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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
  • C12N2310/141—MicroRNAs, miRNAs
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
  • C12N2310/3519—Fusion with another nucleic acid

Definitions

• hybrid tRNA/pre-microRNA and tRNA/shRNA molecules and methods of making and using.
• ncRNAs genomically-encoded, functional noncoding RNAs
• miRNAs or miRs microRNAs
• IncRNAs long noncoding RNAs
• the tumor suppressive ncRNAs e.g., miR-34a
• the tumor suppressive ncRNAs may be reintroduced into cancer cells to manage tumor progression (He, et al. (2007) Nature, 447, 1130-1134; Welch, et al, (2007) Oncogene, 26, 5017-5022; and Liu, et al.
• miRNA agents such as the mimics, precursors and antagomirs are mainly produced via chemical synthesis (Kelnar, et al., (2014) Anal Chem. 86(3): 1534-42; Ling, et al, (2013) Nature Reviews. Drug Discovery, 12, 847-865;
• RNAs have been successfully employed as scaffolds for the production of RNAs, given the fact that tRNAs and rRNAs are present as stable RNA molecules in the cells.
• the recombinant RNA chimeras are thus isolated, and the target RNAs may be released in demand for structural and biophysical analyses.
• This recombinant RNA technology provides a novel way for cost-effective and fast production of large quantities of recombinant RNAs (e.g., multimilligrams of RNA species from 1 L bacterial culture). Nevertheless, it has not been shown whether recombinant RNAs comprise any natural modifications (e.g., methylation of a base and pseudouridylation), and whether they are biologically functional in human cells.
• RNA coli that offers the versatility to carry various types of functional small RNAs of interests such as miRNAs, siRNAs, RNA aptamers, guide RNAs, catalytic RNAs and riboswitches, where the OnRS may be a highly expressed tRNA fusion pre-miRNA or modified/artificial short hairpin RNA (shRNA).
• shRNA modified/artificial short hairpin RNA
• polynucleotides comprising a tRNA operably linked to a pre-microRNA (pre-miRNA) or short hairpin RNA (shRNA) are provided.
• pre-miRNA pre-microRNA
• shRNA short hairpin RNA
• the tRNA is a methionyl tRNA.
• the tRNA has a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91 >, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 1.
• the pre-miRNA or shRNA is naturally or artificially/synthetically derived.
• the pre-miRNA is selected from pre- miRNA- 1291, pre-miRNA-34a, pre-miRNA- 125b, pre-miRNA- 124, pre-miRNA-27b, and pre-miRNA-22. [0006] In varying embodiments:
• the pre-miRNA (or shRNA)- 1291 comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the miRBase Accession No. MI0006353;
• the pre-miRNA (or shRNA)-34a comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to miRBase Accession No. MI0000268;
• the pre-miRNA (or shRNA)- 125b- 1 comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to miRBase Accession No. MI0000446;
• the pre-miRNA (or shRNA)- 124 comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to miRBase Accession No. MI0000443;
• the pre-miRNA (or shRNA)-27b comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to miRBase Accession No. MI0000440; and/or
• the pre-miRNA (or shRNA)-22 comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to miRBase Accession No. MI0000078.
• sequence identity e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to miRBase Accession No. MI0000078.
• the pre-miR A (or shRNA)-34a comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:2;
• the pre-miRNA (or shRNA)-1291 comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:9;
• the pre-miRNA (or shRNA)- 125-1 comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO : 17;
• the pre-miRNA (or shRNA)-124 comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:26;
• the pre-miRNA (or shRNA)-27b comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:29; and/or
• the pre-miRNA (or shRNA)-22 comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:32.
• the polynucleotide comprises a methionyl tRNA operably linked to a pre-miRNA (or shRNA), or mutants or variants thereof, selected from the group consisting of pre-miRNA- 1291, pre-miRNA-34a, pre-miRNA- 125b, pre-miRNA- 124, pre-miRNA-27b and pre-miRNA-22.
• the polynucleotide comprises:
• the methionyl tRNA operably linked to the pre-miRNA (or shRNA)-34a has a nucleic acid sequence having at least 90%> sequence identity, e.g., at least 91 >, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a polynucleotide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39 and SEQ ID NO:40;
• the methionyl tRNA operably linked to the pre-miRNA (or shRNA)- 1291 has a nucleic acid sequence having at least 90%> sequence identity, e.g., at least 91 >, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a polynucleotide selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13; SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16;
• the methionyl tRNA operably linked to the pre-miRNA (or shRNA)-125 has a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a polynucleotide selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21; SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25;
• the methionyl tRNA operably linked to the pre-miRNA (or shRNA)-124 has a nucleic acid sequence having at least 90%> sequence identity, e.g., at least 91 >, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a polynucleotide selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:28;
• the methionyl tRNA operably linked to the pre-miRNA (or shRNA)-27b has a nucleic acid sequence having at least 90%> sequence identity, e.g., at least 91 >, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a polynucleotide selected from the group consisting of SEQ ID NO : 30 and SEQ ID NO : 31 ; and/or
• the methionyl tRNA operably linked to the pre-miRNA (or shRNA)-155 has a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a polynucleotide selected from the group consisting of SEQ ID NO:33 and SEQ ID NO:34.
• the tRNA comprises or comprised a stem-loop anticodon and all or part of the stem-loop anticodon of the tRNA is replaced with the pre- miRNA (or shRNA).
• the tRNA operably linked to the short hairpin RNA comprises a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to a polynucleotide selected from the group consisting of SEQ ID NOs:41-45.
• the tRNA and/or pre-miRNA are further operably linked to one or more inserted RNA molecules.
• the inserted RNA molecule is inserted at, abutted with or operably linked to: a) the 5' end of the pre-miRNA (or shRNA); b) the 3' end of the pre-miRNA (or shRNA); c) 5 Of a dicer cleavage site of the pre-miRNA (or shRNA); or d) 3 Of a dicer cleavage site of the pre-miRNA (or shRNA).
• the inserted RNA has at least about 18 nucleotides and up to about 200 nucleotides, e.g., at least about 18 nucleotides and up to about 50 nucleotides.
• the inserted RNA is selected from the group consisting of a noncoding RNA (ncRNA), mature microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a guide RNA (gRNA), a catalytic RNA, a riboswitch, and an RNA aptamer.
• the inserted RNA is a noncoding RNA.
• the noncoding RNA is HOX antisense intergenic RNA (HOT AIR).
• the inserted RNA is a mature miRNA selected from the group consisting of miR-21 , miR-22, miR-27b, miR-33, miR-34a, miR 122, miR 124-1 , miR-125-1 , miR-1291 and let-7a.
• the inserted RNA prevents, reduces or inhibits the expression of a target polypeptide.
• the inserted RNA is an aptamer that binds to a target molecule or a target polypeptide.
• the target polypeptide is selected from the group consisting of a fluorescent protein, a cytokine, a growth factor, a hormone, an enzyme, an ion channel, a kinase, a nuclear receptor, a G protein-coupled receptor, an epigenetic regulator, a transcription factor.
• the fluorescent protein is selected from the group consisting of a violet fluorescent protein, a blue fluorescent protein (BFP), a cyan fluorescent protein, a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), an orange fluorescent protein (OFP), a red fluorescent protein (RFP) and a sapphire-type protein.
• the cytokine is selected from the group consisting of interleukin (IL)-l , IL- 1 ⁇ , tumor necrosis factor (TNF)a, interferon (IFN)a, ⁇ , IFNy, TGFpi , IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, IL-22, IL-23 and migration inhibitory factor (MIF).
• the nuclear receptor is Peroxisome proliferator-activated receptor gamma (PPAR- ⁇ or PPARG).
• the growth factor is vascular endothelial growth factor (VEGF).
• the kinase is epidermal growth factor receptor (EGFR).
• the polynucleotide has a nucleic acid sequence having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o, 99% or 100% sequence identity, to a polynucleotide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: l l , SEQ ID NO: 12, SEQ ID NO: 13; SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16; SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 ; SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:28; SEQ ID NO:30, SEQ ID NO:31
• expression cassettes comprising a polynucleotide comprising a tR A operably linked to a pre-microRNA (pre-miRNA) or short-hairpin RNA (shRNA) as described above and herein
• pre-miRNA pre-microRNA
• shRNA short-hairpin RNA
• a liposome comprising a polynucleotide or an expression cassette comprising a tRNA operably linked to a pre-microRNA (pre-miRNA) or short-hairpin RNA (shRNA) as described above and herein
• pre-miRNA pre-microRNA
• shRNA short-hairpin RNA
• a viral vector comprising a polynucleotide or an expression cassette comprising a tRNA operably linked to a pre -microRNA (pre- miRNA) or short-hairpin RNA (shRNA) as described above and herein.
• pre- miRNA pre-microRNA
• shRNA short-hairpin RNA
• host cells transfected or transformed with a polynucleotide encoding a hybrid tRNA/pre-microRNA or tRNA/shRNA molecule, as described above and herein, or an expression cassette, liposome, polymer, nanoparticle, viral vector comprising a hybrid tRNA/pre-microRNA or tRNA/shRNA molecule.
• the host cell is a prokaryotic cell or a eukaryotic cell.
• the host cell is selected from a bacterial cell (e.g., E. coli), a mammalian cell (e.g., a human cell), an insect cell or a plant cell.
• tRNA/pre- microRNA or tRNA/shRNA molecule comprising expressing in a population of host cells the hybrid tRNA/pre-microRNA or tRNA/shRNA molecule from a polynucleotide, expression cassette, liposome, polymer, nanoparticle or viral vector as described above and herein.
• At least 1 mg e.g., at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, or more, of the hybrid tRNA/pre-microRNA or tRNA/shRNA molecule are produced from a 1 liter culture comprising the population of host cells.
• the host cell is selected from a bacterial cell, a mammalian cell, an insect cell or a plant cell.
• At least about 1 mg, e.g., at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, or more, of the hybrid tRNA/pre-microRNA or tRNA/shRNA molecule are produced from a 1 liter culture of E. coli host cells over a period of about 24 hours.
• At least about 1 mg, e.g., at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, or more, of the hybrid tRNA/pre-microRNA or tRNA/shRNA molecule are produced from a 1 liter culture of yeast host cells, e.g., over a period of about 24 hours.
• the hybrid tR A/pre-microR A or tRNA/shRNA molecule produced comprises at least about 5%, e.g., at least about 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, or more, of the total RNA.
• RNA molecules comprising expressing the RNA molecule from a a hybrid tRNA/pre-microRNA or tRNA/shRNA polynucleotide, expression cassette, liposome, polymer, nanoparticle or viral vector as described above and herein.
• at least 1 mg e.g., at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, or more, of the RNA molecule are produced from a 1 liter culture comprising the population of host cells.
• the host cell is selected from a bacterial cell, a mammalian cell, an insect cell or a plant cell.
• At least about 1 mg, e.g., at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, or more, of the RNA molecule are produced from a 1 liter culture of E. coli host cells over a period of about 24 hours.
• RNA molecule 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, or more, of the RNA molecule are produced from a 1 liter culture yeast host cells, e.g., over a period of about 24 hours.
• the RNA molecule produced comprises at least about 5%, e.g., at least about 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, or more, of the total RNA.
• At least about 1 mg, e.g., at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, or more, of the hybrid tRNA/pre-microRNA or tRNA/shRNA molecule are produced from a 1 liter culture of E. coli host cells over a period of about 24 hours.
• At least about 1 mg, e.g., at least about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, or more, of the hybrid tRNA/pre-microRNA or tRNA/shRNA molecule are produced from a 1 liter culture yeast host cells, e.g., over a period of about 24 hours.
• the hybrid tRNA/pre-microRNA or tRNA/shRNA molecule produced comprises at least about 5%, e.g., at least about 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, or more, of the total R A.
• kits for preventing, mitigating, reducing and/or inhibiting the growth, proliferation, and/or progression of a cancer in a subject in need thereof comprising administering to the subject a polynucleotide, expression cassette, liposome, polymer, nanoparticle or viral vector as described above and herein.
• the cancer is selected from the group consisting of breast cancer, lymphoma, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, and lung cancer.
• RNA binding methods comprising contacting a sample suspected of containing the target molecule under conditions that allow binding an inserted RNA expressed from the a polynucleotide, expression cassette, liposome, polymer, nanoparticle or viral vector as described above and herein, wherein binding of the inserted RNA to the target molecule in the sample identifies its presence.
• kits comprising a polynucleotide, expression cassette, liposome, polymer, nanoparticle or viral vector as described above and herein.
• polynucleotide refers to polymers composed of
• nucleotide refers to a chemical moiety having a sugar (modified, unmodified, or an analog thereof), a nucleotide base (modified, unmodified, or an analog thereof), and a phosphate group (modified, unmodified, or an analog thereof).
• Nucleotides include deoxyribonucleotides, ribonucleotides, and modified nucleotide analogs including, for example, locked nucleic acids (“LNAs”), peptide nucleic acids (“PNAs”), L-nucleotides, ethylene-bridged nucleic acids (“EN As”), arabinoside, and nucleotide analogs (including abasic nucleotides).
• LNAs locked nucleic acids
• PNAs peptide nucleic acids
• EN As ethylene-bridged nucleic acids
• nucleotide analogs including abasic nucleotides.
• nucleic acid refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof and to naturally occurring or synthetic molecules.
• RNA may be used in the methods described herein and/or may be converted to cDNA by reverse-transcription and/or RNA for use in the methods described herein.
• a "microRNA,” “miR,” or “miRNA” refer to the unprocessed or processed RNA transcript from a miRNA gene.
• the unprocessed miRNA gene transcript is also called a “miRNA precursor,” and typically comprises an RNA transcript of about 70-100 nucleotides in length.
• the miRNA precursor can be processed by digestion with an RNAse (for example, Dicer, Argonaut, or RNAse III) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the "processed" miRNA gene transcript or "mature" miRNA.
• pre-microRNA or "pre-miR” or pre-miRNA” interchangeably refer to an RNA hairpin comprising within its polynucleotide sequence at least one mature micro RNA sequence and at least one dicer cleavable site.
• pre-miRNA-1291 or “hsa-mir-1291” or “HGNC:MIR1291” interchangeable refer to an RNA polynucleotide having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to miRBase Accession No. MI0006353 (www.mirbase.org) or SEQ ID NO:9.
• pre-miR A-34a or “hsa-mir-34a” or “HGNC :MIR34A” interchangeable refer to an R A polynucleotide having at least 90% sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to miRBase Accession No. MI0000268 (www.mirbase.org) or SEQ ID NO:2.
• pre-miRNA- 125-1 or “hsa-mir-125b-l” or “HGNC:MIR125B1” interchangeable refer to an RNA polynucleotide having at least 90%> sequence identity, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to miRBase Accession No. MI0000446 (www.mirbase.org) or SEQ ID NO: 17.
• nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., share at least about 80%> identity, for example, at least about 85%, 90%>, 91%>, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a specified region to a reference sequence, e.g., the tRNA, pre-microRNA and tRNA/microRNA hybrid polynucleotide molecules described herein, e.g, SEQ ID NOs: 1-45, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms (e.g.
• sequences are then said to be “substantially identical.” This definition also refers to the compliment of a test sequence. Preferably, the identity exists over a region that is at least about 10, 15, 20, 25, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120 nucleotides in length, or over the full-length of a reference sequence.
• siRNA refers to any nucleic acid molecule capable of down regulating ⁇ i.e., inhibiting) gene expression in a mammalian cells (preferably a human cell).
• siRNA includes without limitation nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA).
• siRNA short interfering RNA
• dsRNA double-stranded RNA
• miRNA micro-RNA
• shRNA short hairpin RNA
• the sense strand of a siRNA molecule may also include additional nucleotides not complementary to the antisense region of the siRNA molecule.
• the term "antisense region” refers to a nucleotide sequence of a siRNA molecule complementary (partially or fully) to a target nucleic acid sequence.
• the antisense strand of a siRNA molecule may include additional nucleotides not complementary to the sense region of the siRNA molecule.
• PiRNA and "Piwi-interacting RNA” are interchangeable and refer to a class of small RNAs involved in gene silencing. PiRNA molecules typically are between 26 and 31 nucleotides in length.
• snRNA and "small nuclear RNA” are interchangeable and refer to a class of small RNAs involved in a variety of processes including RNA splicing and regulation of transcription factors.
• the subclass of small nucleolar RNAs (snoRNAs) is also included.
• the term is also intended to include artificial snRNAs, such as antisense derivatives of snRNAs comprising antisense sequences directed against the ncRNA.
• operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
• a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present.
• Expression is meant to include the transcription of any one or more of transcription of a microRNA, siRNA, piRNA, snRNA, lncRNA, antisense nucleic acid, or mRNA from a DNA or RNA template and can further include translation of a protein from an mRNA template.
• the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
• homologous region refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a "homologous region” is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term “homologous, region,” as used herein, refers to the ability of nucleic acid molecules to hybridize to each other. For example, a single-stranded nucleic acid molecule can have two homologous regions which are capable of hybridizing to each other. Thus, the term
• homologous region includes nucleic acid segments with complementary sequence.
