


343250
403
AAACCCCTATCACGATTAGCA
PS
hypothetical miRNA-071


343251
404
GCCCCATTAATATTTTAACCA
PS
hypothetical miRNA-075


343252
405
CCCAATATCAAACATATCA
PS
hypothetical miRNA-079


343253
406
TATGATAGCTTCCCCATGTAA
PS
hypothetical miRNA-083


343254
407
CCTCAATTATTGGAAATCACA
PS
hypothetical miRNA-088


343255
408
ATTGATGCGCCATTTGGCCTA
PS
hypothetical miRNA-090


343256
409
CTGTGACTTCTCTATCTGCCT
PS
hypothetical miRNA-099


343257
410
AAACTTGTTAATTGACTGTCA
PS
hypothetical miRNA-101


343258
411
AAAGAAGTATATGCATAGGAA
PS
hypothetical miRNA-105


343259
412
GATAAAGCCAATAAACTGTCA
PS
hypothetical miRNA-107


343260
413
TCCGAGTCGGAGGAGGAGGAA
PS
hypothetical miRNA-111


343261
414
ATCATTACTGGATTGCTGTAA
PS
hypothetical miRNA-120


343262
415
CAAAAATTATCAGCCAGTTTA
PS
hypothetical miRNA-137


343263
416
AATCTCATTTTCATACTTGCA
PS
hypothetical miRNA-138


343264
417
AGAAGGTGGGGAGCAGCGTCA
PS
hypothetical miRNA-142


343265
418
CAAAATTGCAAGCAAATTGCA
PS
hypothetical miRNA-143


343266
419
TCCACAAAGCTGAACATGTCT
PS
hypothetical miRNA-144


343267
420
TATTATCAGCATCTGCTTGCA
PS
hypothetical miRNA-153


343268
421
AATAACACACATCCACTTTAA
PS
hypothetical miRNA-154


343269
422
AAGAAGGAAGGAGGGAAAGCA
PS
hypothetical miRNA-156


343270
423
ATGACTACAAGTTTATGGCCA
PS
hypothetical miRNA-161


343271
424
CAAAACATAAAAATCCTTGCA
PS
hypothetical miRNA-164


343272
425
TTACAGGTGCTGCAACTGGAA
PS
hypothetical miRNA-166


343273
426
AGCAGGTGAAGGCACCTGGCT
PS
hypothetical miRNA-168


343274
427
TATGAAATGCCAGAGCTGCCA
PS
hypothetical miRNA-169


343275
428
CCAAGTGTTAGAGCAAGATCA
PS
hypothetical miRNA-170


343276
429
AACGATAAAACATACTTGTCA
PS
hypothetical miRNA-171


343277
430
AGTAACTTCTTGCAGTTGGA
PS
hypothetical miRNA-172


343278
431
AGCCTCCTTCTTCTCGTACTA
PS
hypothetical miRNA-173


343279
432
ACCTCAGGTGGTTGAAGGAGA
PS
hypothetical miRNA-175


343280
433
ATATGTCATATCAAACTCCTA
PS
hypothetical miRNA-176


343281
434
GTGAGAGTAGCATGTTTGTCT
PS
hypothetical miRNA-177


343282
435
TGAAGGTTCGGAGATAGGCTA
PS
hypothetical miRNA-178


343283
436
AATTGGACAAAGTGCCTTTCA
PS
hypothetical miRNA-179


343284
437
ACCGAACAAAGTCTGACAGGA
PS
hypothetical miRNA-180


343285
438
AACTACTTCCAGAGCAGGTGA
PS
hypothetical miRNA-181


343286
439
GTAAGCGCAGCTCCACAGGCT
PS
hypothetical miRNA-183


343287
440
GAGCTGCTCAGCTGGCCATCA
PS
hypothetical miRNA-185


343288
441
TACTTTTCATTCCCCTCACCA
PS
hypothetical miRNA-188


343289
236
TAGCTTATCAGACTGATGTTGA
PS
miR-104 (Mourelatos)


343290
1780
ACAGGAGTCTGAGCATTTGA
PS
miR-105 (Mourelatos)


343291
1882
GGAACTTAGCCACTGTGAA
PS
miR-27 (Mourelatos)


343292
848
CTACCTGCACGAACAGCACTTT
PS
miR-93 (Mourelatos)


343293
855
TGCTCAATAAATACCCGTTGAA
PS
miR-95 (Mourelatos)


343294
1821
CACAAGATCGGATCTACGGGTT
PS
miR-99 (Mourelatos)


343295
1903
TCAGACCGAGACAAGTGCAATG
PS
miR-25 (Tuschl)


343296
1853
CTCAATAGACTGTGAGCTCCTT
PS
miR-28 (Tuschl)


343297
1825
CAGCTATGCCAGCATCTTGCC
PS
miR-31 (Tuschl)


343298
1865
GCAACTTAGTAATGTGCAATA
PS
miR-32 (Tuschl)


343299
854
GGAGTGAAGACACGGAGCCAGA
PS
miR-149


343300
1845
CGCAAGGTCGGTTCTACGGGTG
PS
miR-99b


343301
852
CACAGGTTAAAGGGTCTCAGGGA
PS
miR-125a


343302
853
AGCCAAGCTCAGACGGATCCGA
PS
miR-127


343303
1909
TCCATCATCAAAACAAATGGAGT
PS
miR-136


343304
1843
CGAAGGCAACACGGATAACCTA
PS
miR-154


343305
1880
GCTTCCAGTCGAGGATGTTTACA
PS
miR-30a-s


343306
1911
TCCGTGGTTCTACCCTGTGGTA
PS
miR-140-as


343307
1836
CCATAAAGTAGGAAACACTACA
PS
miR-142-as


343308
1761
AACAGGTAGTCTGAACACTGGG
PS
miR-199-s


343309
1762
AACCAATGTGCAGACTACTGTA
PS
miR-199-as


343310
1904
TCATACAGCTAGATAACCAAAGA
PS
miR-9


343311
1773
ACAAGTGCCTTCACTGCAGT
PS
miR-17


343312
1871
GCATTATTACTCACGGTACGA
PS
miR-126a


343313
1787
ACCTAATATATCAAACATATCA
PS
miR-190


343314
1766
AAGCCCAAAAGGAGAATTCTTTG
PS
miR-186


343315
1839
CCTATCTCCCCTCTGGACC
PS
miR-198a


343316
1806
AGCTGCTTTTGGGATTCCGTTG
PS
miR-191c


343317
760
CCACACACTTCCTTACATTCCA
PS
miR-206d


343318
761
ATCTGCACTGTCAGCACTTT
PS
miR-94


343319
762
ACCCTTATCAGTTCTCCGTCCA
PS
miR-184


343320
763
GCCAATATTTCTGTGCTGCTA
PS
miR-195


343321
764
CTGGGACTTTGTAGGCCAGTT
PS
miR-193


343322
1861
GAACTGCCTTTCTCTCCA
PS
miR-185


343323
1786
ACCCTCCACCATGCAAGGGATG
PS
miR-188


343324
1879
GCTGGGTGGAGAAGGTGGTGAA
PS
miR-197a


343325
1906
TCCACATGGAGTTGCTGTTACA
PS
miR-194


343326
1771
ACAAGCTTTTTGCTCGTCTTAT
PS
miR-208


343327
938
AGACACGTGCACTGTAGA
PS
miR-139


343328
1887
GTCATCATTACCAGGCAGTATTA
PS
miR-200b


343329
1831
CATCGTTACCAGACAGTGTTA
PS
miR-200a


344290
1774
ACACAAATTCGGTTCTACAGGG
PO
miR-10b (Tuschl)


344292
1867
GCACGAACAGCACTTTG
PO
miR-93 (Tuschl)


344293
1770
ACAAGATCGGATCTACGGGT
PO
miR-99a (Tuschl)


344297
1912
TCTAGTGGTCCTAAACATTTCA
PO
miR-203 (Tuschl)


344298
1828
CAGGCATAGGATGACAAAGGGAA
PO
miR-204 (Tuschl)


344299
1767
AATACATACTTCTTTACATTCCA
PO
miR-1d (Tuschl)


344300
1769
ACAAATTCGGATCTACAGGGTA
PS
miR-10 (Tuschl)


344301
1774
ACACAAATTCGGTTCTACAGGG
PS
miR-10b (Tuschl)


344302
1890
TAACCGATTTCAAATGGTGCTA
PS
miR-29c (Tuschl)


344303
1867
GCACGAACAGCACTTTG
PS
miR-93 (Tuschl)


344304
1770
ACAAGATCGGATCTACGGGT
PS
miR-99a (Tuschl)


344305
1816
CAAACACCATTGTCACACTCCA
PS
miR-122a,b (Tuschl)


344306
1920
TGTCAATTCATAGGTCAG
PS
miR-192 (Tuschl)


344307
1832
CCAACAACATGAAACTACCTA
PS
miR-196 (Tuschl)


344308
1912
TCTAGTGGTCCTAAACATTTCA
PS
miR-203 (Tuschl)


344309
1828
CAGGCATAGGATGACAAAGGGAA
PS
miR-204 (Tuschl)


344310
1767
AATACATACTTCTTTACATTCCA
PS
miR-1d (Tuschl)


344354
1812
ATGCCCTTTTAACATTGCACTG
PO
mir-130 (Kosik)


344356
1921
TGTCCGTGGTTCTACCCTGTGGTA
PO
mir-239* (Kosik)


344358
1814
ATGCTTTTTGGGGTAAGGGCTT
PO
mir-129as/mir-258*






(Kosik)


344359
1811
ATGCCCTTTCATCATTGCACTG
PO
mir-266* (Kosik)


344360
1918
TGGCATTCACCGCGTGCCTTA
PS
mir-124a (Kosik)


344361
1754
AAAGAGACCGGTTCACTGTGA
PS
mir-128 (Kosik)


344362
1812
ATGCCCTTTTAACATTGCACTG
PS
mir-130 (Kosik)


344363
1854
CTCACCGACAGCGTTGAATGTT
PS
mir-178 (Kosik)


344364
1921
TGTCCGTGGTTCTACCCTGTGGTA
PS
mir-239* (Kosik)


344365
1823
CACATGGTTAGATCAAGCACAA
PS
mir-253* (Kosik)


344366
1814
ATGCTTTTTGGGGTAAGGGCTT
PS
mir-129as/mir-258*






(Kosik)


344367
1811
ATGCCCTTTCATCATTGCACTG
PS
mir-266* (Kosik)


344625
1785
ACATTTTTCGTTATTGCTCTTGA
PO
mir-240* (Kosik)


344626
1790
ACGGAAGGGCAGAGAGGGCCAG
PO
mir-232* (Kosik)


344627
1775
ACACCAATGCCCTAGGGGATGCG
PO
mir-227* (Kosik)


344628
1834
CCAGCAGCACCTGGGGCAGT
PO
mir-226* (Kosik)


344629
1900
TCAACAAAATCACTGATGCTGGA
PO
mir-244* (Kosik)


344630
1800
AGAGGTCGACCGTGTAATGTGC
PO
mir-224* (Kosik)


344631
1862
GACGGGTGCGATTTCTGTGTGAGA
PO
mir-248* (Kosik)


344632
1785
ACATTTTTCGTTATTGCTCTTGA
PS
mir-240* (Kosik)


344633
1790
ACGGAAGGGCAGAGAGGGCCAG
PS
mir-232* (Kosik)


344634
1775
ACACCAATGCCCTAGGGGATGCG
PS
mir-227* (Kosik)


344635
1834
CCAGCAGCACCTGGGGCAGT
PS
mir-226* (Kosik)


344636
1900
TCAACAAAATCACTGATGCTGGA
PS
mir-244* (Kosik)


344637
1800
AGAGGTCGACCGTGTAATGTGC
PS
mir-224* (Kosik)


344638
1862
GACGGGTGCGATTTCTGTGTGAGA
PS
mir-248* (Kosik)


345527
1827
CAGCTTTCAAAATGATCTCAC
PO
miR-Bantam


345529
1897
TAGGAGAGAGAAAAAGACTGA
PS
miR-14


345531
1827
CAGCTTTCAAAATGATCTCAC
PS
miR-Bantam


345708
1897
TAGGAGAGAGAAAAAGACTGA
PO
miR-14


346721
1884
GGCGGAACTTAGCCACTGTGAA
PO
miR-27a (RFAM-Human)


346722
1857
CTTCAGTTATCACAGTACTGTA
PO
miR-101 (RFAM-Human)


346727
1802
AGCAAGCCCAGACCGCAAAAAG
PO
miR-129b (RFAM-Human)


346728
1898
TAGTTGGCAAGTCTAGAACCA
PO
miR-182* (RFAM-Human)


346729
1830
CATCATTACCAGGCAGTATTAGAG
PO
miR-200a (RFAM-Human)


346730
1792
ACTGATATCAGCTCAGTAGGCAC
PO
miR-189 (RFAM-Human)


346731
1870
GCAGAAGCATTTCCACACAC
PO
miR-147 (RFAM-Human)


346732
1889
TAAACGGAACCACTAGTGACTTG
PO
miR-224 (RFAM-Human)


346733
1838
CCCTCTGGTCAACCAGTCACA
PO
miR-134 (RFAM-Human)


346734
1763
AACCCATGGAATTCAGTTCTCA
PO
miR-146 (RFAM-Human)


346735
1824
CACTGGTACAAGGGTTGGGAGA
PO
miR-150 (RFAM-Human)


346736
1893
TACCTGCACTATAAGCACTTTA
PS
mir-20


346737
1788
ACCTATCCTGAATTACTTGAA
PS
mir-26b


346738
1884
GGCGGAACTTAGCCACTGTGAA
PS
miR-27a (RFAM-Human)


346739
1857
CTTCAGTTATCACAGTACTGTA
PS
miR-101 (RFAM-Human)


346740
1793
ACTGATTTCAAATGGTGCTA
PS
mir-29b


346741
1847
CGGCTGCAACACAAGACACGA
PS
miR-187 (RFAM-Human)


346742
1844
CGACCATGGCTGTAGACTGTTA
PS
miR-132 (RFAM-Human)


346743
1901
TCACATAGGAATAAAAAGCCATA
PS
miR-135 (RFAM-Human)


346744
1802
AGCAAGCCCAGACCGCAAAAAG
PS
miR-129b (RFAM-Human)


346745
1898
TAGTTGGCAAGTCTAGAACCA
PS
miR-182* (RFAM-Human)


346746
1830
CATCATTACCAGGCAGTATTAGAG
PS
miR-200a (RFAM-Human)


346747
1792
ACTGATATCAGCTCAGTAGGCAC
PS
miR-189 (RFAM-Human)


346748
1870
GCAGAAGCATTTCCACACAC
PS
miR-147 (RFAM-Human)


346749
1889
TAAACGGAACCACTAGTGACTTG
PS
miR-224 (RFAM-Human)


346750
1838
CCCTCTGGTCAACCAGTCACA
PS
miR-134 (RFAM-Human)


346751
1763
AACCCATGGAATTCAGTTCTCA
PS
miR-146 (RFAM-Human)


346752
1824
CACTGGTACAAGGGTTGGGAGA
PS
miR-150 (RFAM-Human)


346939
1907
TCCAGTCAAGGATGTTTACA
PO
miR-30e (RFAM-M. musculus)


346940
1781
ACAGGATTGAGGGGGGGCCCT
PO
miR-296 (RFAM-M. musculus)


346941
1815
ATGTATGTGGGACGGTAAACCA
PO
miR-299 (RFAM-M. musculus)


346942
1881
GCTTTGACAATACTATTGCACTG
PO
miR-301 (RFAM-M. musculus)


346943
1902
TCACCAAAACATGGAAGCACTTA
PO
miR-302 (RFAM-M. musculus)


346944
1866
GCAATCAGCTAACTACACTGCCT
PO
miR-34a (RFAM-M. musculus)


346945
1776
ACACTGATTTCAAATGGTGCTA
PO
miR-29b (RFAM-M. musculus)


