WO2014106012A1 - Methods of modulating proliferation and uses thereof - Google Patents

Methods of modulating proliferation and uses thereof Download PDF

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Publication number
WO2014106012A1
WO2014106012A1 PCT/US2013/077930 US2013077930W WO2014106012A1 WO 2014106012 A1 WO2014106012 A1 WO 2014106012A1 US 2013077930 W US2013077930 W US 2013077930W WO 2014106012 A1 WO2014106012 A1 WO 2014106012A1
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cell
trna
subject
seq
lymphoma
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PCT/US2013/077930
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French (fr)
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Andrea Califano
Riccardo Dalla-Favera
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The Trustees Of Columbia University In The City Of New York
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    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications

Definitions

  • MicroRNAs are 20-23 nucleotides (nt) RNA molecules that are produced by the processing of a larger enclosing stem- loop structure (>50bp), called precursors, by cellular enzymes. miRNAs are processed from hairpin precursors of 70 nt (pre-miRNA) which are derived from primary transcripts (pri-miRNA) through sequential cleavage by the RNAse III enzymes drosha and dicer. miRNAs target the messenger RNA of other genes by binding to their 3' UTR and interfering with their translation or causing degradation by enzyme targeting double-stranded RNA.
  • pre-miRNA hairpin precursors of 70 nt
  • pri-miRNA primary transcripts
  • miRNAs target the messenger RNA of other genes by binding to their 3' UTR and interfering with their translation or causing degradation by enzyme targeting double-stranded RNA.
  • miRNAs are non-coding RNAs (ncRNAs) that exist in a variety of organisms, including mammals, and are conserved in evolution. Many miRNAs tend to be ciusiered and transcribed as poiycistrons and often have similar spatial temporal expression patterns. miRNAs have been implicated in various biological processes including developmental timing, differentiation, apoptosis, cell proliferation, organ development, and metabolism.
  • the invention is based, at least in part, on the discovery of a miRNA composition (GUI 276) that is useful for the treatment of a blood cancer (e.g., a lymphoma) and/or the amelioration of symptoms associated with the blood cancer.
  • a blood cancer e.g., a lymphoma
  • one aspect of the invention features a method for treating a blood cancer in a subject in need thereof. Tn one embodiment, the method comprises administering to the subject an effective amount of a nucleic acid composition comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , thereby treating the blood cancer in the subject.
  • the nucleic acid is a fragment of SEQ ID NO: 1.
  • the blood cancer is a lymphoma.
  • the blood cancer is a B cell lymphoma.
  • One aspect of the invention provides a method for decreasing proliferation of a blood cancer cell.
  • the method comprises delivering to a cell an effective amount of a nucleic acid composition comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1, thereby decreasing proliferation of the cancer cell.
  • the nucleic acid is a fragment of SEQ ID NO: I .
  • the blood cancer is a lymphoma.
  • the blood cancer is a B cell lymphoma.
  • the proliferation comprises cell invasion, cell migration, or a combination of either activity.
  • An aspect of the invention features a method for reducing the number of blood cancer cells.
  • the method comprises delivering to a cell an effective amount of a nucleic acid composition comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , thereby reducing the number of cancer cells.
  • the nucleic acid is a fragment of SEQ ID NO: 1
  • the blood cancer is a lymphoma.
  • the blood cancer is a B cell lymphoma.
  • An aspect of the invention provides for a method of decreasing growth of a solid tumor in a subject in need thereof.
  • the method comprises administering to the subject an effective amount of a nucleic acid composition comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , wherein the nucleic acid composition decreases the size of the solid tumor.
  • the nucleic acid is a fragment of SEQ ID NO: 1.
  • the solid tumor is a lymphoma.
  • An aspect of the invention features a method for treating a B cell lymphoma in a subject in need thereof.
  • the method comprises administering to the subject an effective amount of a nucleic acid composition comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , thereby treating B ceil lymphoma in the subject.
  • the nucleic acid is a fragment of SEQ ID NO: 1.
  • the kit comprises at least one oligonucleotide that specifically hybridizes to a nucleic acid comprising about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , or a portion thereof.
  • the cancer-associated tRNA fragment comprises SEQ ID NO: 1 .
  • the cancer-associated tRNA fragment comprises CU1276.
  • the oligonucleotide comprises a set of nucleic acid primers. In some embodiments, the oligonucleotide comprises a probe. In further embodiments, the probe that detects the presence of a tRN A-derived microRNA comprising CUS 1276. In other embodiments, the probe comprises about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID N O: 2. In one embodiment, the oligonucleotide comprises at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, or at least 15 consecutive nucleotides comprising SEQ ID NO: 3 or 4. In another embodiment, the absence of cancer-associated tRNA is indicative if a blood cancer. In a further embodiment, the blood cancer is a lymphoma. In yet another embodiment, the blood cancer is a B cell lymphoma.
  • An aspect of the invention features a method for detecting the presence of a cancer-associated tRNA fragment in a human subject.
  • the method comprises obtaining a biological sample from a human subject; and detecting whether or not a nucleic acid sequence comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , or a portion thereof, is present in the subject.
  • the cancer- associated tRNA fragment comprises CU1276.
  • the absence of cancer- associated tRNA is indicative if a blood cancer.
  • the blood cancer is a lymphoma.
  • the blood cancer is a B cell lymphoma.
  • the detecting comprises using hybridization, amplification, or sequencing techniques to detect the presence of absence of a cancer-associated tRNA fragment.
  • the amplification uses primers comprising at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, or at least 15 consecutive nucleotides comprising SEQ ID NO: 3 or 4.
  • FIG. 1A-1B Figure 1A-1B.
  • CU1276 is a Dicer-dependent tRNA fragment expressed in mature B cells.
  • IB FASTA sequences of annotated human tRNAs with perfect match to the CU 1276 small RNA. CU1276 sequence is highlighted in bold.
  • FIG. 1C-1D Figure 1C-1D.
  • CU1276 is a Dicer-dependent tRNA fragment expressed in mature B cells.
  • (lC) Northern blot analysis of total R from GC B cells, Ramos, and 293T cells transiently transfected with empty vector or a vector encoding for the Gfy(GCC) chri .tRNA68.
  • the three primary bands correspond to the 22nt tRNA fragment CU1276, the 74nt mature tRN A, and a high molecular weight tRNA primary transcript. See also FIG. 6.
  • FIG. 2A-2D CU1276 is bound by all four rusman Argonaute proteins and functions as a miRNA.
  • IP immunoprecipitaiion
  • FIG. 3A-3B OJ1276 is downregulated in lymphoma cell lines and primary biopsies.
  • FIG. 4A ClJl 276 directly represses RPA1.
  • FIG. 4B-4C CU1276 directly represses RPAl
  • the GC sample was obtained by pooling cells from two independent donors. Ail samples are identical to those used for CU1276 expression analysis in Figure 3.4.
  • Figure SB CU1276 modulates proliferation and DNA damage signaling in an RPAl-dependent manner.
  • SB Western blot analysis of RPAl, total H2AFX, and yH2AFX in Etoposide-treated control cells and cells expressing CU1276. ACTB was used as loadmg control. Image is representative of three independent experiments, for which average yH2AFX quantifications are indicated in bar chart format. Error bars represent the standard deviation of three independent experiments.
  • Figure SC €111276 modulates proliferation and DNA damage signaling in an RPAl-dependent manner
  • (5C Western blot analysis of control cells, ceils expressing CU1276, and cells simultaneously expressing CU 1276 and exogenous RPAl.
  • FIG. 6 shows CU 1276 is expressed from two independent tRNA loci.
  • GC Germinal Center
  • FIG. 7 shows Normal Germinal Center B cells and B cell lymphoma lines express similar levels of Gly(GCC) tRNA.
  • BL Burkitt's lymphoma
  • ABS-DLBCL Activated B Cell-like Diffuse Large B Cell Lymphoma
  • GCB-DLBCL GC-like Diffuse Large B Ceil Lymphoma
  • FIGS. 8A-8B show an experimental schematic of CU 1276 target candidate identification, and validation of CU1276 targets.
  • (HA) 293T ceils were transiently transfected with empty vector, chri .tRNA68, or CUT 276 hairpin-encoding vectors. 48 hours post- transfection, ceils were harvested, and their extracted total RNA was used for gene expression profiling with Affymetrix HG-U133Plus2.0 arrays. Genes that were significantly downregulated (threshold p ⁇ 0.05) in tR A-, and/or in hairpin-expressing cells relative to empty vector transfected cells, were considered for further analysis.
  • FiGS. 8C-8D are bar graphs. (8C) WHSC 1L1 3 'UTR reporter activity in response to Gly(GCC) chrl .tRNA68-delivered or hairpin-delivered CU1276. The
  • WHSC1 L1 3 'UTR was not sensitive to repression by either tRNA-mediated or hairpin- mediated expression of GUI 276. Mutational analysis of the predicted binding site for CU1276 was not pursued. Firefly luciferase values were normalized to a Renilla luciferase control, and plotted relative to reporter activity upon co-transfeetton of empty vector. Error bars represent the standard deviation of three independent experiments, each performed in duplicate. (8D) STAG2 3 'UTR reporter activity in response to Gly(GCC) chrl.tRNA68- or hairpin-delivered CU1276.
  • Firefly luciferase values were normalized to a Renilla luciferase control, and plotted relative to reporter activity upon co-transfection of empty vector. Error bars represent the standard deviation of ten independent experiments, each performed in duplicate.
  • FIG. A-9B Western Blot quantifications and qRT-PCR afRPAl upon CU1276 expression.
  • FIG. 9C-9D Western Blot quantifications and qRT-PCR of RPAl upon CU1276 expression.
  • FiG. 18 shows Germinal Center cells and Diffuse Large B Cell Lymphoma cell lines express similar levels of RPAl mRNA.
  • This invention provides for the discovery of cancer-associated tRNA fragments.
  • One of these fragments is a tRNA -derived microRNA (e.g., CU1276) that is downregulated in the blood cell cancer B cell lymphoma.
  • a tRNA -derived microRNA e.g., CU1276
  • SEQ ID NO: 1 is the nucleic acid sequence of GUI 276:
  • a lymphoma is a type of blood cancer that begins in cells of the immune system (e.g., lymphocytes such as B cells and T cells). Lymphomas are classified according to certain histological characteristics: the site the cell arises from; the presence of a Reed- Sternberg cell; and whether the cell that is replication is a B cell or T cell.
  • NHL Hodgkin lymphoma
  • HL Hodgkin lymphoma
  • NDL non-Hodgkin lymphoma
  • NHL can be further divided into cancers that have an indolent (slow- growing) course and those that have an aggressive (fast-growing) course. Both Hodgkin and non-Hodgkin lymphomas can occur in children and adults, and prognosis and treatment depend on the stage and the type of cancer.
  • Non-limiting examples of lymphomas include, but are not limited to Precursor T-cell leukemia/lymphoma. Follicular lymphoma, Diffuse large B cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia/lymphoma, MALT lymphoma, Burkitt's lymphoma, Mycosis fungoides, Peripheral T-cell iymphoma-Not-Otherwise-Specified, Nodular sclerosis form of Hodgkin lymphoma, and Mixed-eeiiuiarity subtype of Hodgkin lymphoma.
  • MicroRNAs are naturally-occurring 19 to 25 nucleotide transcripts found in over one hundred distinct organisms (such as nematodes, fruit files, and humans). miRNAs can be processed from 60- to 70-nucleotide foldback RNA precursor structures, which are transcribed from the miRNA gene.
  • the miRNA precursor processing reaction requires Dicer RNase 111 and Argonaute family members (Sasaki et al., 2003 Genomics 82, 323-330).
  • the miRNA precursor or processed miRN A products are easily detected, and an alteration in the levels of these molecules within a cell can indicate a perturbation in the chromosomal region containing the miRNA gene.
  • the unprocessed miRN A gene transcript is called a miRN A precursor (pre- miRNA) and comprises an RNA transcript of about 70 nucleotides in length.
  • the pre-miRNA can be processed by digestion with an RNAse (such as, Dicer, Argonaut, or RNAse 111, e.g., E. coli R Ase III)) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the processed miRNA gene transcript.
  • RNAse such as, Dicer, Argonaut, or RNAse 111, e.g., E. coli R Ase III
  • the active 19-25 tRNA-derived micro RNA can be obtained from the miRNA precursor through natural processing routes (for example, using intact cells or cell lysates) or by synthetic processing routes (for example, using isolated processing enzymes, such as isolated Dicer, Argonaut, or RNAase III).