• Homologous regions may vary in length, but will typically be between 4 and 40 nucleotides (e.g., from about 4 to about 40, from about 5 to about 40, from about 5 to about 35, from about 5 to about 30, from about 5 to about 20, from about 6 to about 30, from about 6 to about 25, from about 6 to about 15, from about 7 to about 18, from about 8 to about 20, from about 8 to about 15, etc.).
• nucleotides e.g., from about 4 to about 40, from about 5 to about 40, from about 5 to about 35, from about 5 to about 30, from about 5 to about 20, from about 6 to about 30, from about 6 to about 25, from about 6 to about 15, from about 7 to about 18, from about 8 to about 20, from about 8 to about 15, etc.
• complementary and complementarity are interchangeable and refer to the ability of polynucleotides to form base pairs with one another.
• Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands or regions.
• Complementary polynucleotide strands or regions can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G).
• 100% complementary refers to the situation in which each nucleotide unit of one polynucleotide strand or region can hydrogen bond with each nucleotide unit of a second polynucleotide strand or region.
• Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands or two regions can hydrogen bond with each other and can be expressed as a percentage.
• a "target site” or “target sequence” is the nucleic acid sequence recognized
• an antisense oligonucleotide or inhibitory R A molecule (i.e., sufficiently complementary for hybridization) by an antisense oligonucleotide or inhibitory R A molecule .
• the term "subject” refers to a mammal, such as a human, but can also be another animal such as a domestic animal (e.g., a dog, cat, or the like), a farm animal (e.g., a cow, a sheep, a pig, a horse, or the like) or a laboratory animal (e.g., a monkey, a rat, a mouse, a rabbit, a guinea pig, or the like).
• the term "patient” refers to a subject who is, or is suspected to be, afflicted with a disease.
• the terms "effective amount” or “pharmaceutically effective amount” or “therapeutically effective amount” of a composition is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in, the symptoms associated with a disease that is being treated.
• the amount of a composition of the invention administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
• the compositions of the present invention can also be administered in combination with one or more additional therapeutic
• cancer-associated antigen or “tumor-associated antigen” or
• tumor-specific marker or “tumor marker” interchangeably refers to a molecule (typically protein, carbohydrate or lipid) that is preferentially expressed on the surface of a cancer cell in comparison to a normal cell, and which is useful for the preferential targeting of a pharmacological agent to the cancer cell.
• a cancer-associated antigen is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, 1-fold over expression, 2-fold overexpression, 3 -fold overexpression or more in comparison to a normal cell.
• a cancer-associated antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. Oftentimes, a cancer-associated antigen will be expressed exclusively on the cell surface of a cancer cell and not synthesized or expressed on the surface of a normal cell.
• Exemplified cell surface tumor markers include the proteins c-erbB-2 and human epidermal growth factor receptor (HER) for breast cancer, PSMA for prostate cancer, and
• carbohydrate mucins in numerous cancers including breast, ovarian and colorectal.
• FIGS 1 A-D illustrate the establishment of OnRS for high-level expression of recombinant RNAs in E. coli.
• A and
• B When only the tRNA scaffold was used, the majority of pre -miRNAs were not expressed or at a limited level despite that mir-34a, mir- 1291 and mir-125 etc. were expressed at very high levels.
• C and (D) Therefore, the tRNA/mir-34a, tRNA/mir-1291 and tRNA/mir-125, etc. were developed as new scaffolds that allow a consistent, high-level production of miRNAs or siRNAs (or other small RNAs; please see the following figures).
• FIGS 2 A-D illustrate the design of recombinant RNAs using the tRNA/pre-miRNA or tRNA/shRNA non-coding RNA (ncRNA) scaffolds (OnRS). Shown are the secondary structures of OnRS such as tRNA/pre-miRNA hybrid carrying a miRNA or siRNA (A), or tRNA/shRNA OnRS bearing a siRNA or miRNA (B), the OnRS carrying a small RNA (e.g., an aptamer) or other RNA species at the 5' of the pre-miRNA or shRNA (C), and the OnRS carrying a small RNA (e.g., an aptamer) or other target RNA species at the 3 ' of the pre-miRNA or shRNA (D).
• OnRS such as tRNA/pre-miRNA hybrid carrying a miRNA or siRNA (A), or tRNA/shRNA OnRS bearing a siRNA or miRNA (B), the OnRS carrying a small RNA (e
• Figures 3A-D illustrate the consistently high-level expression of recombinant miRNAs using OnRS, which are also indicated by their FPLC traces during purification. Approximately the same amount of total RNAs were loaded onto the column.
• the level of tRNA/pre-mir-34a scaffold (A) is about 5- to 10-fold higher than tRNA scaffold.
• Figures 4A-C illustrate the effectiveness of recombinant mir-34a in suppressing cancer cell proliferation and target gene expression, and controlling xenograft tumor progression.
• tRNA/mir-34a inhibited the growth of human carcinoma A549 cells in a dose dependent manner and to a much greater degrees than the control tRNA/MSA (P ⁇ 0.001 , two-way ANOVA).
• Cell viability was determined using MTT assay at 72 h post- transfection. Values are mean ⁇ SD of triplicate cultures.
• tRNA/mir-34a Compared to the control tRNA/MSA, recombinant tRNA/mir-34a sharply reduced the protein levels of a number of miR-34a target genes including CDK6, MET and SIRT1 in A549 cells. Western blots were conducted using protein selective antibodies. GAPDH was used as a loading control.
• Figure 5A-D illustrate the fate of recombinant ncRNAs in human cells
• (a and b) Unbiased deep sequencing study revealed that OnRS-carried miR-124 and GFP- siRNA were precisely processed to target small RNAs, leading to 3 orders of magnitude increase in miR-124 in A549 cells and GFP siRNA in ES-2/GFP cells, respectively. Note the presence of miRNA and siRNA isoforms as well as corresponding passenger strands and other small RNAs at much lower levels. In contrast, the levels of other cellular miRNAs showed no or minor changes.
• Values are mean ⁇ SD of triplicated treatments that were sequenced separately, (c and d) Mapping major cellular tRFs derived from OnRS/miR-124 versus OnRS (tRNA/mir-34a) in A549 cells and OnRS/GFP-siRNA versus OnRS/Neg in ES-2/GFP cells, respectively. Shown are the mean numbers of reads of triplicated treatments. 3,000 reads was used as a cut off.
• FIGS 6A-E illustrate OnRS-carried miRNA is biologically/ pharmacologically active in regulating target gene expression and controlling cellular processes in human cells
• RT-qPCR analysis revealed that mature miR-124 levels retained 3 orders of magnitude higher in A549 cells for 4 days since transfection with OnRS/miR-124, as compared with OnRS/Neg.
• Western blots showed that OnRS/miR- 124 was effective in reducing the protein expression level of miR-124 target gene STAT3 in A549 cells at 72 h post-transfection.
• OnRS/miR-124 significantly suppressed the proliferation of A549 cells at 72 h post- treatment, as compared to OnRS/Neg. (e) Inhibition of A549 cell proliferation by
• FIGS 7A-F illustrate OnRS-carried siRNA is effective for RNAi in vitro and in vivo. GFP fluorescence intensity was sharply reduced in ES-2/GFP cells in vitro at 72 h after transfected with OnRS/GFP-siRNA (a), which was associated with (b) 70-80% lower GFP mRNA levels and (c) 1000-fold higher GFP siRNA levels. Following i.v.
• hepatic GFP fluorescence was significantly suppressed in the GFP-transgenic mouse models in vivo, as demonstrated by microscopic examination of (d) non-fixed and (e) fixed liver slices, as well as (f) RT-qPCR analysis of hepatic GFP mRNA levels. Fixed liver slices were stained with DAPI, and GFP
• Figure 8 illustrates the basic principle of the herein described RNase assay method using a malachite green aptamer (MGA) sensor.
• Figures 9A-E illustrate high-yield large-scale production of chimeric MGA sensor that produces strong and selective fluorescence upon binding to MG.
• MGA may be inserted at the 5 Or 3' end of the OnRS scaffold to offer OnRS/MGA5 and
• OnRS/MGA3 respectively.
• the heat color gradation indicates the base-pairing probability from 0 to 1.
• (b) A consistent high-level expression of chimeric MGA in E. coli, e.g., over 50% of OnRS/MGA5 and OnRS/MGA3 in total RNAs.
• (c) Representative FPLC traces of OnRS/MGA5 during FPLC purification. Insert is urea-PAGE analysis of the collected RNA fractions (1, 2 and 3) eluted at 10.6 min.
• OnRS/MGA3 led to a shift of the wavelength of MG maximum absorbance from 618 to 630 nm. The same shift was observed when FPLC-purified OnRS/MGA and total RNAs isolated from OnRS/MGA-expressing bacteria were used.
• the SEPHADEXTM-purified aptamer (OnRS/Seph) and corresponding total RNAs were used as additional controls, (e) Strong and selective fluorescence was shown when MG bound to OnRS/MGA5 or
• OnRS/MGA3 The same results were obtained when using FPLC-purified OnRS/MGA and OnRS/MGA-containing total RNAs.
• Figure 10A-E illustrate methods to determine RNase activity using chimeric
• OnRS/MGA was much more susceptible to human RNase A (10 min incubation) than angiogenin (RNase 5; 30 min incubation), (e) Human pancreatic cancer patients showed significantly higher serum RNase activities than benign/normal patients, as determined by the decrease in MGA-bound MG fluorescence intensity ( ⁇ . ⁇ ./ ⁇ / ⁇ ). N 10 in each group. OnRS/MGA5 was used in this study.
• Figures 11 A-D illustrate design, expression and purification of recombinant chimeric miR-1291.
• A Secondary structures of tRNA/MSA, pre-miR-1291 and chimeric tRNA/mir- 1291-123nt were predicted with CentroidFold.
• B Target pre-miR- 1291 inserts were cloned into the pBSMrna vector linearized with endonucleases Sal I and Aat II.
• C Successful expression of recombinant tRNA/mir- 1291 in E. coli was demonstrated by the appearance of new RNA band (indicated by the arrow) at expected size/mobility after urea- PAGE separation.
• ncRNAs were determined by ESI-MS analyses, and the differences between measured and predicted MWs suggest the presence of post-transcriptionally- modified nucleosides.
• B Post-transcriptionally modified nucleosides were identified by LC-UV-MS analyses of the hydrolysates of recombinant ncRNAs. Shown are LC-UV traces of the hydrolysates of tRNA/MSA and tRNA/mir-1291, and individual peaks were annotated according to their retention times and mass spectra.
• C Mapping and sequencing of tRNA/MSA and tRNA/mir-1291 was achieved by LC-MS/MS analyses of RNase Tl digestions. Modified nucleosides were also localized, based upon their MS/MS
• FIGS 13A-D illustrate tRNA-carried pre-miR-1291 is processed to mature miR-1291 in in human MCF-7 breast cancer cells.
• A-B The levels of pre-miR-1291 and mature miR-1291 were increased in a dose dependent manner in MCF-7 cells after transfection with purified tRNA/mir-1291.
• C-D The time courses of pre-miR-1291 and mature miR-1291 were monitored in MCF-7 cells at 6, 24, 48 and 72 h post-transfection with 20 nM recombinant tRNA/mir-1291. Cells without treatment or treated with the same doses of tRNA/MSA or vehicle were used as controls. RNA levels were determined by selective qPCR assays. Values are mean ⁇ SD of triplicate treatments. *P ⁇ 0.05, compared to cells treated with the same doses of tRNA/MSA or vehicle or cells without treatment that were harvested at the same time points.
• FIGS 14A-C illustrate recombinant tRNA/mir-1291 is effective to regulate miR-1291 target gene expression in human carcinoma cells.
• MRPl (A), FOXA2 (B) and MeCP2 (C) protein levels were significantly reduced in human carcinoma cells at 48 h post- transfection with 20 nM of tRNA/mir-1291.
• Western blot analyses were conducted with selective antibodies. GAPDH was used as a loading control. Values are mean ⁇ SD of triplicate treatments. *P ⁇ 0.05 and **P ⁇ 0.01, compared to the control tRNA/MSA treatment.
• Figures 15 A-C illustrate chimeric miR-1291 is effective to suppress human carcinoma cell proliferation.
• PANC-1 (A) and MCF-7 (B) cells were sensitive to recombinant tRNA/mir-1291 in a dose dependent manner, to a significantly (P ⁇ 0.01, two- way ANOVA) greater degree than tRNA/MSA. This is also indicated by the estimated EC50 and Hill slope values (C).
• Figures 16 A-B illustrate tRNA-carried pre-miR- 1291 enhances the chemosensitivity of PANC-1 cells.
• A tRNA/mir-1291 significantly (P ⁇ 0.01, two-way ANOVA) sensitized PANC-1 cells to doxorubicin, as compared with tRNA/MSA or vehicle treatment.
• Figures 17A-E illustrate production and structural characterization of recombinant tRNA/mir-34a agents.
• A The secondary structure of chimeric tRNA/mir-34a (233 nt) predicted by CentroidFold showed that the stem-loop structure of mir-34a retained within the chimeric ncRNA. The heat color gradation indicates the base-pairing probability from 0 to 1.
• B Expression of tRNA/mir-34a in various strains of E. coli. Among those tested, the highest levels of recombinant ncRNAs were found in HST08.
• C FPLC traces of tRNA/mir-34a and tRNA/MSA during the purification.
• RNAs were separated using anion-exchange FPLC and monitored at 260 nm.
• D HPLC analysis confirmed the high homogeneity (>98%) of purified tRNA/mir-34a and tRNA/MSA.
• E LC-MS/MS mapping/sequencing of purified tRNA/mir-34a and tRNA/MSA after the digestion with RNase Tl . All posttranscriptional modifications except deoxyadenosine identified by nucleoside analysis ( Figure 18) could be mapped to RNase Tl digestion products and assigned to specific sites. [0057] Figures 18 A-E illustrate expression and affinity purification of recombinant tRNA/mir-34a.
• tRNA/mir-34a-129nt (233 nt in total) and tRNA/mir-34a-l 12nt (216 nt in total) chimeras were expressed at comparably high levels in HST08 E. coli.
• the tRNA/mir-34a-129nt was chosen for further investigation which is simply named tRNA/mir-34a.
• B Time course of tRNA/mir-34a levels accumulated within HST08 cells after transformation indicated that higher ncRNA levels were achieved at 9-14 hr post- transformation. Chimeric tRNA/mir-34a levels were determined by qPCR assay, normalized to bacterial 16S, and then multiplied by the quantities of total RNAs isolated from corresponding cultures.
• Figures 19A-C illustrate structural characterization of recombinant tRNA/mir-34a and tRNA/MSA purified by anion-exchange FPLC.
• A The molecular weight (MW) of intact ncRNA was determined by electrospray ionization mass
• C RNase Tl mapping tRNA/mir- 34a and tRNA/MSA. Shown are the total ion LC-MS chromatograms of RNase Tl -digested tRNA/MSA and tRNA/mir-34a, and the identification of an example fragment
• FIG. 20A-C illustrate tRNA-carried pre-miR-34a was selectively processed to mature miR-34a in human carcinoma cells while tRNA scaffold was degraded to tRFs, as revealed by unbiased deep sequencing studies. Mature miR-34a levels were over 70-fold higher in A549 cells treated with 5 nM tRNA/mir-34a, as compared to cells treated with the control tRNA/MSA (A) or vehicle (B). This was associated with a 60- to 65-fold increase in the numbers of reads of 15-nt miR-34a-p3 fragment (5'-
• Figures 21A-D illustrate cellular stability and RNase susceptibility of chimeric tRNA/mir-34a.
• tRNA/mir-34a or tRNA/MSA The levels of chimeric ncRNA (tRNA/mir-34a or tRNA/MSA), pre-miRNA mir-34a, and mature miRNA miR-34a were increased in a dose dependent manner (P ⁇ 0.001, one-way ANOVA) in A549 cells at 24 h after transfection with 1, 5 or 25 nM of FPLC-purified tRNA/mir-34a or tRNA/MSA. Note that mir-34a and miR-34a levels were only elevated in cells treated with tRNA/mir- 34a.
• C Chimeric ncRNA (tRNA/mir-34a or tRNA/MSA; 5 nM) exhibited a good stability in A549 cells.
• FIGS 22A-B that recombinant mir-34a was biologically/pharmacologically active in suppressing cancer cell proliferation and target gene expression.
• tRNA/mir- 34a inhibited the growth of human carcinoma A549 and HepG2 cells in a dose dependent manner and to much greater degrees than the control tRNA/MSA (P ⁇ 0.001, two-way ANOVA).
• Antiproliferative activities against H460 and Huh-7 cells are shown in
• Figures 23A-B illustrate recombinant tRNA/mir-34a inhibited the growth of lung H460 (A) and liver Huh-7 (B) human carcinoma cells in a dose dependent manner and to greater degree than the control tRNA/MSA (P ⁇ 0.001, two-way ANOVA). Cells transfected with the same doses of tRNA/MSA were used as controls. Cell viability was determined using MTT assay at 72 h post-transfection. Values are mean ⁇ SD of triplicate treatments. [0063] Figures 24A-B illustrate that recombinant mir-34a was equally or more effective than synthetic miR-34a mimics to reduce human carcinoma cell proliferation and target gene expression.