346947
1795
AGAAAGGCAGCAGGTCGTATAG
PO
let-7d* (RFAM-M. musculus)


346948
1810
ATCTGCACTGTCAGCACTTTA
PO
miR-106b (RFAM-M. musculus)


346949
1784
ACATCGTTACCAGACAGTGTTA
PO
miR-200a (RFAM-M. musculus)


346950
1874
GCGGAACTTAGCCACTGTGAA
PO
miR-27a (RFAM-M. musculus)


346951
1826
CAGCTATGCCAGCATCTTGCCT
PO
miR-31 (RFAM-M. musculus)


346954
1801
AGCAAAAATGTGCTAGTGCCAAA
PO
miR-96 (RFAM-M. musculus)


346955
1759
AACAACCAGCTAAGACACTGCCA
PO
miR-172 (RFAM-M. musculus)


346956
1907
TCCAGTCAAGGATGTTTACA
PS
miR-30e (RFAM-M. musculus)


346957
1781
ACAGGATTGAGGGGGGGCCCT
PS
miR-296 (RFAM-M. musculus)


346958
1815
ATGTATGTGGGACGGTAAACCA
PS
miR-299 (RFAM-M. musculus)


346959
1881
GCTTTGACAATACTATTGCACTG
PS
miR-301 (RFAM-M. musculus)


346960
1902
TCACCAAAACATGGAAGCACTTA
PS
miR-302 (RFAM-M. musculus)


346961
1866
GCAATCAGCTAACTACACTGCCT
PS
miR-34a (RFAM-M. musculus)


346962
1776
ACACTGATTTCAAATGGTGCTA
PS
miR-29b (RFAM-M. musculus)


346963
1851
CTAGTGGTCCTAAACATTTCA
PS
miR-203 (RFAM-M. musculus)


346964
1795
AGAAAGGCAGCAGGTCGTATAG
PS
let-7d* (RFAM-M. musculus)


346965
1810
ATCTGCACTGTCAGCACTTTA
PS
miR-106b (RFAM-M. musculus)


346966
1784
ACATCGTTACCAGACAGTGTTA
PS
miR-200a (RFAM-M. musculus)


346967
1874
GCGGAACTTAGCCACTGTGAA
PS
miR-27a (RFAM-M. musculus)


346968
1826
CAGCTATGCCAGCATCTTGCCT
PS
miR-31 (RFAM-M. musculus)


346969
1829
CAGGCCGGGACAAGTGCAATA
PS
miR-92 (RFAM-M. musculus)


346970
1849
CTACCTGCACGAACAGCACTTTG
PS
miR-93 (RFAM-M. musculus)


346971
1801
AGCAAAAATGTGCTAGTGCCAAA
PS
miR-96 (RFAM-M. musculus)


346972
1759
AACAACCAGCTAAGACACTGCCA
PS
miR-172 (RFAM-M. musculus)


348169
1922
TTCGCCCTCTCAACCCAGCTTTT
PO
miR-320


348170
1860
GAACCCACAATCCCTGGCTTA
PO
miR-321-1


348172
1908
TCCATAAAGTAGGAAACACTACA
PO
miR-142as (Michael et






al)


348175
1905
TCATCATTACCAGGCAGTATTA
PO
miR-200b (Michael et






al)


348177
1820
CACAAATTCGGTTCTACAGGGTA
PO
miR-10b (Michael et






al)


348178
1878
GCTGGATGCAAACCTGCAAAACT
PO
miR-19b (Michael et






al)


348180
1869
GCAGAACTTAGCCACTGTGAA
PO
miR-27* (Michael et






al)


348181
1858
CTTCCAGTCAAGGATGTTTACA
PO
miR-97 (Michael et al)


348182
1855
CTGGCTGTCAATTCATAGGTCA
PO
miR-192 (Michael et






al)


348183
1922
TTCGCCCTCTCAACCCAGCTTTT
PS
miR-320


348184
1860
GAACCCACAATCCCTGGCTTA
PS
miR-321-1


348185
1886
GTAAACCATGATGTGCTGCTA
PS
miR-15b (Michael et






al)


348186
1908
TCCATAAAGTAGGAAACACTACA
PS
miR-142as (Michael et






al)


348188
1883
GGATTCCTGGGAAAACTGGAC
PS
miR-145 (Michael et






al)


348189
1905
TCATCATTACCAGGCAGTATTA
PS
miR-200b (Michael et






al)


348190
1791
ACTATACAATCTACTACCTCA
PS
let-7f (Michael et al)


348191
1820
CACAAATTCGGTTCTACAGGGTA
PS
miR-10b (Michael et






al)


348192
1878
GCTGGATGCAAACCTGCAAAACT
PS
miR-19b (Michael et






al)


348193
1873
GCCTATCCTGGATTACTTGAA
PS
miR-26a (Michael et






al)


348194
1869
GCAGAACTTAGCCACTGTGAA
PS
miR-27* (Michael et






al)


348195
1858
CTTCCAGTCAAGGATGTTTACA
PS
miR-97 (Michael et al)


348196
1855
CTGGCTGTCAATTCATAGGTCA
PS
miR-192 (Michael et






al)


354168
1751
AAACCACACAACCTACTACCTCA
PS
let-7b-Ruvkun


354169
1752
AAACCATACAACCTACTACCTCA
PS
let-7c-Ruvkun


354170
1764
AACTATGCAACCTACTACCTCT
PS
let-7d-Ruvkun


354171
1765
AACTGTACAAACTACTACCTCA
PS
let-7gL-Ruvkun


354172
1760
AACAGCACAAACTACTACCTCA
PS
let-7i-Ruvkun


354173
1924
TTGGCATTCACCGCGTGCCTTAA
PS
mir-124a-Ruvkun


354174
1833
CCAAGCTCAGACGGATCCGA
PS
mir-127-Ruvkun


354175
1896
TACTTTCGGTTATCTAGCTTTA
PS
mir-131-Ruvkun


354176
1846
CGGCCTGATTCACAACACCAGCT
PS
mir-138-Ruvkun


354177
1768
ACAAACCATTATGTGCTGCTA
PS
mir-15-Ruvkun


354178
1789
ACGCCAATATTTACGTGCTGCTA
PS
mir-16-Ruvkun


354179
1852
CTATCTGCACTAGATGCACCTTA
PS
mir-18-Ruvkun


354180
1779
ACAGCTGCTTTTGGGATTCCGTTG
PS
mir-191-Ruvkun


354181
1891
TAACCGATTTCAGATGGTGCTA
PS
mir-29a-Ruvkun


354182
1813
ATGCTTTGACAATACTATTGCACTG
PS
mir-301-Ruvkun


354183
1805
AGCTGAGTGTAGGATGTTTACA
PS
mir-30b-Ruvkun


354184
1804
AGCTGAGAGTGTAGGATGTTTACA
PS
mir-30c-Ruvkun


354185
1807
AGCTTCCAGTCGGGGATGTTTACA
PS
mir-30d-Ruvkun


354186
1835
CCAGCAGCACCTGGGGCAGTGG
PS
mir-324-3p-Ruvkun


354187
1899
TATGGCAGACTGTGATTTGTTG
PS
mir-7-1*-Ruvkun


354188
1850
CTACCTGCACTGTAAGCACTTTG
PS
mir-91-Ruvkun


354189
1822
CACATAGGAATGAAAAGCCATA
PS
mir-135b (Ruvkun)


354190
1895
TACTAGACTGTGAGCTCCTCGA
PS
mir-151* (Ruvkun)


354191
1885
GGCTATAAAGTAACTGAGACGGA
PS
mir-340 (Ruvkun)


354192
1923
TTCTAGGATAGGCCCAGGGGC
PS
mir-331 (Ruvkun)


354193
1892
TACATACTTCTTTACATTCCA
PS
miR-1 (RFAM)


354194
1817
CAATCAGCTAACTACACTGCCT
PS
miR-34c (RFAM)


354195
1837
CCCCTATCACGATTAGCATTAA
PS
miR-155 (RFAM)


354196
1910
TCCATCATTACCCGGCAGTATT
PS
miR-200c (RFAM)


354197
1818
CAATCAGCTAATGACACTGCCT
PS
miR-34b (RFAM)


354198
1753
AAACCCAGCAGACAATGTAGCT
PS
mir-221 (RFAM-M. musculus)


354199
1796
AGACCCAGTAGCCAGATGTAGCT
PS
mir-222 (RFAM-M. musculus)


354200
1917
TGAGCTCCTGGAGGACAGGGA
PS
mir-339-1 (RFAM)


354201
1925
TTTAAGTGCTCATAATGCAGT
PS
miR-20* (human)


354202
1926
TTTTCCCATGCCCTATACCTCT
PS
miR-202 (human)


354203
1856
CTTCAGCTATCACAGTACTGTA
PS
miR-101b


354204
1894
TACCTGCACTGTTAGCACTTTG
PS
miR-106a


354205
1772
ACAAGTGCCCTCACTGCAGT
PS
miR-17-3p


354206
1859
GAACAGGTAGTCTAAACACTGGG
PS
miR-199b (mouse)


354207
1915
TCTTCCCATGCGCTATACCTCT
PS
miR-202 (mouse)


354208
1808
AGGCAAAGGATGACAAAGGGAA
PS
miR-211 (mouse)


354209
1809
ATCCAGTCAGTTCCTGATGCAGTA
PS
miR-217 (mouse)


354210
1888
TAAACGGAACCACTAGTGACTTA
PS
miR-224 (RFAM-mouse)


354211
1758
AACAAAATCACAAGTCTTCCA
PS
miR-7b


354212
1919
TGTAAGTGCTCGTAATGCAGT
PS
miR-20* (mouse)


354213
1778
ACACTTACTGGACACCTACTAGG
PS
mir-325 (human)


354214
1777
ACACTTACTGAGCACCTACTAGG
PS
mir-325 (mouse)


354215
1877
GCTGGAGGAAGGGCCCAGAGG
PS
mir-326 (human)


354216
1794
ACTGGAGGAAGGGCCCAGAGG
PS
mir-326 (mouse)


354217
1755
AAAGAGGTTAACCAGGTGTGTT
PS
mir-329-1 (human)


354218
1750
AAAAAGGTTAGCTGGGTGTGTT
PS
mir-329-1 (mouse)


354219
1914
TCTCTGCAGGCCGTGTGCTTTGC
PS
mir-330 (human)


354220
1913
TCTCTGCAGGCCCTGTGCTTTGC
PS
mir-330 (mouse)


354221
1757
AAAGGCATCATATAGGAGCTGGA
PS
mir-337 (human)


354222
1756
AAAGGCATCATATAGGAGCTGAA
PS
mir-337 (mouse)


354223
1872
GCCCTGGACTAGGAGTCAGCA
PS
mir-345 (human)


354224
1868
GCACTGGACTAGGGGTCAGCA
PS
mir-345 (mouse)


354225
1799
AGAGGCAGGCATGCGGGCAGACA
PS
mir-346 (human)


354226
1798
AGAGGCAGGCACTCGGGCAGACA
PS
mir-346 (mouse)


354228
1841
CCTCAAGGAGCCTCAGTCTAGT
PS
miR-151 (rat)


354229
1797
AGAGGCAGGCACTCAGGCAGACA
PS
miR-346 (rat)


354230
1819
CAATCAGCTAATTACACTGCCTA
PS
miR-34b (mouse)


354231
1842
CCTCAAGGAGCTTCAGTCTAGT
PS
miR-151 (human)


354232
1751
AAACCACACAACCTACTACCTCA
PO
let-7b-Ruvkun


354234
1764
AACTATGCAACCTACTACCTCT
PO
let-7d-Ruvkun


354235
1765
AACTGTACAAACTACTACCTCA
PO
let-7gL-Ruvkun


354236
1760
AACAGCACAAACTACTACCTCA
PO
let-7i-Ruvkun


354238
1833
CCAAGCTCAGACGGATCCGA
PO
mir-127-Ruvkun


354239
1896
TACTTTCGGTTATCTAGCTTTA
PO
mir-131-Ruvkun


354240
1846
CGGCCTGATTCACAACACCAGCT
PO
mir-138-Ruvkun


354242
1789
ACGCCAATATTTACGTGCTGCTA
PO
mir-16-Ruvkun


354243
1852
CTATCTGCACTAGATGCACCTTA
PO
mir-18-Ruvkun


354244
1779
ACAGCTGCTTTTGGGATTCCGTTG
PO
mir-191-Ruvkun


354245
1891
TAACCGATTTCAGATGGTGCTA
PO
mir-29a-Ruvkun


354246
1813
ATGCTTTGACAATACTATTGCACTG
PO
mir-301-Ruvkun


354248
1804
AGCTGAGAGTGTAGGATGTTTACA
PO
mir-30c-Ruvkun


354250
1835
CCAGCAGCACCTGGGGCAGTGG
PO
mir-324-3p-Ruvkun


354251
1899
TATGGCAGACTGTGATTTGTTG
PO
mir-7-1*-Ruvkun


354253
1822
CACATAGGAATGAAAAGCCATA
PO
mir-135b (Ruvkun)


354254
1895
TACTAGACTGTGAGCTCCTCGA
PO
mir-151* (Ruvkun)


354255
1885
GGCTATAAAGTAACTGAGACGGA
PO
mir-340 (Ruvkun)


354256
1923
TTCTAGGATAGGCCCAGGGGC
PO
mir-331 (Ruvkun)


354258
1817
CAATCAGCTAACTACACTGCCT
PO
miR-34c (RFAM)


354259
1837
CCCCTATCACGATTAGCATTAA
PO
miR-155 (RFAM)


354260
1910
TCCATCATTACCCGGCAGTATT
PO
miR-200c (RFAM)


354261
1818
CAATCAGCTAATGACACTGCCT
PO
miR-34b (RFAM)


354264
1917
TGAGCTCCTGGAGGACAGGGA
PO
mir-339-1 (RFAM)


354265
1925
TTTAAGTGCTCATAATGCAGT
PO
miR-20* (human)


354266
1926
TTTTCCCATGCCCTATACCTCT
PO
miR-202 (human)


354267
1856
CTTCAGCTATCACAGTACTGTA
PO
miR-101b


354268
1894
TACCTGCACTGTTAGCACTTTG
PO
miR-106a


354269
1772
ACAAGTGCCCTCACTGCAGT
PO
miR-17-3p


354270
1859
GAACAGGTAGTCTAAACACTGGG
PO
miR-199b (mouse)


354271
1915
TCTTCCCATGCGCTATACCTCT
PO
miR-202 (mouse)


354272
1808
AGGCAAAGGATGACAAAGGGAA
PO
miR-211 (mouse)


354273
1809
ATCCAGTCAGTTCCTGATGCAGTA
PO
miR-217 (mouse)


354274
1888
TAAACGGAACCACTAGTGACTTA
PO
miR-224 (RFAM-mouse)


354275
1758
AACAAAATCACAAGTCTTCCA
PO
miR-7b


354276
1919
TGTAAGTGCTCGTAATGCAGT
PO
miR-20* (mouse)


354277
1778
ACACTTACTGGACACCTACTAGG
PO
mir-325 (human)


354278
1777
ACACTTACTGAGCACCTACTAGG
PO
mir-325 (mouse)


354279
1877
GCTGGAGGAAGGGCCCAGAGG
PO
mir-326 (human)


354280
1794
ACTGGAGGAAGGGCCCAGAGG
PO
mir-326 (mouse)


354281
1755
AAAGAGGTTAACCAGGTGTGTT
PO
mir-329-1 (human)


354282
1750
AAAAAGGTTAGCTGGGTGTGTT
PO
mir-329-1 (mouse)


354283
1914
TCTCTGCAGGCCGTGTGCTTTGC
PO
mir-330 (human)


354284
1913
TCTCTGCAGGCCCTGTGCTTTGC
PO
mir-330 (mouse)


354285
1757
AAAGGCATCATATAGGAGCTGGA
PO
mir-337 (human)


354286
1756
AAAGGCATCATATAGGAGCTGAA
PO
mir-337 (mouse)


354287
1872
GCCCTGGACTAGGAGTCAGCA
PO
mir-345 (human)


354288
1868
GCACTGGACTAGGGGTCAGCA
PO
mir-345 (mouse)


354289
1799
AGAGGCAGGCATGCGGGCAGACA
PO
mir-346 (human)


354290
1798
AGAGGCAGGCACTCGGGCAGACA
PO
mir-346 (mouse)


354292
1841
CCTCAAGGAGCCTCAGTCTAGT
PO
miR-151 (rat)


354293
1797
AGAGGCAGGCACTCAGGCAGACA
PO
miR-346 (rat)


354294
1819
CAATCAGCTAATTACACTGCCTA
PO
miR-34b (mouse)


354295
1842
CCTCAAGGAGCTTCAGTCTAGT
PO
miR-151 (human)









It is also understood that, although many of the oligomeric compounds listed in Tables 64-66 have been designed to target or mimic a particular miRNA from humans, for example, that oligomeric compound may also target or mimic other miRNAs from mammals, such as those from rodent species, for example. It is also understood that these miRNAs and mimics can serve as the basis for several variations of nucleic acid oligomeric compounds, including compounds with chemical modifications such as uniform or chimeric 2′-MOE oligomeric compounds, as well as LNAs and PNAs; such oligomeric compounds are also within the scope of the invention. One such non-limiting example is ISIS Number 351104 (CTAGTGGTCCTAAACATTTCAC; SEQ ID NO: 296), which is a PNA oligomeric compound targeted to the human mir-203 miRNA.
Example 35
Targeting miRNAs in Introns and Exons
By mapping the coding sequences of miRNAs onto genomic contigs (which sequence information is available from public databases, such as GenBank and Locus Link), and identifying loci at which other reported gene coding sequences also co-map, it was observed that miRNAs can be encoded within the exons or introns of other genes. The oligomeric compounds of the present invention can be designed to target introns and exons of these genes. For example, the oligomeric compounds of the present invention can be designed to target introns or exons of the genes listed in Table 67. More specifically, these oligomeric compounds can target the miRNAs encoded within the exons or introns of these genes listed in Table 67.