  • the active 19-25 tRNA-derived microRNA can also be produced directly by biological or chemical syntheses, without having been processed from the miRNA precursor.
  • the nucleic acid is the tRNA-derived microRNA. CU1276. In some embodiments, the nucleic acid is about 70%, about 75%, about 80%, about 85%, about 90%, about 93%, about 95%, about 97%, about 98%, or about 99% identical to SEQ ID O: 1.
  • an isolated nucleic acid such as tRNA-derived microRNA CU1276, can be synthesized, or altered, or removed from the natural state through human intervention.
  • An isolated miRNA can exist in substantially purified form, or can exist in a cell into which the miRNA has been delivered.
  • an isolated nucleic acid such as a miRNA of the invention
  • a miRNA of the invention can be obtained using a number of standard techniques utilized in the art.
  • the miRNA gene products can be chemically s nthesized or recombinantly produced using methods known in the art.
  • a miRN A can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dhannacon Research (Lafayette, Colo., USA), Rosetta Genomics (North
  • a eukaryotic expression vector can be used to transfect cells.
  • Mammalian c can contain an expression vector (for example, one thai contains the tRNA-derived microRNA CUI276) via introducing the expression vector into an appropriate host cell methods known in the art.
  • miRN A can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing RNA from a plasmid include, e.g., the U6 or HI RNA pol III promoter sequences, or the cytomegalovirus promoters.
  • Recombinant plasmids can comprise inducible or regulatable promoters for expression of the miRNA in cancer cells (such as hematopoietic ceils, i.e., B cells).
  • cancer cells such as hematopoietic ceils, i.e., B cells.
  • the tRNA-derived microRNA CU 12.76 can be placed under the control of the CM V intermediate-early promoter, whereby the nucleic acid sequences encoding the miRNA molecule are located 3 ' of the promoter, so that the promoter can initiate transcription of the miRNA gene product coding sequences.
  • Plasmids suitable for expressing a miRNA, methods for inserting nucleic acid sequences into the plasmid to express the miRNA of interest, and methods of delivering the recombmant plasmid to cells of interest are well-established and practiced in the art. See, for example, Zeng et al. (2002), Molecular Cell 9: 1327-1333; Tuschl (2002), Nat Biotechnol, 20:446-448; Brammelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat Biotechnol 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosures of which are herein incorporated by reference,
  • tRNA-derived microRNAs can also be expressed from recombinant viral vectors.
  • the RNA expressed from the recombinant viral vectors can either be isolated from cultured cell expression systems by standard techniques, or can be expressed directly in cancer cells (such as hematopoietic cells, i.e., B ceils).
  • the recombinant viral vectors can comprise sequences that encode the miRNA molecule of interest and any suitable promoter for expressing the RNA sequences.
  • Vectors can also comprise inducible or regulatable promoters for expression of the miRNA molecule in cells, such as cancer ceil.
  • suitable promoters include the U6 or HI RNA pol 111 promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is practiced by those of ordinary skill in the art.
  • Any viral vector that can harbor the nucleotide sequences for the miRNA molecules can be used.
  • Non-limiting examples of such vectors include: vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.
  • AV adenovirus
  • AAV adeno-associated virus
  • retroviruses e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus
  • herpes virus and the like.
  • the tropism of the viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
  • leniiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
  • VSV vesicular stomatitis virus
  • AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes.
  • An AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2.
  • This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.
  • Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J.
  • Useful viral vectors can be those derived from A.V and AAV.
  • A. suitable AV vector for expressing an mRNA molecule of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells are described in Xia et al. (2002), Nat. Biotech. 20: 1006- 1010, the entire disclosure of which is herein incorporated by reference.
  • Suitable AAV vectors for expressing a miRNA molecule having SEQ ID NO: 1 methods for constructing the recombinant AAV vector, and methods for delivering the vectors into target cells are described in Samulski ei al. (1987), J. Virol.
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences.
  • host cells include CHO, HeLa, MDCK, HEK293, WT38, as well as various lymphoma cell lines, which are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va. 201 10-2209).
  • ATCC American Type Culture Collection
  • Human leukemia and lymphoma cell lines that can also be used as host cells have been described by MacLeod et al, (2008) Curr Med Chem. ! 5(4):339-59, which is incorporated by reference in its entirety.
  • An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextran-niediated iransfeciion, or eleetroporation.
  • Eleetroporation is earned out at approximate voltage and capacitance to result in entry of the nucleic acid constructs ) into cells of interest (such as lymphoma cells (e.g., cell line DB, ATCC CRL-2289; cell line HX, ATCC CRL-2260; cell line BC-3, ATCC CRL-2277; cell line CA46, ATCC CRL-1648; cell line Raji, ATCC CCL-86; cell line Daudi, ATCC CCL- 213; cell line GA-lO-Cfone-4, ATCC CRL-2393; cell line HH, ATCC CRL-2105; cell line H9, ATCC HTB- 176)).
  • Other transfeetion methods also include modified calcium phosphate precipitation, poiybrene precipitation, liposome fusion, and receptor-mediated gene delivery.
  • Various culturing parameters can be used with respect to the host cell being cultured.
  • Appropriate culture conditions for mammalian cells are well known in the art (Cleveland WL, et ai., J Immunol Methods, 1983, 56(2): 221-234) or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford University Press: New York, 1992)).
  • Cell culturing conditions can vary according to the type of host cell selected.
  • Commercially available medium can be utilized. Non-limiting examples of medium include, for example, Minimal Essential Medium (MEM, Sigma, St.
  • CD-CHO Medium (Invitrogen, Carlsbad, Calif.).
  • the cell culture media can be supplemented as necessary with supplementary components or ingredients, including optional components, in appropriate concentrations or amounts, as necessar or desired.
  • Ceil culture medium solutions provide at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; (3) vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that can be required at very low concentrations, usually in the micromolar range.
  • the medium also can be supplemented electively with one or more components from any of the following categories: ( 1) salts, for example, magnesium, calcium, and phosphate; (2) hormones and other growth factors such as, serum, insulin, transferrin, and epidermal growth factor; (3) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (4) nucleosides and bases such as, adenosine, thymidine, and hypoxanthine; (5) buffers, such as HEPES; (6) antibiotics, such as gentamycin or ampicillin; (7) cell protective agents, for example pluromc polyoi; and (8) galactose.
  • soluble factors can be added to the culturing medium.
  • the mammalian cell culture that can be used with the present invention is prepared in a medium suitable for the type of cell being cultured.
  • the cell culture medium can be any one of those previously discussed (for example, MEM) that is supplemented with serum from a mammalian source (for example, fetal bovine serum (FBSY).
  • the medium can be a conditioned medium to sustain the growth of host cells.
  • a blood cancer can be treated by restoring the level of miR A expression associated with that cancer to normal levels. For example, if the level of miR expression is down-regulated in cancer cells of a subject, then the cancer can be treated by increasing the miRNA expression level. For example, the level of a miRNA in a cancerous blood cell of a subject is first determined relative to normal control ceils. Techniques suitable for determining the relative level of a miRNA molecule in cells have been described above. If miRN A expression is down-regulated in the blood cancer cell relative to normal control cells, then the cancer cells are treated with an effective amount of a composition comprising an isolated miRNA molecule which is down-regulated (such as, a tRNA-derived microRNA (e.g., CU 1276)).
  • a composition comprising an isolated miRNA molecule which is down-regulated (such as, a tRNA-derived microRNA (e.g., CU 1276)).
  • the invention provides a method of decreasing the growth of a solid tumor in a subject.
  • the tumor is associated with, but not limited to, a lymphoma.
  • the method comprises detecting the presence or absence of a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU 1276)) in a sample obtained from a subject, in some embodiments, the sample displaying downregulation of a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)) is provided with tRNA- derived microRNA according to known delivery methods described herein.
  • the method comprises administering to the subject an effective amount of a tRNA-derived microRNA (e.g., CU1276), wherein the miRNA decreases the size of the solid tumor.
  • the invention also provides a method for treating a blood cancer in a subject.
  • the blood cancer comprises a lymphoma.
  • the blood cancer comprises a B cell lymphoma.
  • the method comprises deteciing the presence or absence of a cancer- associated tRNA fragment (such as a tRNA- derived microRNA (e.g., CU 12.76)) in a sample obtained from a subject
  • the method further comprises administering to the subject in need (e.g., the subject who exhibits a downregulation of a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)), a therapeutic treatment of a tRNA-derived microRNA (e.g., CU1276).
  • the sample displaying downregulation of a cancer-associated tRNA fragment such as a tRNA-derived microRNA (e.g., CU1276)
  • tRNA-derived microRNA e.g., CU1276
  • the invention provides a method of decreasing proliferation of a blood cancer cell.
  • the blood cancer is a lymphoma.
  • the blood cancer is a B ceil lymphoma.
  • the method comprises detecting the presence or absence of a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)) in a sample obtained from a subject.
  • a cancer-associated tRNA fragment such as a tRNA-derived microRNA (e.g., CU1276)
  • the sample displaying downregulation of a cancer-associated tRNA fragment is provided with tRNA-derived microRNA according to known delivery methods described herein.
  • the method comprises administering to the subject an effective amount of a tRNA -derived microRNA (e.g., CU1276), wherein the presence of the miRNA in the ceil decreases proliferation of the blood cancer ceils.
  • proliferation comprises cell invasion, cell migration, or a combination of the two.
  • Cell migration and invasion assays are commercially available, e.g., Cell Biolabs Inc. (San Diego, CA), R&D Systems (Minneapolis, MN), and EMD Millipore (Bilierica, MA).
  • the invention provides a method of reducing the number of blood cancer cells.
  • the blood cancer is a lymphoma.
  • the blood cancer is a B cell lymphoma.
  • the method comprises detecting the presence or absence of a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)) in a sample obtained from a subject.
  • the sample displaying downregulation of a cancer-associated tRNA fragment such as a tRNA- derived microRNA (e.g., CU 12.76)
  • the method comprises administering to the subject an effective amount of a tRNA-derived microRNA (e.g., CU1276), wherein the presence of the miRNA in the ceil reduces the number of blood cancer cells.
  • the administering or delivery step in the claimed methods can comprise a drag administration, such as a composition comprising a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)).
  • a cancer-associated tRNA fragment such as a tRNA-derived microRNA (e.g., CU1276)
  • the therapeutic molecule to be administered comprises a nucleic acid comprising at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% of the nucleic acid sequence of SEQ ID NO: 1.
  • administration of the therapeutic molecule decreases the size of the solid tumor associated with a lymphoma.
  • administration of the therapeutic molecule treats a blood cancer in a subject, for example a lymphoma (such as a B cell lymphoma).
  • administration of the therapeutic molecule decreases proliferation of blood cancer cells (e.g., a lymphoma).
  • administration of the therapeutic molecule reduces the number of blood cancer cells (e.g., a lymphoma).
  • the biological sample comprises serum, bone marrow, blood, peripheral blood, lymph nodes, urine, a saliva sample, a buccal swab, a a sputum sample, a lacrimal secretion sample, a semen sample, a vaginal secretion sample, a fetal tissue sample, or a combination thereof.
  • Non-limiting examples of ly mphoma animal models to be used with the methods of the invention include JAX mice strains for lymphomas (e.g., B cell and T cell
  • lymphomas such as B6.Cg-Tg(IghMyc)22Bri/J, B6.129S l-Ingltml Avg/J, B6.129S4(Cg)- Trp53tm2.1Tyj/J, B6J2984-Trp53tm3 JTyj/J, and B6.Cg-Tg(Cd79b-TCLlA)BKTeit/J, ail of which are commercially available from the Jackson Laboratory (Bar Harbor, ME).
  • Other murine models useful with the methods described herein include, but are not limited, to those described by Donnou et al. (2012) Adv Hematol, 12:701 704, which is incorporated by reference in its entirety. These animal models are known in the art to correspond to the human condition.
  • a cancer-associated tRNA fragment (such as a tRNA-derived mieroRNA (e.g., CU12.76)) can be determined at the nucleic acid level.
  • detection can be determined by performing an oligonucleotide ligation assay, a confirmation based assay, a hybridization assay, a sequencing assay, an allele-specific amplification assay, a
  • microsequencing assay a melting curve analysis
  • DPLC denaturing high performance liquid chromatography
  • the detection or determination comprises nucleic acid sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof.