• (A) The effects of 5 nM biologic tRNA/mir-34a, and synthetic miR- 34a precursor and mimics on the growth of A549 and HepG2 carcinoma cells. Cell viability was determined using MTT assay at 72 h post-transfection. Values are mean ⁇ SD of triplicate treatments. *P ⁇ 0.05, compared to corresponding control.
• (B) The effects of 25 nM recombinant mir-34a and synthetic miR-34a mimics on the protein levels of miR-34a target genes CDK6, MET, and SIRT1 in A549 and HepG2 carcinoma cells, as determined by Western blots at 72 h post-transfection.
• FIGS 25A-B illustrate that recombinant mir-34a was effective to control xenograft tumor progression in mouse models.
• A Compared to the same dose of tRNA/MSA or vehicle control, tRNA/mir-34a (100 ⁇ g) significantly (P ⁇ 0.001, unpaired t test) suppressed the growth of A549 xenograft tumors. Note that the tumors in three mice completely disappeared after the treatment with 100 ⁇ g of tRNA/mir-34a.
• Figures 26A-C illustrate that biologic ncRNAs were well tolerated in mouse models.
• A In vivo-jetPEI-loaded ncRNAs were protected against the degradation by serum RNases.
• B Compared with vehicle control treatment, in vivo-jetPEI formulated recombinant ncRNAs (100 ⁇ g, i.v.) did not cause significant change in mouse serum IL-6 levels while LPS did (two-way ANOVA with Bonferroni post-tests).
• ncRNAs 100 ⁇ g, i.v.
• ALT alanine aminotransferase
• AST aspartate aminotransferase
• BUN blood urea nitrogen
• ALP alkaline phosphatase
• creatinine total bilirubin, albumin and total protein.
• tRNA/pre-microRNA and tRNA/shRNA scaffolds are provided, based in part, on the unexpected observation that the majority pre -miRNAs and tRNA/shRNAs are not expressed or at limited levels only using a tRNA scaffold.
• the present tRNA/pre- microRNA and tRNA/shRNA scaffolds find use for high-level production of recombinant RNAs bearing target small RNAs (e.g., miRNAs, siRNAs, RNA aptamers, catalytic RNAs, riboswitches, guide RNAs and other RNA species).
• target small RNAs e.g., miRNAs, siRNAs, RNA aptamers, catalytic RNAs, riboswitches, guide RNAs and other RNA species.
• tRNA/pre-miRNA and tRNA/shRNA hybrid molecules based non-coding RNA (ncRNA) scaffolds for a consistent high-level expression of recombinant ncRNAs consisting of target RNAs on a large scale.
• ncRNA non-coding RNA
• the OnRS-carried RNAs are biologically active in regulating target gene expression and controlling cellular processes, even to a greater degree than the same concentration of chemically modified RNAs (e.g., miRNA mimics or pre-microRNAs).
• the tRNA/pre-microRNA and tRNA/shRNA scaffolds allow for the consistent high-level expression of biologically-active target miRNAs, siRNA and RNA aptamers (as well as a scrambled small RNA). Consequently, the OnRS-carried recombinant RNAs find use as therapeutic, diagnostic or prognostic agents and used as research materials.
• RNA molecule e.g., a siRNA, an aptamer or diagnostic
• RNA agent may be attached to an appropriate site of the OnRS such as the Dicer-cleavable site, 5 ' and 3 ' flanking region.
• OnRS such as the Dicer-cleavable site, 5 ' and 3 ' flanking region.
• ncRNAs expressed from the present tRNA/pre-microRNA and tRNA/shRNA scaffolds as the precursors or carriers for the delivery of RNA therapeutics, and thus the recombinant OnRS/RNA themselves may be used as "pre-RNAdrugs" and/or research agents.
• design, preparation and application of the present tRNA/pre-microRNA and tRNA/shRNA scaffolds for the production and delivery of a siRNA, miRNA and/or miRNA antagomir are examples of the present tRNA/pre-microRNA and tRNA/shRNA scaffolds for the production and delivery of a siRNA, miRNA and/or miRNA antagomir.
• RNA molecule such as RNA aptamer against a drug target.
• tRNA/pre-microRNA and tRNA/shRNA scaffolds for the production of a druggable RNA target for the identification of new therapeutic agents, as well as diagnostic/prognostic RNA molecule for the development of new diagnostic/prognostic agent or kit.
• RNA sensors for the quantification of specific small molecule compounds (e.g., drug, hormones, etc.) or biological molecules (e.g., proteins).
• biological molecules e.g., proteins.
• OnRS for the production of RNA species for selective or nonselective knocking out of knocking down of specific genes or blocking particular proteins or other biological molecules.
• RNA materials as diagnostic or prognostic biomarkers as well as assay kits.
• OnRS for the production of RNA species as catalysts for the production of organic compounds and/or other biological molecules.
• RNA species as riboswitch for the control of gene expression and cellular processes and use as therapeutics or research agents.
• OnRS for the production of RNA species as guide RNAs for the genome editing and disease treatment.
• RNA molecules including consequent chemical modifications and/or formulations as research materials, reagents, catalysts, diagnostic, prognostic and/or therapeutic treatments.
• microRNAs such as mir-125-1 and mir-33, etc. for the development of new treatment, diagnosis, and/or prognosis.
• the polynucleotides comprise a tRNA operably linked to a pre- microRNA.
• the anticodon of the tRNA is replaced with a pre- microRNA molecule.
• the 3 '-terminus and the 5'- terminus of the pre-microRNA are ligated or fused to the 3 '-terminus and the 5 '-terminus of the tRNA that are created when the anticodon is removed.
• the tRNA molecule and the pre- microRNA molecule can be, but need not be directly ligated or fused to one another to be operably linked.
• the pre-microRNA can contain one or more dicer cleavable sites to allow for the high level expression and efficient cleavage of an inserted RNA molecule desired to be expressed from the hybrid tRNA/pre-microRNA
• the hybrid tRNA/pre-microRNA molecules can be produced by standard recombinant methods, or can be synthetically prepared.
• the polynucleotides can have one or more chemical modifications, including without limitation, e.g., internucleotide linkages, internucleoside linkages, dideoxyribonucleotides, 2 '-sugar modification, 2 '-amino groups, 2'-fiuoro groups, 2'-methoxy groups, 2'-alkoxy groups, 2'- alkyl groups, 2'-deoxyribonucleotides, 2'-0-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, universal base nucleotides, acyclic nucleotides, 5-C-methyl nucleotides, biotin groups, terminal glyceryl incorporation, inverted deoxy abasic residue incorporation, sterically hindered molecules,
• the hybrid tRNA/pre-microR A molecules comprise analog ribonucleotide bases.
• analog defines possible derivatives of the ribonucleotide originating from the activity of tR A post-transcriptional modification enzymes of the cell in which they are produced.
• the analogs of the ribonucleotides A, C, G and U which may be found in a tRNA depend on the cell in which that tRNA is produced and on the position of the ribonucleotide in question in the tRNA. A large number of analogs are given in SRocl et al. (1998) "Compilation of tRNA sequences and sequences of tRNA genes”.
• RNA modification database data (http://medstat.med.utah.edu/RNAmods/).
• the analogs of A may be selected more particularly from the group constituted by 1 -methyl- A, inosine and 2'- O-methyl-A.
• the analogs of C may be selected more particularly from the group constituted by 5-methyl-C and 2'-0-methyl-C.
• the analogs of G may be selected more particularly from the group constituted by 7-methyl-G and 2'-0-methyl-G.
• the analogs of U may be selected more particularly from the group constituted by pseudouridine, ribothymidine, 2'-0-methyl-ribothymidine, dihydrouridine, 4-thiouridine and 3-(3-amino-3- carboxypropyl)-uridine.
• a tRNA is formed of a single ribonucleotide chain which is capable of folding to adopt a characteristic, so-called cloverleaf secondary structure.
• This characteristic secondary structure comprises:
• an acceptor stem composed of the first 7 ribonucleotides of the 5' end of the ribonucleotide chain and the 7 ribonucleotides that precede the last 4 ribonucleotides of the 3' end of the ribonucleotide chain, thus forming a double-stranded structure comprising 6 or 7 pairs of ribonucleotides, it being possible for the ribonucleotides constituted by the first ribonucleotide of the 5' end of the ribonucleotide chain and the ribonucleotide that precedes the last 4 ribonucleotides of the 3' end of the ribonucleotide chain not to be paired;
• a D arm constituted by 4 pairs of ribonucleotides and a D loop constituted by 8 to 10 ribonucleotides, formed by the folding of a part of the ribonucleotide chain that follows the first 7 ribonucleotides of the 5' end of the ribonucleotide chain;
• variable loop constituted by from 4 to 21 ribonucleotides and formed by a part of the ribonucleotide chain that follows the stem of the anticodon and the loop of the anticodon;
• a T arm constituted by 5 pairs of ribonucleotides, and a T loop constituted by 8 ribonucleotides, formed by the folding of a part of the ribonucleotide chain that follows the variable loop and precedes the ribonucleotides of the 3' end of the ribonucleotide chain which are involved in the constitution of the acceptor stem.
• the hybrid tRNA/pre-microRNA polynucleotides can contain any tRNA known in the art, e.g., for encoding any amino acid.
• the selection of an appropriate tRNA molecule may be, in part, driven by the host cells to be used for expression of the inserted RNA.
• the tRNA selected can be from a tRNA encoding for codon preferred by the species of host cell rather than from a rare codon in that species of host cell.
• the tRNA is a methionyl-tRNA.
• the tRNA is derived from the host cell used for expression.
• the tRNA is a mammalian tRNA. In varying embodiments, the tRNA is a human tRNA.
• the chimeric tRNA defined above does not comprise the substantially intact stem of the anticodon of the tRNA from which it is derived. For example, in the chimeric tRNA, between the ribonucleotide that precedes the stem-loop of the anticodon in the tRNA before modification and the ribonucleotide that follows the stem- loop of the anticodon in the tRNA before modification, the stem of the anticodon of the tRNA before modification is no longer present.
• the hybrid tRNA/pre-microRNA and tRNA/shRNA polynucleotides can contain any pre-microRNA molecule known in the art, and can be obtained from naturally occurring sources (e.g., pre-miRNAs or miRNAs), or artificially derived (e.g., shRNAs).
• the pre-microRNA is selected from human pre-miRNA-1291, human pre-miRNA-34a, human pre-miRNA- 125-1, human pre-miRNA-124, human pre-miRNA- 27b, human pre-miRNA-22, and mutants or variants thereof.
• pre-microRNA molecules that can be used in the hybrid tRNA/pre-microRNA polynucleotides include pre- microRNA molecules that express in the host cell (e.g., E. coli host cell) at or above the levels of expression of human pre-miRNA-1291, human pre-miRNA-34a, human pre- miRNA-125-1, human pre-miRNA-124, human pre-miRNA-27b, human pre-miRNA-22, in the same host cell (e.g., E. coli host cell).
• the pre-microRNA molecule is from a mammalian pre-microRNA molecule.
• the pre- microRNA molecule is from a human pre-microRNA molecule.
• the pre-microRNA component of the hybrid tRNA/pre-microRNA polynucleotides is from about 80 nucleotides to about 120 nucleotides in length, e.g., from about 80 nucleotides to about 100 nucleotides in length, e.g., about 80, 85, 90, 95, 100, 105, 110, 115 or 120 nucleotides in length.
• the pre-microRNA is a human pre-microRNA selected from the group consisting of hsa-let-7a-l (miRBase.org accession no.:
• MI0000060 hsa-let-7a-2 (MI0000061), hsa-let-7a-3 (MI0000062), hsa-let-7b
• MI0000063 hsa-let-7c (MI0000064), hsa-let-7d (MI0000065), hsa-let-7e (MI0000066), hsa-let-7f-l (MI0000067), hsa-let-7f-2 (MI0000068), hsa-let-7g (MI0000433), hsa-let-7i (MI0000434), hsa-mir-1-1 (MI0000651), hsa-mir-1-2 (MI0000437), hsa-mir-7-1
• MI0000466 hsa-mir-9-2 (MI0000467), hsa-mir-9-3 (MI0000468), hsa-mir-lOa
• MI0000266 hsa-mir-lOb (MI0000267), hsa-mir-15a (MI0000069), hsa-mir-15b
• MI0000073 hsa-mir-19b-l (MI0000074), hsa-mir-19b-2 (MI0000075), hsa-mir-20a (MI0000076), hsa-mir-20b (MI0001519), hsa-mir-21 (MI0000077), hsa-mir-22
• MI0000082 hsa-mir-26a-l
• MI0000083 hsa-mir-26a-2
• MI0000750 hsa-mir-26b
• MI0000084 hsa-mir-27a
• MI0000440 hsa-mir-28
• MI0000086 hsa-mir-29a (MI0000087), hsa-mir-29b-l (MI0000105), hsa-mir-29b-2 (MI0000107) , hsa- -mir- 29c (MI0000735), hsa-mir-30a (MI0000088), hsa-mir-30b
• MI0000100 hsa- -mir- 99a (MI0000101), hsa-mir-99b (MI0000746), hsa-mir-100
• MI0000102 hsa- -mir- 101-1
• MI0000103 hsa-mir-101-2
• MI0000739 hsa-mir-103a-l
• MI0000109 hsa- -mir- 103a-2
• MI0000108 hsa-mir-103b-l
• MI0007261 hsa-mir-103b-2
• MI0000113 hsa- -mir- 106b
• MI0000734 hsa-mir-107
• MI0000114 hsa-mir-122
• MI0000442 hsa- -mir- 124-1 (MI0000443), hsa-mir- 124-2 (MI0000444), hsa-mir- 124-3
• MI0000445 hsa- -mir- 125a (MI0000469), hsa-mir- 125b- 1 (MI0000446), hsa-mir- 125b-2
• MI0000470 hsa- -mir- 126
• MI0000472 hsa-mir- 127
• MI0000472 hsa-mir- 128-1
• MI0000447) hsa- -mir- 128-2 (MI0000727), hsa-mir- 129-1 (MI0000252), hsa-mir- 129-2
• MI0000449 hsa- -mir- 133a-l (MI000045), hsa-mir- 133a-2 (MI0000451), hsa-mir-133b
• MI0000822 hsa- -mir- 134
• MI0000452 hsa-mir- 135a-2
• MI0000454 hsa- -mir- 138-1 (MI0000476), hsa-mir-138-2 (MI0000455), hsa-mir-139
• MI0000261 hsa- -mir- 140 (MI0000456), hsa-mir-141 (MI0000457), hsa-mir-142
• MI0000458 hsa- -mir- 143 (MI0000459), hsa-mir-144 (MI0000460), hsa-mir-145
• MI0000461 hsa- -mir- 146a (MI0000477), hsa-mir- 146b (MI0003129), hsa-mir- 147a
• MI0000811) hsa- -mir- 149 (MI0000478), hsa-mir-150 (MI0000479), hsa-mir-151a
• MI0000463 hsa- -mir- 153-2 (MI0000464), hsa-mir- 154 (MI0000480), hsa-mir- 155
• MI0000681 hsa- -mir- 181a-l (MI0000289), hsa-mir-181a-2 (MI0000269), hsa-mir- 18 lb- 1
• MI0000481) hsa- -mir- 185 (MI0000482), hsa-mir- 186 (MI0000483), hsa-mir- 187
• MI0000274 hsa- -mir- 188 (MI0000484), hsa-mir-190a (MI0000486), hsa-mir-190b
• MI0000732 hsa-mi r-195 (MI0000489), hsa-mir-196a-l (MI0000238), hsa-mir-196a-2
• MI0000240 hsa-mi r-199a-l
• MI0000281 hsa-mir-199b
• MI0000282 hsa-mi r-200a (MI0000737), hsa-mir-200b (MI0000342), hsa-mir-200c
• hsa-mi r-208a MI0000251
• hsa-mir-208b MI0005570
• hsa-mir-210 MI0000490
• MI0000286 hsa-mi r-211
• MI0000288 hsa-mir-212
• MI0000288 hsa-mir-214
• MI0000290 hsa-mi r-215
• MI0000292 hsa-mir-216a
• MI0000292 hsa-mir-216b
• MI0000808 hsa-mi r-328 (MI0000804), hsa-mir-329-1 (MI0001725), hsa-mir-329-2
• MI0000816) hsa-mi r-337 (MI0000806), hsa-mir-338 (MI0000814), hsa-mir-339
• MI0000815) hsa-mi r-340 (MI0000802), hsa-mir-342 (MI0000805), hsa-mir-345
• MI0000825) hsa-mi r-346 (MI0000826), hsa-mir-361 (MI0000760), hsa-mir-362
• MI0000762 hsa-mi r-363 (MI000076), hsa-mir-365a (MI0000767), hsa-mir-365b
• MI0000769 hsa-mi r-367 (MI0000775), hsa-mir-369 (MI0000777), hsa-mir-370
• MI0000776) hsa-mi r-377 (MI0000785), hsa-mir-378a (MI0000786), hsa-mir-378b (MI0014154) , hsa- -mi Lr-378c (MI0015825), hsa-mir-378d- l (MI0016749), hsa-mir-378d-2
• MI0021273 hsa- -mi Lr-379
• MI0000787 hsa-mir-380
• MI0000788 hsa-mir-381
• MI0001444 hsa- -mi Lr-423 (MI0001445), hsa-mir-424 (MI0001446), hsa-mir-425
• hsa- -mi Lr-433 MI0001723
• hsa-mir-448 MI0001637
• MI0001652 hsa- -mi Lr-450a-2 (MI0003187), hsa-mir-450b (MI0005531), hsa-mir-451a
• hsa- -mi Lr-451b MIOO 17360
• hsa-mir-452 MI0001733
• hsa- -mi Lr-455 MI0003513
• hsa-mir-466 MI0014157
• hsa- -mi Lr-484 MI0002468
• hsa-mir-485 MI0002469
• MI0003132 hsa- -mi Lr-494
• MI0003134 hsa-mir-495
• MI0003135 hsa-mir-496
• MI0003136 hsa- -mi Lr-497 (MI0003138), hsa-mir-498 (MI0003142), hsa-mir-499a
• MI0003193 hsa- -mi Lr-507
• MI0003194 hsa-mir-508
• MI0003195 hsa-mir-509-1
• MI0003197 hsa- -mi Lr-511
• MI0003127 hsa-mir-512-1
• MI0003140 hsa-mir-512-2
• MI0003144 hsa- -mi Lr-515-2 (MI0003147), hsa-mir-516a- 1 (MI0003180), hsa-mir-516a-2
• MI0003181 hsa- -mi Lr-516b-l
• MI0003172 hsa-mir-516b-2
• MI0003167 hsa-mir-517a
• MI0003161 hsa- -mi Lr-517b (MI0003165), hsa-mir-517c (MI0003174), hsa-mir-518a- 1
• MI0003170 hsa- -mi Lr-518a-2 (MI0003173), hsa-mir-518b (MI0003156), hsa-mir-518c
• MI0003159 hsa- -mi Lr- 18d (MI0003171), hsa-mir-518e (MI0003169), hsa-mir-518f (MI0003154), hsa-mir-519a-l (MI0003178), hsa-mir-519a-2 (MI0003182), hsa-mir-519b (MI0003151), hsa-mir-519c (MI0003148), hsa-mir-519d (MI0003162), hsa-mir-519e (MI0003145), hsa-mir-520a (MI0003149), hsa-mir-520b (MI0003155), hsa-mir-520c (MI0003158), hsa-mir-520d (MI0003164), hsa-mir-520e (MI0003143), hsa-mir-520f (MI0003146), hsa-mir-520g (MI0003166)
• MI0005539 hsa-mir-542 (MI0003686), hsa-mir-543 (MI0005565), hsa-mir-544a (MI0003515), hsa-mir-544b (MI0014159), hsa-mir-545 (MI0003516), hsa-mir-548a-l (MI0003593.