TABLE 67







Oligomeric compounds targeting miRNAs found within introns or exons











SEQ ID

Locus SEQ


ISIS #
NO:
Locus containing miRNA
ID NO













327873
291
Ubiquitin protein ligase WWP2 containing mir-
1928




140



327874
292
hypothetical protein FLJ13189
1929


327877
295
deleted in lymphocytic leukemia, 2 containing
1930




mir-16-1 and mir-15a-1



327877
295
SMC4 (structural maintenance of chromosomes 4,
1931




yeast)-like 1 containing mir-16-3 and mir-15b



327879
297
heterogeneous nuclear ribonucleoprotein K
1932




containing mir-7-1



327879
297
pituitary gland specific factor 1a containing
1933




mir-7-3



327881
299
R3H domain (binds single-stranded nucleic
1934




acids) containing containing mir-128a



327882
300
protein tyrosine phosphatase, receptor type, N
1935




polypeptide 2 containing mir-153-2



327882
300
protein tyrosine phosphatase, receptor type, N
1936




containing mir-153-1



327883
301
chromosome 9 ORF3 containing mir-23b, mir-24-2
1937




and mir-27b



327892
310
Transcriptional activator of the c-fos
1938




promoter containing mir-131-1/miR-9



327896
314
hypothetical protein MGC14376 containing mir-
1939




22



327906
324
hypothetical protein FLJ11729 containing mir-
1940




103-2



327906
324
hypothetical protein FLJ12899 containing mir-
1941




103-1



327907
325
conserved gene amplified in osteosarcoma
1942




containing miR-26a-2



327907
325
HYA22 protein containing miR-26a-1
1943


327908
326
Sterol regulatory element binding
1944




transcription factor 2 containing mir-33a



327910
328
pantothenate kinase containing mir-107
1945


327912
330
upstream regulatory element binding protein 1
1946




containing mir-98 and let-7f-2



327915
333
slit (Drosophila) homolog 3 containing mir-
1947




218-2



327915
333
slit (Drosophila) homolog 2 containing mir-
1948




218-1



327923
341
cyclic AMP-regulated phosphoprotein, 21 kD
1949




containing mir-128b



327932
350
transient receptor potential cation channel,
1950




subfamily M, member 3 containing mir-204



327946
364
melastatin 1 containing mir-211
1951


327947
365
RNA cyclase homolog containing mir-101-3
1952


327954
372
CGI-120 protein containing mir-148b
1953


327963
381
nuclear LIM interactor-interacting factor
1954




containing mir-26b



327964
382
COPZ2 for nonclathrin coat protein zeta-COP
1955




containing mir-152



327967
385
hypothetical protein PRO2730 containing let-7g
1956


327968
386
sterol regulatory element-binding protein-1/
1957




mir-33b



328089
391
talin 2 containing hypothetical miR-13/miR-190
1958


328091
393
calcitonin receptor containing hypothetical
1959




miRNA 30



328092
394
glutamate receptor, ionotrophic, AMPA 3/
1960




hypothetical miRNA-033



328093
395
myosin, heavy polypeptide 7B, cardiac muscle,
1961




beta containing hypothetical miRNA 039



328101
403
LOC 114614/hypothetical miRNA-071
1962


328104
406
dachshund (Drosophila) homolog containing
1963




hypothetical miRNA 083



328105
407
DiGeorge syndrome critical region gene 8/
1964




hypothetical miRNA-088



328111
413
hypothetical protein FLJ21016, containing
1965




hypothetical miRNA 111



328117
419
collagen, type I, alpha 1/hypothetical miRNA-
1966




144



328119
421
hypothetical protein HH114 containing
1967




hypothetical miRNA 154



328120
422
sprouty (Drosophila) homolog 4 containing
1968




hypothetical miRNA 156



328124
426
ribosomal protein L5/hypothetical miRNA 168-2
1969


328125
427
forkhead box P2/hypothetical miRNA 169
1970


328127
429
glutamate receptor, ionotropic, AMPA 2/
1971




hypothetical miRNA 171



328128
430
potassium large conductance calcium-activated
1972




channel, subfamily M, alpha member 1





containing hypothetical miRNA 172



328131
433
hypothetical protein FLJ20307
1973


328135
437
cezanne 2/hypothetical miRNA-180
1974


328137
439
tight junction protein 1 (zona occludens 1)/
1975




hypothetical miRNA-183



340343
1780
gamma-aminobutyric acid (GABA) A receptor,
1976




alpha 3 containing miR-105 (Mourelatos) and





miR-105-2



340348
848
Minichromosome maintenance deficient (S. cerevisiae)
1977




7 containing miR-93 (Mourelatos)





and miR-25 and miR-94



340350
855
KIAA1808 protein containing miR-95
1978




(Mourelatos)



340356
1853
LIM domain-containing preferred translocation
1979




partner in lipoma containing miR-28



340360
1865
chromosome 9 open reading frame 5 containing
1980




miR-32



341785
854
glypican 1 containing miR-149
1981


341798
1871
Notch 4 like containing mir-123/mir-126
1982


341800
1766
zinc finger protein 265 containing miR-186
1983


341801
1839
follistatin-like 1 containing miR-198
1984


341802
1806
hypothetical protein FLJ10496 containing miR-
1985




191



341808
1861
hypothetical protein DKFZp761P1121, containing
1986




miR-185



341809
1786
chloride channel 5 (nephrolithiasis 2, X-
1987




linked, Dent disease) containing miR-188



341812
1771
myosin, heavy polypeptide 6, cardiac muscle,
1988




alpha (cardiomyopathy, hypertrophic 1)





containing miR-208



341813
938
phosphodiesterase 2A, cGMP-stimulated
1989




containing miR-139



344611
1785
mesoderm specific transcript (mouse) homolog
1990




containing mir-240* (Kosik)



344615
1900
Apoptosis-associated tyrosine kinase
1991




containing mir-244* (Kosik)



344617
1862
RNB6 containing mir-248* (Kosik)
1992


346692
1889
gamma-aminobutyric acid (GABA) A receptor,
1993




epsilon, containing miR-224 (Sanger)



348128
1858
Nuclear transcription factor Y, gamma
1994




containing miR-30c-2 and miR-30e









Example 36
Oligomeric Compounds Targeting Components of the RNAi Pathway
In one step of miRNA processing, the pre-miRNAs, approximately 70 to 110 nucleotides in length, are processed by the human Dicer RNase into mature miRNAs. The Dicer enzyme is conserved from fungi to vertebrates. The helicase-moi gene is the human homolog of Dicer from Drosophila. Human Dicer is required for the production of active small non-coding RNAs involved in repressing gene expression by the RNA interference pathway; targeted destruction in cultured human cells of the mRNA encoding human Dicer leads to accumulation of the let-7 pre-miRNA (Hutvagner, et al., 2001, Science 293(5531):834-8). Furthermore, the zebrafish Dicer1 ortholog was cloned and its expression disrupted by target-selected gene inactivation; in homozygous dicer 1 mutants, an initial build-up of miRNA levels produced by maternal Dicer1 was observed, but miRNA accumulation halted after a few days, and a developmental arrest was observed at around day 10, indicating that miRNA-producing Dicer1 is essential for vertebrate development (Wienholds, et al., 2003, Nat Genet., 35(3):217-8). The Dicer gene has also been disrupted in mice. Loss of Dicer1 led to lethality early in development, with Dicer1-null embryos depleted of stem cells. Coupled with the inability to generate viable Dicer1-null embryonic stem cells, this suggests a role for Dicer and, by implication, the RNAi machinery in maintaining the stem cell population during early mouse development (Bernstein, et al., 2003, Nat Genet., 35(3):215-7).
Thus, it was predicted that treatment of cells with oligomeric compounds targeting human Dicer would result in an increase in expression levels of miRNA precursor structures, and thus would be useful in increasing the sensitivity of or enabling the detection of certain pre-miRNAs and/or pri-miRNAs otherwise beneath the limits of detection. It was also predicted that treatment of cells with oligomeric compounds targeting human Dicer would result in a decrease in mature miRNAs, leading to dysregulation of miRNA-regulated targets. Thus, a transcriptomics- or proteomics-based approach could be used to compare and identify target RNAs or proteins for which changes in expression levels correlate directly or inversely with the changes in mature miRNA levels. Target RNAs or their downstream protein products which are being misregulated upon treatment with oligomeric compounds targeting human Dicer, can thereby lead to the identification of any potential miRNA-regulated targets.
The present invention provides methods of maintaining a pluripotent stem cell comprising contacting the cell with an effective amount of an oligomeric compound targeting human Dicer. The pluripotent stem cell can be present in a sample of cord blood or bone marrow, or may be present as part of a cell line. In addition, the pluripotent stem cell can be an embryonic stem cell.
In some embodiments, oligomeric compounds ISIS Number 138648 (GCTGACCTTTTTGCTTCTCA; herein incorporated as SEQ ID NO: 1995) and ISIS Number 138678 (CATAAACATTTCCATCAGTG; herein incorporated as SEQ ID NO:-1996), both 5-10-5 2′-MOE gapmers with phosphorothioate backbones, were designed to target the human Dicer mRNA. These oligomeric compounds were used to transfect the A549, T-24, HepG2, HMEC, T47D, HuVEC, and MCF7 cell lines, as well as human primary dendritic cells, preadipocytes, differentiated adipocytes, and human spleen tissue, and the effects of treatment with the oligomeric compounds on phenotypic parameters, such as caspase activity and expression of markers of adipocyte differentiation (aP2, HSL, Glut4) was assessed as described in Examples 11 and 13, respectively.
Interestingly, treatment of T47D breast adenocarcinoma (p53 mutant) cells with the oligomeric compound ISIS 138648 targeting human Dicer was observed to result in a 41% increase in caspase activity. This phenotype is similar to the effect of treatment of T47D cells with oligomeric compound ISIS Number 328645 (SEQ ID NO: 554), targeting mir-124a-1 described in Example 11. It is believed that treatment of T47D cells with the oligomeric compound ISIS 138648 inhibits expression of human Dicer, which results in reduced production of mature miRNAs. Inadequate levels of miRNAs or inappropriately elevated levels of miRNA precursors may disrupt important cellular events, such as regulation of the cell cycle, and lead cells to trigger apoptotic pathways.
In adipocyte differentiation assays performed as described in Example 13, treatment of human white preadipocytes with ISIS Number 138648 targeting human Dicer was observed to result in decreased triglyceride production. An increase in triglyceride content is a well-established marker of adipocyte differentiation; treatment of adipocytes with oligomeric compound ISIS 138648 resulting in a decrease in triglyceride levels indicates an apparent inhibition of adipocyte differentiation. Thus, the oligomeric compound ISIS 138648 targeting human Dicer may be useful as a pharmaceutical agent with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as in the maintenance of the undifferentiated, pluripotent phenotype of stem or precursor cells. The inhibition of expression of human Dicer by ISIS 138648 is believed to result in decreased production of miRNAs, and some of these miRNAs may be critical for proper regulation of the cell cycle (such as is predicted for the regulation of ERK5 by mir-143); treatment of preadipocytes with this inhibitor of human Dicer and the resulting decrease in production of mature miRNAs, as well as the concommitant accumulation of pre-miRNAs or pri-miRNAs may upset the balance between cellular proliferation and differentiation, predisposing cells to an undifferentiated state.
Example 37
Design of Additional Double-Stranded miRNA Mimics
As described supra, a reporter vector system employing, for example, the pGL3-bulge(x3) plasmid or the pGL3-mir-143 sensor plasmids can be used to assess the ability of miRNA mimics to bind target sites or to assess their effects on the expression of miRNAs, pre-miRNAs or pri-miRNAs. Various chemically modified miRNA mimics have been designed and synthesized for this purpose. The oligomeric compounds of the present invention can be designed to mimic a pri-miRNA, pre-miRNA or a single- or double-stranded miRNA while incorporating certain chemical modifications that alter one or more properties of the mimic, thus creating a construct with superior qualities over the endogenous precursor or miRNA.
In accordance with the present invention, a series of oligomeric compounds was designed and synthesized to mimic double-stranded miRNAs. In some embodiments, various oligomeric compounds representing the sense strand of the mir-143 miRNA, were synthesized, incorporating various chemically modified sugars and/or internucleoside linkages. Similarly, various oligomeric compounds representing the antisense strand complementary to the mir-143 miRNA were synthesized, incorporating various chemically modified sugars and/or internucleoside linkages. The antisense and sense oligomeric compounds designed to mimic mir-143 are shown in Table 68 and 69, respectively. All of the sugar moieties of the oligomeric compounds listed in Tables 68 and 69 are ribonucleotides unless otherwise indicated, and the 3′-terminal nucleosides each have a 3′-OH group unless otherwise specified. The sequences are written in the 5′ to 3′ direction. All antisense oligomeric compounds in Table 68 have the nucleotide sequence GAGCUACAGUGCUUCAUCUCA (herein incorporated as SEQ ID NO: 1864). The sense oligomeric compounds in Table 69 have one of three nucleotide sequences which only differ in that there is a thymidine substitution in place of uridine in two of the sequences; these are: UGAGAUGAAGCACUGUAGCUC (herein incorporated as SEQ ID NO: 1088), UGAGATGAAGCACUGUAGCUC (herein incorporated as SEQ ID NO: 1088), and UGAGAUGAAGCACUGTAGCUC (herein incorporated as SEQ ID NO: 1088). In Tables 68 and 69, the column “Chemical modification” lists the general class and type of chemical modification for the respective oligomeric compounds. The column “Sequence” indicates the nucleobase sequence with symbols indicating sugar and linkage modifications. In the Sequence columns of Tables 68 and 69, internucleoside linkages are assumed to be phosphodiester unless otherwise indicated; phosphorothioate internucleoside linkages are indicated by “s” after the letter indicating the nucleobase (for example, “GsC” indicates a guanosine linked to a cytidine with a 3′,5′-phosphorothioate (PS) internucleoside linkage). Other symbols used to indicate sugar and linkage modifications in the Sequence columns of Tables 68 and 69 are as follows: “mC” indicates that the cytidine residue at the specified position is a 5-methylcytidine; replacement of the 2′-OH of the ribosyl sugar with a 2′-O-methoxyethyl (2′-MOE) is indicated by “e” after the letter indicating the nucleobase (for example, “GAe” indicates a guanosine linked to a 2′-MOE adenosine with a 3′,5′-phosphodiester internucleoside linkage); replacement or substitution of the 2′-OH of the ribosyl sugar with a 2-O-methyl (2′-OMe) is indicated by “m” after the letter indicating the nucleobase (for example, “CmA” indicates a 2′-O-methyl cytidine linked to an adenosine with a 3′,5′-phosphodiester internucleoside linkage); nucleosides having a 2′-Fluoro (2′-F) substituent group are indicated with a “f” after the letter indicating the nucleobase (for example, “GfAm” indicates a 2′-F guanosine linked to a 2′-O-Methyl-adenosine with a 3′,5′-phosphodiester internucleoside linkage); 4′-Thio (4′-S) residues are indicated by “4s” (for example, “GC4s” indicates a guanosine linked to a 4′-S cytidine with a 3′,5′-phosphodiester internucleoside linkage).
In the “Chemical modification” column of Tables 68 and 69, “unmodified” indicates a native strand. “Full” indicates a fully modified oligomeric compound where the chemical modification occurs at each nucleoside or internucleoside linkage. For example each nucleoside of the oligomeric compound could have a modified sugar selected from one of 4′-S, 2′-MOE, 2′-F, 2′-O-Methyl, LNA or ENATM or could have uniformly modified internucleoside linkages such as uniform phosphorothioate internucleoside linkages.
In the “Chemical modification” column of Tables 68 and 69, “Alt” indicates that the nucleosides and or the internucleoside linkages have an alternating motif. The alternating motif can be the result of different sugar modifications that alternate (for example, 2′-ribose alternating with a 2′-modification other than ribose such as MOE, 2′-F or 2′-O-Methyl, or alternating fully modified sugars such as 2′-O-Methyl alternating with 2′-F), or can be the result of alternating internucleoside linkages (for example alternating phosphodiester and phosphorothioate internucleoside linkages). Oligomeric compounds having alternating modifications are described in the chemical modification column with the modification at the first 5′-nucleoside or the first internucleoside linkage at the 5′-end of the nucleoside listed first. For example, oligomeric compounds described as “Alt 2′-F/2′-OMe” have a 2′-F modified sugar at the 5′-terminal nucleoside with the next nucleoside having a 2′-F modified sugar and this alternating pattern is repeated through to the 3′-terminal nucleoside.
In the “Chemical modification” column of Tables 68 and 69, “gapmer” indicates that the oligomeric compound is divided into three distinct regions. The wings are the regions located externally at the 3′ and the 5′-end with the gap being the internal region. Gapmers can be the result of differences in linkage (PO vs. PS) or nucleoside (modified sugar moiety or heterocyclic base). Gapmers also include chimeric gapped oligomeric compounds such as when the wings and the gapped regions are all distinct one from each other. Examples of chemistries that can be used to prepare gapped oligomeric compounds include 2′-MOE, 2′-F, 2′-O-Methyl, LNA and ENATM
In the “Chemical modification” column of Tables 68 and 69, “hemimer” indicates an oligomeric compound that has two distinct regions resulting from differences in the nucleoside or the internucleoside linkage or both. Examples include oligomeric compounds having two regions wherein one region has modified internucleoside linkages such as PS or modified sugar moieties such as 2′-MOE, 2′-F, 2′-O-Methyl, LNA and ENATM.
In the “Chemical modification” column of Tables 68 and 69, “blockmer” indicates an oligomeric compound that has at least one block of modified nucleosides or internucleoside linkages that are located internally. The blocks are generally from two to about five nucleosides in length and are not located at one of the ends as that could be a hemimer Examples of blockmers include oligomeric compounds having from two to about five internally modified nucleosides such as 2′-MOE, 2′-F, 2′-O-Methyl, LNA and ENATM.
In the “Chemical modification” column of Tables 68 and 69, “point modification” indicates an oligomeric compound having a single modified nucleoside located in the oligomeric compound at any position.