  • the detection is performed by sequencing all or part of a cancer-associated tRNA fragment (such as a tRN A-derived mieroRNA (e.g., CU1276)), or by selective hybridization or amplification of ail or part of the tRNA-derived mieroRNA.
  • a nucleic acid specific amplification can be carried out before the fusion identification step.
  • the detecting comprises using a northern blot; real time PCR and primers directed to SEQ ID NO: 1 ; a ribonuclease protection assay; a hybridization, amplification, or sequencing technique to detect a tRNA-derived mieroRNA (e.g., CU1276): or a combination thereof.
  • the PCR primers comprise at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 3, 4, or a combination of the primers.
  • Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid.
  • a detection technique involves the use of a nucleic acid probe specific for the presence of a tRNA-derived mieroRN A (e.g., CO 1276), followed by the detection of the presence of a hybrid.
  • the probe can be in suspension or immobilized on a substrate or support (for example, as in nucleic acid array or chips technologies).
  • the probe can be labeled to facilitate detection of hybrids.
  • the probe according to the invention can comprise a nucleic acid directed to SEQ ID NO: 1 .
  • the probe that detects the presence of a tRNA-derived mieroRNA comprises SEQ ID NO: 2.
  • Sequencing can be carried out using techniques well known in the art, using automatic sequencers.
  • the sequencing can be performed on a tRNA-derived microRNA (e.g., CU1276).
  • Amplification is based on the formation of specific hybrids between
  • Amplification can be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), iigase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based ampiification (NASBA). These techniques can be performed using commercially available reagents and protocols. Useful techniques in the art encompass real-time PCR, allele- specific PCR, or PCR based single- strand conformational polymorphism (SSCP). Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction. In one embodiment, ampiification comprises using forward and reverse PCR primers directed to SEQ ID NO: 1.
  • amplification can comprise using forward and reverse PCR primers comprising at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at feast 18, at feast 19, at feast 20 consecutive nucleotides comprising SEQ ID NO: 3 or 4.
  • Non-limiting amplification methods include, e.g., polymerase chain reaction, PCR (PCR Protocols, A Guide To Methods And Applications, ed. Innis, Academic Press, N.Y., 1990 and PCR Strategies, 1995, ed.
  • RNA polymerase mediated techniques e.g., NASBA, Cangene, Mississauga, Ontario; see also Berger (1987) Methods Enzymol. 152:307-316; U.S. Pat. os. 4,683,195 and 4,683,202; and Sooknanan (1995) Biotechnology 13:563-564). All the references stated above are incorporated by reference in their entireties,
  • the invention provides for a nucleic acid primer, wherein the primer can be complementary to and hybridize specifically to a portion of a tRNA-derived microR A (e.g., CU1276),
  • the blood cancer comprises a lymphoma, such as a B cell lymphoma.
  • Primers can be specific for a tRNA-derived microRNA (e.g., CU1276). By using such primers, the detection of an amplification product indicates the presence of a tRNA-derived microRN A (e.g., CU1276).
  • primers of this invention can be single-stranded nucleic acid molecules of about 8 to about 1 5 nucleotides in length.
  • a nucleic acid primer or a pair of nucleic acid primers as descr ibed above can be used in a method for detecting the presence of a blood cancer in a subject.
  • primers can be used to detect the absence of reduced level of a cancer- associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)).
  • the primers are directed to SEQ ID NO: 1.
  • the PGR primers comprise at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 3 or 4.
  • the invention provides for a diagnostic kit comprising products and reagents for detecting in a sample from a subject the presence or absence of a cancer-associated tRNA fragment (such as a tRNA-derived microRN A (e.g., CU1276)).
  • a cancer-associated tRNA fragment such as a tRNA-derived microRN A (e.g., CU1276)
  • the kit can be useful for determining whether a sample from a subject exhibits decreased or reduced expression of a cancer-associated tRNA fragment.
  • the diagnostic kit according to the present invention comprises any primer, or any pair of primers directed specifically to a cancer- associated tRNA fragment.
  • the diagnostic kit according to the present invention can further comprise reagents and/or protocols for performing a hybridization, or amplification.
  • the kit can comprise nucleic acid primers that specifically hybridize to and can prime a polymerase reaction from a tRN A-derived microRN A (e.g., CU1276) comprising at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 3 or 4, or a combination of the primers.
  • primers can be used to detect the absence of reduction of a tRNA-derived microRNA (e.g., GUI 276), such as a primer directed to SEQ ID NO: i .
  • the PCR primer comprises at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 3 or 4.
  • the kit comprises a probe for detecting a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276).
  • the probe comprises SEQ ID NO: 2.
  • the diagnosis methods can be performed in vitro, ex vivo, or in vivo. These methods utilize a sample from the subject in order to assess the status of a tRNA-derived microRNA (e.g., CU1276).
  • the sample can be any biological sample derived from a subject, which contains nucleic acids or polypeptides. Examples of such samples include, but are not limited to, fluids, tissues, cell samples, organs, and tissue biopsies. Non-limiting examples of samples include blood, li ver, plasma, serum, sali va, urine, or seminal fluid.
  • the sample can be collected according to conventional techniques and used directly for diagnosis or stored.
  • the sample can be treated prior to performing the method, in order to render or improve av ailability of nucleic acids or polypeptides for testing.
  • Treatments include, for instance, lysis (e.g., mechanical, physical, or chemical), centrifugation.
  • the nucleic acids and/or polypeptides can be pre-purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides can also be treated with enzymes or other chemical or physical treatments to produce fragments thereof.
  • the sample is contacted with reagents, such as probes or primers, in order to assess the absence or presence of a tRNA-derived microRNA (e.g., CU1276).
  • a tRNA-derived microRNA e.g., CU1276
  • Contacting can be perfonned in any suitable device, such as a plate, tube, well, or glass.
  • the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array.
  • the substrate can be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, or polymers.
  • the substrate can be of various forms and sizes, such as a slide, a membrane, a bead, a column, or a gel.
  • the contacting can be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids or polypeptides of the sample.
  • nucleic acids into viable cells can be effected ex vivo, in situ, or in vivo by use of vectors, such as viral vectors (e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).
  • vectors such as viral vectors (e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).
  • Non-limiting techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, eiectroporation, microinjection, cell fusion, DEAE-dextran, and the calcium phosphate precipitation method (See, for example, Anderson, Nature, (1998) supplement to 392(6679):25.
  • introduction of a nucleic acid can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).
  • Cells can also be cultured ex vivo in the presence of therapeut ic compositions of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
  • Nucleic acids can be inserted into vectors and used as gene therapy vectors.
  • viruses include papovaviruses, e.g., SV40 (Madzak et al., (1992) J Gen Virol 73( Pi 6):1533-6); adenovirus (U.S. Patent No. 8,460,932; Berkner (1992) Curr Top Microbiol 7WMW?K>/.158:39-66; Berkner (1988) Biolechniques, 6(7):616-29; Gorziglia and Kapikian (1992) ./ Virol. 66(7):4407-12; Quantin et al., (1992) Proc Nail Acad Sci USA. 89(7):2581-4; Rosenfeld et aL, (1992) Cell.
  • Non-limiting examples of in vivo gene transfer techniques include transfection with viral (e.g., retroviral) vectors (see U.S. Pat. Nos. 5,252,479; 8,460,932; 8,529,885, each of which is incorporated by reference in its entirety) and viral coat protein-liposome mediated transfection (Dzau et al., (1993) Trends in Biotechnology 1 1 :205-210), incorporated entirely by reference).
  • viral e.g., retroviral vectors
  • viral coat protein-liposome mediated transfection Dzau et al., (1993) Trends in Biotechnology 1 1 :205-210
  • naked DNA vaccines are generally known in the art; see Brower, (1998) Nature Biotechnology, 16: 1304- 1305, which is incorporated by reference in its entirety.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. Nos. 5,328,470; 8,529,885; 8,460,932) or by stereotactic injection (see, e.g., Chen, et al, (1994) Proc. Natl, Acad. Sci. USA 91 :3054- 3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • a tRNA-derived microR A (e.g., CU1276) can also be delivered in a controlled release system.
  • the molecule can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of
  • a pump can be used (see Sefton ( 1987) Biomed. Eng. 14:201 ; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med.
  • polymeric materials can be used (see Medical Imaging
  • a controlled release system can be placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical
  • a tRNA-derived microRNA (e.g., CU1276) can be incorporated into
  • compositions suitable for administration for example the rniRNA and a pharmaceutically acceptable carrier.
  • A. tRNA-derived microRNA. (e.g., CO 1276) can be administered to the subject once (e.g., as a single injection or deposition).
  • a tRNA-derived microRNA e.g., CU1276
  • a tRN A-derived microRN A can also be administered once or twice daily to a subject for a period of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 times per year, or a combination thereof.
  • a tRNA-derived microRNA e.g., CU1276
  • another therapeutic such as a chemotherapy drug
  • the effective amount of the tRNA-derived microRNA (e.g., CU1276) administered to the subject can comprise the total amount of gene product administered over the entire dosage regimen.
  • a tRNA-derived microRNA (e.g., CU1276) can be administered to a subject by any means suitable for delivering the composition to cells of the subject, such as cancer cells, e.g., a blood cancer.
  • a tRNA-derived microRNA e.g., CU1276
  • Transtection methods for eukarvoiic cells include direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, biobalHstic or particle acceleration; calcium phosphate precipitation, and transtection mediated by viral vectors.
  • compositions can be formulated and administered to reduce the symptoms associated with a blood cancer, e.g., a lymphoma, by any means that produces contact of the active ingredient with the agent's site of action in the body of a subject, such as a human or animal (e.g., a dog, cat, or horse). They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • a therapeutically effective dose of tRNA-derived microRNA can depend upon a number of factors known to those or ordinary skill in the art.
  • the dose(s) of the composition can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the tRNA-derived microRNA (e.g., CU1276) to have. These amounts can be readily determined by a skilled artisan.
  • any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.
  • a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.
  • compositions can be formulated and administered to inhibit, reduce, or ameliorate a variety of cancer states by any means that produces contact of the active ingredient with the agent's site of action in the body of the subject (e.g., a mammal). They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • compositions for use in accordance with the invention can be formulaied in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration.
  • compositions of the invention can be formulated in liquid solutions, for example, in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use.
  • a pharmaceutically acceptable carrier can comprise any and ail solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used.
  • Supplementary active compounds can also be incorporated into the compositions.
  • the compositions can be administered alone or in combination with at least one other agent such as a stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions can be administered to a subject alone, or in combination with other agents and/or drugs,
  • the present pharmaceutical formulations comprise a tRN A-derived microRNA
  • the pharmaceutical formulations can also comprise the miRNA, which is encapsulated by liposomes and a pharmaceutically-acceptable carrier.
  • Useful pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid, and the like.
  • compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives.
  • suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTP A or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • physiologically biocompatible buffers e.g., tromethamine hydrochloride
  • additions of chelants such as, for example, DTP A or DTPA-bisamide
  • calcium chelate complexes as for example calcium DTPA, CaNaDTPA-bisamide
  • calcium or sodium salts for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate.
  • Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
  • Sterile injectable solutions can be prepared by incorporating the miR A in the required amount in an appropriate sol vent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein.
  • examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solid pharmaceutically-acceptable carriers for solid pharmaceutical compositions of the invention, conventional nontoxic solid pharmaceutically-acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • the therapeutic compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregeiatinised maize starch,
  • polyvinylpyrrolidone or hydroxypropyl methylcellulose e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose,
  • microcrys tailine cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch giycoiaie); or wetting agents (e.g., sodium lauryl sulphate).
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch or sodium starch giycoiaie
  • wetting agents e.g., sodium lauryl sulphate.
  • the tablets can be coated by methods well known in the art.
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with waf er or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils
  • preservatives e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid.
  • compositions for oral administration can be suitably formulated to give controlled release of the active agent.
  • buccal administration the therapeutic compositions can take the form of tablets or lozenges formulated in a conventional manner.
  • the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellent, e.g., dichlorodifluoromethane,
  • the dosage unit can be determined by providing a valve to deliver a metered amount.
  • compositions can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.
  • Subcutaneous administration can refer to administration just beneath the skin (i.e., beneath the dermis).