• hsa-mir-548a-2 (MI000359), hsa-mir-548a-3 (MI0003612), hsa-mir-548aa-l (MIOO 16689), hsa-mir-548aa-2 (MIOO 16690), hsa-mir-548ab (MIOO 16752), hsa-mir-548ac (MIOO 16762), hsa-mir-548ad (MIOO 16770), hsa-mir-548ae-l (MIOO 16779), hsa-mir-548ae- 2 (MIOO 16780), hsa-mir-548ag- 1 (MIOO 16793), hsa-mir-548ag-2 (MIOO 16794), hsa-mir- 548ah (MI0016796), hsa-mir-548ai (MI0016813), hsa-mir-548aj-l (MI0016814), hsa-mir- 548
• MI0003600 hsa- -mi ⁇ 550a-2 (MI0003601), hsa-mir-550a-3 (MI0003762), hsa-mir-550b-l
• MI0003575 hsa- -mi 552 (MI0003557) , hsa- -mi Lr-553 (MI0003558 , hsa-mir-554
• MI0003559 hsa- -mi 555 (MI0003561) , hsa- -mi Lr-556 (MI0003562 , hsa-mir-557
• MI0003563 hsa- -mi 558
• MI0003564 hsa- -mi Lr-559
• MI0003565 hsa-mir-561
• MI0003567 hsa- -mi 562
• MI0003568 hsa- -mi Lr-563
• MI0003569 hsa-mir-564
• MI0003570 hsa- -mi 566
• MI0003572 hsa- -mi Lr-567
• MI0003573 ⁇ hsa-mir-568
• MI0003574 hsa- -mi 569
• MI0003576 hsa- -mi Lr-570
• MI0003577 ⁇ hsa-mir-571
• MI0003578 hsa- -mi 572
• MI0003579 hsa- -mi Lr-573
• MI0003580 ⁇ hsa-mir-574
• MI0003581 hsa- -mi 575
• MI0003582 hsa- -mi Lr-576
• MI0003583 ⁇ hsa-mir-577
• MI0003584 hsa- -mi 578
• MI0003585 hsa- -mi Lr-579
• MI0003586 hsa-mir-580
• MI0003587 hsa- -mi 581
• MI0003588 hsa- -mi Lr-582
• MI0003589 ⁇ hsa-mir-583
• MI0003590 hsa- -mi 584 (MI0003591) , hsa- -mi Lr-585 (MI000359), hsa-mir-586
• MI0003594 hsa- -mi 587 (MI0003595) , hsa- -mi Lr-588 (MI0003597 ⁇ , hsa-mir-589
• MI0003604 hsa- -mi 593 (MI0003605) , hsa- -mi Lr-595 (MI0003607 , hsa-mir-596
• MI0003608 hsa- -mi 597 (MI0003609) , hsa- -mi Lr-598 (MI0003610 162), hsa-mir-599
• MI0003615 hsa- -mi 603 (MI0003616) , hsa- -mi Lr-604 (MI0003617 ⁇ , hsa-mir-605
• MI0003621 hsa- -mi 609 (MI0003622) , hsa- -mi Lr-610 (MI0003623 , hsa-mir-611
• MI0003624 hsa- -mi 612 (MI0003625) , hsa- -mi Lr-613 (MI0003626 ⁇ , hsa-mir-614
• MI0003627 hsa- -mi 615 (MI0003628) , hsa- -mi Lr-616 (MI0003629 ⁇ , hsa-mir-617
• MI0003631 hsa- -mi 618 (MI0003632) , hsa- -mi Lr-619 (MI0003633 ⁇ , hsa-mir-620
• MI0003634 hsa- -mi 621 (MI0003635) , hsa- -mi Lr-622 (MI0003636 ⁇ , hsa-mir-623
• MI0003637 hsa- -mi 624 (MI0003638) , hsa- -mi Lr-625 (MI0003639 , hsa-mir-626
• MI0003640 hsa- -mi 627 (MI0003641) , hsa- -mi Lr-628 (MI0003642 ⁇ , hsa-mir-629
• MI0003643 hsa- -mi 630
• MI0003645 hsa-mir-632
• MI0003647 hsa- -mi 633
• MI0003648 hsa- -mi Lr-634
• MI0003649 ⁇ hsa-mir-635
• MI0003650 hsa- -mi 636 (MI0003651) , hsa- -mi Lr-637 (MI0003652 ⁇ , hsa-mir-638
• MI0003653 hsa- -mi 639 (MI0003654) , hsa- -mi Lr-640 (MI0003655 , hsa-mir-641
• hsa- -mi 642a MI0003657
• hsa-mir-642b MIOO 16685
• MI0003658 hsa- -mi 644a (MI0003659), hsa-mir-645 (MI0003660), hsa-mir-646 (MI0003661) , hsa- -mi Lr-647 (MI0003662), hsa-mir-648 (MI0003663), hsa-mir-649
• MI0003664 hsa- -mi Lr-650
• MI0003666 hsa-mir-651
• MI0003666 hsa-mir-652
• MI0003682 hsa- -mi Lr-659
• MI0003683 hsa-mir-660
• MI0003684 hsa-mir-661
• MI0003669 hsa- -mi Lr-662 (MI0003670), hsa-mir-663a (MI0003672), hsa-mir-663b
• hsa- -mi Lr-668 MI0003761
• hsa-mir-670 MI0003933
• hsa- -mi Lr-675 MI0005416
• hsa-mir-676 MIOO 16436
• hsa- -mi Lr-758 MI0003757
• hsa-mir-759 MI0004065
• hsa- -mi Lr-761 MI0003941
• hsa-mir-762 MI0003892
• hsa- -mi Lr-765 MI0005116
• hsa-mir-766 MI0003836
• hsa- -mi Lr-769 MI0003834
• hsa-mir-770 MI0005118
• MI0005541 hsa- -mi Lr-876
• MI0005542 hsa-mir-877
• MI0005561 hsa-mir-885
• MI0005560 hsa- -mi Lr-887 (MI0005562), hsa-mir-888 (MI0005537), hsa-mir-889
• MI0005540 hsa- -mi Lr-890
• MI0005533 hsa-mir-891a
• MI0005524 hsa-mir-891b
• hsa- -mi Lr-924 MI0005716
• hsa-mir-933 MI0005755
• MI0005756 hsa- -mi Lr-935 (MI0005757), hsa-mir-936 (MI0005758), hsa-mir-937
• MI0005762 hsa- -mi Lr-941-1 (MI0005763), hsa-mir-941-2 (MI0005764), hsa-mir-941-3
• MI0006272 hsa- -mi Lr-1180
• MI0006273 hsa-mir-1181
• MI0006274 hsa-mir-1182
• MI0006275 hsa- -mi Lr-1183
• MI0006276 hsa-mir-1184-1
• MI0006277 hsa-mir-1184-2
• MI0015971 hsa- -mi Lr-1184-3 (MI0015972), hsa-mir-1 185-1 (MI0003844), hsa-mir-1185-2
• MI0020340 hsa- -mi Lr-1200 (MI0006332), hsa-mir-1202 (MI0006334), hsa-mir-1203
• MI0006335 hsa- -mi Lr-1204
• MI0006338 hsa-mir-1205
• MI0006338 hsa-mir-1206
• MI0006339 hsa- -mi Lr-1207
• MI0006341 hsa-mir-1208
• MI0006341 hsa-mir-1224
• hsa- -mir 1233-1 MI0006323
• hsa-mir- 1233 -2 MI0015973
• MI0006324 hsa- -mir 1236 (MI0006326), hsa-mir-1237 (MI0006327), hsa-mir-1238
• MI0006328 MI0006328
• hsa- -mir 1243 MI0006373
• hsa-mir- 1244-1 MI0006379
• hsa- -mir 1244-3 MI0015975
• hsa-mir-1245a MI0006380
• hsa-mir-1245b MIOO 15974
• MIOO 17431 MIOO 17431
• hsa- -mir 1246 MI0006381
• hsa-mir- 1247 MI0006382
• MI0006383 hsa- -mir 1249 (MI0006384), hsa-mir-1250 (MI0006385), hsa-mir-1251
• MI0006386 hsa- -mir 1252 (MI0006434), hsa-mir-1253 (MI0006387), hsa-mir- 1254-1
• MI000638 hhssaa--mmiirr-- 1254-2 (MI0016747), hsa-mir-1255a (MI0006389), hsa-mir- 1255b- 1
• MI0006391 hsa- -mir 1258
• MI0006394 hsa-mir-1260b
• hsa- -mir 1261 MI0006396
• hsa-mir-1262 MI0006397
• hsa- -mir 1264 MI0003758
• hsa-mir-1265 MI0006401
• MI0006403 hsa- -mir 1267 (MI0006404), hsa-mir- 1268a (MI0006405), hsa-mir- 1268b
• MIOO 16748 hsa- -mir 1269a (MI0006406), hsa-mir-1269b (MI0016888), hsa-mir- 1270-1
• MI0006407 hsa- -mir 1270-2 (MI0015976), hsa-mir-1271 (MI0003814), hsa-mir-1272
• MI0006408 hsa- -mir 1273a (MI0006409), hsa-mir-1273c (MI0014171), hsa-mir-1273d
• hsa- -mir 1273e MI0016059
• hsa-mir-1273f MI0018002
• hsa-mir-1273g MI0014254
• hsa- -mir 1273h MI0025512
• hsa-mir-1275 MI0006415
• hsa- -mir 1277 MI0006419
• hsa-mir- 1278 MI0006425
• hsa- -mir 1281 MI0006428
• hsa-mir-1282 MI0006429
• hsa- -mir 1283-2 MI0006430
• hsa-mir-1284 MI0006431
• MI0006346 MI0006346
• hsa- -mir 1285-2 MI0006347
• hsa-mir-1286 MI0006348
• MI0006351 MI0006352
• hsa-mir-1291 MI0006353
• hsa- -mir 1293 MI0006355
• hsa-mir-1294 MI0006356
• hsa- -mir 1295b MI0019146
• hsa-mir-1296 MI0003780
• MI0006358 hsa- -mir 1298 (MI0003938), hsa-mir-1299 (MI0006359), hsa-mir-1301
• MI0003815 hsa- -mir 1302-1 (MI0006362), hsa-mir- 1302- 10 (MI0015979), hsa-mir-1302- 1 (MI0015980), hsa-mir- 1302-2 (MI0006363), hsa-mir- 1302-3 (MI0006364), hsa-mir- 302-4 (MI0006365), hsa-mir- 1302-5 (MI0006366), hsa-mir- 1302-6 (MI0006367), hsa- mir- 1302-7 (MI0006368), hsa-mir- 1302-8 (MI0006369), hsa-mir- 1302-9 (MI0015978), hsa-mir-1303 (MI0006370), hsa-mir-1304 (MI0006371), hsa-mir-1305 (MI0006372), hsa- mir-1306 (MI0006443), hsa-mir-1307 (MI0006444), hsa-mir
• MI0007075 hsa-mir-1471 ( MI0007076), hsa-mir-1537 MI0007258), hsa-mir-1538
• MI0008330 hsa-mir-1910 ( MI0008331), hsa-mir-1911 MI0008332), hsa-mir-1912
• MI0008333 hsa-mir-1913 ( MI0008334), hsa-mir-1914 MI0008335), hsa-mir-1915
• MIOO 10487) hsa-mir-2054 ( MI0010488), hsa-mir-2110 MIOO 10629), hsa-mir-2113
• hsa-mir-2114 MI0010633
• hsa-mir-2115 MIOO 10634
• hsa-mir-2116
• MIOO 16870 hsa-mir-2467 ( MIOO 17432), hsa-mir-2681 MIOO 12062), hsa-mir-2682
• MI0014136 hsa-mir-3121 ( MI0014137), hsa-mir-3122 (MI0014138), hsa-mir-3123
• MI0014139 hsa-mir-3124 ( MIOO 14140), hsa-mir-3125 (MIOO 14142), hsa-mir-3126
• MIOO 14143 MIOO 14143
• hsa-mir-3127 MIOO 14144
• hsa-mir-3128 MIOO 14145
• MI0014151 hsa-mir-3132
• MI0014152 hsa-mir-3133
• MI0014153 hsa-mir-3134
• hsa-mir-3137 MI0014160
• hsa-mir-3138 MI0014161
• MIOO 14166 MIOO 14166
• hsa-mir-3143 MIOO 14167
• hsa-mir-3144 MIOO 14169
• MIOO 16839 hsa-mir-3156- ⁇ (MI0014184), hsa-mir-3156-2 (MI0014230), hsa-mir-3156-3 (MI0014242), hsa-mir-3157 (MI0014185), hsa-mir-3158-1 (MI0014186), hsa-mir-3158-2 (MI0014187), hsa-mir-3159 (MI0014188), hsa-mir-3160-1 (MI0014189), hsa-mir-3160-2 (MI0014190), hsa-mir-3161 (MI0014191), hsa-mir-3162 (MI0014192), hsa-mir-3163 (MI0014193), hsa-mir-3164 (MI0014194), hsa-mir-3165 (MI0014195), hsa-mir-3166 (MI0014196), hsa-mir-3167 (MI0014198), hsa-mir-3168 (MI
• MIOO 16412 hsa- -mi Lr-3909
• MIOO 16413 hsa-mir- -3910-1
• MI0016414 hsa-mir-3910-2
• MIOO 16431 hsa- -mi Lr-3911
• MI0016415 hsa-mir- -3912
• MI0016416 hsa-mir-3913-1
• MIOO 16417 hsa- -mi Lr-3913-2
• MI0016418 hsa-mir-3914-1
• MI0016419 hsa-mir-3914-2
• MIOO 16421 hsa- -mi Lr-3915 (MIOO 16420), hsa-mir- -3916 (MIOO 16422), hsa-mir-3917
• MIOO 16423 hsa- -mi Lr-3918
• MIOO 16424 hsa-mir- -3919
• MIOO 16425 hsa-mir-3920
• MIOO 16427 hsa- -mi Lr-3921
• MIOO 16428 hsa-mir- -3922
• MIOO 16429 hsa-mir-3923
• MIOO 16430 hsa- -mi Lr-3924
• MIOO 16432 hsa-mir- -3925
• MIOO 16433 hsa-mir-3926-1
• MIOO 16434 hsa- -mi Lr-3926-2
• MIOO 16437 hsa-mir-3927
• MIOO 16435 hsa-mir-3928
• MIOO 16438 hsa- -mi Lr-3929
• MIOO 16439 hsa-mir- -3934
• MIOO 16590 hsa-mir-3935
• MI0016591 hsa- -mi Lr-3936
• MIOO 16592 hsa-mir- -3937
• MI0016593 hsa-mir-3938
• MIOO 16594 hsa- -mi Lr-3939
• MIOO 16596 hsa-mir- -3940
• MIOO 16597 hsa-mir-3941
• MIOO 16598 hsa- -mi Lr-3942
• MIOO 16599 hsa-mir- -3943
• MIOO 16600 hsa-mir-3944
• MIOO 16601 hsa- -mi Lr-3945
• MIOO 16602 hsa-mir- -3960
• MIOO 16964 MIOO 16964
• MIOO 15860 MIOO 15860
• hsa- -mi Lr-4254 MIOO 15862
• hsa-mir- -4255 MI0015863
• MI0015855 hsa- -mi Lr-4257
• MI0015858 hsa-mir- -4259
• MI0015858 hsa-mir-4260
• MIOO 15876 hsa- -mi Lr-4264 (MIOO 15877), hsa-mir- -4265 (MIOO 15869), hsa-mir-4266
• MIOO 15870 hsa- -mi Lr-4267
• MIOO 15871 hsa-mir- -4268
• MIOO 15874 hsa-mir-4269
• MI0015875 hsa- -mi Lr-4270 (MIOO 15878), hsa-mir- -4271 (MIOO 15879), hsa-mir-4272
• MI0015880 hsa- -mi Lr-4273
• MI0015881 hsa-mir- -4274
• MI0015884 hsa-mir-4275
• MI0015883 hsa- -mi Lr-4276
• MI0015886 hsa-mir-4277
• MI0015885 hsa- -mi Lr-4282 (MIOO 15890) , hsa-mir-4283-1 (MI0015892), hsa-mir-4283-2
• MI0015830 hsa- -mi Lr-4299 (MIOO 15829) , hsa-mir-4300 (MI0015831), hsa-mir-4301
• MI0015828 hsa- -mi Lr-4302
• MI0015833 hsa-mir-4303
• MI0015834 hsa-mir-4304
• MI0015832 hsa- -mi Lr-4305
• MI0015835 hsa-mir-4306
• MI0015836 hsa-mir-4307
• MIOO 15840 hsa- -mi ir-4311
• MIOO 15841 hsa-mir-4312
• MI0015842 hsa-mir-4313
• MI0015843 hsa- -mi Lr _4314
• MI0015846 hsa-mir-4315-1
• MI0015844 hsa-mir-4315-2