TABLE 68







Antisense oligomeric compounds mimicking mir-143











SEQ




ISIS
ID
Chemical



NO:
NO
modification
Sequence





348173
1864
Unmodified
GAGCUACAGUGCUUCAUCUCA


348187
1864
Full PS
GsAsGsCsUsAsCsAsGsUsGsCsUsUsCsAsUsCsUsCsA


362972
1864
Alt ribose/2′-
GAeGCeUAeCAeGUeGCeUUeCAeUCeUCeA




MOE



366179
1864
Alt ribose/2′-
GAmGCmUAmCAmGUmGCmUUmCAmUCmUCmA




OMe



366181
1864
Alt 2′-
GmAGmCUmACmAGmUGmCUmUCmAUmCUmCAm




OMe/ribose



366182
1864
Full 2′-OMe
GmAmGmCmUmAmCmAmGmUmGmCmUmUmCmAmUmCmUmCmAm


366188
1864
2′-MOE 3-15-3
GeAeGeCUACAGUGCUUCAUCUeCeAe




gapmer



366189
1864
Full 2′-MOE
GeAeGeCeUeAeCeAeGeUeGeCeUeUeCeAeUeCeUeCeAe


366190
1864
Alt 2′-
GeAGeCUeACeAGeUGeCUeUCeAUeCUeCAe




MOE/ribose



366198
1864
Alt 2′-F/2′-OMe
GfAmGfCmUfAmCfAmGfUmGfCmUfUmCfAmUfCmUfCmAf
















TABLE 69







Sense oligomeric compounds mimicking mir-143











SEQ




ISIS
ID
Chemical



NO:
NO
modification
Sequence





348201
1088
Unmodified
UGAGAUGAAGCACUGUAGCUC


342199
 220
Unmodified
UGAGAUGAAGCACUGUAGCUCA


348215
1088
Full PS
UsGsAsGsAsUsGsAsAsGsCsAsCsUsGsUsAsGsCsUsC


366175
1088
PO/PS/PO
UGAGAUGAAGsCsAsCsUsGUAGCUC




gapmer



366176
1088
5′ PS hemimer
UsGsAsGsAsUGAAGCACUGUAGCUC


366177
1088
3′ PS hemimer
UGAGAUGAAGCACUGUsAsGsCsUsC


366178
1088
Alt 2′-
UmGAmGAmUGmAAmGCmACmUGmUAmGCmUCm




OMe/ribose



366180
1088
Alt
UGmAGmAUmGAmAGmCAmCUmGUmAGmCUmC




ribose/2′-OMe



366183
1088
2′-OMe
UGAGAUmGmAAGmCmACUGUAGCmUmCm




blockmer



366184
1088
2′-OMe
UGAGAUGAmAmGCAmCmUGUAGCmUmCm




blockmer



366185
1088
2′-MOE
UGAGAUGAAGCAeCeUGUAGCUC




blockmer



366186
1088
2′-MOE
UGAGeAeUeGAAGCACUGUAGCUC




blockmer



366187
1088
2′-MOE
UGAGAUGAAGCACUGUeAeGeCUC




blockmer



366191
1088
4′s gapmer
U4sGAGAUGAAGCACUGUAGC4sU4sC4s


366192
1088
4′s 2′-OMe
U4sGAGAUGAAGCACUGUAGCmUmCm




gapmer



366193
1088
2′-F blockmer
UGfAfGfAfUfGfAfAfGCACUGUAGCUC


366194
1088
LNA blockmer
UGAGlAlUlGAAGCACUGUAGCUC


366195
1088
LNA blockmer
UGAGAUGAAGCACUGUlAlGlCUC


366196
1088
LNA blockmer
UGAGAUGAAGCAlClUGUAGCUC


366197
1088
Alt 2′-
UmGfAmGfAmUfGmAfAmGfCmAfCmUfGmUfAmGfCmUfCm




OMe/2′-F



366209
1088
LNA blockmer
UGAGlAlTlGAAGCACUGUAGCUC


366210
1088
LNA blockmer
UGAGAUGAAGCACUGTlAlGlCUC


366211
1088
LNA point
UGAGAUGAAGCAlmClUGUAGCUC




modification









Oligomeric compounds representing mimics of the antisense and the sense strands of a double-stranded miRNA can be hybridized, and various combinations of synthetic, modified or unmodified double-stranded oligomeric compounds, each representing a double-stranded miRNA mimic, may be formed. With the various chemical modifications, many permutations of such double-stranded miRNA mimics can be achieved. These double-stranded oligomeric compounds can be blunt-ended or can comprise two strands differing in length such that the resulting double-stranded oligomeric compound has a 3′- and/or a 5′-overhang of one to five nucleotides on either the sense and/or antisense strands. The compounds can be analyzed for their ability to mimic miRNAs, pre-miRNAs, or pri-miRNAs and to bind to nucleic acid targets (for example, RNA transcripts, mRNAs, reporter contructs), for their effects on miRNA, pre-miRNA, or pri-miRNA expression levels by quantitative real-time PCR, or they can be used in other in vivo or in vitro phenotypic assays to investigate the role of miRNAs in regulation of downstream nucleic acid targets, as described in other examples herein. These oligomeric compounds of the present invention may disrupt pri-miRNA and/or pre-miRNA structures, and sterically hinder cleavage by Drosha-like and/or Dicer-like Rnase III enzymes, respectively. Oligomeric compounds capable of binding to the mature miRNA are also predicted to prevent the RISC-mediated binding of a miRNA to its mRNA target, either by cleavage or steric occlusion of the miRNA.
In some embodiments, HeLa cells transiently expressing the pGL3-mir-143 sensor reporter vector and the pRL-CMV Renilla luciferase plasmids, as described in Example 27, -were also treated with double-stranded oligomeric compounds produced by hybridizing an antisense oligomeric compound from Table 68 with a sense oligomeric compound from Table 69, as described herein. HeLa cells were routinely cultured and passaged and on the day before transfection, the HeLa cells were seeded onto 96-well plates 3,000 cells/well. Cells were transfected according to standard published procedures with plasmids using 2 μg LipofectamineTM 2000 Reagent (Invitrogen) per μg of plasmid DNA, or, when transfecting double-stranded oligomeric compounds, 1.25 μg of LipofectamineTM 2000 Reagent was used per 100 nM oligonucleotide. Cells were treated at 10 nM and 100 nM with the double-stranded oligomeric compound mimics A double-stranded oligomeric compound representing a 10-base mismatched sequence antisense to the unrelated PTP1B mRNA, composed of ISIS Number 342427 (SEQ ID NO: 863) hybridized to its perfect complement ISIS Number 342430 (SEQ ID NO: 864) was used as a negative control (“ds-Control”). The pGL3-mir-143 sensor reporter plasmid was transfected at 0.025 μg per well. The luciferase signal in each well was normalized to the Renilla luciferase (RL) activity produced from the co-transfected pRL-CMV plasmid, which was transfected at 2.5 μg per well. In accordance with methods described in Example 12 and 27, a luciferase assay was performed 48-hours after transfection. Briefly, cells were lysed in passive lysis buffer (PLB; Promega), and 20 ul of the lysate was then assayed for RL activity using a Dual Luciferase Assay kit (Promega) according to the manufacturer's protocol. The results below are an average of two trials and are presented as percent pGL3-Control luciferase expression normalized to pRL-CMV expression (RL). The data are shown in Table 70.







TABLE 70







Luciferase assays showing effects of double-stranded


compounds mimicking mir-143









ISIS Numbers
luciferase expression



hybridized to form ds
(% lucif. only control)