  • the subcutaneous tissue is a layer of fat and connective tissue that houses larger blood vessels and nerves. The size of this layer varies throughout the body and from person to person.
  • the interface between the subcutaneous and muscle layers can be encompassed by subcutaneous administration. This mode of administration can be feasible where the subcutaneous layer is sufficiently thin so that the factors present in the compositions can migrate or diffuse from the locus of administration.
  • the bolus of composition administered is localized proximate to the subcutaneous layer.
  • Suitable enteral administration routes for the present methods include oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include intravascular administration (e.g. intravenous bolus injection, intravenous infusion, mtra-arterial bolus injection, intra -arterial infusion and catheter instillation into the vasculature); peri- and intra- tissue injection (e.g., peri-tumoral and intra-tumoral injection, intra-retinal injection, or subretinaf injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps): direct application to the tissue of interest, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation.
  • the miR A can be administered by injection or infusion.
  • the therapeutic compositions can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneous! ⁇ ' or intramuscularly) or by intramuscular injection.
  • the therapeutic compositions can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt,
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives, in addition, detergents can be used to facilitate permeation,
  • Transmucosal administration can be through nasal sprays or using suppositories.
  • the compositions of the invention are formulated into ointments, salves, gels, or creams as generally kno wn in the art, A.
  • wash solution can be used locally to treat an injury or inflammation to accelerate healing.
  • the therapeutic compositions are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
  • the miRNA can be applied via transdermal delivery systems, which slowly releases the active compound for percutaneous absorption.
  • Permeation enhancers can be used to facilitate transdermal penetration of the active factors in the conditioned media.
  • Transdermal patches are described in for example, U.S. Pat. No. 5,407,713; U.S. Pat, No. 5,352,456; U.S. Pat No. 5,332,213; U.S. Pat, No. 5,336,168; U.S. Pat. No. 5,290,561 ; U.S. Pat. No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No.
  • a miRNA composition can also be formulated as a sustained and/or timed release formulation.
  • sustained and/or timed release formulations can be made by sustained release means or delivery devices that are well known, to those of ordinary skill in the art, such as those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809;
  • compositions of the present invention can be used to provide slow or susta ined release of one or more of the active ingredients using, for example, hydropropylm ethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions.
  • Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the invention.
  • single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gel caps, caplets, powders, and the like, that are adapted for sustained release are encompassed by the present invention.
  • the iR A-derived microRNA (e.g., CU1276) can be administered to the subject either as naked RN A, in conjunction with a delivery reagent, or as a nucleic acid (e.g., a recombinant plasmid or viral vector) comprising sequences which expresses the gene product.
  • a delivery reagent e.g., a nucleic acid comprising sequences which expresses the gene product.
  • Suitable delivery reagents for administration of the miRNA molecule include the Minis Transit TKO lipophilic reagent; lipofectm;
  • lipofectamine e.g., celifectm; or polycations (e.g., pofylysine), or liposomes.
  • the dosage administered will be a therapeutically effective amount of the composition sufficient to result in treatment and/or amelioration of symptoms of a blood cancer (e.g., a ceil lymphoma) and can vary depending upon known factors such as the pharmacodynamic characteristics of the particular active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired.
  • a blood cancer e.g., a ceil lymphoma
  • Toxicity and therapeutic efficacy of therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (The Dose Lethal To 50% of the Population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Therapeutic agents that exhibit large therapeutic indices are useful.
  • Therapeutic compositions that exhibit some toxic side effects can be used.
  • doses depend upon a number of factors known to those or ordinary skill in the art, e.g., a physician.
  • the dose(s) will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the composition to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight.
  • the effective amount of the administered tRNA-derived microRNA is at least about 0.0001 ⁇ ig/kg body weight, at least about 0.00025 ug kg body weight, at least about 0.0005 ⁇ 2 / ⁇ body weight, at least about 0.00075 ug kg body weight, at least about 0.001 ⁇ 3 ⁇ 43 ⁇ 4 body weight, at least about 0.0025 g/kg body weight, at least about 0.005 ⁇ ig/kg body weight, at least about 0.0075 ug kg body- weight, at least about 0.01 body weight, at least about 0.025 ⁇ g/ g body weight, at least about 0.05 ⁇ g kg body weight, at least about 0.075 ⁇ g/kg body weight, at least about 0.1 ug/kg body weight, at least about 0.25 ⁇ g kg body weight, at least about 0.5 ⁇ g/kg body weight, at least about 0.75 ⁇ g/kg body weight, at least about 1 ⁇ g/kg body weight, at least about 5 ⁇ g l
  • compositions can be further approximated through analogy to compounds known to exert the desired effect.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.
  • Example 1 A tRNA-derivcd microRNA modulates proliferation and the DNA damage response, and is downregulated in B cell lymphoma
  • CU 1276 is abundant in normal Germinal Center B cells but absent in Germinal Center-derived lymphomas, suggesting a role in the pathogenesis of this disease.
  • CU1276 represses endogenous RPAI, an essential gene involved in many aspects of DNA dynamics, and consequently, expression of this tRNA-derived microRNA in a lymphoma cell line suppresses proliferation and modulates the molecular response to DNA damage.
  • miRNAs guide the binding of Argonaute- containing induced silencing complexes (miRISC) to the 3' untranslated region (3'UTR) of genes bearing partially complementary sites, and are typically expressed from within the introns of protein coding genes or as part of long non-coding RNA transcripts (1).
  • miRISC Argonaute- containing induced silencing complexes
  • 3'UTR 3' untranslated region
  • miRNAs can also arise from previously unanticipated non- canonical pathways. Specifically, miRNAs can be generated in a DROSHA- (2, 3) or
  • RNA molecules such as small nucleolar RNA (5).
  • Cloning and sequencing of small RNAs purified from human Naive, Germinal Center (GC), and Memory B cells, as well as from the Burkitt Lymphoma cell line Ramos (6) have been reported.
  • GC Germinal Center
  • Ramos Burkitt Lymphoma cell line Ramos
  • an interesting class of abundantly expressed small RNAs were noted whose sequences perfectly matched either to mature or to precursor tRNA transcripts.
  • Several other groups have reported similar small RNA species expressed in a variety of human cell types (7- 1 1 ) as well as in other organisms (12-14). However, the functional role of these small RNAs, and in particular the possibility that they may act as miRNAs, has not yet been adequately addressed.
  • ai leasi one such sequence, designated CO 1276, is shown to function as a niiRNA.
  • CU1276 is a DfCERl -dependent tRNA fragment expressed in mature B cells.
  • previous reports (8, 9, 1 1) define a minimum of three distinct categories of tRNA fragment: those matching to the 5' end of mature tRNA (tRF-5), those matching to the 3' end of mature tRNA (tRF-3), and those matching to the 3' end of precursor iRNA transcripts (tRF - 1).
  • tRF-5 those matching to the 5' end of mature tRNA
  • tRF-3 those matching to the 3' end of mature tRNA
  • tRF - 1 those matching to the 3' end of precursor iRNA transcripts
  • CU1276 is a 22nt small R A (5'-TCGATTCCCGGCCAATGCACCA-3'; SEQ D NO: 1) differentially expressed in three stages of mature B cell differentiation and one GC-derived lymphoma cell line, and cloned most frequently in normal GC B cells (FiglA). Despite its roiRNA-like size, CU1276 is a perfect match to the post-transcriptionally modified 3' end of at leasi five annotated human tRNAs (15) (FiglB).
  • a CU1276-matching tRN A locus was cloned into an expression vector, and transiently transfected this vector into HEK-293T cells (293T).
  • Northern Biot analy sis of these cells re vealed a clear increase in both the mature tRNA and a 22nt band co-migrating with the endogenous fragment observed in B cells (FiglC), suggesting that CU1276 is indeed tRNA-derived.
  • CU1276 expression was confirmed from a second, independent Gly(GCC) tRNA locus, and the possibility of expression from a candidate precursor genomic locus (6) closely matching the CU 1276 sequence, but not encoding a tRN A (Fig6) was ruled out.
  • tRNA do not meet the structural criteria of a classical DICERl substrate ( 16): nonetheless, due to the observed similarities in size between CU1276 and DICER /-dependent miRNA, this enzyme could be involved in CUT276 biogenesis. Therefore, a CU.1276- matching tRNA was transiently overexpressed in 293T cell lines stably expressing either a control shRNA or a pool of three DICERl -targe ting shR A.
  • CU1276 associates with all four human Argonaute proteins, and functions as a miRNA. As a prerequisite for investigating a possible miRNA-like function of CU1276, it was determined whether this small RNA was physically associated with Argonaute proteins. Utilizing a monoclonal antibody with reactivity against all four human Argonaute proteins (17), Argonaute-associated RNAs were purified from the B cell line R1VA; qRT- PCR analysis of the co-precipitated RNA confirms that CU1276 is indeed enriched in the pan-Ago immunoprecipitation (IP) fraction relative to a control IP (Fig2A).
  • IP pan-Ago immunoprecipitation
  • IP of HA-tagged versions of human AGOl , AG02, AG03, and AG04 transiently expressed in 293T cells were performed; the results demonstrate that CU1276 is enriched in the IP fractions of each Argonaute protein relative to that of the HA-EGFP control, indicating that it is specifically incorporated into silencing complexes containing each of the four human Argonautes (Fig2B).
  • the dynamics of this interaction can be influenced by the availability of unoccupied Argonaute complexes, given that the magnitude of CU 1276 enrichment in AGO I complexes increased proportionally with the total levels of this protein, meeting and eventually exceeding the enrichment of the canonical miRNA miR- 16 at high levels of AGO l expression (Fig2C).
  • CU1276 Given its demonstrated binding to the functional effectors of miRNA signaling, the effect of CU1276 was tested on a firefly luciferase reporter bearing two antisertse binding sites in its 3 'UTR. Because overexpression of full-length tRNA inevitably produces a complex mixture of RNA molecules, including a previously reported ⁇ -34nt 5' tRN A fragment capable of broadly repressing translation (18), CUI276 was also cloned into a miRNA hairpin in order to investigate its activity in a context free from confounding factors. Expression of either the tRNA or hairpin was indeed sufficient to repress the antisense reporter (Fig2D).
  • CU1276 is downregulated m lymphoma cell lines and primary biopsies.
  • GC structures are the cell of origin for the majority of B ceil lymphomas (20).
  • initial small R A sequencing and Northern Blot analysis suggested that CU1276 is abundantly expressed in normal GC B cells, but is low in at least one GC-derived lymphoma cell line (FiglA, C).
  • GUI 276 was assessed by qRT-PCR in normal GC B ceils and a panel of GC-derived lymphoma cell lines. Strikingly, only normal GC B cells efficiently expressed this small RNA., while the entire pane! of cell lines displayed low CU1276 levels (Fig3A).
  • the Gly(GCC) tRN A from which CU1276 is derived can be found in at least five distinct genomic loci (FiglB), making it an unlikely candidate for genetic deletion. Indeed, each ceil line tested expresses roughly equal levels of the mature, post- transcriptionally modified form of the tRNA from which CU1276 is derived (Fig7), indicating that the observed decrease in CU1276 production in lymphomas is probably regulated at the level of tRNA cleavage.
  • G UI 276 represses a set of endogenous genes, including RPA1. Genes were identified both significantly downregulated by CU1276 expression and computationally predicted to contain CU 1276 binding sites in their 3'UTR (21). Without being bound by theory, expression of mature Gly(GCC) tRN A can induce changes in gene expression due to CU1276-independent effects on translation; therefore, in order to focus on the most physiologically relevant CU1276-specific targets, expression profiles were compared from 293T cells tratisfected with empty, tRNA-expressing, or GUI 276 hairpin-expressing vectors. This analysis revealed a modest, but statistically significant overlap between the
  • a stable B cell lymphoma line was constructed carrying a vector with a doxycycline-mducible bidirectional promoter encoding for GFP alone, or GFP plus GUI 276 hairpin; induction of GUI 276 repressed both endogenous RPAl protein and RPAl mRNA relative to control eelis (Fig4B, Fig9C-D).
  • RPAl is a bona fide target of the tR A-derived miRNA GUI 276.
  • CU1276 suppresses proliferation and modulates the molecular response to DNA damage in an HAll-dependent manner.