• MI0015852 hsa- -mi Lr-4322 (MIOO 15851) , hsa-mir-4323 (MI0015853), hsa-mir-4324
• MI0015854 hsa- -mi Lr-4325 (MIOO 15865) , hsa-mir-4326 (MIOO 15866), hsa-mir-4327
• MIOO 15867 hsa- -mi Lr-4328 (MIOO 15904) , hsa-mir-4329 (MI0015901), hsa-mir-4330
• MIOO 16766 MIOO 16766
• hsa- -mi Lr-4428 MIOO 16767
• hsa-mir-4429 MIOO 16768
• MIOO 16798 hsa- -mi Lr-4453 (MIOO 16799) , hsa-mir-4454 (MIOO 16800), hsa-mir-4455 (MI0016801) , hsa- -mi Lr-4456 (MIOO 16802), hsa-mir -4457 (MI0016803), hsa-mir-4458
• MIOO 16860 MIOO 16860
• hsa- -mi Lr-4499 MIOO 16862
• hsa-mir -4500 MIOO 16863
• MIOO 16864 hsa- -mi Lr-4502 (MIOO 16865), hsa-mir -4503 (MIOO 16866), hsa-mir-4504
• MIOO 16867 hsa- -mi Lr-4505 (MIOO 16868), hsa-mir -4506 (MIOO 16869), hsa-mir-4507
• MI0016883 hsa- -mi Lr-4518
• MIOO 16884 hsa-mir -4519
• MIOO 16885 hsa-mir-4520a
• MIOO 17277 hsa- -mi Lr-4650-2 (MIOO 17278), hsa-mir-4651 (MIOO 17279), hsa-mir-4652
• MIOO 17297 hsa- -mi Lr-4668 (MIOO 17298), hsa-mir-4669 (MIOO 17300), hsa-mir-4670
• MIOO 17304 hsa- -mi Lr-4674 (MIOO 17305), hsa-mir-4675 (MIOO 17306), hsa-mir-4676
• MIOO 17307 hsa- -mi Lr-4677 (MIOO 17308), hsa-mir-4678 (MIOO 17309), hsa-mir-4679-1
• MIOO 17326 MIOO 17326
• hsa- -mi Lr-4694 MIOO 17327
• hsa-mir-4695 MIOO 17328
• MIOO 17332 hsa- -mi Lr-4700 (MIOO 17333), hsa-mir-4701 (MIOO 17334), hsa-mir-4703
• MIOO 17339) hsa- -mi Lr-4707 (MIOO 17340), hsa-mir-4708 (MIOO 17341), hsa-mir-4709
• MIOO 17346 hsa- -mi Lr-4713
• MIOO 17348 hsa-mir-4714
• MIOO 17348 hsa-mir-4715
• MIOO 17349 hsa- -mi Lr-4716 (MIOO 17350), hsa-mir-4717 (MIOO 17352), hsa-mir-4718
• MIOO 17356 hsa- -mi Lr-4722 (MIOO 17357), hsa-mir-4723 (MIOO 17359), hsa-mir-4724
• MIOO 17364 hsa- -mi Lr-4728 (MIOO 17365), hsa-mir-4729 (MIOO 17366), hsa-mir-4730
• MIOO 17367 hsa- -mi Lr-4731 (MIOO 17368), hsa-mir-4732 (MIOO 17369), hsa-mir-4733
• MIOO 17373 hsa- -mi Lr-4737 (MI0017374), hsa-mir-4738 (MI0017376), hsa-mir-4739 (MIOO 17377), hsa-mir-4740 (MIOO 17378), hsa-mir-4741 (MIOO 17379), hsa-mir-4742 (MIOO 17380), hsa-mir-4743 (MIOO 17381), hsa-mir-4744 (MIOO 17382), hsa-mir-4745 (MIOO 17384), hsa-mir-4746 (MIOO 17385), hsa-mir-4747 (MIOO 17386), hsa-mir-4748 (MI0017387), hsa-mir-4749 (MI0017388), hsa-mir-4750 (MI0017389), hsa-mir-4751 (MI0017390), hsa-mir-4752 (MI
• MI0017410 hsa-mir-4770 (MI0017411), hsa-mir-4771-1 (MI0017412), hsa-mir-4771-2 (MI0017413), hsa-mir-4772 (MI0017414), hsa-mir-4773-1 (MI0017415), hsa-mir-4773-2 (MIOO 17416), hsa-mir-4774 (MIOO 17417), hsa-mir-4775 (MIOO 17418), hsa-mir-4776-1 (MIOO 17419), hsa-mir-4776-2 (MIOO 17420), hsa-mir-4777 (MIOO 17421), hsa-mir-4778 (MIOO 17422), hsa-mir-4779 (MIOO 17423), hsa-mir-4780 (MIOO 17424), hsa-mir-4781 (MIOO 17426), hsa-mir-4782 (MIOO 17424
• MI0019115 hsa- mir- -5572
• MI0019117 hsa-mir-5579
• MI0019133 hsa-mir-5580
• MI0019135 hsa- mir- -5581 (MI0019136), hsa-mir-5582 (MI0019138), hsa-mir-5583-1
• MI0019139 hsa- mir- -5583-2 (MI0019140), hsa-mir-5584 (MI0019141), hsa-mir-5585 (MI0019142), hsa- mir- -5586 (MI0019143), hsa-mir-5587 (MI0019144), hsa-mir-5588
• MI0019147 hsa- mir- -5589
• MI0019148 hsa-mir-5590
• MI0019150 hsa-mir-5591
• MI0019151 hsa- mir- -5680 (MI0019280), hsa-mir-5681a (MI0019281), hsa-mir-5681b
• MIOO 19285 hsa- mir- -5685 (MI0019287), hsa-mir-5687 (MI0019291), hsa-mir-5688 (MIOO 19292), hsa- mir- -5689 (MIOO 19294), hsa-mir-5690 (MIOO 19295), hsa-mir-5691
• MIOO 19296 hsa- mir- -5692a-l (MI0019297), hsa-mir-5692a-2 (MI0019298), hsa-mir-
• MIOO 19306 hsa- -mir- -5700 (MIOO 19307), hsa-mir-5701- 1 (MIOO 19308), hsa-mir-5701-2
• MI0020347 hsa- -mir- -6071
• MI0020348 hsa-mir-6072
• MI0020349 hsa-mir-6073
• MI0020350 hsa- -mir- -6074
• MI0020351 hsa-mir-6075
• MI0020352 hsa-mir-6076
• MI0020353 hsa- -mir- -6077- 1 (MI0020354), hsa-mir-6077-2 (MI0023562), hsa-mir-6078
• MI0020355 hsa- -mir- -6079
• MI0020356 hsa-mir-6080
• MI0020357 hsa-mir-6081
• MI0020358 hsa- -mir- -6082 (MI0020359), hsa-mir-6083 (MI0020360), hsa-mir-6084
• MI0020361 hsa- -mir- -6085
• MI0020362 hsa-mir-6086
• MI0020363 hsa-mir-6087
• MI0020364 hsa- -mir- -6088 (MI0020365), hsa-mir-6089- 1 (MI0020366), hsa-mir-6089-2
• MI0023563 hsa- -mir- -6090
• MI0020367 hsa-mir-6124
• MI0021258 hsa-mir-6125
• MI0021259 hsa- -mir- -6126 (MI0021260), hsa-mir-6127 (MI0021271), hsa-mir-6128
• MI0021272 hsa- -mir- -6129 (MI0021274), hsa-mir-6130 (MI0021275), hsa-mir-6131
• MI0021276 hsa- -mir- -6132
• MI0021277 hsa-mir-6133
• MI0021278 hsa-mir-6134
• MI0022211 hsa- -mir- -6501 (MI0022213), hsa-mir-6502 (MI0022214), hsa-mir-6503
• MI0022215 hsa- -mir- -6504
• MI0022216 hsa-mir-6505
• MI0022217 hsa-mir-6506
• MI0022218 hsa- -mir- -6507 (MI0022219), hsa-mir-6508 (MI0022220), hsa-mir-6509 (MI0022221), hsa-mir-6510 (MI0022222), hsa-mir-651 la-1 (MI0022223), hsa-mir-651 la-2 (MI0023564), hsa-mir-651 la-3 (MI0023565), hsa-mir-651 la-4 (MI0023566), hsa-mir- 651 lb-1 (MI0022552), hsa-mir-651 lb-2 (MI0023431), hsa-mir-6512 (MI0022224), hsa- mir-6513 (MI0022225), hsa-mir-6514 (MI0022226), hsa-mir-6515 (MI0022227), hsa-mir- 6516 (MI0025513), hsa-mir-6715a
• MI0022554 hsa- -mi Lr-6720
• MI0022555 hsa-mir- -6721
• MI0022556 hsa-mir-6722
• MI0022557 hsa- -mi Lr-6723
• MI0022558 hsa-mir- -6724
• MI0022559 hsa-mir-6726
• MI0022571 hsa- -mi Lr-6727
• MI0022572 hsa-mir- -6728
• MI0022573 hsa-mir-6729
• MI0022574 hsa- -mi Lr-6730
• MI0022575 hsa-mir- -6731
• MI0022576 hsa-mir-6732
• MI0022577 hsa- -mi Lr-6733
• MI0022578 hsa-mir- -6734
• MI0022579 hsa-mir-6735
• MI0022580 hsa- -mi Lr-6736 (MI0022581 ⁇ , hsa-mir- -6737 (MI0022582), hsa-mir-6738
• MI0022583 hsa- -mi Lr-6739
• MI0022584 hsa-mir- -6740
• MI0022585 hsa-mir-6741
• MI0022586 hsa- -mi Lr-6742
• MI0022587 hsa-mir- -6743
• MI0022588 hsa-mir-6744
• MI0022589 hsa- -mi Lr-6745
• MI0022590 hsa-mir- -6746
• MI0022591 hsa-mir-6747
• MI0022601 hsa- -mi Lr-6757
• MI0022602 hsa-mir- -6758
• MI0022603 hsa-mir-6759
• MI0022604 hsa- -mi Lr-6760 (MI0022605 ⁇ , hsa-mir- -6761 (MI0022606), hsa-mir-6762
• MI0022607 hsa- -mi Lr-6763 (MI0022608 ⁇ , hsa-mir- -6764 (MI0022609), hsa-mir-6765
• MI0022610 hsa- -mi Lr-6766
• MI0022611 ⁇ hsa-mir- -6767
• MI0022612 hsa-mir-6768
• MI0022613 hsa- -mi Lr-6769a (MI0022614), hsa-mir-6769b (MI0022706), hsa-mir-6770-1
• MI0022616 hsa- -mi Lr-6772 (MI0022617 , hsa-mir- -6773 (MI0022618), hsa-mir-6774
• MI0022619 hsa- -mi Lr-6775 (MI0022620 ⁇ , hsa-mir- -6776 (MI0022621), hsa-mir-6777
• MI0022622 hsa- -mi Lr-6778 (MI0022623 ⁇ , hsa-mir- -6779 (MI0022624), hsa-mir-6780a
• MI0022625 hsa- -mi Lr-6780b (MI002268 l), hsa-mir-6781 (MI0022626), hsa-mir-6782
• MI0022627 hsa- -mi Lr-6783 (MI0022628 ⁇ , hsa-mir- -6784 (MI0022629), hsa-mir-6785
• MI0022630 hsa- -mi Lr-6786 (MI0022631 , hsa-mir- -6787 (MI0022632), hsa-mir-6788
• MI0022636 hsa- -mi Lr-6792 (MI0022637 ⁇ , hsa-mir- -6793 (MI0022638), hsa-mir-6794
• MI0022642 hsa- -mi Lr-6798 (MI0022643 ⁇ , hsa-mir- -6799 (MI0022644), hsa-mir-6800 (MI0022645) , hsa- -mir- -6801 (MI0022646) , hsa-mir- -6802 (MI0022647) , hsa-mir-6803
• MI0022648 hsa- -mir- -6804 (MI0022649) , hsa-mir- -6805 (MI0022650) , hsa-mir-6806
• MI0022651 hsa- -mir- -6807
• MI0022653 hsa-mir- -6808
• MI0022654 hsa- -mir- -6810 (MI0022655) , hsa-mir- -6811 (MI0022656) , hsa-mir-6812
• MI0022657 hsa- -mir- -6813 (MI0022658) , hsa-mir- -6814 (MI0022659) , hsa-mir-6815
• MI0022660 hsa- -mir- -6816 (MI0022661) , hsa-mir- -6817 (MI0022662) , hsa-mir-6818
• MI0022663 hsa- -mir- -6819 (MI0022664) , hsa-mir- -6820 (MI0022665) , hsa-mir-6821
• MI0022666 hsa- -mir- -6822 (MI0022667) , hsa-mir- -6823 (MI0022668) , hsa-mir-6824
• MI0022669 hsa- -mir- -6825 (MI0022670) , hsa-mir- -6826 (MI0022671) , hsa-mir-6827
• MI0022672 hsa- -mir- -6828 (MI0022673) , hsa-mir- -6829 (MI0022674) , hsa-mir-6830
• MI0022675 hsa- -mir- -6831
• MI0022676 hsa-mir- -6832
• MI0022677 hsa-mir-6833
• MI0022678 hsa- -mir- -6834 (MI0022679) , hsa-mir- -6835 (MI0022680) , hsa-mir-6836
• MI0022682 hsa- -mir- -6837 (MI0022683) , hsa-mir- -6838 (MI0022684) , hsa-mir-6839
• MI0022685 hsa- -mir- -6840
• MI0022686 hsa-mir- -6841
• MI0022687 hsa-mir-6842
• MI0022688 hsa- -mir- -6843 (MI0022689) , hsa-mir- -6844 (MI0022690) , hsa-mir-6845
• MI0022691 hsa- -mir- -6846 (MI0022692) , hsa-mir- -6847 (MI0022693) , hsa-mir-6848
• MI0022694 hsa- -mir- -6849 (MI0022695) , hsa-mir- -6850 (MI0022696) , hsa-mir-6851
• MI0022697 hsa- -mir- -6852 (MI0022698) , hsa-mir- -6853 (MI0022699) , hsa-mir-6854
• MI0022700 hsa- -mir- -6855 (MI0022701) , hsa-mir- -6856 (MI0022702) , hsa-mir-6857
• MI0022703 hsa- -mir- -6858 (MI0022704) , hsa-mir- -6859 -1 (MI0022705), hsa-mir-6859-2
• MI0026420 hsa- -mir- -6859- 3 (MI0026421), hsa-mir-6860 (MI0022707), hsa-mir-6861
• MI0022708 hsa- -mir- -6862- 1 (MI0022709), hsa-mir-6862-2 (MI0026415), hsa-mir-6863
• MI0022710 hsa- -mir- -6864 (MI0022711) , hsa-mir- -6865 (MI0022712) , hsa-mir-6866
• MI0022713 hsa- -mir- -6867 (MI0022714) , hsa-mir- -6868 (MI0022715) , hsa-mir-6869
• MI0022716 hsa- -mir- -6870 (MI0022717) , hsa-mir- -6871 (MI0022718) , hsa-mir-6872
• MI0022722 hsa- -mir- -6876 (MI0022723) , hsa-mir- -6877 (MI0022724) , hsa-mir-6878
• MI0022728 hsa- -mir- -6882 (MI0022729) , hsa-mir- -6883 (MI0022730) , hsa-mir-6884
• MI0022731 hsa- -mir- -6885 (MI0022732) , hsa-mir- -6886 (MI0022733) , hsa-mir-6887
• MI0022734 hsa- -mir- -6888 (MI0022735) , hsa-mir- -6889 (MI0022736) , hsa-mir-6890
• MI0022737 hsa- -mir- -6891 (MI0022738) , hsa-mir- -6892 (MI0022739) , hsa-mir-6893
• MI0022957 hsa- -mir- -7107 (MI0022958) , hsa-mir- -7108 (MI0022959) , hsa-mir-7109 (MI0022960 hsa-mir-7110 (MI0022961), hsa-mir-7111 (MI0022962), hsa-mir-7112-1 (MI0022963 hsa-mir-7112-2 (MI0026414), hsa-mir-7113 (MI0022964), hsa-mir-7114 (MI0022965 hsa-mir-7150 (MI0023610), hsa-mir-7151 (MI0023611), hsa-mir-7152 (MI0023612 hsa-mir-7153 (MI0023613), hsa-mir-7154 (MI0023614), hsa-mir-7155 (MI0023615 hsa-mir-7156 (MI0023616), hsa-mir-7
• the pre-microRNA is not pre-miRNA-22, pre- miRNA-122, pre-miRNA- 124-2, pre-miRNA-125-2, pre-miRNA-155 or pre-miRNA-221.