compound
10 nM treatment
100 nM treatment












pGL3-mir-143 sensor +
79.4
94.1


pRL-CMV only




pGL3-mir-143 sensor +
120.6
105.9


pRL-CMV only




342430 + 342427
75.0
86.1


ds-Control




348215 + 348173
23.1
37.5


348215 + 362972
28.6
32.4


366175 + 348173
20.0
25.0


366175 + 362972
56.9
33.4


366176 + 348173
42.6
30.0


366176 + 362972
63.4
98.5


366177 + 348173
35.7
33.6


366177 + 362972
32.8
29.1


366183 + 348173
29.2
24.5


366183 + 362972
54.3
36.8


366184 + 348173
35.6
27.7


366184 + 362972
47.3
31.9


366185 + 348173
22.2
18.5


366185 + 362972
27.2
28.7


366186 + 348173
34.8
26.8


366186 + 362972
50.2
60.8


366187 + 348173
34.6
32.4


366187 + 362972
25.5
27.9


366209 + 348173
112.9
85.4


366209 + 362972
111.3
97.5


366210 + 348173
37.1
28.2


366210 + 362972
51.8
41.1


366211 + 348173
32.1
28.7


366211 + 362972
46.6
36.7


366193 + 348173
20.0
17.6


366193 + 362972
24.4
22.6


366191 + 348173
27.3
26.9


366191 + 362972
37.5
25.8


366192 + 348173
22.3
27.9


366192 + 362972
28.9
25.7


366197 + 348173
37.0
22.2


366197 + 362972
42.0
32.7


366197 + 366198
30.2
28.7


366178 + 348173
75.0
74.0


366178 + 362972
98.6
104.0


366178 + 366179
63.5
75.4


366178 + 366181
74.1
70.6


366180 + 366179
97.0
38.5


366180 + 366181
43.5
50.2


pGL3-mir-143 sensor +
100.0
112.9


pRL-CMV only




342430 + 342427
81.2
165.9


ds-Control




348201 + 348187
44.0
55.4


348201 + 366182
138.9
89.2


348201 + 366179
76.2
68.5


348201 + 366181
92.2
340.0


348201 + 362972
65.2
67.3


348201 + 366198
47.3
58.8


342199 + 348173
40.3
122.0


342199 + 348187
91.3
55.5


342199 + 366182
47.4
84.1


342199 + 366179
76.5
45.9


342199 + 366181
86.1
34.2


342199 + 362972
50.8
78.7


342199 + 366189
26.7
45.2


342199 + 366190
93.0
37.9


342199 + 366198
52.5
45.5









From these data, it was observed that treatment of HeLa cells expressing the pGL3-mir-143 sensor reporter vector with many of the double-stranded oligomeric compounds mimicking mir-143 at both the 10 nM and 100 nM concentrations resulted in inhibition of luciferase activity. For example, the double stranded oligomeric compounds comprising ISIS Number 348173 as an unmodified antisense strand in combination with ISIS Number 366177 (a hemimer with phosphorothioate modified residues at the 3′ end) or ISIS Number 366185 (a 2′-MOE blockmer) as the modified sense strand resulted in significant reductions in luciferase activity. Furthermore, double stranded oligomeric compounds comprising, as the antisense strand, either ISIS Number 366189 (a fully modified 2′-MOE compound) or ISIS Number 366198 (with alternating 2′-Fluoro and 2′-O-Methyl residues), in combination with ISIS Number 342199 as the unmodified sense strand resulted in significant reductions in luciferase activity, indicating that these compounds are effective mir-143 mimics Taken with the previous observations that the mir-143 miRNA is involved in adipocyte differentiation, these double-stranded mir-143 mimics may be useful as therapeutic agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells.
Example 38
Design of Oligomeric Compounds Targeting Pri-miRNAs
As described above, mature miRNAs originate from pri-miRNAs, which are believed to be processed into pre-miRNAs by the Drosha RNase III enzyme, and subsequently exported from the nucleus to the cytoplasm, where the pre-miRNAs are processed by human Dicer into double-stranded intermediates resembling siRNAs, which are then processed into mature miRNAs.
Some oligomeric compounds of the present invention are believed to bind to pri-miRNA molecules and interfere with their processing into a mature miRNA. These oligomeric compounds were observed to affect a decrease in expression levels of mature miRNA, presumably due, at least in part, to steric interference with their processing into mature miRNAs by human Dicer. Furthermore, as described above, some oligomeric compounds of the present invention have been observed to affect an increase in expression levels of pri-miRNAs; it is believed that the decrease in levels of mature miRNAs cells treated with these oligomeric compounds may trigger a feedback mechanism that signals these cells to increase production of the pri-miRNA molecule. This increase may be the result, at least in part, of a stimulation of transcription of the pri-miRNAs in response to the decrease in mature miRNAs. Not mutually exclusive with the processing interference and the feedback mechanisms is the possibility that treatment with oligomeric compounds could stimulate the activity of an RNA-dependent RNA polymerase (RdRP) that amplifies pre-miRNAs or pri-miRNAs.
In one embodiment, several nested series of single-stranded oligomeric compounds, 15-nucleotides in length, composed of 2′-methoxyethoxy (2′-MOE) modified nucleotides and phosphorothioate (P═S) internucleoside linkages throughout the compound, were designed and synthesized to target several pri-miRNAs, to test the effects of these compounds on the expression levels of small non-coding RNAs. These compounds are shown in Table 71, below. “Pri-miRNA” indicates the particular pri-miRNA which contains the miRNA that the oligomeric compound was designed to target. The “Region” column describes the general region of the pri-miRNA that is being targeted. The following features of the stemloop structures of pri-miRNA were targeted: 1) “5′-stem side mir start” means the 5′-stem side at the 5′-end of the sequence representing the mature miRNA, with the oligomeric compounds targeting and spanning sequences completely outside of the mature miRNA to completely within it; 2) “5′-stem side mir end” means the 5′-stem side at the 3′-end of the sequence representing the mature miRNA, with the oligomeric compounds targeting and spanning sequences completely within the mature miRNA to spanning and extending beyond the 3′-end of it; 3) “loop start” means the 5′-side of the loop region; 4) “loop end” means with the oligomeric compounds targeting and ending at the 3′-side of the loop region; 5) “3′-stem side mir start” means the 3′-stem side at the 5′-end of the sequence representing the mature miRNA, with the oligomeric compounds targeting and completely within the mature miRNA to a few nucleotides outside of it; 6) “3′-stem side mir end” means the 3′-stem side at the 3′-end of the sequence representing the mature miRNA, with the oligomeric compounds targeting and spanning sequences completely within the mature miRNA to completely outside of it.







TABLE 71







Uniform 2′-MOE oligomeric compounds targeting pri-miRNAs















SEQ






ID


pri-miRNA
Region
Isis #
Sequence
NO:





mir-182
mir-182 5′-stem side mir start
366888
AAACGGGGGGAGGCA
1997


mir-182
mir-182 5′-stem side mir start
366889
GCCAAAAACGGGGGG
1998


mir-182
mir-182 5′-stem side mir start
366890
ATTGCCAAAAACGGG
1999


mir-182
mir-182 5′-stem side mir start
366891
ACCATTGCCAAAAAC
2000


mir-182
mir-182 5′-stem side mir start
366892
TCTACCATTGCCAAA
2001


mir-182
mir-182 5′-stem side mir end
366893
TGTGAGTTCTACCAT
2002


mir-182
mir-182 5′-stem side mir end
366894
CAGTGTGAGTTCTAC
2003


mir-182
mir-182 5′-stem side mir end
366895
CACCAGTGTGAGTTC
2004


mir-182
mir-182 5′-stem side mir end
366896
CCTCACCAGTGTGAG
2005


mir-182
mir-182 loop start
366897
TCCTGTTACCTCACC
2006


mir-182
mir-182 loop start
366898
GATCCTGTTACCTCA
2007


mir-182
mir-182 loop start
366899
CGGATCCTGTTACCT
2008


mir-182
mir-182 loop end
366900
TGTTACCTCACCAGT
2009


mir-182
mir-182 loop end
366901
CCTGTTACCTCACCA
2010


mir-182
mir-182 loop end
366902
ATCCTGTTACCTCAC
2011


mir-182
mir-182 loop end
366903
GGATCCTGTTACCTC
2012


mir-182
mir-182 loop end
366904
CCGGATCCTGTTACC
2013


mir-182
mir-182 3′-stem side mir start
366905
GAACCACCGGATCCT
2014


mir-182
mir-182 3′-stem side mir start
366906
CTAGAACCACCGGAT
2015


mir-182
mir-182 3′-stem side mir start
366907
AGTCTAGAACCACCG
2016


mir-182
mir-182 3′-stem side mir start
366908
GCAAGTCTAGAACCA
2017


mir-182
mir-182 3′-stem side mir end
366909
ATAGTTGGCAAGTCT
2018


mir-182
mir-182 3′-stem side mir end
366910
CGCCCCATAGTTGGC
2019


mir-182
mir-182 3′-stem side mir end
366911
CCTCGCCCCATAGTT
2020


mir-182
mir-182 3′-stem side mir end
366912
AGTCCTCGCCCCATA
2021


mir-182
mir-182 3′-stem side mir end
366913
CTGAGTCCTCGCCCC
2022


mir-216
mir-216 5′-stem side mir start
366914
AAGCCAACTCACAGC
2023


mir-216
mir-216 5′-stem side mir start
366915
AGATTAAGCCAACTC
2024


mir-216
mir-216 5′-stem side mir start
366916
CTGAGATTAAGCCAA
2025


mir-216
mir-216 5′-stem side mir start
366917
CAGCTGAGATTAAGC
2026


mir-216
mir-216 5′-stem side mir start
366918
TGCCAGCTGAGATTA
2027


mir-216
mir-216 5′-stem side mir end
366919
TCACAGTTGCCAGCT
2028


mir-216
mir-216 5′-stem side mir end
366920
ATCTCACAGTTGCCA
2029


mir-216
mir-216 5′-stem side mir end
366921
AACATCTCACAGTTG
2030


mir-216
mir-216 5′-stem side mir end
366922
ATGAACATCTCACAG
2031


mir-216
mir-216 loop start
366923
ATTGTATGAACATCT
2032


mir-216
mir-216 loop start
366924
GGATTGTATGAACAT
2033


mir-216
mir-216 loop start
366925
AGGGATTGTATGAAC
2034


mir-216
mir-216 loop end
366926
TGTATGAACATCTCA
2035


mir-216
mir-216 loop end
366927
TGAGGGATTGTATGA
2036


mir-216
mir-216 3′-stem side mir start
366928
ACTGTGAGGGATTGT
2037


mir-216
mir-216 3′-stem side mir start
366929
ACCACTGTGAGGGAT
2038


mir-216
mir-216 3′-stem side mir start
366930
GAGACCACTGTGAGG
2039


mir-216
mir-216 3′-stem side mir start
366931
CCAGAGACCACTGTG
2040


mir-216
mir-216 3′-stem side mir end
366932
CATAATCCCAGAGAC
2041


mir-216
mir-216 3′-stem side mir end
366933
GTTTAGCATAATCCC
2042


mir-216
mir-216 3′-stem side mir end
366934
TCTGTTTAGCATAAT
2043


mir-216
mir-216 3′-stem side mir end
366935
TGCTCTGTTTAGCAT
2044


mir-216
mir-216 3′-stem side mir end
366936
AATTGCTCTGTTTAG
2045


mir-143
mir-143 5′-stem side mir start
366937
AGGCTGGGAGACAGG
2046


mir-143
mir-143 5′-stem side mir start
366938
ACCTCAGGCTGGGAG
2047


mir-143
mir-143 5′-stem side mir start
366939
TGCACCTCAGGCTGG
2048


mir-143
mir-143 5′-stem side mir start
366940
CACTGCACCTCAGGC
2049


mir-143
mir-143 5′-stem side mir start
366941
CAGCACTGCACCTCA
2050


mir-143
mir-143 5′-stem side mir end
366942
AGAGATGCAGCACTG
2051


mir-143
mir-143 5′-stem side mir end
366943
ACCAGAGATGCAGCA
2052


mir-143
mir-143 5′-stem side mir end
366944
CTGACCAGAGATGCA
2053


mir-143
mir-143 5′-stem side mir end
366945
CAACTGACCAGAGAT
2054


mir-143
mir-143 loop start
366946
CAGACTCCCAACTGA
2055


mir-143
mir-143 loop start
366947
CTCAGACTCCCAACT
2056


mir-143
mir-143 loop start
366948
ATCTCAGACTCCCAA
2057


mir-143
mir-143 loop end
366949
AACTGACCAGAGATG
2058


mir-143
mir-143 loop end
366950
CCAACTGACCAGAGA
2059


mir-143
mir-143 loop end
366951
TCCCAACTGACCAGA
2060


mir-143
mir-143 loop end
366952
ACTCCCAACTGACCA
2061


mir-143
mir-143 3′-stem side mir start
366953
TTCATCTCAGACTCC
2062


mir-143
mir-143 3′-stem side mir start
366954
TGCTTCATCTCAGAC
2063


mir-143
mir-143 3′-stem side mir start
366955
CAGTGCTTCATCTCA
2064


mir-143
mir-143 3′-stem side mir end
366956
TGAGCTACAGTGCTT
2065


mir-143
mir-143 3′-stem side mir end
366957
TCTTCCTGAGCTACA
2066


mir-143
mir-143 3′-stem side mir end
366958
CTCTCTTCCTGAGCT
2067


mir-143
mir-143 3′-stem side mir end
366959
CTTCTCTCTTCCTGA
2068


mir-143
mir-143 3′-stem side mir end
366960
CAACTTCTCTCTTCC
2069


mir-23b
mir-23b 5′-stem side mir start
366961
AGCAGCCAGAGCACC
2070


mir-23b
mir-23b 5′-stem side mir start
366962
ACCCAAGCAGCCAGA
2071


mir-23b
mir-23b 5′-stem side mir start
366963
GGAACCCAAGCAGCC
2072


mir-23b
mir-23b 5′-stem side mir start
366964
CCAGGAACCCAAGCA
2073


mir-23b
mir-23b 5′-stem side mir start
366965
ATGCCAGGAACCCAA
2074


mir-23b
mir-23b 5′-stem side mir end
366966
AATCAGCATGCCAGG
2075


mir-23b
mir-23b 5′-stem side mir end
366967
ACAAATCAGCATGCC
2076


mir-23b
mir-23b 5′-stem side mir end
366968
GTCACAAATCAGCAT
2077


mir-23b
mir-23b 5′-stem side mir end
366969
TAAGTCACAAATCAG
2078


mir-23b
mir-23b loop start
366970
AATCTTAAGTCACAA
2079


mir-23b
mir-23b loop start
366971
TTAATCTTAAGTCAC
2080


mir-23b
mir-23b loop start
366972
TTTTAATCTTAAGTC
2081


mir-23b
mir-23b loop end
366973
CTTAAGTCACAAATC
2082


mir-23b
mir-23b loop end
366974
ATCTTAAGTCACAAA
2083


mir-23b
mir-23b loop end
366975
TAATCTTAAGTCACA
2084


mir-23b
mir-23b loop end
366976
TTTAATCTTAAGTCA
2085


mir-23b
mir-23b loop end
366977
ATTTTAATCTTAAGT
2086


mir-23b
mir-23b 3′-stem side mir start
366978
TGTGATTTTAATCTT
2087


mir-23b
mir-23b 3′-stem side mir start
366979
CAATGTGATTTTAAT
2088


mir-23b
mir-23b 3′-stem side mir start
366980
TGGCAATGTGATTTT
2089


mir-23b
mir-23b 3′-stem side mir start
366981
CCCTGGCAATGTGAT
2090


mir-23b
mir-23b 3′-stem side mir end
366982
TGGTAATCCCTGGCA
2091


mir-23b
mir-23b 3′-stem side mir end
366983
GTTGCGTGGTAATCC
2092


mir-23b
mir-23b 3′-stem side mir end
366984
GTGGTTGCGTGGTAA
2093


mir-23b
mir-23b 3′-stem side mir end
366985
GTCGTGGTTGCGTGG
2094


mir-23b
mir-23b 3′-stem side mir end
366986
AAGGTCGTGGTTGCG
2095


mir-203
mir-203 5′-stem side mir start
366987
GACCCAGCGCGCGAG
2096


mir-203
mir-203 5′-stem side mir start
366988
CACTGGACCCAGCGC
2097


mir-203
mir-203 5′-stem side mir start
366989
AACCACTGGACCCAG
2098


mir-203
mir-203 5′-stem side mir start
366990
AAGAACCACTGGACC
2099


mir-203
mir-203 5′-stem side mir start
366991
GTTAAGAACCACTGG
2100


mir-203
mir-203 5′-stem side mir end
366992
TTGAACTGTTAAGAA
2101


mir-203
mir-203 5′-stem side mir end
366993
CTGTTGAACTGTTAA
2102


mir-203
mir-203 5′-stem side mir end
366994
GAACTGTTGAACTGT
2103


mir-203
mir-203 5′-stem side mir end
366995
ACAGAACTGTTGAAC
2104


mir-203
mir-203 loop start
366996
AATTGCGCTACAGAA
2105


mir-203
mir-203 loop start
366997
ACAATTGCGCTACAG
2106


mir-203
mir-203 loop start
366998
TCACAATTGCGCTAC
2107


mir-203
mir-203 loop end
366999
TACAGAACTGTTGAA
2108


mir-203
mir-203 loop end
367000
GCTACAGAACTGTTG
2109


mir-203
mir-203 loop end
367001
GCGCTACAGAACTGT
2110


mir-203
mir-203 loop end
367002
TTGCGCTACAGAACT
2111


mir-203
mir-203 3′-stem side mir start
367003
TTTCACAATTGCGCT
2112


mir-203
mir-203 3′-stem side mir start
367004
ACATTTCACAATTGC
2113


mir-203
mir-203 3′-stem side mir start
367005
TAAACATTTCACAAT
2114


mir-203
mir-203 3′-stem side mir start
367006
TCCTAAACATTTCAC
2115


mir-203
mir-203 3′-stem side mir end
367007
CTAGTGGTCCTAAAC
2116


mir-203
mir-203 3′-stem side mir end
367008
CCGGGTCTAGTGGTC
2117


mir-203
mir-203 3′-stem side mir end
367009
CCGCCGGGTCTAGTG
2118


mir-203
mir-203 3′-stem side mir end
367010
CGCCCGCCGGGTCTA
2119


mir-203
mir-203 3′-stem side mir end
367011
CCGCGCCCGCCGGGT
2120


mir-21
mir-21 5′-stem side mir start
367012
GCTACCCGACAAGGT
2121


mir-21
mir-21 5′-stem side mir start
367013
AAGCTACCCGACAAG
2122


mir-21
mir-21 5′-stem side mir start
367014
GATAAGCTACCCGAC
2123


mir-21
mir-21 5′-stem side mir start
367015
TCTGATAAGCTACCC
2124


mir-21
mir-21 5′-stem side mir start
367016
CAGTCTGATAAGCTA
2125


mir-21
mir-21 5′-stem side mir end
367017
TCAACATCAGTCTGA
2126


mir-21
mir-21 5′-stem side mir end
367018
CAGTCAACATCAGTC
2127


mir-21
mir-21 5′-stem side mir end
367019
CAACAGTCAACATCA
2128


mir-21
mir-21 5′-stem side mir end
367020
ATTCAACAGTCAACA
2129


mir-21
mir-21 loop start
367021
GCCATGAGATTCAAC
2130


mir-21
mir-21 loop start
367022
TTGCCATGAGATTCA
2131


mir-21
mir-21 loop start
367023
TGTTGCCATGAGATT
2132


mir-21
mir-21 loop end
367024
TTCAACAGTCAACAT
2133


mir-21
mir-21 loop end
367025
GATTCAACAGTCAAC
2134


mir-21
mir-21 loop end
367026
GAGATTCAACAGTCA
2135


mir-21
mir-21 loop end
367027
ATGAGATTCAACAGT
2136


mir-21
mir-21 loop end
367028
CCATGAGATTCAACA
2137


mir-21
mir-21 3′-stem side mir start
367029
GTGTTGCCATGAGAT
2138


mir-21
mir-21 3′-stem side mir start
367030
CTGGTGTTGCCATGA
2139


mir-21
mir-21 3′-stem side mir start
367031
CGACTGGTGTTGCCA
2140


mir-21
mir-21 3′-stem side mir start
367032
CATCGACTGGTGTTG
2141


mir-21
mir-21 3′-stem side mir end
367033
GACAGCCCATCGACT
2142


mir-21
mir-21 3′-stem side mir end
367034
ATGTCAGACAGCCCA
2143


mir-21
mir-21 3′-stem side mir end
367035
AAATGTCAGACAGCC
2144


mir-21
mir-21 3′-stem side mir end
367036
CAAAATGTCAGACAG
2145


mir-221
mir-221 5′-stem side mir start
367037
CATGCCCCAGACCTG
2146


mir-221
mir-221 5′-stem side mir start
367038
AGGTTCATGCCCCAG
2147


mir-221
mir-221 5′-stem side mir start
367039
GCCAGGTTCATGCCC
2148


mir-221
mir-221 5′-stem side mir start
367040
TATGCCAGGTTCATG
2149


mir-221
mir-221 5′-stem side mir start
367041
TTGTATGCCAGGTTC
2150


mir-221
mir-221 5′-stem side mir end
367042
ATCTACATTGTATGC
2151


mir-221
mir-221 5′-stem side mir end
367043
GAAATCTACATTGTA
2152


mir-221
mir-221 5′-stem side mir end
367044
ACAGAAATCTACATT
2153


mir-221
mir-221 5′-stem side mir end
367045
AACACAGAAATCTAC
2154


mir-221
mir-221 loop start
367046
CTGTTGCCTAACGAA
2155


mir-221
mir-221 loop start
367047
AGCTGTTGCCTAACG
2156


mir-221
mir-221 loop start
367048
GTAGCTGTTGCCTAA
2157


mir-221
mir-221 loop end
367049
GAACACAGAAATCTA
2158


mir-221
mir-221 loop end
367050
ACGAACACAGAAATC
2159


mir-221
mir-221 loop end
367051
TAACGAACACAGAAA
2160


mir-221
mir-221 loop end
367052
CCTAACGAACACAGA
2161


mir-221
mir-221 loop end
367053
TGCCTAACGAACACA
2162


mir-221
mir-221 3′-stem side mir start
367054
AATGTAGCTGTTGCC
2163


mir-221
mir-221 3′-stem side mir start
367055
GACAATGTAGCTGTT
2164


mir-221
mir-221 3′-stem side mir start
367056
GCAGACAATGTAGCT
2165


mir-221
mir-221 3′-stem side mir end
367057
AAACCCAGCAGACAA
2166


mir-221
mir-221 3′-stem side mir end
367058
AGCCTGAAACCCAGC
2167


mir-221
mir-221 3′-stem side mir end
367059
GGTAGCCTGAAACCC
2168


mir-221
mir-221 3′-stem side mir end
367060
CCAGGTAGCCTGAAA
2169


mir-221
mir-221 3′-stem side mir end
367061
TTTCCAGGTAGCCTG
2170









These modified oligomeric compounds targeting pri-miRNAs can be transfected into preadipocytes or other undifferentiated cells, which are then induced to differentiate, and it can be determined whether these modified oligomeric compounds act to inhibit or promote cellular differentiation. These compounds can be transfected into differentiating adipocytes and their effects on expression levels of the pri-miRNA molecules assessed in pre-adipocytes vs. differentiated adipocytes. By using a primer/probe set specific for the pri-miRNA or the pre-miRNA, real-time RT-PCR methods can be used to determine whether modified oligomeric compounds targeting pri-miRNAs can affect the expression or processing of the mature miRNAs from the pri-miRNA or pre-miRNA molecules.
Example 39
Effects of Oligomeric Compounds Targeting miRNAs in the Immune Response
To investigate the role of miRNAs in the immune response, oligomeric compounds of the present invention targeting miRNAs were tested for their effects upon lipopolysaccharide (LPS)-activated primary murine macrophages. Macrophages participate in the immune response, for example, in the recognition and phagocytosis of microorganisms, including bacteria. Interferon-gamma (IFN-gamma) released by helper T cells is one type of signal required for macrophage activation, and LPS can serve as an additional stimulus. LPS is a component of the gram-negative bacterial cell wall and acts as an agonist for toll-like receptor 4 (TLR4), the primary LPS receptor expressed by macrophages. The proinflammatory cytokines interleukin-12 (IL-12) and interleukin-6 (IL-6) are induced by LPS treatment of macrophages, thus the expression of the mRNAs encoding these cytokines was used to evaluate the response of macrophages to LPS following treatment with oligomeric compounds targeting miRNAs.
Macrophages were isolated as follows. Female C57B1/6 mice (Charles River Laboratories, Wilmington, Mass.) were injected intraperitoneally with 1 ml 3% thioglycollate broth (Sigma-Aldrich, St. Louis, Mo.), and peritoneal macrophage cells were isolated by peritoneal lavage 4 days later. The cells were plated in 96-well plates at 40,000 cells/well for one hour in serum-free RPMI adjusted to contain 10 mM HEPES (Invitrogen Life Technologies, Carlsbad, Calif.), allowed to adhere, then non-adherent cells were washed away and the media was replaced with RPMI containing 10 mM HEPES, 10% FBS and penicillin/streptomycin (“complete” RPMI; Invitrogen Life Technologies, Carlsbad, Calif.).
Oligomeric compounds were introduced into the cells using the non-liposomal transfection reagent FuGENE 6 Transfection Reagent (Roche Diagnostics Corp., Indianapolis, Ind.). Oligomeric compound was mixed with FuGENE 6 in 1 mL of serum-free RPMI to achieve a concentration of 10 μL FuGENE per 1000 nM oligomeric compound. The oligomeric compound/FuGENE complex was allowed to form at room temperature for 20 minutes. This mixture was diluted to the desired concentration of oligomeric compound by the addition of the appropriate volume of complete RPMI. The final ratio of FuGENE 6 to oligomeric compound was 1 μL of FuGENE 6 per 100 nM oligomeric compound. A volume of 100 μL of oligomeric compound/FuGENE/RPMI was added to each well of the 96-well plate in which the macrophages were cultured. Each oligomeric compound treatment was repeated in triplicate.
Following oligomeric compound treatment, cells were stimulated with LPS. Cells were cultured in the presence of the transfection complex for approximately 24 to 28 hours at 37° C. and 5% CO2, after which the medium containing the transfection complex was removed from the cells, and complete RPMI containing 100 ng/mL LPS (Sigma-Aldrich Corp., St. Louis, Mo.) was added to the cells for a period of approximately 24 hours. Control samples included (1) cells receiving no oligomeric compound, stimulated with LPS and (2) cells receiving neither oligomeric compound nor LPS treatment.
In another embodiment, following oligomeric compound treatment, cells were first activated by IFN-gamma, to amplify the response to LPS. Cells were cultured in the presence of the transfection complex for approximately 24 hours at 37° C. and 5% CO2, at which point the medium containing the transfection complex was removed from the cells, and complete RPMI containing 100 ng/mL recombinant mouse IFN-gamma (R&D Systems, Minneapolis, Minn.) was added to the cells. After the 4 hour treatment with INF-gamma, cells were treated with 100 ng/mL LPS for approximately 24 hours. Control samples included (1) cells receiving no oligomeric compound, stimulated with LPS and (2) cells receiving neither oligomeric compound nor LPS treatment.
Oligomeric compounds used as negative controls included ISIS 129690 (SEQ ID NO: 907), a universal scrambled control; ISIS 342673 (SEQ ID NO: 758), an oligomeric compound containing 15 mismatches with respect to the mature mir-143 miRNA; ISIS 342683 (SEQ ID NO: 790), an oligomeric compound representing the scrambled nucleotide sequence of an unrelated PTP1B antisense oligonucleotide; and ISIS 289606 (CCTTCCCTGAAGGTTCCTCC, incorporated herein as SEQ ID NO: 863), an oligomeric compound representing the scrambled nucleotide sequence of an unrelated PTP1B antisense oligonucleotide. ISIS 289606 is uniformly composed of 2′-MOE nucleotides, with phosphorothioate internucleoside linkages throughout the compound. All cytidines are 5-methyl cytidines. Used as a positive control was ISIS 229927 (CCACATTGAGTTTCTTTAAG, incorporated herein as SEQ ID NO: 2171), targeting the mouse toll-like receptor 4 (TLR4) mRNA, which is the primary LPS receptor on macrophages. ISIS 229927 is a chimeric oligomeric compound (“gapmer”) composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five nucleotide “wings,” wherein the wings are composed of 2′-methoxyethoxy (2′-MOE) nucleotides. Internucleoside linkages are phosphorothioate throughout the compound, and all cytidines are 5-methylcytidines. Treatments with control oligomeric compounds were performed as described for oligomeric compounds targeting miRNAs.
Following the 24 hour treatment with LPS, the cells were lysed and RNA was isolated using the RNEASY 96TM kit, as described herein. mRNA expression was quantitated by real-time PCR, performed as described herein, using primer and probe sets to amplify and quantitate TLR4, IL-12 and IL-6 mRNA expression levels. Primers and probe for TLR4, designed using GenBank Accession number NM—021297.1, were: forward primer, 5′-CATGGAACACATGGCTGCTAA-3′ (SEQ ID NO: 2172), reverse primer, 5′-GGAAAGGAAGGTGTCAGTGCTACT-3′ (SEQ ID NO: 2173), probe 5′-FAM-TAGCATGGACCTTACCGGGCAGAAGG-TAMRA-3′ (SEQ ID NO: 2174). Primers and probe for IL-12, designed using GenBank Accession number M86671.1, were: forward primer, 5′-GCCAGTACACCTGCCACAAA-3′ (SEQ ID NO: 2175), reverse primer, 5′-GACCAAATTCCATTTTCCTTCTTG-3′ (SEQ ID NO: 2176), probe 5′-FAM-AGGCGAGACTCTGAGCCACTCACATCTG-TAMRA-3′ (SEQ ID NO: 2177). Primers and probe for IL-6, designed using GenBank Accession number X54542.1, were: forward primer, 5′-CCTAGTGCGTTATGCCTAAGCA-3′ (SEQ ID NO: 2178), reverse primer, 5′-TTCGTAGAGAACAACATAAGTCAGATACC-3′ (SEQ ID NO: 2179), probe 5′-FAM-TTTCTGACCACAGTGAGGAATGTCCACAA-TAMRA-3′ (SEQ ID NO: 2180). The amount of total RNA in each sample was determined using a Ribogreen Assay (Molecular Probes, Eugene, Oreg.), and expression levels of TLR4, IL-12 and IL-6 were normalized to total RNA.
TLR4 is the primary macrophage receptor for LPS. Thus, ISIS Number 229927, targeted to TLR4, was tested for its ability to inhibit TLR4 expression and interfere with the response of macrophages to LPS, both with and without pretreatment with IFN-gamma. The treatment of primary murine macrophages with ISIS Number 229927 at reduced the expression of TLR4 in a dose-dependent manner, in both LPS-stimulated and LPS- and IFN-gamma-stimulated cells. As judged by the dose-dependent reduction in IL-12, the response of macrophages to LPS was reduced following inhibition of the TLR4 receptor expression, in both LPS-stimulated and LPS- and IFN-gamma-stimulated cells. These results demonstrated that ISIS 229927 can be used as a positive control for the inhibition of IL-12 expression in macrophages responding to LPS.
Primary mouse macrophages were treated with a selected group of oligomeric compounds targeting various miRNAs. These compounds and their miRNA targets are shown in Table 72. Table 72 shows IL-12 mRNA expression following treatment with 300 nM of oligomeric compounds and LPS (−IFN), and IL-12 mRNA expression following treatment with 300 nM of oligomeric compounds and stimulation with IFN-gamma and LPS (+IFN). The “−IFN” data represents a single experiment, and the “+IFN” data represents the average of 2 experiments. Data were normalized to values from cells receiving no oligomeric compound that were treated with LPS. IL-12 expression in cells receiving neither oligomeric compound nor LPS treatment was 2% of the control, both with and without IFN-gamma pretreatment, demonstrating that IL-12 mRNA expression was not stimulated in the absence of LPS treatment. Where present, “N.D.” indicates “not determined”.







TABLE 72







IL-12 mRNA expression in primary macrophages treated with


oligomeric compounds targeting miRNAs and stimulated with


LPS











ISIS
SEQ ID

−IFN
+IFN


NO:
NO:
pri-miRNA
% UTC
% UTC














289606
863
Scrambled control
N.D.
129


342673
758
mismatch to mir-143
91
N.D.


129690
907
Universal control
73
129


229927
2171
TLR4
92
145


327874
292
mir-30a
202
15


327876
294
mir-29b-1
194
9


327883
301
mir-27b
266
39


327887
305
mir-132
287
33


327889
307
mir-23b
153
10


327890
308
let-7i
183
94


327893
311
let-7b
117
52


327899
317
mir-183
164
7


327901
319
mir-143
225
9


327903
321
let-7a-3
200
23


327912
330
let-7f-1
206
39


327913
331
mir-29c
276
73


327917
335
mir-21
225
35


327919
337
mir-221
179
37


327920
338
mir-222
171
68


327921
339
mir-30d
325
24


327923
341
mir-128b
269
134


327924
342
mir-129-2
171
88


327925
343
mir-133b
302
60


327927
345
mir-15b
164
33


327928
346
mir-29a-1
201
61


327931
349
let-7c
105
48


327935
353
mir-20
254
24


327936
354
mir-133a-1
221
55


327940
358
mir-199a-2
228
107


327941
359
mir-181b
89
34


327945
363
mir-24-2
202
68


327956
374
mir-216
212
59


327958
376
mir-187
188
60


327959
377
mir-210
183
20


327961
379
mir-223
203
10


327963
381
mir-26b
224
23


327967
385
let-7g
203
43


327971
389
mir-23a
146
17


328105
407
hypothetical miRNA-088
108
57


328110
412
hypothetical miRNA-107
221
8


328117
419
hypothetical miRNA-144
162
72


328123
425
hypothetical miRNA-166
176
14


328129
431
hypothetical miRNA-173
87
10


328133
435
hypothetical miRNA-178
165
62


328137
439
hypothetical miRNA-183
213
12


328138
440
hypothetical miRNA-185
277
31


340341
236
mir-104 (Mourelatos)
139
13


340345
1882
miR-27 (Mourelatos)
104
78


341786
1845
miR-149
266
99


341790
1843
miR-154
318
84


341793
1836
miR-142-as
202
147


341800
1766
miR-186
180
100


341811
1906
miR-194
154
88


341815
1831
miR-200a
190
157









A comparison of the data from IFN-gamma-stimulated and unstimulated cells reveals that many of the oligomeric compounds targeting miRNAs attenuated the response of macrophages to LPS, as judged by IL-12 mRNA expression, when the cells were activated with IFN-gamma prior to LPS treatment. When macrophages were pretreated with IFN-gamma, treatment with several of the oligomeric compounds, such as ISIS Number 328110, ISIS Number 327901, ISIS Number 327899, ISIS Number 327876 and ISIS Number 327961 resulted in a reduction in IL-12 mRNA expression ranging from 20-fold to 30-fold. Other oligomeric compounds, such as ISIS Number 341800, ISIS Number 341811, ISIS Number 341793, ISIS Number 340345 and ISIS Number 341815 resulted in a less pronounced reduction in IL-12 mRNA expression ranging from 1.2-fold to 2-fold.
In a further embodiment, oligomeric compounds ISIS Number 327941 targeting mir-181b and ISIS Number 327921 targeting mir-30d were selected for a dose response study in LPS-stimulated primary macrophages, with and without IFN-gamma pre-treatment. Cells were treated as described herein, with oligomeric compound doses of 75, 150, 300 and 600 nM. Untreated control cells received no oligomeric compound treatment but did receive LPS treatment. ISIS 229927 (SEQ ID NO: 2171) was used as a positive control and ISIS 342683 (SEQ ID NO: 790), ISIS 126690 (SEQ ID NO: 907) and ISIS 289606 (SEQ ID NO: 863) were used as negative controls. IL-12 and IL-6 mRNA expression levels were measured by real-time PCR and normalized to untreated control cells that received LPS treatment. The IL-12 expression data, shown in Table 73, represent the average of 3 treatments. In cells receiving neither oligomeric compound nor LPS treatment, IL-12 expression was undetectable in IFN-gamma stimulated cells and was 1% of the untreated control in unstimulated cells.