  • RPAl has a number of well -characterized roles in DNA dynamics, including in replication and DNA. repair (23). Without being bound by theory, through repression of RPAl, CU1276 might influence cellular proliferation and the response to DNA damage.
  • CU1276 expression is also capable of modulating the molecular response to DNA. damage.
  • Robust expression of CU1276 is sufficient to sensitize a Burkitt Lymphoma cell line to Etoposide-induced DNA damage, as measured by an increase in H2AFX phosphorylation relative to controls (FigSB). Similar to the CU1276-induced reduction in cellular proliferation, this sensitization can be fully rescued by restoration of RPAl protein to wild-type levels (FigSC), confirming that RPAl is also the critical CUI276 target responsible for this effect. [80125] Discussion
  • tRNAs and tRNA fragments have the capacity to regulate a surprising range of cellular processes, including translational efficiency under stress conditions (18), mitochondrial-mediated apoptosis (24), and oncogenic transformation (25).
  • the data presented further extend the regulatory repertoire of tRNA, and greatly expand the pool of candidate miRNAs, by demonstrating that a tRNA fragment can post-transcriptionally regulate endogenous genes in a sequence-specific, miRNA-like fashion.
  • Gly(GCC) tRNA expression is largely constant between samples with highly discordant GUI 276 levels (Fig7 and Fig3A), ihe defect in GUI 276 biogenesis is likeiy to lie at the level ofDiCERl cleavage.
  • HBLI highly discordant GUI 276 levels
  • Fig4C DiCERl protein
  • GUI 276 Because of its ability to regulate endogenous target genes, GUI 276 was shown to repress endogenous RPA1 (Fig4A, B).
  • RPA1 is an essential gene for many aspects of DNA dynamics, including genome replication. Consequently, stable CU1276 expression in a Burkitt Lymphoma-derived cell line results in an RPA1 -dependent suppression of their proliferation rate (FigSA).
  • FigSA RPA1 -dependent suppression of their proliferation rate
  • GC B-ceils undergo several physiological processes of somatic mutation and DNA rearrangement (20).
  • RPAl is a required component for some types of DNA repair, and additionally has a OC-specific role in facilitating AICDA-mcdiated mutagenic processes (27, 28).
  • Physiological expression of CU 1276 in the GC can contribute to fine -tuning of RPAl levels in GC B cells, and can thereby indirectly influence the efficiency of DNA repair, somatic hypermutation, and class-switch recombination.
  • CU1276 expression in a Burkitt Lyraphoraa-derived cell line results in an RPA /-dependent sensitization of the molecular response of these cells to DNA damage, as indicated by phosphorylation of H2AFX upon Etoposide treatment (FigSB, C).
  • the tRNA-derived miRNA CU1276 can be a new genetic participant in the modulation of DNA damage response pathways in the GC.
  • loss of CU1276 expression in lymphomas may decrease their sensitivity to ongoing DNA damage, thereby helping them to tolerate the accumulation of mutations and genomic aberrations during tumor evolution.
  • 293T-shCtrl and 293T-shDICER stable cell lines were established by transfection with pLKO-based vectors (see Supplementary Methods for details of plasmids and cloning information) followed by selection for 4 days with 2 ⁇ tg/ml puromycin.
  • P3HR1 stable cells were established by electroporation of exponentially growing cells with 5pmol of pRTS l -GLS VP-based vectors according to standard protocol. After a 48hr recovery in IMDM supplemented with 20% FBS, cells were selected with 0.5fig/nil puromycin for 4 days.
  • Induction of expression from stable P3HR1 cells was achieved by addition of doxycyciine to growth media at a concentration of lOOng/ml.
  • DNA damage response of stable P3HR1 ceil lines was assayed by pre-induction with doxycyciine for 24 hours, followed by treatment with ⁇ , ⁇ ⁇ , 2 ⁇ , or ⁇ concentrations of Etoposide (Sigma) for 3 hours.
  • QRTPCR was also performed as previously described (Basso et al., 2009), starting from 2 ⁇ g total RNA or a percentage of RNA from IP fraction.
  • QPCR analysis of cDNA was performed with ABsoIute Blue Sybr Green Master Mix (Thermo Scientific) using an AB7300 thermocycler (Applied Biosystems).
  • Argonaute Immunoprecipitation was performed as previously described (17), starting from 1X10 s exponentially growing RIVA cells or -5X10' 293T cells, with anti-pan- AGO antibody (MABE56, Millipore), or control IgG overnight with rotation at 4°C.
  • Protein G magnetic beads New England Biosciences
  • HA- tagged proteins were imm tioprecipitated by overnight incubation of lysate with EZ vie lM HA affinity beads (Sigma). Beads were washed and resuspended in Trizol Reagent (lnvitrogen) or lysis buffer for downstream RNA and protein analysis, respectively.
  • Gene expression profiling and data analysis were generated from total RNA using the HG-U133Plus2.0 platform (Affymetrix) according to the manufacturer's indications. Differential expression was determined by t-test using the geWorkbench software suite (30), with a significance cut-off of p ⁇ 0.05.
  • RNA libraries were generated using the SOLiD Small RNA Expression Kit (Applied Biosystems) following the manufacturer's indications. SOLID sequencing was performed on 4 libraries of purified GC B cells and 25 libraries of DLBCL.
  • Tetrahymena Piwi protein carries tRNA fragment cargo. Genes & development 24(24):2742-2747.
  • Argonaute2 is the catalytic engine of mammalian RNAi. Science 305(5689): 1437- 1441.
  • Plasmids and shRNA Expression vectors for transient transfection of tRNA were generated by PCR amplification of Gly(GCC) chrl .tRNA68 (chrl : 161493501- 161493953), Gly(GCC) chrI7.tRNA5 (chrl 7:8028941-8029251 ), and a CU1276 predicted genomic precursor (chrl 9:3 1 15779-351 16077) followed by insertion into the multiple cloning site of pcDNA.3 expression vector (Invitrogen).
  • CU1276 hairpin-expressing vector was generated by restriction digestion of pcDNA3 vector containing the human miR-26a-l locus (chr3:38()10684-3801 1069) with ael and Btgl to remove the miR-26a-l hairpin; annealed oligos with sequences 5"-
  • GCACTTGGTGCATTGGCCGGGAATCGACGAGGCC-3' (SEQ ID NO: 4) were ligated in its place.
  • An inducible CU1276 hairpin-expressing vector was created by restriction digest of the pc ' DNA3-CUl 276_hairpin vector and subsequent subcloning into the Sfil sites of pRTSl- GLSVP vector (SI ).
  • Self-ligated pRTSl-GLSVP vector containing no insert was used as an empty vector control.
  • i-pui -shDICER-61 and pLKO.
  • l-puro-shDICER-62 were purchased from Open Biosystems.
  • pIRESneo-HA-FLAG- AGO I , -AG02, -AG03, -AG04, and -EGFP were obtained from Addgene (S2).
  • the RPA1, WHSC1L1 , and STAG2 3'UTR reporter constructs were generated by PGR amplification of human genomic DNA, followed by insertion into the multiple cloning site of the pmiRGLO vector (Promega).
  • RPA1 3'UTR- MUT reporter was generated by site-directed mutagenesis with primers 5'- CAAATAGGCATAAlTTCCTATATTTCCTCCCACCTCCG-3' (SEQ ID NO: 5) and 5'- GGAAATATAGGAAATTATGCCTATTTGCAAACTTCTGC-3' (SEQ ID NO: 6); STAG2 3'UTR-MUT reporter was generated by site-directed mutagenesis with primers 5'- GCTGTTAGTTGGCTTTTTCCTATATTATTTCATGCTT-3 ' (SEQ ID NO: 7) and 5'- GAAATAATATAGGAAAAAGCCAACTAACAGCGCATAAATAAAATA-3' (SEQ ID NO: 8).
  • RNA samples were reverse-transcribed with the Superscript II First Strand S nthesis Kit (Invitrogen) in the presence of 0.2.uM RTFS primer (5'-TGTCAGGCAACCGTATTCACCGTGAGTGGTTGGTGCATTG-3'; (SEQ ID NO: 9)), random hexamers, or Oligo d(T) primer according to the manufacturer's indications.
  • 1/10TM of the cDNA volume was used as a template for PGR amplification in the presence of 70nM SS primer (5 ' -CGTCAGATGTCCGAGTAGAGGGGG AACGGCGTCGATTCCCGGC-3 ' ; (SEQ ID NO: 10», and 70nM each of MPF (5 ' -TGTCAGGC AACCGTATTC ACC-3 ' ; (SEQ ID NO: 1 1)) and MPR (5'-CGTCAGATGTCCGAGTAGAGG-3'; (SEQ ID NO: 12)) universal primers, or gene specific primers, as appropriate.
  • 5s rRNA and RNU66 were detected from cDNA generated by random hexamer reverse transcription, according to gene-specific primers (5srRNAF: 5'-GCCCGATCTCGTCTGATCT-3' (SEQ ID NO: 13), 5srRNAR: 5'-AGCCTACAGCACCCGGTATT-3' (SEQ ID NO: 14), RNU66F: 5' - GGTGATGGAAATGTGTTAGCC-3 ' (SEQ ID NO: 15), RNU66R: 5'- AGGATAGAAAGAACCACCTCA-3 '(SEQ ID NO: 16)).
  • RPA I, RPA2, and GAPDH were detected from cDNA generated by Oligo d(T) reverse transcription, according to gene- specific primers (RPA1F: 5 '-CTTCACGTCC ATCACAGTGG-3 ' (SEQ ID NO: 17), RP .1R: 5 '-TTTCC AGAATGCCAACTTCC-3 ' (SEQ ID NO: 18), RPA2F: 5'- AGGGAGAGCACCTATCAGCA-3' (SEQ ID NO: 19), RPA2R: 5'- TTCAACCCTTCAGGTCTTGG-3 ' (SEQ ID NO: 20), GAPDHF: 5'- CTGACTTCAACAGCGACACC-3 ' (SEQ ID NO: 21 ), GAPDHR: 5'- CCCTGTTGCTGTAGCCAAAT-3' (SEQ ID NO: 22)).
  • HRP- conjugated secondary antibodies anti-mouse IgG HRP (NA931V; GE Healthcare), anti-goat IgG HRP (2020; Santa Cruz), and anti-rabbit IgG HRP (1238850; Boehringer Mannheim) were diluted at 1 : 10000 in PBS with 0.1% Tween20, 5% milk, and used for 30mins-3hr incubations at room temperature. Blots were visualized with ECL substrate or SuperSignal West Dura ECL substrate (Thermo Scientific). Where applicable, blots were quantified using the ImageJ software suite (S3).

Abstract

The invention provides for methods of treating a blood cancer in a subject. The invention further provides methods for treating blood cancer cell invasion, migration, and proliferation.

Description

METHODS OF MODULATING PROLIFERATION AND USES THEREOF 8001] This application claims priority to U.S. Provisional Patent Application No. 61/746,336 filed December 27, 2.012, the contents of which are hereby incorporated by reference in their entireties.
[8002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The discl osures of these publications in their entireties are hereby incorporated by reference into this application.
[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
GOVERNMENT INTERESTS
[8004] The work described herein was supported in whole, or in part, by National Cancer Institute Grant No. R01-CA109755 "Genetic Network Interference with
Combinatorial Phenotypes", and National institute of Allergy and infectious Diseases Grant No. R01 AI0661 16 "Regulatory Modules in Normal and Transformed b-Cell". The
Government has certain rights to the invention,
BACKGROUND OF THE INVENTION
[8005] MicroRNAs (miRNAs, miRs) are 20-23 nucleotides (nt) RNA molecules that are produced by the processing of a larger enclosing stem- loop structure (>50bp), called precursors, by cellular enzymes. miRNAs are processed from hairpin precursors of 70 nt (pre-miRNA) which are derived from primary transcripts (pri-miRNA) through sequential cleavage by the RNAse III enzymes drosha and dicer. miRNAs target the messenger RNA of other genes by binding to their 3' UTR and interfering with their translation or causing degradation by enzyme targeting double-stranded RNA. miRNAs are non-coding RNAs (ncRNAs) that exist in a variety of organisms, including mammals, and are conserved in evolution. Many miRNAs tend to be ciusiered and transcribed as poiycistrons and often have similar spatial temporal expression patterns. miRNAs have been implicated in various biological processes including developmental timing, differentiation, apoptosis, cell proliferation, organ development, and metabolism.