• the hybrid molecules comprise the full-length native pre-micro-RNA.
• the hybrid molecules comprise fragments or subsequences of the native pre-micro-RNA molecules. Fragments or subsequences of the native pre-micro-RNA molecules that find use will have one or more cleavage sites recognized by and accessible to an endoribonuclease (e.g.
• RNA molecules e.g., a noncoding RNA (ncRNA), mature microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), an aptamer
• ncRNA noncoding RNA
• miRNA mature microRNA
• siRNA small interfering RNA
• shRNA short hairpin RNA
• piRNA Piwi-interacting RNA
• snRNA small nuclear RNA
• snoRNA small nucleolar RNA
• the hybrid tRNA/pre -microRNA molecules contain an inserted RNA sequence and serve as a scaffold for the high-level production of the inserted RNA sequence, which can be cleaved from the hybrid tRNA/pre-microRNA molecule, e.g. , by an endoribonuclease, e.g. , by Dicer.
• the inserted RNA molecule can be from about 18 nucleotides and up to about 200 nucleotides, e.g., at least about 18 nucleotides and up to about 150 nucleotides, e.g., at least about 18 nucleotides and up to about 125 nucleotides, e.g., at least about 18 nucleotides and up to about 100 nucleotides, e.g., at least about 18 nucleotides and up to about 75 nucleotides, e.g., at least about 18 nucleotides and up to about 50 nucleotides, e.g., at least about 18 nucleotides and up to about 40 nucleotides, e.g., at least about 18 nucleotides and up to about 30 nucleotides.
• the inserted RNA molecule can be about 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 nucleotides in length.
• the inserted RNA can be an inhibitory nucleic acid, that prevents, reduces or inhibits the transcription or translation of a target nucleic acid or protein.
• the inhibitory nucleic acid is a noncoding RNA
• the inserted RNA is a mature miRNA, e.g., that is derived from (e.g., is homologous to) or is heterologous to the pre- microRNA molecule in the hybrid tRNA/pre-microRNA scaffold.
• the inserted RNA is a mature miRNA selected from the group consisting of miR-21, miR-22, miR-27b, miR-33, miR-34a, miR 122, miR 124-1, miR-125-1, miR-1291 and let-7a.
• the inserted RNA is a noncoding RNA.
• the noncoding RNA is Homeobox (HOX) antisense intergenic RNA
• the inserted RNA is an aptamer that binds to a target molecule or a target polypeptide.
• the target nucleic acid or polypeptide is selected from the group consisting of a fluorescent protein, a cytokine, a growth factor, a hormone, a kinase, a nuclear receptor, a G protein-coupled receptor, an epigenetic regulator, a transcription factor.
• the target nucleic acid or polypeptide is a fluorescent protein selected from a violet fluorescent protein, a blue fluorescent protein (BFP), a cyan fluorescent protein, a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), an orange fluorescent protein (OFP), a red fluorescent protein (RFP) and a sapphire -type protein.
• the target nucleic acid or polypeptide is a cytokine selected from interleukin (IL)-l , IL- ⁇ , tumor necrosis factor (TNF)a, interferon (IFN)a, ⁇ ⁇ , IFNy, TGFpi, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, IL-22, IL-23 and migration inhibitory factor (MIF).
• IL interleukin
• TNF tumor necrosis factor
• IFN interferon
• the target nucleic acid or polypeptide is a nuclear receptor selected from Peroxisome proliferator-activated receptor gamma (PPAR- ⁇ or PPARG), retinoic acid receptor (RAR), vitamin D receptor, estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), thyroid hormone receptor (THR), farnesoid X receptor (FXR) or NR1H4 (nuclear receptor subfamily 1, group H, member 4), a liver X receptor (LXR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR).
• PPAR- ⁇ or PPARG Peroxisome proliferator-activated receptor gamma
• RAR retinoic acid receptor
• vitamin D receptor vitamin D receptor
• ER estrogen receptor
• AR glucocorticoid receptor
• TTR thyroid hormone receptor
• FXR farnesoid X receptor
• NR1H4 nuclear receptor subfamily 1,
• the target nucleic acid or polypeptide is a growth factor selected from vascular endothelial growth factor (VEGF), Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF),
• VEGF vascular endothelial growth factor
• AM Adrenomedullin
• Ang Angiopoietin
• BMPs Bone morphogenetic proteins
• BDNF Brain-derived neurotrophic factor
• EGF Epidermal growth factor
• EPO Erythropoietin
• FGF Fibroblast growth factor
• G-CSF Granulocyte colony-stimulating factor
• G-CSF Granulocyte macrophage colony-stimulating factor
• GDF9 Growth differentiation factor-9
• HGF Hepatocyte growth factor
• HDGF Hepatoma-derived growth factor
• IGF Insulin- like growth factor
• GDF-8 Nerve growth factor
• NGF Nerve growth factor
• PDGF Platelet-derived growth factor
• TPO Thrombopoietin
• TGF-a Transforming growth factor alpha
• TGF- ⁇ Transforming growth factor beta
• TNF-a Tumor necrosis factor-alpha
• WNT wingless-type MMTV integration site
• PEF placental growth factor
• GPF Somatotrophin
• IL-2 IL-3
• IL-4 Somatotrophin
• IL-5 IL-6
• IL-7 IL-7
• the target nucleic acid or polypeptide is a biomarker associated with the progression or causative of cancer.
• a target miRNA may be selected from human miRNAs including but not limited to miR-lOb, miR-21, miR-29b, miR-17-5p, miR-125b, miR-145, miR-146, and miR-155.
• a target miRNA may be selected from human miRNAs including but not limited to miR-155, miR-17, miR- 18a, miR-19a, miR-20a, miR-19b, and miR-92.
• Breast tumors moreover, comprise heterogeneous miRNA profiles and miRNA signature of, e.g., let-7 family, mir-lOb, mir - 18a, mir-106a, mirl25-a, mirl25-b, mir-126, mir-130a, mir-145, mir-155, mir-141, mir-214, mir-205, mir-206, mir-210, mir-126, mir-335, mir-213, mir-203, 17-5p, miR-30, mir-34, and mir-342, have been proposed to affect breast cancer outcomes. See, e.g., Wiemer, Eur. J Cancer 43: 1529-1544 (2007).
• a target miRNA may be selected from human miRNAs including but not limited to the let-7 family, miR-lOa, miR-20a, miR-24, miR-29b, miR-31, miR-96, miR-133b, miR-135b, miR-143, miR-145, miR-183, miR-17, miR-18a, miR-19a, miR-19b and miR-92.
• the target miRNA may be selected from human miRNAs including but not limited to miR-18, miR-125a, miR-195, miR-199a, miR- 200a, and miR-224.
• miR-16, miR-199a, and/or miR-195 can serve as target miRNA for various liver cancers.
• the target miRNA may be selected from human miRNAs including but not limited to miR-21 , miR-24, miR-34a, miR- 100, miR- 101, miR- 103, miR-107, miR-125b, miR-143, miR-145, miR-148b, miR-200, and miR-155. See Wiemer, Eur. J Cancer 43: 1529-1544 (2007); Zhao et al, Mol Cancer Ther. 2013
• the target miRNA may be selected from human miRNAs including but not limited to let-7d, miR-128a, miR-195, and miR-203.
• the target miRNA may be selected from human miRNAs including but not limited to the let-7 family, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR 34, miR-92, miR-21, miR-126*, miR-155, miR-200b, miR-205, and miR- 210.
• miRNAs are differentially expressed in melanoma cells, and several of the over-expressed miRNAs appear to regulate melanoma cell invasiveness (Ma et al, 2009; Mueller and Bosserhoff, 2009; Mueller et al, 2009;
• miRNAs miR-221/222 down-regulate p27Kipl/CDKNlB and the c-KIT receptor mRNA levels, thereby controlling the progression of neoplasia, leading to enhanced proliferation and reduced differentiation in such cancers cells (Felicetti et al, 2008).
• miR-137 moreover, down-regulates the expression of MITF, a master regulator of cell growth, maturation, and pigmentation in melanoma (Bemis et al., 2008). It has recently been shown that several miRNA genes are differentially regulated in melanoma cells, and therefore, lead to cancer.
• miRNA-211 is consistently reduced in melanoma (see Mazar et al, 2010), which is associated with increased invasiveness and high proliferation rates in susceptible cells.
• a group of epigenetically regulated miRNA genes has been associated with melanomas, e.g., miR-34b, -489, -375, -132, -142-3p, -200a, -145, -452, -21, -34c, -496, -let7e, -654, and -519b.
• the target nucleic acid or polypeptide is selected from the group consisting of miR- 1291; AKT2; Cyclin Bl; MeCP2; FOXA2; AMPKal; Anterior gradient homolog 2 (AGR2); Argininosuccinate synthase (ArSS); Chain C, structure of the H3-H4 chaperone ASF1; Ornithine aminotransferase (OAT); Keratin, type II cytoskeletal 8 (KRT8); Phosphoenolpyruvate carboxykinase 2 (PEPCK2); Enoyl-coenzyme A (Co A) hydratase ( ECHS1); Phosphoserine aminotransferase isoform 1 (PSAT1);
• Dihydrolipoamide acetyltransferase DLAT
• Peroxiredoxin 3 isoform CRA a (PRDX3)
• Cysteine— rich protein 2 CRIP2
• Chain C human PCNA
• Fascin homolog 1, actin- bundling protein isoform CRA a (FSCN1)
• Serpin HI precursor Protein disulfide— isomerase precursor
• Chain A disulfide isomerase related chaperone ERP29;
• Triosephosphate isomerase isoform 2 Triosephosphate isomerase isoform 2 (TPII); Peroxiredoxin-4 (PRDX4); and Isocitrate dehydrogenase [NAD] subunit beta (IDH3B); a-fetoprotein (AFP); AFP-L3%, des-gamma- carboxyprothrombin (DCP); CDH1 (E-cadherin); trimethylated lysine 27 of H3 histone (H3K27me3); histone deacetylase -1; histone deacetylase -2; SIRT1 ; CD44; aldehyde dehydrogenase; KRAS2; or RREB1, an ABC transporter (e.g., ABCC1, ABCG2, ABCB1, ABCC2, ABCC3, and ABCC4) or any combination thereof.
• an ABC transporter e.g., ABCC1, ABCG2, ABCB1, ABCC2, ABCC3, and ABCC4 or any combination thereof.
• the hybrid tRNA/pre-microRNA and tRNA/shRNA molecules can be produced by recombinant expression in a host cell, or can be synthetically prepared. Such recombinant and synthetic methods are well known in the art.
• the host cell can be eukaryotic or prokaryotic.
• the host cell is of the same species of cell as that of the tRNA molecule or the pre-microRNA or shRNA molecule used in the hybrid tRNA/pre- microRNA or tRNA/shRNA molecule.
• a host cell that does not comprise an endoribonuclease that may cleave out the inserted RNA (e.g., Dicer) can be used.
• the host cell for the recombinant expression of a hybrid tRNA/pre-microRNA molecule is a prokaryotic cell, e.g., a bacterial cell, e.g., an
• the host cell for the recombinant expression of a hybrid tRNA/pre-microRNA or tRNA/shRNA molecule is a eukaryotic cell, e.g. , a mammalian cell, a human cell, an insect cell, a plant cell or a yeast cell.
• Eukaryotic (e.g., mammalian or human) host cells which are deficient for Dicer are known in the art and find use for the high level expression for production of the hybrid tRNA/pre-microRNA and/or tRNA/shRNA molecules in a eukaryotic host cell, e.g., as described in Commins, et al.
• hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds facilitate stable, consistent and reliable high level expression of a desired inserted RNA molecule in vivo and in vitro, as described herein.
• high levels of the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds are produced in vitro by a host cell that does not comprise an endoribonuclease that may cleave out the inserted RNA (e.g., Dicer).
• at least about 5-100 mg, e.g., at least about 10-50 mg, of hybrid tRNA/pre-microRNA and tRNA/shRNA scaffold molecules can be produced in vitro from 1 liter of E.
• hybrid tRNA/pre-microRNA and tRNA/shRNA scaffold molecules can be produced in vitro from 1 liter of yeast cell culture.
• the tRNA/pre-microRNA or tRNA/shRNA molecule produced comprises at least about 5%, e.g., at least about 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, or more, of the total RNA.
• the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds are purified as part of the total RNA from the production host cells. Such methods of isolating or purifying total RNA from a host cell are established in the art. In some embodiments, the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds are further substantially isolated or purified from the other RNA molecules and components of the production host cell. This can be done using any method in the art, including, e.g., separation by separation by gel electrophoresis, affinity chromatography, chromatography, FPLC and/or HPLC.
• the substantially isolated and/or purified hybrid tRNA/pre- microRNA and tRNA/shRNA scaffolds can then be transfected or delivered into a eukaryotic cell, which will then process the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds to cleave or release the inserted RNA.
• the hybrid tRNA/pre-microRNA scaffolds are contacted with or exposed to an endoribonuclease ⁇ e.g., Dicer) in vitro, under conditions sufficient to allow cleave or release of the inserted RNA.
• an endoribonuclease ⁇ e.g., Dicer
• the efficiency of in vitro cleavage or release of the inserted RNA from the hybrid tRNA/pre- microRNA scaffolds can be facilitated by adding a RNase, ribozyme or DNAzyme site to the tRNA-pre-miRNA molecule.
• the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds can be administered to a subject in need thereof for delivery of an inserted RNA of interest ⁇ e.g. , an inhibitory nucleic acid, an aptamer) to interior of a target cell.
• an inserted RNA of interest e.g. , an inhibitory nucleic acid, an aptamer
• the subject is a mammal and therefore comprises eukaryotic cells which express endoribonucleases ⁇ e.g. , Dicer).
• the target eukaryotic cells of the subject have been transfected or transformed with the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds, the endoribonucleases (e.g. , Dicer) within the target cell cleave out or release the inserted RNA of interest.
• the inserted RNA is an inhibitory nucleic acid (e.g., a noncoding RNA (ncRNA), mature microRNA (miRNA), a small interfering RNA
• ncRNA noncoding RNA
• miRNA mature microRNA
• the inhibitory RNA once released from the hybrid scaffold in a eukaryotic cell reduces the amount and/or activity of the target nucleic acid or polypeptide by at least about 10% to about 100%, 20% to about 100%, 30% to about 100%, 40% to about 100%, 50% to about 100%, 60% to about 100%, 70% to about 100%, 10% to about 90%, 20% to about 85%, 40%) to about 84%o, 60%> to about 90%>, including any percent within these ranges, such as but not limited to 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%.
• the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds are expressed in vivo from a vector.
• a "vector” is a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus.
• viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
• An expression construct can be replicated in a living cell, or it can be made synthetically.
• the terms "expression construct,” “expression vector,” and “vector,” are used interchangeably to demonstrate the application in a general, illustrative sense, and are not intended to limit the invention.
• an expression vector for expressing the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds comprises a promoter "operably linked" to a polynucleotide encoding the hybrid tRNA/pre -microRNA and tRNA/shRNA scaffolds (e.g., containing an inserted RNA).
• the phrase "operably linked” or “under transcriptional control” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
• the nucleic acid encoding a polynucleotide of interest is under transcriptional control of a promoter.
• a "promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
• the term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase I, II, or III.
• Illustrative promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter (see, U.S. Pat. Nos.
• mice mammary tumor virus LTR promoter the mouse mammary tumor virus LTR promoter
• Ad MLP adenovirus major late promoter
• herpes simplex virus promoter among others.
• Other nonviral promoters such as a promoter derived from the murine
• Enhancer elements may be used in association with the promoter to increase expression levels of the constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al, Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et al, Cell (1985) 41 :521, such as elements included in the CMV intron A sequence.
• LTR long terminal repeat
• transcription terminator/polyadenylation signals will also be present in the expression construct.
• sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al, supra, as well as a bovine growth hormone terminator sequence (see, e.g., U.S. Pat. No. 5,122,458).
• 5'-UTR sequences can be placed adjacent to the coding sequence in order to enhance expression of the same.
• Such sequences include UTRs which include an Internal Ribosome Entry Site (IRES) present in the leader sequences of picornaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63: 1651-1660.
• IRES Internal Ribosome Entry Site
• EMCV encephalomyocarditis virus
• Other picornavirus UTR sequences that will also find use in the present invention include the polio leader sequence and hepatitis A virus leader and the hepatitis C IRES.
• the expression construct comprises a virus or engineered construct derived from a viral genome.
• One of the available methods for in vivo delivery involves the use of an adenovirus expression vector.
• Adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express a polynucleotide that has been cloned therein.
• the expression vector comprises a genetically engineered form of adenovirus.
• Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992).
• retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
• adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage.
• Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
• ITRs inverted repeats
• the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
• Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
• the typical vector according to the present invention is replication defective and will not have an adenovirus El region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the El -coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
• the polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors, as described by Karlsson et al. (1986), or in the E4 region where a helper cell line or helper virus complements the E4 defect.
• Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991 ; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991 ; Stratford-Perricaudet et al, 1990; Rich et al., 1993).
• Retroviral vectors are also suitable for expressing the hybrid tRNA/pre- microRNA and tRNA/shRNA scaffolds (e.g., containing an inserted RNA) in cells.
• the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription (Coffin, 1990).
• the resulting DNA then stably integrates into cellular chromosomes as a pro virus and directs synthesis of viral proteins.
• the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
• the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
• a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
• Two long terminal repeat (LTR) sequences are present at the 5 ' and 3 ' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
• LTR long terminal repeat
• a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
• a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983).
• a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example)
• the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983).
• the media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer.
• Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).
• Other viral vectors may be employed as expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988), adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).
• the expression construct In order to effect cleavage and expression of inserted RNA, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
• the nucleic acid encoding the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds may be positioned and expressed at different sites.
• the nucleic acid encoding the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
• the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA.
• nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
• the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
• Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.
• a naked DNA expression construct may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads. [0132] In a further embodiment, the expression construct may be entrapped in a liposome.
• Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self- rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ohosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes. [0133] In certain embodiments, the liposome may be complexed with a
• HVJ hemagglutinating virus
• HMG-I nuclear non-histone chromosomal proteins
• HMG-I nuclear non-histone chromosomal proteins
• the liposome may be complexed or employed in conjunction with both HVJ and HMG-I.
• expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
• a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
• receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a particular IncRNA or inhibitor into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
• Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor- specific ligand and a DNA-binding agent.
• ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990).
• ASOR asialoorosomucoid
• transferrin Wang a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al, 1993; Perales et al, 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).
• the delivery vehicle may comprise an encapsulating particle and an external targeting ligand, e.g., that specifically binds to a tumor associated antigen.
• an external targeting ligand e.g., that specifically binds to a tumor associated antigen.
• Nicolau et al. (1987) employed lactosyl-ceramide, a galactose- terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
• a nucleic acid encoding a particular gene also may be specifically delivered into a cell type by any number of receptor-ligand systems with or without liposomes.
• epidermal growth factor may be used as the receptor for mediated delivery of a nucleic acid into cells that exhibit upregulation of EGF receptor.
• Mannose can be used to target the mannose receptor on liver cells.
• antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.
• tumor associated antigens of use for targeting include without limitation, e.g. , include without limitation, melanoma associated antigens (MAGE-1, MAGE-3, TRP-2, melanosomal membrane glycoprotein gplOO, gp75 and MUC-1 (mucin- 1) associated with melanoma); CEA
• cancer which can be associated, e.g., with ovarian, melanoma or colon cancers; folate receptor alpha expressed by ovarian carcinoma; free human chorionic gonadotropin beta (hCGP) subunit expressed by many different tumors, including but not limited to myeloma; HER-2/neu associated with breast cancer; encephalomyelitis antigen HuD associated with small-cell lung cancer; tyrosine hydroxylase associated with neuroblastoma; prostate-specific antigen (PSA) associated with prostate cancer; CA125 associated with ovarian cancer; and the idiotypic determinants of a B cell lymphoma can generate tumor-specific immunity (attributed to idiotype-specific humoral immune response).
• hCGP free human chorionic gonadotropin beta
• antigens of human T cell leukemia virus type 1 have been shown to induce specific CTL responses and antitumor immunity against the virus-induced human adult T cell leukemia (ATL).
• ATL virus-induced human adult T cell leukemia
• Other TAAs are known and find use for the formulation and targeted delivery of the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds.
• the oligonucleotide may be administered in combination with a cationic lipid.
• cationic lipids include, but are not limited to, lipofectin, DOTMA, DOPE, and DOTAP.
• DOTAP cholesterol or cholesterol derivative formulation that can effectively be used for gene therapy.
• Other disclosures also discuss different lipid or liposomal formulations including nanoparticles and methods of administration; these include, but are not limited to, U.S. Patent Publication 20030203865, 20020150626, 20030032615, and 20040048787, which are specifically incorporated by reference to the extent they disclose formulations and other related aspects of administration and delivery of nucleic acids. Methods used for forming particles are also disclosed in U.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901, 6,200,801, and 5,972,900, which are incorporated by reference for those aspects.
• gene transfer may more easily be performed under ex vivo conditions.
• Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues.
• compositions comprising the hybrid tR A/pre-microRNA or tRNA/shRNA scaffolds and a pharmaceutically acceptable carrier.
• pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
• Colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds described herein.
• fat emulsions that are suitable for delivering the nucleic acids to tissues, such as cardiac muscle tissue and smooth muscle tissue, include Intralipid, Liposyn, Liposyn II, Liposyn III, Nutrilipid, and other similar lipid emulsions.
• a preferred colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. No. 5,981,505; U.S. Pat. No. 6,217,900; U.S. Pat. No. 6,383,512; U.S. Pat. No. 5,783,565; U.S. Pat. No. 7,202,227; U.S. Pat. No. 6,379,965; U.S. Pat. No. 6,127,170; U.S. Pat. No. 5,837,533; U.S. Pat. No.
• Aqueous compositions of the present invention comprise an effective amount of the delivery vehicle, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
• pharmaceutically acceptable or “pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
• pharmaceutically acceptable carrier includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans.
• the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the nucleic acids of the compositions.
• the pharmaceutical forms suitable for injectable use or catheter delivery include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above.
• the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• compositions of the present invention generally may be formulated in a neutral or salt form.
• Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like).
• inorganic acids e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like.
• Salts formed with the free carboxyl groups of the protein can also be
• solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
• the formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
• aqueous solution for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose.
• aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
• sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure.
• a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15 th Edition, pages 1035-1038 and 1570-1580).
• Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
• the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
• preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards.
• the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds may be administered to a subject with cancer to enhance or increase the responsiveness to chemotherapy comprising a platinum coordination complex.
• the cancer is resistant to treatment with a chemotherapy regime.
• resistant to chemotherapy is meant that the cancer does not substantially respond to treatment with the chemotherapy. Identification of such resistant cancers and cancer
• the hybrid tR A/pre-microRNA and tRNA/shR A scaffolds may be used to treat subjects who have failed (relapsed) after standard chemotherapy or bone marrow transplantation or other emerging or novel targeted therapies.
• treat By “treat,” “treatment” or “treating” is meant ameliorating symptoms associated with cancer, including preventing or delaying the onset of the disease symptoms and/or lessening the severity or frequency of the disease symptoms and/or prolonging remission and/or decreasing the frequency or severity of relapse.
• the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds can be administered to the subject in conjunction with chemotherapy comprising a platinum coordination complex (e.g., prior to or concurrently with chemotherapy comprising a platinum coordination complex.
• the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds may be provided alone or in combination with other compounds (for example, chemotherapeutics), in the presence of a liposome, an adjuvant, or any pharmaceutically acceptable carrier, in a form suitable for administration to mammals, for example, humans, cattle, sheep, etc.
• treatment with the hybrid tRNA/pre- microRNA and tRNA/shRNA scaffolds may be combined with traditional and existing, or emerging, therapies for cancer, e.g., targeted chemotherapies using cancer-specific peptides described, e.g., in Intl. Publ. No.
• the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds may be administered chronically or intermittently.
• Chronic administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
• Intermittent administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
• the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds are administered to a subject in need of such inhibitors, e.g., a subject diagnosed with or suspected of having a cancer.
• a hybrid tRNA/pre-microRNA or tRNA/shRNA scaffold may be effectively delivered to cancer cells, by a variety of methods known to those skilled in the art. Such methods include but are not limited to liposomal encapsulation/delivery, vector-based gene transfer, fusion to peptide or immunoglobulin sequences (peptides described, e.g., in Intl. Publ. No. 2011/038142) for enhanced cell targeting and other techniques.
• Suitable viral vectors include retroviral vectors such as lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, etc.
• a hybrid tRNA/pre-microRNA or tRNA/shRNA scaffold may also be formulated in pharmaceutical compositions well known to those in the field. These include liposomal formulations and combinations with other agents or vehicles/excipients such as cyclodextrins which may enhance delivery of the inhibitory nucleic acid.
• suitable carriers include lipid-based carriers such as a stabilized nucleic acid-lipid particle (e.g., SNALP or SPLP), cationic lipid or liposome nucleic acid complexes (i.e., lipoplexes), a liposome, a micelle, a virosome, or a mixture thereof.
• the carrier system is a polymer-based carrier system such as a cationic polymer-nucleic acid complex (i.e., polyplex).
• the carrier system is a cyclodextrin-based carrier system such as a
• the carrier system is a protein-based carrier system such as a cationic peptide-nucleic acid complex.
• Suitable carriers are known in the art and are described in, without limitation,
• the present invention contemplates a nucleic acid-lipid particle comprising a hybrid tRNA/pre-microRNA or tRNA/shRNA scaffold (e.g., containing an inserted RNA).
• a nucleic acid-lipid particle comprising a hybrid tRNA/pre-microRNA or tRNA/shRNA scaffold (e.g., containing an inserted RNA).
• suitable nucleic acid-lipid particles and their use are described in U.S. Pat. Nos. 6,815,432, 6,586,410, and 6,534,484.
• Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the hybrid tRNA/pre-microRNA or
• tRNA/shRNA scaffolds e.g., containing an inserted RNA
• Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose
• any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular,
• Therapeutic formulations may be in the form of liquid solutions or suspensions.
• the compound may be administered in a tablet, capsule or dissolved in liquid form.
• the table or capsule may be enteric coated, or in a formulation for sustained release.
• intranasal formulations in the form of powders, nasal drops, or aerosols.
• a hybrid tRNA/pre-microRNA or tRNA/shRNA scaffold may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non- water soluble hybrid tRNA/pre-microRNA or tRNA/shRNA scaffold (e.g., containing an inserted RNA) such as those used for vitamin K.
• Suitable formulations include those that have desirable pharmaceutical properties, such as targeted delivery to cancer cells, improved serum half-life/stability of a hybrid tRNA/pre-microRNA or tRNA/shRNA scaffold (e.g., containing an inserted RNA), improved intracellular penetration and cytoplasmic delivery, improved persistence of in-vivo activity, reduction in dose required for efficacy, reduction in required dosing frequency, etc.
• desirable pharmaceutical properties such as targeted delivery to cancer cells, improved serum half-life/stability of a hybrid tRNA/pre-microRNA or tRNA/shRNA scaffold (e.g., containing an inserted RNA), improved intracellular penetration and cytoplasmic delivery, improved persistence of in-vivo activity, reduction in dose required for efficacy, reduction in required dosing frequency, etc.
• a liposomal nanoparticle-based dosing formulation of a hybrid tRNA/pre- microRNA or tRNA/shRNA scaffold may be prepared using methods well known to those skilled in the art and currently practiced for the preparation pharmaceutical formulations of other oligonucleotide -based
• reagents/therapeutics including anti-sense oligonucleotides and/or RNAi (siRNA)-based agents.
• siRNA RNAi
• a gene therapy approach for transduction of hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds (e.g., containing an inserted RNA) to target cells (e.g. cancer cells) using for example lentiviral-based vectors may be used.
• Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
• Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
• Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
• the hybrid tR A/pre-microR A and tRNA/shRNA scaffolds are administered to an individual in an amount sufficient to stop or slow a cancer, or to promote differentiation, or inhibit or decrease self-renewal, or inhibit or decrease engraftment or metastasis of cancer cells.
• An "effective amount" of a hybrid tRNA/pre-microRNAor tRNA/shRNA scaffold (e.g., containing an inserted RNA) according to the invention includes a
• a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as treatment of a cancer or promotion of differentiation, or inhibition or decrease of self-renewal or inhibition or decrease of engraftment or metastasis of a cancer cell.
• the increase or decrease may be between 10% and 90%, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or may be over 100%), such as 200%, 300%, 500%) or more, when compared with a control or reference subject, sample or compound.
• a therapeutically effective amount of a hybrid tRNA/pre-microRNA or tRNA/shRNA scaffold may vary according to factors such as the disease state, age, sex, and weight of the individual subject, and the ability of the hybrid tRNA/pre-microRNA and tRNA/shRNA scaffolds (e.g., containing an inserted RNA) to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the hybrid tRNA/pre-microRNA or tRNA/shRNA scaffold (e.g., containing an inserted RNA) are outweighed by the therapeutically beneficial effects.
• a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as prevention or protection against a cancer or promotion of differentiation, inhibition or decrease of self- renewal or inhibition or decrease of engraftment or metastasis of cancer cells.
• a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.
• dosages may be adjusted depending on whether the subject is in remission from cancer or not.
• a preferred range for therapeutically or prophylactically effective amounts of a hybrid tRNA/pre-microR A and tRNA/shRNA scaffolds may be any integer from 0.1 nM-O.lM, 0.1 nM-0.05M, 0.05 nM-15 ⁇ or 0.01 nM-10 ⁇ .
• a therapeutically or prophylactically effective amount that is administered to a subject may range from about 5 to about 3000 micrograms/kg if body weight of the subject, or any number therebetween.
• tRNA/shRNA scaffold (e.g., containing an inserted RNA) is provided in an amount that is from 10% to 99% greater than the amount of target nucleic acid or polypeptide present in cancer cells, or more generally at least 10%, 20%, 30%, 40%, 50, 55% or 60%, or at least 65%, 75%, 80%, 85%, 90%, or 95%, or as much as 96%, 97%, 98%, or 99% greater than the amount present in cancer cells.
• the hybrid tRNA/pre- microRNA or tRNA/shRNA scaffold (e.g., containing an inserted RNA) is provided in an amount that is 0.5 to 50 fold greater than the amount present in cancer cells, or more generally at least 0.5, 1, 1.5, 2, 5, 10, 20, 25, 30, 35, 40, 45 fold greater than the amount present in cancer cells.
• the hybrid tRNA/pre-microRNA or tRNA/shRNA scaffold (e.g., containing an inserted RNA) is provided in an amount that is equivalent to the amount present in non-cancerous bladder cells or the amount present in normal bladder cells. [0159] It is to be noted that dosage values may vary with the severity of the condition to be alleviated.
• dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.
• the amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual.
• Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
• kits comprising the hybrid tRNA/pre- microRNA and/or tRNA/shRNA scaffolds (e.g., containing an inserted RNA) described herein.
• suitable formulations may be provided in a kit including one or more hybrid tRNA/pre-microRNA and/or tRNA/shRNA scaffolds (e.g., containing an inserted RNA), together with instructions for using the hybrid tRNA/pre- microRNA scaffolds (e.g., containing an inserted RNA) to treat a cancer.
• the kit may contain additional agents such as a pharmaceutical carrier e.g, a liposomal carrier or additional active ingredients such as a chemotherapeutic agent.