TABLE 73







IL-12 mRNA expression following treatment of primary


mouse macrophages with oligomeric compounds targeting


mir-181b and mir-30d and LPS: dose response study











IL-12 mRNA expression, % UTC



SEQ
Dose of oligomeric compound












ISIS
ID
75 nM
150 nM
300 nM
600 nM
















NO:
NO:
−IFN
+IFN
−IFN
+IFN
−IFN
+IFN
−IFN
+IFN



















327941
359
49
4
45
2
34
3
41
3


327921
339
109
14
88
7
67
5
53
5


229927
2171
67
46
53
35
45
16
46
8


342683
790
121
92
165
76
147
65
130
64


129690
907
114
66
109
54
101
66
128
81


289606
863
89
59
99
46
80
52
98
66









These data reveal that ISIS Number 327941 inhibited IL-12 expression in cells stimulated with LPS alone, where the percentage of untreated control ranged from 34% to 49%. ISIS Number 327921 inhibited IL-12 mRNA expression in a dose-dependent manner in cells stimulated with LPS alone, with the lowest IL-12 expression at 53% of untreated control. In cells pretreated with IFN-gamma and subsequently treated with LPS, ISIS Number 327941 markedly reduced IL-12 mRNA expression to less than 5% of the untreated control at all doses. ISIS Number 327921 reduced IL-12 expression to 14% of the control at all 75 nM and to less than 10% of the untreated control at all other doses. Thus, ISIS Number 327941, targeting mir-181b, and ISIS Number 327921, targeting mir-30d, resulted in a greater reduction in IL-12 expression than ISIS 229927, which is targeted to TLR4.
The IL-6 expression data, shown in Table 74, represents the average of 3 treatments. In cells receiving neither oligomeric compound nor LPS treatment, IL-12 expression was undetectable in IFN-gamma stimulated cells and was 2% of the untreated control in unstimulated cells.







TABLE 74







IL-6 mRNA expression following treatment of primary


mouse macrophages with oligomeric compounds targeting


mir-181b and mir-30d and LPS: dose response study











IL-6 mRNA expression, % UTC



SEQ
Dose of oligomeric compound












ISIS
ID
75 nM
150 nM
300 nM
600 nM
















NO:
NO:
−IFN
+IFN
−IFN
+IFN
−IFN
+IFN
−IFN
+IFN



















327941
359
293
181
325
197
271
197
501
301


327921
339
223
122
294
144
522
287
632
313


229927
2171
57
54
52
39
44
40
104
69


342683
790
135
115
161
86
156
110 
311
149


129690
907
98
92
99
86
109
94
258
203


289606
863
77
78
68
69
65
70
77
59









These data reveal that, in contrast to IL-12 expression, IL-6 expression is increased in a dose-dependent manner following treatment with ISIS Number 327941 and ISIS Number 327921, in both IFN-gamma-stimulated and unstimulated cells. This is in contrast to treatment with ISIS 229927, which exhibited some reduction in IL-6 expression in both IFN-gamma-stimulated and unstimulated cells.
Abnormalities in the signaling pathways controlling the expression of cytokines and cytokine receptors have been implicated in a number of diseases. Compounds that modulate the activity of macrophages, for example, the response to foreign antigens such as LPS, are candidate therapeutic agents with application in the treatment of conditions involving macrophage activation, such as septic shock and toxic shock
The expression of mir-181 in mouse cells and tissues was evaluated by Northern blot. Mouse tissues RNA was purchased from Ambion, Inc. (Austin, Tex.). RNA was prepared from macrophages were prepared and stimulated with LPS as described herein. Northern blotting was performed as described herein, and mir-181 levels were normalized to U6 levels, both of which were quantitated by Phosphorimager analysis. Expression levels are presented in arbitrary units. mir-181 was found to be most highly expressed in lung and kidney, at approximately equal levels. The next highest expression levels were found in brain, heart and liver. For example, as compared to kidney mir-181 expression levels, mir-181 was expressed approximately 2.5-fold lower in brain, approximately 2.2-fold lower in heart and approximately 1.8-fold lower in liver. mir-181 levels in both naïve and LPS-stimulated macrophages were 4.5-fold and 4.9-fold lower than in kidney, respectively. The lowest expression levels were found in thymus and spleen, which were 12.9-fold and 14.7-fold less as compared to kidney.
Example 40
Adipocyte Assay of Oligomeric Compounds
The effect of several oligomeric compounds of the present invention targeting miRNA target nucleic acids on the expression of markers of cellular differentiation was examined in differentiating adipocytes.
As described in Example 13, some genes known to be upregulated during adipocyte differentiation include HSL, aP2, Glut4 and PPARγ. These genes play important roles in the uptake of glucose and the metabolism and utilization of fats. An increase in triglyceride content is another well-established marker for adipocyte differentiation.
For assaying adipocyte differentiation, expression of the four hallmark genes, HSL, aP2, Glut4, and PPARγ, as well as triglyceride (TG) accumulation were measured as previously described in adipocytes transfected with oligomeric compounds targeting miRNAs. Triglyceride levels as well as mRNA levels for each of the four adipocyte differentiation hallmark genes are expressed as a percentage of untreated control (UTC) levels. In this experiment, the negative control oligomeric compound was ISIS Number 342672 (SEQ ID NO: 789) or ISIS Number 342673 (SEQ ID NO: 758). Results are shown in Table 75. Each value represents at least one oligomeric compound treatment; data from more than one oligomeric compound treatment were averaged. Where present, “N.D.” indicates “not determined”.







TABLE 75







Effects of oligomeric compounds targeting miRNAs


on expression of adipocyte differentiation markers















SEQ








Isis
ID





PPAR


Number
NO
Pri-miRNA
TG
HSL
aP2
GLUT4
gamma

















UTC
N/A
N/A
100
100
100
100
100


327873
291
mir-140
105
116
113
106
104


327879
297
mir-7-1/mir-7-1*
59
103
103
99
81


327881
299
mir-128a
91
93
95
97
98


327885
303
mir-17/mir-91
29
57
69
40
59


327886
304
mir-123/mir-126
12
22
19
13
25


327887
305
mir-132
54
53
60
43
81


327891
309
mir-212
22
52
56
47
50


327895
313
mir-122a
76
88
90
76
86


327896
314
mir-22
22
37
43
35
52


327897
315
mir-92-1
28
39
62
32
66


327898
316
mir-142
102
92
96
82
101


327899
317
mir-183
25
27
47
14
62


327900
318
mir-214
26
21
32
12
55


327902
320
mir-192-1
55
56
58
15
56


327906
324
mir-103-1
25
37
46
14
50


327907
325
mir-26a-1
19
21
29
6
49


327910
328
mir-107
24
32
35
16
39


327911
329
mir-106
59
71
76
48
75


327912
330
let-7f-1
112
95
101
79
78


327916
334
mir-124a-2
56
64
67
51
71


327917
335
mir-21
26
26
32
15
54


327918
336
mir-144
65
85
91
66
74


327920
338
mir-222
20
14
22
0
34


327921
339
mir-30d
56
76
76
36
75


327923
341
mir-128b
88
64
65
54
77


327929
347
mir-199b
65
68
62
49
71


327935
353
mir-20
41
61
60
47
67


327936
354
mir-133a-1
23
40
40
6
47


327940
358
mir-199a-2
62
67
62
43
64


327943
361
mir-18
112
109
106
87
98


327944
362
mir-220
38
55
71
28
64


327945
363
mir-24-2
48
41
43
26
51


327946
364
mir-211
82
76
73
68
81


327949
367
mir-10a
43
49
52
20
54


327950
368
mir-19a
125
94
95
104
93


327952
370
mir-137
93
64
56
61
84


327957
375
mir-100-1
29
15
23
11
68


327958
376
mir-187
28
5
10
5
55


327959
377
mir-210
33
11
24
152
65


327961
379
mir-223
77
88
91
101
95


327962
380
mir-30c-1
64
77
75
58
80


327963
381
mir-26b
124
89
75
91
91


327964
382
mir-152
60
102
96
114
93


327965
383
mir-135-1
116
84
67
88
91


327966
384
mir-217
52
56
53
43
77


327968
386
sterol regulatory
94
79
67
85
79




element-binding









protein-1/









mir-33b







327969
387
mir-182
34
45
44
36
67


327970
388
mir-148a
48
25
29
27
46


327971
389
mir-23a
45
38
49
60
69


327972
390
mir-181c
67
70
70
75
85


328089
391
hypothetical 
67
55
50
59
79




miR-13/miR-190







328090
392
hypothetical 
128
81
68
86
95




miRNA-023







328091
393
hypothetical 
48
40
46
26
85




miRNA-30







328092
394
glutamate 
134
80
74
78
86




receptor,









ionotrophic, 









AMPA 3/









hypothetical 









miRNA-033







328094
396
hypothetical 
65
74
68
83
94




miRNA-040







328095
397
hypothetical 
110
83
70
98
92




miRNA-041







328096
398
hypothetical 
74
76
71
79
89




miRNA-043







328097
399
hypothetical 
65
54
48
62
63




miRNA-044







328098
400
hypothetical 
39
28
23
25
54




miRNA-055







328099
401
hypothetical 
57
74
80
61
72




miRNA-058







328100
402
hypothetical 
20
49
47
39
48




miRNA-070







328101
403
LOC 114614 
67
78
83
57
70




containing









miR-155/









hypothetical









miRNA-071







328102
404
hypothetical 
70
99
96
58
94




miRNA-075







328103
405
hypothetical 
113
87
96
86
83




miRNA-079







328104
406
hypothetical 
64
81
94
83
73




miRNA-083







328105
407
DiGeorge 
82
95
102
75
85




syndrome









critical region 









gene 8/









hypothetical 









miRNA-088







328106
408
hypothetical 
70
86
91
79
81




miRNA-090







328107
409
hypothetical 
51
55
68
52
71




miRNA-099







328108
410
hypothetical 
79
75
87
65
72




miRNA-101







328109
411
hypothetical 
23
62
68
55
69




miRNA-105







328110
412
hypothetical 
96
84
89
77
80




miRNA-107







328111
413
hypothetical 
65
77
79
50
65




miRNA-111







328113
415
hypothetical 
74
83
87
78
85




miRNA-137







328115
417
hypothetical 
53
75
74
84
80




miRNA-142







328116
418
hypothetical 
107
91
99
105
95




miRNA-143







328117
419
collagen, type I,
16
18
28
13
42




alpha 1/









hypothetical









miRNA-144







328118
420
hypothetical 
69
67
74
57
72




miRNA-153







328119
421
hypothetical 
109
101
119
104
102




miRNA-154







328120
422
hypothetical 
80
67
80
68
73




miRNA-156







328121
423
hypothetical 
119
110
119
115
105




miRNA-161







328122
424
hypothetical 
97
89
99
91
103




miRNA-164







328123
425
hypothetical 
54
91
119
129
88




miRNA-166







328124
426
hypothetical 
108
96
118
105
92




miRNA-168-1/









similar to









ribosomal 









protein L5







328125
427
forkhead box
44
48
75
65
68




P2/hypothetical 









miRNA-169







328126
428
hypothetical 
108
135
120
107
98




miRNA-170







328127
429
glutamate 
81
93
95
75
85




receptor,









ionotropic, 









AMPA 2/









hypothetical 









miRNA-171







328128
430
hypothetical 
61
72
90
73
86




miRNA-172







328129
431
hypothetical 
19
34
54
36
59




miRNA-173







328130
432
hypothetical 
91
64
72
55
77




miRNA-175







328131
433
hypothetical
74
51
63
56
55




miRNA-176







328133
435
hypothetical 
43
49
66
59
53




miRNA-178







328134
436
hypothetical 
107
109
97
109
86




miRNA-179







328135
437
cezanne 2/
29
20
34
19
33




hypothetical 









miRNA-180







328136
438
hypothetical
26
37
57
35
54




miRNA-181







328137
439
tight junction 
37
25
45
29
36




protein 1









(zona occludens 









1)/hypothetical 









miRNA-183







328138
440
hypothetical 
80
56
52
52
63




miRNA-185







328139
441
hypothetical 
90
116
100
85
91




miRNA-188







340341
236
mir-104 
46
49
62
48
71




(Mourelatos)







340343
1780
mir-105
35
46
60
33
59




(Mourelatos)







340348
848
mir-93 
48
57
68
52
78




(Mourelatos)







340350
855
mir-95 
38
45
64
53
59




(Mourelatos)







340352
1821
mir-99
110
123
107
97
102




(Mourelatos)







340354
1903
mir-25
64
56
72
61
74


340356
1853
mir-28
43
59
73
54
62


340358
1825
mir-31
23
24
47
21
42


340360
1865
mir-32
106
102
102
91
96


341791
1880
mir-30a
50
72
80
47
75


341795
1762
mir-199a-2
57
74
76
55
74


341796
1904
mir-131-1/mir-9
59
67
74
58
66


341797
1773
mir-17/mir-91
20
29
45
17
50


341798
1871
mir-123/mir-126
62
77
84
55
70


341799
1787
hypothetical 
98
103
101
89
89




miR-13/miR-190







341800
1766
mir-186
18
42
50
28
61


341801
1839
mir-198
65
89
90
77
82


341802
1806
mir-191
155
121
98
85
127


341803
760
mir-206
N.D.
79
85
73
68


341804
761
mir-94/mir-106b
N.D.
75
78
62
71


341805
762
mir-184
N.D.
86
90
74
77


341806
763
mir-195
N.D.
77
83
58
70


341807
764
mir-193
N.D.
102
82
101
83


344268
1774
mir-10b
57
44
46
22
53


344269
1890
mir-29c
42
35
41
28
48


344275
1912
mir-203
36
39
36
21
46


344276
1828
mir-204
66
68
72
49
72


344277
1767
mir-1d-2
75
57
61
45
68


344338
1812
mir-130a
103
89
86
66
91


344340
1921
mir-140
60
47
82
16
67


344341
1823
mir-218-1
50
33
42
14
49


344342
1814
mir-129-2
88
87
88
71
83


344343
1811
mir-130b
32
22
25
4
30


344611
1785
mir-240* (Kosik)
43
31
34
3
34


344612
1790
mir-232* (Kosik)
69
59
72
40
62


344613
1775
mir-227* (Kosik)/
47
46
55
38
57




mir-226* (Kosik)







344614
1834
mir-227* (Kosik)/
89
71
78
61
86




mir-226* (Kosik)







344615
1900
mir-244* (Kosik)
149
154
166
145
144


344616
1800
mir-224* (Kosik)
32
23
26
2
36


344617
1862
mir-248* (Kosik)
52
55
59
42
72


346685
1884
mir-27 
164
172
181
233
138




(Mourelatos)







346686
1857
mir-101-1
73
80
83
73
83


346687
1802
mir-129-1
55
53
56
35
60


346688
1898
mir-182
33
39
48
12
55


346689
1830
mir-200b
59
63
79
45
64


346691
1870
mir-147 (Sanger)
56
69
69
64
79


346692
1889
mir-224 (Sanger)
35
18
26
11
28


346693
1838
mir-134 (Sanger)
69
66
77
65
81


346694
1763
mir-146 (Sanger)
31
18
41
5
32


346695
1824
mir-150 (Sanger)
69
73
72
58
78


346906
1781
mir-296 
83
70
77
70
80




(RFAM/mmu)