SUMMARY OF THE INVENTION
[8(506] The invention is based, at least in part, on the discovery of a miRNA composition (GUI 276) that is useful for the treatment of a blood cancer (e.g., a lymphoma) and/or the amelioration of symptoms associated with the blood cancer. Accordingly, one aspect of the invention features a method for treating a blood cancer in a subject in need thereof. Tn one embodiment, the method comprises administering to the subject an effective amount of a nucleic acid composition comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , thereby treating the blood cancer in the subject. In another embodiment, the nucleic acid is a fragment of SEQ ID NO: 1. In a further embodiment, the blood cancer is a lymphoma. In yet another embodiment, the blood cancer is a B cell lymphoma.
[8007] One aspect of the invention provides a method for decreasing proliferation of a blood cancer cell. In one embodiment, the method comprises delivering to a cell an effective amount of a nucleic acid composition comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1, thereby decreasing proliferation of the cancer cell. In another embodiment, the nucleic acid is a fragment of SEQ ID NO: I . In a further embodiment, the blood cancer is a lymphoma. In yet another embodiment, the blood cancer is a B cell lymphoma. In some embodiments, the proliferation comprises cell invasion, cell migration, or a combination of either activity.
[0008] An aspect of the invention features a method for reducing the number of blood cancer cells. In one embodiment, the method comprises delivering to a cell an effective amount of a nucleic acid composition comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , thereby reducing the number of cancer cells. In another embodiment, the nucleic acid is a fragment of SEQ ID NO: 1 , In a further embodiment, the blood cancer is a lymphoma. In yet another embodiment, the blood cancer is a B cell lymphoma.
[8009] An aspect of the invention provides for a method of decreasing growth of a solid tumor in a subject in need thereof. In one embodiment, the method comprises administering to the subject an effective amount of a nucleic acid composition comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , wherein the nucleic acid composition decreases the size of the solid tumor. In another embodiment, the nucleic acid is a fragment of SEQ ID NO: 1. In a further embodiment, the solid tumor is a lymphoma.
[8(518] An aspect of the invention features a method for treating a B cell lymphoma in a subject in need thereof. In one embodiment, the method comprises administering to the subject an effective amount of a nucleic acid composition comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , thereby treating B ceil lymphoma in the subject. In another embodiment, the nucleic acid is a fragment of SEQ ID NO: 1.
[8(511] An aspect of the invention provides for a diagnostic kit for determining whether a sample from a subject exhibits a presence or absence of a cancer-associated tRNA. fragment, in one embodiment, the kit comprises at feast one oligonucleotide that specifically hybridizes to a nucleic acid comprising about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , or a portion thereof. In another embodiment, the cancer-associated tRNA fragment comprises SEQ ID NO: 1 . In a further embodiment, the cancer-associated tRNA fragment comprises CU1276. In yet another embodiment, the oligonucleotide comprises a set of nucleic acid primers. In some embodiments, the oligonucleotide comprises a probe. In further embodiments, the probe that detects the presence of a tRN A-derived microRNA comprising CUS 1276. In other embodiments, the probe comprises about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID N O: 2. In one embodiment, the oligonucleotide comprises at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, or at least 15 consecutive nucleotides comprising SEQ ID NO: 3 or 4. In another embodiment, the absence of cancer-associated tRNA is indicative if a blood cancer. In a further embodiment, the blood cancer is a lymphoma. In yet another embodiment, the blood cancer is a B cell lymphoma.
[8012] An aspect of the invention features a method for detecting the presence of a cancer-associated tRNA fragment in a human subject. In one embodiment, the method comprises obtaining a biological sample from a human subject; and detecting whether or not a nucleic acid sequence comprising about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of SEQ ID NO: 1 , or a portion thereof, is present in the subject. In a further embodiment, the cancer- associated tRNA fragment comprises CU1276. In another embodiment, the absence of cancer- associated tRNA is indicative if a blood cancer. In a further embodiment, the blood cancer is a lymphoma. In yet another embodiment, the blood cancer is a B cell lymphoma. In some embodiments, the detecting comprises using hybridization, amplification, or sequencing techniques to detect the presence of absence of a cancer-associated tRNA fragment. In further embodiments, the amplification uses primers comprising at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, or at least 15 consecutive nucleotides comprising SEQ ID NO: 3 or 4.
BRIEF DESCRIPTION OF THE FIGURES
[8(513] To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color. The original color versions of Figures 1- 10 can be viewed in Maute et al, (2013) Proc Natl Acad Set U S A, 1 10(4): 1404-9 (including the accompanying Supplementary Information av ailable in the on-line version of the manuscript available on the Proceedings of the National Academy of Science web site). For the purposes of the PCT, the contents of Maute et al., (2013) Proc Nail Acad Sci U S ,4, 110(4): 1404-9, including the accompanying
"Supplementary Information," are herein incorporated by reference.
[8014] Figure 1A-1B. CU1276 is a Dicer-dependent tRNA fragment expressed in mature B cells. (lA) Cloning frequency of CU 1276 from Naive (N), Germinal Center (GC), and Memory (M) B cells purified from human tonsil, and from the Burkit s Lymphoma cell line Ramos (RA). Data is derived from analysis of previously published small RNA libraries (Basso et al., 2009). (IB) FASTA sequences of annotated human tRNAs with perfect match to the CU 1276 small RNA. CU1276 sequence is highlighted in bold.
[8015] Figure 1C-1D. CU1276 is a Dicer-dependent tRNA fragment expressed in mature B cells. (lC) Northern blot analysis of total R from GC B cells, Ramos, and 293T cells transiently transfected with empty vector or a vector encoding for the Gfy(GCC) chri .tRNA68. The three primary bands correspond to the 22nt tRNA fragment CU1276, the 74nt mature tRN A, and a high molecular weight tRNA primary transcript. See also FIG. 6. (ID) Western blot and Northern blot analyses of 293T cells stably expressing control shRNA (shCTRL) or a pool of three Dicer- targeting shRNA (shDicer), transiently transfected with empty or Gly(GCC) chrLiRNA68 vector. Ethidium Bromide (EtBr) staining and immunoblotting for AC'T'B were used as loading controls for Northern blot and Western blot, respectively.
[0016] Figure 2A-2D. CU1276 is bound by all four rusman Argonaute proteins and functions as a miRNA. (2A) qRT-PCR of CU1276 in pan-Ago and control
immunoprecipitaiion (IP) fractions from RIVA cells, (2B) qRT-PCR of CU1276 in HA IP fractions from 293T cells transiently expressing equivalent levels of HA-tagged EGFP, AGOl , AG02, AG03, or AG04 proteins, and (2C) qRT-PCR ofmiR-16 and CU1276 in IPs from 293T cells expressing HA-tagged EGFP, or increasing amounts of HA-tagged AGOl. In all qRT-PCR graphs, values were normalized to 5s rRNA and plotted relative to control IP levels. Error bars represent the standard deviation of triplicate qRT-PCR reactions. (2D) Antisense 3'UTR reporter activity in response to CU1276, with or without exogenous AG02 expression. Firefly luciferase values were normalized io a Renilla iuciferase control, and plotted relative to reporter co-transfected with empty vector; error bars represent the standard deviation of two independent experiments, each performed in duplicate.
[0017] Figure 3A-3B. OJ1276 is downregulated in lymphoma cell lines and primary biopsies. (3A) qRT-PCR of CU1276 in Germinal Center (GC) samples and B cell lymphoma lines, including Burkitt Lymphoma (BL), Activated B Cell-like Diffuse Large B Cell Lymphoma (ABC-DLBCL), and GC-like Diffuse Large B Cell Lymphoma (GCB- DLBCL); qRT-PCR levels were normalized to RNU66, and graphed relative to GCl. Error bars represent the standard deviation of triplicate PCR reactions. See also FIG. 7. (3B) CU1276 counts from deep sequencing of small RNA libraries from purified GC (n=4) and primary biopsies of DLBCL (n=25). The panel of DLBCL includes A BC-DLBCL (n=13) and GCB-DLBCL (n=12) subtypes.
[8018] Figure 4A. ClJl 276 directly represses RPA1. (4Λ) (Top) Schematic representation of the region in the 3'UTR of RPA1 targeted by CU1276. The mutations introduced into this region are highlighted in gray. (Bottom) RPA1 3'UTR reporter activity in response to CU1276 expression. Firefly luciferase values were normalized to a Renilla Luciferase control, and plotted relative to reporter co-transfected with empty vector; error bars represent the standard deviation of seven independent experiments, each performed in duplicate. The reporter is significantly repressed by either Gly(GCC) chrl .iR A68- (Student's t-test, p=3.9E-5) or hairpin-mediated delivery (Student's t-test, p=8.2E-5) of CU1276.
[8019] Figure 4B-4C. CU1276 directly represses RPAl, (4B) Western blot analyses of RPAl and RPA2 from 293T cells transiently transfected with empty, Gly(GCC) chrI .tRNA68-expressing~, or CUI276 hairpin-expressing vector, and from stable lines of the Burkitt Lymphoma cell line P3HR1 engineered to inducibly express GFP (empty ) or GFP plus GUI 276 (GUI 276), GFP indicates successful doxycycline induction of vector, ACTB was used as loading control. Images are representative of four independent experiments each; See also FIG. 9. (4C) Western blot analysis and graphical quantification of RPAl , RPA2, and DICERl, with ACTB used as loadmg control, from normal germinal center (GC) B cells and a panel of GC-derived lymphoma cell lines, including Burkitt lymphoma (BL), activated B cell-like diffuse large B Cell Lymphoma (ABC-DLBCL), and GC-like diffuse large B cell lymphoma (GCB-DLBCL). The GC sample was obtained by pooling cells from two independent donors. Ail samples are identical to those used for CU1276 expression analysis in Figure 3.4.
[8028] Figure SA. CLI1276 modulates proliferation and DNA damage signaling in an RPAl-dependent manner, (SA) (Top) Growth curves of P3HR1 stable cell lines containing bidirectional, doxycycline-iiiducible vectors expressing GFP alone (blue line), GFP plus the CU 1276 hairpin (red line), or RPAl plus the CU1276 hairpin (orange line), and (Bottom) corresponding Western blot analysis of RPAl protein levels from these cell lines, with ACTB used as loading control. Growth curve data is compiled from 8 independent experiments, with each genotype represented by 4 independently derived bulk populations. Error bars represent the 95% confidence intervals of each cell type, calculated according to a normal distribution. CU1.276 expression is sufficient to significantly reduce cellular proliferation relative to the GFP control at 96 hours (Student's t-test, *p=1.8E-3). At 96 hours, RPAl rescue restores growth completely to wild-type levels.
[8021] Figure SB. CU1276 modulates proliferation and DNA damage signaling in an RPAl-dependent manner. (SB) Western blot analysis of RPAl, total H2AFX, and yH2AFX in Etoposide-treated control cells and cells expressing CU1276. ACTB was used as loadmg control. Image is representative of three independent experiments, for which average yH2AFX quantifications are indicated in bar chart format. Error bars represent the standard deviation of three independent experiments. [8(522] Figure SC. €111276 modulates proliferation and DNA damage signaling in an RPAl-dependent manner, (5C) Western blot analysis of control cells, ceils expressing CU1276, and cells simultaneously expressing CU 1276 and exogenous RPAl. Restoration of RPAl protein levels rescues CU1276-mediated sensitization of H2AFX phosphoiylation upon Etoposide treatment. ACTB was used as loading control Image is representative of three independent experiments, for which average vH2AFX quantifications are indicated in bar chart format. Error bars represent standard deviations.
[0023] FIG. 6 shows CU 1276 is expressed from two independent tRNA loci. Northern blot of total RNA from purified Germinal Center (GC) B cells, the Burkitt's Lymphoma cell line Ramos, and 293T ceils transiently transfected with empty vector, a vector encoding for Gly(GCC) chrl7.tRNA5, a vector encoding for a predicted genomic precursor for GUI 276, or a vector encoding for Gly(GCC) chrl .tRNA68. Both t NA-encoding vectors were sufficient to express an ~22nt band which co-migrates with endogenous GC-expressed CU1276.