• the additional agents may be provided in the same container as that containing the hybrid tRNA/pre-microRNA and/or tRNA/shRNA scaffolds (e.g., containing an inserted RNA) or may be provided in a container separate from that containing the hybrid tRNA/pre-microRNA and/or
• tRNA/shRNA scaffolds e.g., containing an inserted RNA
• RNA research and therapy relies primarily on synthetic RNAs.
• OnRS noncoding RNA scaffold
• tRNA/mir-34a multi-milligrams of chimeric RNAs
• OnRS/miR-124, OnRS/ GFP-siRN A, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 L bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and
• OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP-transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum Rnase activities in pancreatic cancer patients.
• RNA interference (RNAi) technologies have been widely utilized for genome function studies.
• RNAi-based therapies under clinical trials 1-3
• an RNA aptamer Pegaptanib
• U.S. Food and Drug Administration for the treatment of age-related macular degeneration (4).
• RNAi agents and noncoding RNA (ncRNA) materials used for basic, translational and clinical research such as small interfering RNAs (siRNAs), short hairpin RNAs
• RNA molecules in vitro transcription (shRNAs), RNA aptamers, and microRNAs (miRs or miRNAs) are mainly produced through chemical synthesis (5-9), while other virus and non-virus-vector based strategies literally utilize DNA agents.
• organic synthesis of oligonucleotides may be automated, a multi-milligram dose of 22-nt double-stranded siRNA or miRNA agents for in- vivo testing or projected therapy is very costly. It is also unclear to what extent chemical modifications would alter the structures, biological activities and safety profiles of these ncRNAs, despite that synthetic ncRNAs exhibit some favorable pharmacokinetic properties such as a longer half-life.
• In vitro transcription (10,11) is another way to produce RNA agents in variable lengths. However, in vitro transcription generally produces RNA molecules in a test tube on micrograms scale, thus the production of larger quantities of RNAs requires considerably more of the costly RNA polymerases.
• RNA chimeras are thus isolated, and the target RNAs may be released in demand by corresponding Rnase (13,14), Ribozyme (15) or DNAzyme (16) for structural and biophysical analyses.
• Rnase 13,14
• Ribozyme 15
• DNAzyme 16
• the human carcinoma cell line A549 was purchased from American Type Culture Collection (Manassas, VA), and ES-2/GFP was from Cell Biolabs (San Diego, CA). Both cell lines were maintained in RPMI 1640 with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% C0 2 and 95% air.
• RNA secondary structures were predicted using the CentroidFold (http://www.ncrna.org/centroidfold) (18) and CentroidHomfold
• Trna/mir-34a was used as a template for the amplification of target sequences using the oligonucleotides (Table 1), and then the amplicons were inserted into pBSMrnaSeph vector linearized by SacII and Eagl (New England Biolabs) which removed the SEPHADEXTM aptamer from Trna scaffold at the same time. All inserts were confirmed by Sanger sequencing analyses at UC Davis Genome Center.
• the underlined are the sequences of tRNA scaffold.
• the rest is the has-miR-34a precursor with ⁇ 9 nt flanking sequence at each prime.
• sequence in red indicates the target sequence and the green part is the complementary sequence. Sequence in bold indicates the MGA.
• RNAs were isolated from E. coli using the Tris-HCl-saturated phenol extraction method, quantitated with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Rockford, IL) and analyzed by denaturing urea (8 M) polyacrylamide (8%) gel electrophoresis (PAGE). All images were acquired with ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA). Intensities of bands were used to provide a rough estimation of relative levels of recombinant ncRNAs present in the total RNAs.
• Buffer B Buffer A plus 1 M sodium chloride
• FPLC traces were monitored at 260 nm using a UV/Vis detector. Peak areas were employed to estimate the relative levels of recombinant ncRNAs within the total RNAs, which agrees with those determined by urea-PAGE analyses. After analyzed on a denaturing PAGE gel, the fractions containing pure ncRNAs were pooled. Recombinant ncRNAs were precipitated with ethanol, reconstituted with nuclease-free water, and then desalted and concentrated with Amicon ultra-2 mL centrifugal filters (30 KD; EMD
• RNAs were isolated using a Direct-zol RNA extraction kit (Zymo Research, Irvine, CA) at 48 h post-transfection, and small RNA libraries were generated using the Illumina TruseqTM Small RNA Preparation kit (Illumina, San Diego, CA) according to the instructions.
• the purified cDNA library was used for cluster generation on Illumina' s Cluster Station and then sequenced on Illumina GAIIx following vendor's instructions.
• Raw sequencing reads (40 nt) were obtained using Illumina's Sequencing Control Studio software version 2.8 (SCS v2.8) following real-time sequencing image analysis and base-calling by Illumina's Real-Time Analysis version 1.8.70 (RTA vl .8.70).
• the extracted sequencing reads were used for the standard sequencing data analysis by following a proprietary pipeline script, ACGTlOl-miR v4.2 (LC Sciences, Houston, TX)(20,21). Cells were treated in triplicate and sequenced separately.
• RT-qPCR Reverse transcription quantitative real-time PCR
• Apoptosis assay The apoptosis assay was performed by using a FACS
• Annexin V assay kit (Trevigen, Inc., Gaithersburg, MD, USA) following the manufacturer's protocol. Briefly, A549 cells were transfected with 100 nM recombinant ncRNAs, harvested at 72 h post-transfection, incubated with Annexin V-FITC conjugate and propidium iodide solution, and then the samples were analyzed on a FACScan flow cytometer (BD Biosciences, San Jose, CA). Data analysis was performed using Flowjo (Ashland, OR). Cells were treated in triplicate and similar results were obtained when the whole experiment was repeated.
• A549 cells were transfected with 20 or 100 nM chimeric RNAs.
• ES-2/GFP cells were seeded 8,000 cells/well on a 24-well plate and transfected with 5 or 15 nM FPLC-purified chimeric RNAs at 24 h later. The fluorescence was monitored with an Olympus 1X81 microscope (Olympus, Center Valley, PA) at 24h, 48h and 72h post-transfection. All images were acquired using the same settings at the same time. At the end of the study, total RNAs were isolated from the cells and subject to RT-qPCR evaluation of GFP mRNA and siRNA levels. Cells were treated in triplicate, and similar results were obtained when the whole experiment was repeated.
• DAPI diamidino-2-phenylindole
• liver tissues were subject to RNA isolation and RT-qPCR analyses for GFP mRNA and siRNA levels against 18S and U74 was used as control, respectively.
• Gene specific primers were presented in Table 1.
• Serum Rnase activity assay Serum specimens from IRB-approved, prospectively-collected UC Davis Pancreas Registry bank were utilized. The serum has been processed uniformly within 4 h of blood collection, aliquoted and stored in a -80 0C freezer till usage with minimal freeze-thaw cycle. A total of 20 patients' serum from 10 pancreatic ductal adenocarcinoma (PDAC) (5 early-stage PDAC (American Joint
• the PDAC cases consisted of 4 males and 6 females, benign/normal of 5 males and 5 females. Age ranges were 51 to 80
• RNAi agents An OnRS is developed to achieve high-yield production of recombinant RNAi agents.
• Figure la A series of plasmids were created and employed to transform E. coli.
• the levels of recombinant pre-miRNA chimeras expressed/accumulated in HST08 E. coli were largely variable when the same tRNA scaffold was used.
• OnRS was able to assemble other miRNAs (e.g., 21-nt miR-27b and 22-nt miR-22, etc.) and a 22-nt scrambled RNA sequence (chimeric RNA was named OnRS/Neg and used as a control in the following studies; Table 1), which were all consistently produced in HST08 E. coli at high yields and on a large scale, i.e., >15% of OnRS/miRNAs in total RNAs and >1.5 mg of FPLC-purified OnRS/miRNAs from 0.5 L bacterial culture at all times.
• OnRS/GFP-siRNA from 0.5 L bacterial culture every time. These results indicate that target miRNA/siRNA agents can be assembled by using OnRS-based platform to offer a consistent high-level production of chimeric ncRNAs in bacteria.
• Target miRNAs/siRNAs are selectively released from chimeric ncRNAs in human cells while tRNA scaffold is processed to tRNA fragments (tRFs).
• tRFs tRNA fragments
• An unbiased deep sequencing study was conducted after the preparation of small RNAs library from human lung carcinoma A549 cells at 48 h post-transfection with OnRS/miR-124 and OnRS (tRNA/mir-34a). The data showed that OnRS/miR-124 was selectively processed to large numbers (5,613 ⁇ 975 reads) of 20-nt miR-124 in A549 cells ( Figure 2a).
• OnRS/ GFP-siRN A and OnRS/Neg The data showed that GFP-siRNA levels were about 1 ,000-fold higher in ES-2/GFP cells treated with chimeric OnRS/ GFP-siRNA than the control OnRS/Neg ( Figure 2b), which was mainly attributable to the increase in 22-, 23- and 21-nt isoforms and accompanied by lower levels of passenger strands. It was also noted that OnRS/Neg was indeed processed to a number of scrambled RNAs at 22-23 nt in length, but at much lower levels, which might be related to a lower stability of the scrambled RNAs or insufficient processing. Nevertheless, there were no or minimal differences in the levels of other cellular miRNAs between OnRS/GFP-siRNA- and OnRS/Neg-treated cells.
• OnRS/Neg-treated cells ( Figure 2d), despite that overall tRF levels were much lower in ES- 2/GFP cells than A549 cells. Together, these results support the utility of OnRS for "stealth delivery" of target miRNAs and siRNAs into human cells beyond high-yield production of the chimeric ncRNAs in bacteria and the use of OnRS/Neg as a control to assess
• OnRS-carried miR-124 is biologically/pharmacologically active in controlling target gene expression and cancer cellular processes. Then we evaluated the bioactivities of OnRS-carried miR-124, as miR-124 is known to regulate a number of target genes such as the oncogenic signal transducer and activator of transcription 3 (STAT3), enhance apoptosis, and inhibit cell proliferation (26-28). Consistent with deep sequencing data, selective stem- loop RT-qPCR analyses showed that mature miR-124 levels were around 1000-fold higher in A549 cells from day 1 to 4 after transfection with OnRS/miR- 124, compared with OnRS/Neg (Figure 3a).
• STAT3 oncogenic signal transducer and activator of transcription 3
• OnRS-carried GFP siRNA is effective in knocking down GFP expression in vitro and in vivo.
• OnRS/GFP-siRNA significantly suppressed the GFP fluorescence intensity and mRNA levels at 72 h post-transfection ( Figure 4a-4b), which was associated with 3 orders of magnitude increase in GFP siRNA levels ( Figure 4c).
• OnRS-carried MGA was further demonstrated by a strong fluorescent intensity at 630/650 nm (excitation/emission) upon binding MG ( Figure 6e).
• label-free OnRS/MGA itself did not exhibit any fluorescence, and only minimal basal-level MG fluorescent intensity was shown in the absence or presence of non-MGA-containing total RNAs and HPLC-purified OnRS/Seph ( Figure 6e), supporting the specificity of MGA- bound-MG fluorescence.
• OnRS/MGA was degraded by 500-fold higher concentration (0.01 ⁇ g/ ⁇ L) of angiogenin in 30 min. Since Rnase A is the major form of Rnase in human serum (30), this assay would mainly indicate pancreas-derived Rnase A activity in human serum. Therefore, we utilized OnRS/MGA to evaluate the Rnase activities in a set of serum samples prospectively collected from PDAC and benign/normal patients. The data showed that serum Rnase activities (AA.U./min/pL) were significantly (P ⁇ 0.01) higher in PDAC (196 ⁇ 22) than benign/normal (118 ⁇ 8) patients. These results implicate that chimeric MGA sensor produced using the OnRS platform could be useful for determination of Rnase activities.
• DISCUSSION A general approach has been established for a consistent, cost-effective production of multiple to tens of milligrams of chimeric ncRNAs in 1 L culture of a common strain of E. coli, bearing various types of small RNAs of interests.
• the OnRS used in this platform is based upon the tRNA fusion pre-miRNA-34a that is resistant to nucleolytic digestion in bacteria and thus accumulated to significantly high level (e.g., >15% of total RNAs) for an easy purification with the anion-exchange FPLC method.
• the miR-34a-5p/miR-34a-3p duplex within the OnRS cargo may be replaced by any target double-stranded small RNAs such as siRNA or miRNA/miRNA* duplex ( Figure lb) to achieving high-yield production of corresponding chimeric siRNA or miRNA agents, as exemplified by successful production of >1.5 mg of OnRS/miR-124, OnRS/GFP-siRNA and control OnRS/Neg chimeras from 0.5 L bacterial culture in this report.
• any target double-stranded small RNAs such as siRNA or miRNA/miRNA* duplex
• RNA aptamers can be sprouting at particular sites on OnRS to offer the aptamer chimeras (Figure 5a), which are nicely demonstrated by the assembling of OnRS/MGA5 and OnRS/MGA3 sensors.
• OnRS-based platform is also supported by successful production of other target RNA agents (e.g., miR-27b, miR-22, and a vascular endothelial growth factor (VEGF) aptamer, etc.), whereas its application to bioengineer other types of biological RNAs such as catalytic RNAs (ribozymes) for biotransformation and guide RNAs (gRNAs) for genome editing warrants further investigations.
• target RNA agents e.g., miR-27b, miR-22, and a vascular endothelial growth factor (VEGF) aptamer, etc.
• VEGF vascular endothelial growth factor
• Chimeric OnRS/miRNAs and OnRS/siRNAs can act as "pro-drugs" for the "delivery” of target RNAi agents into the cells. Indeed they were selectively processed to large numbers of target miRNAs and siRNAs in human cells, as determined by unbiased deep sequencing studies ( Figure 2a-2b). So was the scrambled RNA from OnRS/Neg. The presence of small RNA isoforms differing in 1- or 2-nt at 5' or 3' within chimeric ncRNA- and vehicle-treated human cells may indicate the flexibilities of endoribonucleases in producing small RNAs from pre -miRNAs or shRNAs (31-33).
• ES-2/GFP cells levels were much lower than A549 cells, which is presumably due to the differences in generating, degrading, excreting and/or retaining tRFs in different types of cells.
• target miRNAs/siRNAs, tRFs and other small RNAs derived from chimeric ncRNA agents in human cells were fully elucidated, the roles of specific ribonucleases such as Dicer in the processes remain undefined.
• RNA aptamers [0201] The utility of OnRS was further extended to consistent high-yield production of RNA aptamers. A priori it is unknown whether aptamer activity would still be present in the tRNA (13-15) and 5S rRNA (16) scaffolds, although ribozyme activity was observed in the context of the tRNA scaffold when Hammerhead ribozyme sequences were inserted together with the target RNA to be produced (15).
• the OnRS-resembled RNA aptamer MGA sensor indeed interacted with MG to produce a specific strong fluorescence at 630/650 nm (excitation/emission; Figure 5e), as it was originally discovered (9), which was further employed for the determination of serum Rnase activities in clinical samples (Figure 7e).
• the Rnase activity assay using label-free MGA sensor developed in this study is different from current methods.
• the Kunitz Rnase activity assay (29,34-36) is based upon the ultraviolet absorbance of label-free nucleic acids at 260 nm or nucleosides at 280 nm, which is relatively less selective and sensitive.
• Recent and current Rnase activity assays including those commercially-available kits rely on isotope- or fluorophore-labeled RNAs or antibodies (37-40), and thus offer greater sensitivities to determine very low levels of Rnase activities or indicate Rnase protein levels.
• human serum is comprised of much higher Rnase activities.
• CENTROIDFOLD a web server for RNA secondary structure prediction. Nucleic acids research, 37, W277-280.
• CentroidHomfold-LAST accurate prediction of RNA secondary structure using
• Cytomegalovirus microRNA expression is tissue specific and is associated with persistence. Journal of virology, 85, 378-389.
• N-methylnicotinamide and nicotinamide N- methyltransferase are associated with microRNA- 1291 -altered pancreatic carcinoma cell metabolome and suppressed tumorigenesis. Carcinogenesis, 35, 2264-2272.
• This Example demonstrates a high-yield expression of chimeric pre-miR- 1291 in common E. coli strains using the same tRNA scaffold.
• tRNA/MSA Chimeric tRNA/mir-1291 was readily processed to mature miR-1291 in human carcinoma MCF-7 and PANC-1 cells. Consequently, recombinant tRNA/mir- 1291 reduced the protein levels of miR- 1291 target genes including ATP-binding cassette transporter ABCC 1 , and forkhead box protein FOXA2, and methyl CpG binding protein MeCP2, as compared with cells transfected with the same doses of control tRNA scaffold (tRNA/MSA).
• tRNA-carried pre- miR-1291 suppressed the growth of MCF-7 and PANC-1 cells in a dose dependent manner, and significantly enhanced the sensitivity of ABCCl-overexpressing PANC-1 cells to doxorubicin.
• ncRNAs such as microRNAs (miRNAs or miRs) and long noncoding RNAs in the control of various cellular processes including drug disposition and cell proliferation has expanded our knowledge of "genes" in the cells.
• miRNAs e.g., miR-519c, -328, -326, -379, - 1291 and -124
• AAC ATP- binding cassette

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