346907
1815
mir-299 
47
36
50
37
51




(RFAM/mmu)







346908
1881
mir-301
75
71
77
65
77




(RFAM/mmu)







346909
1902
mir-302 
66
64
68
64
77




(RFAM/mmu)







346910
1866
mir-34a 
80
69
78
63
83




(RFAM/mmu)







346913
1795
let-7d
63
58
66
40
59


346914
1810
mir-94/mir-106b
41
27
48
16
41


346915
1784
mir-200a
73
67
83
75
90


346917
1826
mir-31
39
27
33
20
31


346919
1849
mir-93 
44
45
64
50
65




(Mourelatos)







346920
1801
mir-96
63
53
70
61
70


346921
1759
mir-34
52
49
69
51
62


348116
1922
mir-320
43
58
79
48
76


348117
1860
mir-321-1
66
55
70
73
65


348119
1908
mir-142
91
76
81
86
90


348124
1820
mir-10b
53
43
59
41
63


348125
1878
mir-19b-1
79
64
67
65
64


348127
1869
mir-27b
155
150
185
201
130









Several compounds were found to have effects on adipocyte differentiation. For example, the oligomeric compounds ISIS Number 340348 (SEQ ID NO: 848), targeted to mir-93 (Mourelatos); ISIS Number 341798 (SEQ ID NO: 1871), targeted to mir-123/mir-126; ISIS Number 344340 (SEQ ID NO: 1921) targeted to mir-140; ISIS Number 346687 (SEQ ID NO: 1802), targeted to mir-129-1 and ISIS Number 348117 (SEQ ID NO: 1860), targeted to mir-321-1 were shown to significantly reduce the expression levels of 3 of the 5 markers of adipocyte differentiation. The effects of ISIS Number 327897 (SEQ ID NO: 315), targeted to mir-92-1, were even more pronounced, as shown by the significant reduction in expression of 4 of the 5 markers of differentiation. These data indicate that these oligomeric compounds have the ability to block adipocyte differentiation. Therefore, these oligomeric compounds may be useful as pharmaceutical agents with applications in the treatment, attenuation or prevention of obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells.
Other compounds were shown to stimulate adipocyte differentiation. For example, the oligomeric compounds ISIS Number 328121 (SEQ ID NO: 423), targeted to hypothetical miRNA-161; ISIS Number 344615 (SEQ ID NO: 1900), targeted to mir-244* (Kosik); ISIS Number 346685 (SEQ ID NO: 1884), targeted to mir-27 (Mourelatos); and ISIS Number 348127 (SEQ ID NO: 1869), targeted to mir-27b resulted in significant increases in all 5 markers of adipocyte differentiation. Other oligomeric compounds, for example ISIS Number 340352 (SEQ ID NO: 1821), targeted to mir-99 (Mourelatos) and ISIS Number 328126 (SEQ ID NO: 428), targeted to hypothetical miRNA-170, resulted in increases in 4 of the 5 markers of adipocyte differentiation. These oligomeric compounds may be useful as a pharmaceutical agents in the treatment of diseases in which the induction of adipocyte differentiation is desirable, such as anorexia, or for conditions or injuries in which the induction of cellular differentiation is desirable, such as Alzheimers disease or central nervous system injury, in which regeneration of neural tissue (such as from pluripotent stem cells) would be beneficial. Furthermore, this oligomeric compound may be useful in the treatment, attenuation or prevention of diseases in which it is desirable to induce cellular differentiation and/or quiescence, for example in the treatment of hyperproliferative disorders such as cancer.
In a further embodiment, oligomeric compounds of the present invention were tested for their effects on insulin signaling in HepG2 cells. As described in Example 18, insulin is known to regulate the expression of hepatic IGFBP-1, PEPCK-c and follistatin. Thus, the IGFBP-1, PEPCK-c and follistatin genes serve as marker genes for which mRNA expression can be monitored and used as an indicator of an insulin-resistant state. Oligomeric compounds with the ability to reduce expression of IGFBP-1, PEPCK-c and follistatin are highly desirable as agents potentially useful in the treatment of diabetes and hypertension.oligomeric compounds of the invention were tested for their effects on insulin signalling in liver-derived cells. For assaying insulin signalling, expression of IGFBP-1, PEPCK-c and follistatin mRNAs were measured as previously described in HepG2 cells transfected with oligomeric compounds targeting miRNAs and treated with either no insulin (“basal” Experiment 1, for identification of insulin-mimetic compounds) or with 1 nM insulin (“insulin treated” Experiment 2, for identification of insulin sensitizers) for four hours. At the end of the insulin or no-insulin treatment, total RNA was isolated and real-time PCR was performed on all the total RNA samples using primer/probe sets for three insulin responsive genes: PEPCK-c, IGFBP-1 and follistatin. Expression levels for each gene are normalized to total RNA, and values are expressed relative to the transfectant only untreated control (UTC). In these experiments, the negative control oligomeric compound was ISIS Number 342672 (SEQ ID NO: 789) or ISIS Number 342673 (SEQ ID NO: 758). Results are shown in Tables 76 and 77. Each value represents at least one oligomeric compound treatment; data from more than one oligomeric compound treatment were averaged.







TABLE 76







Experiment 1: Effects of oligomeric compounds targeting


miRNAs on insulin-repressed gene expression in HepG2 cells












Isis
SEQ ID






Number
NO
Pri-miRNA
Follistatin
IGFBP1
PEPCKc















UTC
N/A
N/A
100
100
100


327873
291
mir-140
97
108
72


327885
303
mir-17/mir-91
74
161
73


327886
304
mir-123/mir-126
82
176
61


327887
305
mir-132
113
119
83


327893
311
let-7b
93
107
81


327895
313
mir-122a
83
108
71


327897
315
mir-92-1
129
163
72


327899
317
mir-183
66
105
42


327900
318
mir-214
111
102
88


327911
329
mir-106
81
157
52


327916
334
mir-124a-2
108
102
88


327918
336
mir-144
75
95
81


327920
338
mir-222
99
165
52


327923
341
mir-128b
86
116
83


327946
364
mir-211
103
108
90


327949
367
mir-10a
112
112
81


327950
368
mir-19a
83
109
65


327952
370
mir-137
93
123
70


327957
375
mir-100-1
69
143
59


327958
376
mir-187
91
119
73


327959
377
mir-210
98
124
139


327961
379
mir-223
113
150
98


327963
381
mir-26b
101
108
92


327964
382
mir-152
97
100
74


327965
383
mir-135-1
95
106
63


341800
1766
mir-186
105
114
71


341801
1839
mir-198
85
99
73


341802
1806
mir-191
136
186
98


341803
760
mir-206
68
107
110


341804
761
mir-94/mir-106b
63
162
44


341805
762
mir-184
63
105
40


341806
763
mir-195
75
128
79


341807
764
mir-193
102
129
97


341808
1861
mir-185
96
113
64









Under “basal” conditions (without insulin), treatments of HepG2 cells with oligomeric compounds of the present invention resulting in decreased mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that the oligomeric compounds have an insulin mimetic effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin repression of mRNA expression of these genes.
From these data, it is evident that the oligomeric compounds, ISIS Number 327886 (SEQ ID NO: 304), targeting mir-123/mir-126; ISIS Number 327899 (SEQ ID NO: 317), targeting mir-183; ISIS Number 327911 (SEQ ID NO: 329), targeting mir-106; ISIS Number 327920 (SEQ ID NO: 338), targeting mir-222; ISIS Number 341804 (SEQ ID NO: 761), targeting mir-94/mir-106b; and ISIS Number 341805 (SEQ ID NO: 762), targeting mir-184, for example, resulted in 39%, 58%, 48%, 48%, 56% and 60% reductions, respectively, in PEPCK-c mRNA, a marker widely considered to be insulin-responsive. Thus, these oligomeric compounds may be useful as pharmaceutic agents comprising insulin mimetic properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.
Conversely, the results observed with the oligomeric compounds targeting mir-92-1 (ISIS Number 327897, SEQ ID NO: 315), mir-10a (ISIS Number 327949, SEQ ID NO: 367), mir-223 (ISIS Number 327961, SEQ ID NO: 379) and mir-191 (ISIS Number 341802, SEQ ID NO: 1806), for example, exhibited increased expression of the IGFBP-1 and follistatin marker genes, suggesting that the mir-92-1, mir-10a, mir-223, and mir-191 miRNA targets may be involved in the regulation of these insulin-responsive genes. When these miRNAs are inactivated by an oligomeric compound, IGFBP-1 and follistatin gene expression is no longer repressed. Similarly, treatment oligomeric compounds targeting mir-210 (ISIS Number 327959, SEQ ID NO: 377)) and mir-206 (ISIS Number 341803, SEQ ID NO: 760) resulted in increases in the IGFBP-1 and PEPCK-c marker genes, suggesting that mir-210 and mir-206 may be involved in the regulation of these insulin-responsive genes.







TABLE 77







Experiment 2: Effects of oligomeric compounds targeting


miRNAs on insulin-sensitization of gene expression in


HepG2 cells













SEQ






Isis
ID






Number
NO
Pri-miRNA
Follistatin
IGFBP1
PEPCKc















UTC +
N/A
N/A
100
100
100


1 nM







insulin







327897
315
mir-92-1
123
243
78


327911
329
mir-106
71
160
78


327916
334
mir-124a-2
98
128
88


327918
336
mir-144
76
81
107


327920
338
mir-222
102
267
59


327923
341
mir-128b
106
119
125


327946
364
mir-211
109
138
99


327949
367
mir-10a
111
172
101


327950
368
mir-19a
89
124
82


327952
370
mir-137
100
103
85


327957
375
mir-100-1
73
184
88


327958
376
mir-187
112
149
106


327959
377
mir-210
92
141
156


327961
379
mir-223
128
160
126


327963
381
mir-26b
95
111
94


327964
382
mir-152
114
121
122


327965
383
mir-135-1
79
105
64


328114
416
hypothetical miRNA-
81
177
41




138





328115
417
hypothetical miRNA-
91
120
59




142





328125
427
forkhead box
107
216
77




P2/hypothetical







miRNA-169





328342
451
mir-203
88
98
39


328343
452
mir-7-1/mir-7-1*
139
135
69


328358
467
mir-123/mir-126
106
165
93


328367
476
mir-212
107
141
85


328377
486
hypothetical miRNA-
159
247
182




30





328396
505
mir-205
135
128
65


328397
506
mir-103-1
75
57
76


328423
532
mir-19b-2
114
69
77


328649
558
mir-20
69
115
86


328702
611
mir-10a
88
83
96


328761
670
hypothetical miRNA-
53
193
64




138





328764
673
hypothetical miRNA-
128
145
68




142





328769
678
mir-26b
84
110
100


328774
683
sterol regulatory
68
100
77




element-binding







protein-1/mir-33b





328776
685
forkhead box
114
86
125




P2/hypothetical







miRNA-169









For HepG2 cells treated with 1 nM insulin, treatments with oligomeric compounds of the present invention resulting in a decrease in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds have an insulin sensitization effect. Treatments with oligomeric compounds of the present invention resulting in an increase in mRNA expression levels of the PEPCK-c, IGFBP-1 and/or follistatin marker genes indicate that these compounds inhibit or counteract the normal insulin response of repression of mRNA expression of these genes.
From these data, it is evident that the oligomeric compounds, ISIS Number 327920 (SEQ ID NO: 338), targeting mir-222; ISIS Number 328114 (SEQ ID NO: 416), targeting hypothetical miRNA-138; ISIS Number 328115 (SEQ ID NO: 417), targeting hypothetical miRNA-142; and ISIS Number 328342 (SEQ ID NO: 451) targeting mir-203, for example, were observed to result in a 41%, a 59%, a 41% and a 61% reduction, respectively, of PEPCK-c mRNA expression, widely considered to be a marker of insulin-responsiveness. Thus, these oligomeric compounds may be useful as pharmaceutic agents with insulin-sensitizing properties in the treatment, amelioration, or prevention of diabetes or other metabolic diseases.
Conversely, the results observed with the oligomeric compounds targeting mir-128b (ISIS Number 327923, SEQ ID NO: 341), mir-223 (ISIS Number 327961, SEQ ID NO: 379), mir-152 (ISIS Number 327964, SEQ ID NO: 382) and hypothetical miRNA-30 (ISIS Number 328377, SEQ ID NO: 486), all exhibiting increased expression of the IGFBP-1, PEPCK-c and follistatin marker genes, support the conclusion that the mir-128b, mir-223, mir-152 and hypothetical miRNA-30 may be involved in the regulation of insulin-responsive genes. When these miRNAs are inactivated by the oligomeric compounds of the present invention, IGFBP-1, PEPCK-c and follistatin gene expression is no longer repressed or insulin-sensitive.
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, and the like) cited in the present application is incorporated herein by reference in its entirety.

## Claims

1. A method of inhibiting the activity, function, or amount of miR-103 (SEQ ID NO: 225) and/or miR-107 (SEQ ID NO: 229), comprising contacting a cell with a compound comprising a modified oligonucleotide, wherein:
the modified oligonucleotide consists of 8, 9, 10, 11, or 12 linked monomeric subunits;
each monomeric subunit of the modified oligonucleotide comprises a modified sugar moiety; and
the modified oligonucleotide is complementary to miR-103 and/or miR-107 with no more than one mismatch.

2. The method of claim 1 wherein the modified oligonucleotide consists of 8 monomeric subunits.

3. The method of claim 1 wherein the modified oligonucleotide consists of 9 monomeric subunits.

4. The method of claim 1 wherein the modified oligonucleotide consists of 10 monomeric subunits.

5. The method of claim 1 wherein the modified oligonucleotide consists of 11 monomeric subunits.

6. The method of claim 1 wherein the modified oligonucleotide consists of 12 monomeric subunits.

7. The method of claim 1, wherein each modified sugar moiety is independently selected from a 2′-F sugar moiety, a 2′-O-methyl sugar moiety, a 2′-O-methoxyethyl sugar moiety, and a bicyclic sugar moiety.

8. The method of claim 7, wherein the bicyclic sugar moiety has a 4′-CH2—O-2′ bridge.

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

10. The method of claim 9, wherein the modified internucleoside linkage is a phosphorothioate linkage.

11. The method of claim 1, wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage.

12. The method of claim 1, wherein the oligonucleotide comprises two or more chemically distinct regions.

13. The method of claim 12, wherein the sugar moiety of each nucleoside of a first chemically distinct region is the same and is selected from a 2′-fluoro nucleoside, a 2′-O-methyl nucleoside, a 2′-O-methoxyethyl nucleoside, and a bicyclic sugar nucleoside.

14. The method of claim 13, wherein the bicyclic nucleoside has a 4′-CH2—O-2′ bridge.

15. The method of claim 1, wherein the oligonucleotide comprises at least one 5-methylcytosine.

16. The method of claim 1, wherein the modified oligonucleotide is attached to a conjugate group.

17. The method of claim 16, wherein the conjugate group is cholesterol.

18. The method of claim 16, wherein the conjugate group comprises a carbohydrate.

19. The method of claim 1, wherein each modified sugar moiety is a bicyclic sugar moiety.

20. The method of claim 19, wherein each bicyclic sugar moiety has a 4′-CH2—O-2′ bridge.

21. The method of claim 1, wherein the modified oligonucleotide is complementary to a target region comprising nucleobases 1 to 8 of miR-103 and/or miR-107.

22. The method of claim 1, wherein the modified oligonucleotide is complementary to a target region comprising nucleobases 2 to 9 of miR-103 and/or miR-107.

23. The method of claim 1, wherein the modified oligonucleotide is complementary to a target region comprising nucleobases 3 to 10 of miR-103 and/or miR107.

24. The method of claim 1, wherein the contacting comprises administering the compound to a subject.