[8024] FIG. 7 shows Normal Germinal Center B cells and B cell lymphoma lines express similar levels of Gly(GCC) tRNA. Northern blot analysis of total RNA from purified Germinal Center (GC) B cells, and from a panel of GC-derived lymphoma cell lines, including Burkitt's lymphoma (BL), Activated B Cell-like Diffuse Large B Cell Lymphoma (ABC-DLBCL), and GC-like Diffuse Large B Ceil Lymphoma (GCB-DLBCL) subtypes. Membranes were blotted with a radioactive probe complementary to the 3' end of mature Gly(GCC) tRNA. Ethidium bromide staining was used as loading control. The majorit '- of cell lines express levels of tRNA similar to those observed in GC B cells, suggesting that any deficiency in CU1276 expression is likely due to mechanisms acting downstream of transcriptional regulation.
[8(525] FIGS. 8A-8B show an experimental schematic of CU 1276 target candidate identification, and validation of CU1276 targets. (HA) 293T ceils were transiently transfected with empty vector, chri .tRNA68, or CUT 276 hairpin-encoding vectors. 48 hours post- transfection, ceils were harvested, and their extracted total RNA was used for gene expression profiling with Affymetrix HG-U133Plus2.0 arrays. Genes that were significantly downregulated (threshold p<0.05) in tR A-, and/or in hairpin-expressing cells relative to empty vector transfected cells, were considered for further analysis. The statistically significant overlap (p<lE-40) between fRNA-downregulated and hairpin-downregulated probes confirms that hairpin delivery of CU1276 recapitulates its physiological activity on at least a subset of transcripts. (SB) O verlap of genes containing Target Scan-predicied CU1276 binding sites in their 3'UTR with those significantly downregulated (threshold p<0.05) upon expression of CU1276. Significant enrichment (Hypergeometric test, p=8.6E-8) was observed for CU1276 TargetScan-predicted targets among the genes downregulated by tRNA and 'or hairpin expression.
[8026] FiGS. 8C-8D are bar graphs. (8C) WHSC 1L1 3 'UTR reporter activity in response to Gly(GCC) chrl .tRNA68-delivered or hairpin-delivered CU1276. The
WHSC1 L1 3 'UTR was not sensitive to repression by either tRNA-mediated or hairpin- mediated expression of GUI 276. Mutational analysis of the predicted binding site for CU1276 was not pursued. Firefly luciferase values were normalized to a Renilla luciferase control, and plotted relative to reporter activity upon co-transfeetton of empty vector. Error bars represent the standard deviation of three independent experiments, each performed in duplicate. (8D) STAG2 3 'UTR reporter activity in response to Gly(GCC) chrl.tRNA68- or hairpin-delivered CU1276. The STAG2 3 'UTR is strongly repressed by hairpin-mediated delivery of CU1276, with high significance (Student's t-test, p=3.8E-7), demonstrating that the CU1276 sequence does indeed interact with this 3 'UTR. However, the effect of tRNA expression was ambiguous, showing robust repression in some experiments, and no repression in others. Although this repression did eventually reach a minimal threshold of significance (Student's t-test, p=3.6E-2), the low magnitude of repression suggests that STAG2 may be only weakly targeted by tRNA-delivered CU1276 in this cellular context. Firefly luciferase values were normalized to a Renilla luciferase control, and plotted relative to reporter activity upon co-transfection of empty vector. Error bars represent the standard deviation of ten independent experiments, each performed in duplicate.
[8027] FIG. A-9B. Western Blot quantifications and qRT-PCR afRPAl upon CU1276 expression. (9A) Western Blot quantification of RPA1 and RPA2 from 293T ceils iransienily transiected with either Gly(GCC) tRNA-expressing (iRNA), or CU1276 hairpin- expressing (Hairpin) vector; values are normalized to ACTB expression; error bars are the standard deviation of four independent experiments. (9B) qRT-PCR of RPAl and RPA2 mRNA levels in 293T cells transiently transiected with iRNA- or Hairpin-expressing vector: values are normalized to GAPDH expression; error bars are the standard deviation of three independent experiments. RPAl is not significantly repressed at the mRNA level in this cellular context.
[8028] FIG. 9C-9D. Western Blot quantifications and qRT-PCR of RPAl upon CU1276 expression. (9C) Western Blot quantification of RPAl and RPA2 from stable P3HR 1 cells that express either GFP alone (Empty) or GFP plus GUI 276 hairpin (GUI 276) upon doxycycline (Dox) treatment; values are normalized to ACTB expression; error bars are the standard deviation of four independent experiments. (9D) qRT-PCR of RPAl and RPA2 mRN A levels in stable P3HR1 cells that express either GFP alone (Empty) or GFP plus CU1276 hairpin (GUI 276) upon doxycycline (Dox) treatment; values are normalized to GAPDH expression; error bars are the standard deviation of four independent experiments. RPAl mRNA is significantly repressed by CU1276 expression (p=1.3E-3).
[8(529] FiG. 18 shows Germinal Center cells and Diffuse Large B Cell Lymphoma cell lines express similar levels of RPAl mRNA. N ormalized RPAl mRN A expression in Germinal Center cells (N=5) and a panel of Diffuse Large B Cell Lymphoma (DLBCL) cell lines (N-8; HBL1, Ly3, SUDHL2, RIVA, Ly7, SUDHL4, SUDHL6, and Val). Expression values were measured by Affymetrix HG-U133p2 GeneChip® expression arrays. There is no significant difference between the two sample groups.
DETAILED DESCRIPTION OF THE INVENTION
[8038] This invention provides for the discovery of cancer-associated tRNA fragments. One of these fragments is a tRNA -derived microRNA (e.g., CU1276) that is downregulated in the blood cell cancer B cell lymphoma.
[8031] SEQ ID NO: 1 is the nucleic acid sequence of GUI 276:
TCGATTCCCGGCCAATG CACCA.
[0032] Lymphomas
[8033] A lymphoma is a type of blood cancer that begins in cells of the immune system (e.g., lymphocytes such as B cells and T cells). Lymphomas are classified according to certain histological characteristics: the site the cell arises from; the presence of a Reed- Sternberg cell; and whether the cell that is replication is a B cell or T cell. These
characteristics are used to further categorize this cancer. [8(534] There are two categories of lymphomas: (1) Hodgkin lymphoma (HL; marked by the presence of a type of cell called the Reed-Stemberg cell); and (2) non-Hodgkin lymphoma (NHL). NHL can be further divided into cancers that have an indolent (slow- growing) course and those that have an aggressive (fast-growing) course. Both Hodgkin and non-Hodgkin lymphomas can occur in children and adults, and prognosis and treatment depend on the stage and the type of cancer.
[8(535] Non-limiting examples of lymphomas include, but are not limited to Precursor T-cell leukemia/lymphoma. Follicular lymphoma, Diffuse large B cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia/lymphoma, MALT lymphoma, Burkitt's lymphoma, Mycosis fungoides, Peripheral T-cell iymphoma-Not-Otherwise-Specified, Nodular sclerosis form of Hodgkin lymphoma, and Mixed-eeiiuiarity subtype of Hodgkin lymphoma.
[0036] microRNAs
[8037] MicroRNAs (miRNAs) are naturally-occurring 19 to 25 nucleotide transcripts found in over one hundred distinct organisms (such as nematodes, fruit files, and humans). miRNAs can be processed from 60- to 70-nucleotide foldback RNA precursor structures, which are transcribed from the miRNA gene. The miRNA precursor processing reaction requires Dicer RNase 111 and Argonaute family members (Sasaki et al., 2003 Genomics 82, 323-330). The miRNA precursor or processed miRN A products are easily detected, and an alteration in the levels of these molecules within a cell can indicate a perturbation in the chromosomal region containing the miRNA gene. A further review of miRN As is provided in U.S. Patent No. 7,232,806, U.S. Patent Application Publication No. 2.006/0105360, and in the references: Landgraf et al., 2007, Cell 129: 1401 -1414; Mendell, XT, 2005 Cell Cycle 4(9): 1179-84; Shivdasani RA, 2006 Blood 108(12):3646-53; Hwang and Mendell, 2006 Br J Cancer 94(6):776-80; Hammond SM, 2006; Curt Opin Genet Dev. 16(l):4-9; Osada and Takahashi, 2007 Carcinogenesis 28(i):2-12; and Zhang et al., 2007 Dev Biol. 302(1):1-12, all of which are hereby incorporated by reference in their entirety.
[8038] The unprocessed miRN A gene transcript is called a miRN A precursor (pre- miRNA) and comprises an RNA transcript of about 70 nucleotides in length. The pre-miRNA can be processed by digestion with an RNAse (such as, Dicer, Argonaut, or RNAse 111, e.g., E. coli R Ase III)) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the processed miRNA gene transcript.
Nucleic Aci M Thereof
[8(539] The practice of aspects of the present invention can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Molecular Cloning A Laboratory Manual, 3rQ Ed., ed. by Sambrook (2001), Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N, Glover ed., 1985); Qli gonucleoti.de Synthesis (M. J. Gait ed., 1984); MuJlis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984): Culture Of Animal Cells (R. I.
Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In Enzymology (Academic Press, Inc., N.Y.), specifically, Methods In Enzymology, Vols. 154 and 155 (Wu ei al. eds.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunoch emicai Methods In Cell And Mo) ecu lar Biology (Caner and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C, Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). All patents, patent applications and references cited herein are incorporated by reference in their entireties.
[8048] The active 19-25 tRNA-derived micro RNA can be obtained from the miRNA precursor through natural processing routes (for example, using intact cells or cell lysates) or by synthetic processing routes (for example, using isolated processing enzymes, such as isolated Dicer, Argonaut, or RNAase III). The active 19-25 tRNA-derived microRNA can also be produced directly by biological or chemical syntheses, without having been processed from the miRNA precursor. In one embodiment, the nucleic acid is the tRNA-derived microRNA. CU1276. In some embodiments, the nucleic acid is about 70%, about 75%, about 80%, about 85%, about 90%, about 93%, about 95%, about 97%, about 98%, or about 99% identical to SEQ ID O: 1. For example, an isolated nucleic acid, such as tRNA-derived microRNA CU1276, can be synthesized, or altered, or removed from the natural state through human intervention. A synthetic miRN A, or a miRNA partially or completely separated from the coexisting materials of its natural state, is considered isolated. An isolated miRNA can exist in substantially purified form, or can exist in a cell into which the miRNA has been delivered.
An isolated nucleic acid, such as a miRNA of the invention, can be obtained using a number of standard techniques utilized in the art. For example, the miRNA gene products can be chemically s nthesized or recombinantly produced using methods known in the art. For example, a miRN A can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dhannacon Research (Lafayette, Colo., USA), Rosetta Genomics (North
Brunswick, NJ), Pierce Chemical (pari of Perbio Science, Rockford, 111., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), Ambion (Foster City, (Ά . USA), and Cruaehem (Glasgow, UK).
A eukaryotic expression vector can be used to transfect cells. Mammalian c can contain an expression vector (for example, one thai contains the tRNA-derived microRNA CUI276) via introducing the expression vector into an appropriate host cell methods known in the art. miRN A can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing RNA from a plasmid include, e.g., the U6 or HI RNA pol III promoter sequences, or the cytomegalovirus promoters.
Selection of other suitable promoters is within the skill in the art. Recombinant plasmids can comprise inducible or regulatable promoters for expression of the miRNA in cancer cells (such as hematopoietic ceils, i.e., B cells). For example, the tRNA-derived microRNA CU 12.76 can be placed under the control of the CM V intermediate-early promoter, whereby the nucleic acid sequences encoding the miRNA molecule are located 3 ' of the promoter, so that the promoter can initiate transcription of the miRNA gene product coding sequences.
[8045] Plasmids suitable for expressing a miRNA, methods for inserting nucleic acid sequences into the plasmid to express the miRNA of interest, and methods of delivering the recombmant plasmid to cells of interest are well-established and practiced in the art. See, for example, Zeng et al. (2002), Molecular Cell 9: 1327-1333; Tuschl (2002), Nat Biotechnol, 20:446-448; Brammelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat Biotechnol 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosures of which are herein incorporated by reference,
[8(546] tRNA-derived microRNAs (e.g., CU1276) can also be expressed from recombinant viral vectors. The RNA expressed from the recombinant viral vectors can either be isolated from cultured cell expression systems by standard techniques, or can be expressed directly in cancer cells (such as hematopoietic cells, i.e., B ceils). For example, the recombinant viral vectors can comprise sequences that encode the miRNA molecule of interest and any suitable promoter for expressing the RNA sequences. Vectors can also comprise inducible or regulatable promoters for expression of the miRNA molecule in cells, such as cancer ceil. As discussed previously, non-limiting examples of suitable promoters include the U6 or HI RNA pol 111 promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is practiced by those of ordinary skill in the art.
[0047] Any viral vector that can harbor the nucleotide sequences for the miRNA molecules can be used. Non-limiting examples of such vectors include: vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of the viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, leniiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. For example, AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes. An AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J. E. et al. (2002), J Virol 76:791-801 , the entire disclosure of which is herein incorporated by reference. [8(548] Recombinant viral vectors suitable for expressing miRNA molecules, methods for inserting nucleic acid sequences for expressing RNA in the vector, methods of delivering the viral vector to cells of interest, and recovery of the expressed RNA molecules are within the skill in the art. See, for example, Dornburg (1995), Gene Therap. 2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum. Gene Therap. 1 :5-14; and Anderson (1998), Nature 392:25-30, the entire disclosures of which are herein incorporated by reference. Useful viral vectors can be those derived from A.V and AAV. A. suitable AV vector for expressing an mRNA molecule of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia et al. (2002), Nat. Biotech. 20: 1006- 1010, the entire disclosure of which is herein incorporated by reference. Suitable AAV vectors for expressing a miRNA molecule having SEQ ID NO: 1, methods for constructing the recombinant AAV vector, and methods for delivering the vectors into target cells are described in Samulski ei al. (1987), J. Virol.
61 :3096-3101; Fisher et al (1996), J. Virol, 70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5, 139,941 ; PCX Application No. WO 94/13788; and PCX Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
[8049] A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences. Nort limiting examples of host cells include CHO, HeLa, MDCK, HEK293, WT38, as well as various lymphoma cell lines, which are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va. 201 10-2209). Human leukemia and lymphoma cell lines that can also be used as host cells have been described by MacLeod et al, (2008) Curr Med Chem. ! 5(4):339-59, which is incorporated by reference in its entirety.
[0050] An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextran-niediated iransfeciion, or eleetroporation. Eleetroporation is earned out at approximate voltage and capacitance to result in entry of the nucleic acid constructs ) into cells of interest (such as lymphoma cells (e.g., cell line DB, ATCC CRL-2289; cell line HX, ATCC CRL-2260; cell line BC-3, ATCC CRL-2277; cell line CA46, ATCC CRL-1648; cell line Raji, ATCC CCL-86; cell line Daudi, ATCC CCL- 213; cell line GA-lO-Cfone-4, ATCC CRL-2393; cell line HH, ATCC CRL-2105; cell line H9, ATCC HTB- 176)). Other transfeetion methods also include modified calcium phosphate precipitation, poiybrene precipitation, liposome fusion, and receptor-mediated gene delivery.
[0051] Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for mammalian cells are well known in the art (Cleveland WL, et ai., J Immunol Methods, 1983, 56(2): 221-234) or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cell culturing conditions can vary according to the type of host cell selected. Commercially available medium can be utilized. Non-limiting examples of medium include, for example, Minimal Essential Medium (MEM, Sigma, St. Louis, Mo.); Dulbecco's Modified Eagles Medium (DMEM, Sigma); Hani's FIO Medium (Sigma); HyClone ceil culture medium (HyClone, Logan, Utah); RPMI-1640 Medium (Sigma); and chemically-defined (CD) media, which are formulated for various cell types, e.g., CD-CHO Medium (Invitrogen, Carlsbad, Calif.).
[0052] The cell culture media can be supplemented as necessary with supplementary components or ingredients, including optional components, in appropriate concentrations or amounts, as necessar or desired. Ceil culture medium solutions provide at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; (3) vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that can be required at very low concentrations, usually in the micromolar range.
[8053] The medium also can be supplemented electively with one or more components from any of the following categories: ( 1) salts, for example, magnesium, calcium, and phosphate; (2) hormones and other growth factors such as, serum, insulin, transferrin, and epidermal growth factor; (3) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (4) nucleosides and bases such as, adenosine, thymidine, and hypoxanthine; (5) buffers, such as HEPES; (6) antibiotics, such as gentamycin or ampicillin; (7) cell protective agents, for example pluromc polyoi; and (8) galactose. In one embodiment, soluble factors can be added to the culturing medium.
[8054] The mammalian cell culture that can be used with the present invention is prepared in a medium suitable for the type of cell being cultured. In one embodiment, the cell culture medium can be any one of those previously discussed (for example, MEM) that is supplemented with serum from a mammalian source (for example, fetal bovine serum (FBSY). In another embodiment, the medium can be a conditioned medium to sustain the growth of host cells.
Assessment an Therapuetic Treatment
[8(555] A blood cancer can be treated by restoring the level of miR A expression associated with that cancer to normal levels. For example, if the level of miR expression is down-regulated in cancer cells of a subject, then the cancer can be treated by increasing the miRNA expression level. For example, the level of a miRNA in a cancerous blood cell of a subject is first determined relative to normal control ceils. Techniques suitable for determining the relative level of a miRNA molecule in cells have been described above. If miRN A expression is down-regulated in the blood cancer cell relative to normal control cells, then the cancer cells are treated with an effective amount of a composition comprising an isolated miRNA molecule which is down-regulated (such as, a tRNA-derived microRNA (e.g., CU 1276)).
[8056] The invention provides a method of decreasing the growth of a solid tumor in a subject. The tumor is associated with, but not limited to, a lymphoma. In one embodiment, the method comprises detecting the presence or absence of a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU 1276)) in a sample obtained from a subject, in some embodiments, the sample displaying downregulation of a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)) is provided with tRNA- derived microRNA according to known delivery methods described herein. In further embodiments, the method comprises administering to the subject an effective amount of a tRNA-derived microRNA (e.g., CU1276), wherein the miRNA decreases the size of the solid tumor.
[8057] The invention also provides a method for treating a blood cancer in a subject. In one embodiment, the blood cancer comprises a lymphoma. In another embodiment, the blood cancer comprises a B cell lymphoma. In one embodiment, the method comprises deteciing the presence or absence of a cancer- associated tRNA fragment (such as a tRNA- derived microRNA (e.g., CU 12.76)) in a sample obtained from a subject In some embodiments, the method further comprises administering to the subject in need (e.g., the subject who exhibits a downregulation of a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)), a therapeutic treatment of a tRNA-derived microRNA (e.g., CU1276). In some embodiments, the sample displaying downregulation of a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)) is provided with tRNA-derived microRNA according to known delivery methods described herein.
[8058] The invention provides a method of decreasing proliferation of a blood cancer cell. In some embodiments, the blood cancer is a lymphoma. In other embodiments, the blood cancer is a B ceil lymphoma. In one embodiment, the method comprises detecting the presence or absence of a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)) in a sample obtained from a subject. In some embodiments, the sample displaying downregulation of a cancer-associated tRNA fragment (such as a tRNA- derived microRNA (e.g., CU1276)) is provided with tRNA-derived microRNA according to known delivery methods described herein. In further embodiments, the method comprises administering to the subject an effective amount of a tRNA -derived microRNA (e.g., CU1276), wherein the presence of the miRNA in the ceil decreases proliferation of the blood cancer ceils. In one embodiment, proliferation comprises cell invasion, cell migration, or a combination of the two. Cell migration and invasion assays are commercially available, e.g., Cell Biolabs Inc. (San Diego, CA), R&D Systems (Minneapolis, MN), and EMD Millipore (Bilierica, MA). Cell migration and invasion assays are well known in the art (e.g., a Matrigel invasion assay), and are described by Kleinman and Jacob ((2001 ) Curr Proloc Cell Biol, Chapter 12: Unit 12.2.); Vaslter et al. ((2005) Methods, 37(2):208- 15); and Kramer et a!., ((2013) Mutal Res. , 752(1 ): 10-24), each of which is incorporated by reference in its entirety.
[8059] The invention provides a method of reducing the number of blood cancer cells. In some embodiments, the blood cancer is a lymphoma. In other embodiments, the blood cancer is a B cell lymphoma. In one embodiment, the method comprises detecting the presence or absence of a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)) in a sample obtained from a subject. In some embodiments, the sample displaying downregulation of a cancer-associated tRNA fragment (such as a tRNA- derived microRNA (e.g., CU 12.76)) is provided with tRNA-derived microRNA according to known delivery methods described herein. In further embodiments, the method comprises administering to the subject an effective amount of a tRNA-derived microRNA (e.g., CU1276), wherein the presence of the miRNA in the ceil reduces the number of blood cancer cells.
[0060] The administering or delivery step in the claimed methods can comprise a drag administration, such as a composition comprising a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)). In one embodiment, the therapeutic molecule to be administered comprises a nucleic acid comprising at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% of the nucleic acid sequence of SEQ ID NO: 1. In one embodiment, administration of the therapeutic molecule decreases the size of the solid tumor associated with a lymphoma. In another embodiment, administration of the therapeutic molecule treats a blood cancer in a subject, for example a lymphoma (such as a B cell lymphoma). In a further embodiment, administration of the therapeutic molecule decreases proliferation of blood cancer cells (e.g., a lymphoma). In yet another embodiment, administration of the therapeutic molecule reduces the number of blood cancer cells (e.g., a lymphoma).
[8061] In one embodiment, the biological sample comprises serum, bone marrow, blood, peripheral blood, lymph nodes, urine, a saliva sample, a buccal swab, a a sputum sample, a lacrimal secretion sample, a semen sample, a vaginal secretion sample, a fetal tissue sample, or a combination thereof.
[8(562] Non-limiting examples of ly mphoma animal models to be used with the methods of the invention include JAX mice strains for lymphomas (e.g., B cell and T cell
lymphomas), such as B6.Cg-Tg(IghMyc)22Bri/J, B6.129S l-Ingltml Avg/J, B6.129S4(Cg)- Trp53tm2.1Tyj/J, B6J2984-Trp53tm3 JTyj/J, and B6.Cg-Tg(Cd79b-TCLlA)BKTeit/J, ail of which are commercially available from the Jackson Laboratory (Bar Harbor, ME). Other murine models useful with the methods described herein include, but are not limited, to those described by Donnou et al. (2012) Adv Hematol, 12:701 704, which is incorporated by reference in its entirety. These animal models are known in the art to correspond to the human condition.
[8063] A cancer-associated tRNA fragment (such as a tRNA-derived mieroRNA (e.g., CU12.76)) can be determined at the nucleic acid level. Optionally, detection can be determined by performing an oligonucleotide ligation assay, a confirmation based assay, a hybridization assay, a sequencing assay, an allele-specific amplification assay, a
microsequencing assay, a melting curve analysis, a denaturing high performance liquid chromatography (DHPLC) assay (for example, see Jones et al, (2.000) Hum Genet.,
106(6):663-8), or a combination thereof. In one embodiment, the detection or determination comprises nucleic acid sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof. In one embodiment, the detection is performed by sequencing all or part of a cancer-associated tRNA fragment (such as a tRN A-derived mieroRNA (e.g., CU1276)), or by selective hybridization or amplification of ail or part of the tRNA-derived mieroRNA. A nucleic acid specific amplification can be carried out before the fusion identification step. In one embodiment, the detecting comprises using a northern blot; real time PCR and primers directed to SEQ ID NO: 1 ; a ribonuclease protection assay; a hybridization, amplification, or sequencing technique to detect a tRNA-derived mieroRNA (e.g., CU1276): or a combination thereof. In another embodiment, the PCR primers comprise at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 3, 4, or a combination of the primers.
[8064] Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid. A detection technique involves the use of a nucleic acid probe specific for the presence of a tRNA-derived mieroRN A (e.g., CO 1276), followed by the detection of the presence of a hybrid. The probe can be in suspension or immobilized on a substrate or support (for example, as in nucleic acid array or chips technologies). The probe can be labeled to facilitate detection of hybrids. In one embodiment, the probe according to the invention can comprise a nucleic acid directed to SEQ ID NO: 1 . In another embodiment, the probe that detects the presence of a tRNA-derived mieroRNA comprises SEQ ID NO: 2.
[8(565] A guide to the hybridization of nucleic acids is found in e.g., Sambrook, ed., Molecular Cloning: A Laboratory Manual (3ld Ed,), Vols, 1-3, Cold Spring Harbor Laboratory, 1989; Current Protocols In Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York, 2001 ; Laboraiorv Techniques in Biochemisirv And Molecular Biology: Hybridizatio With Nucleic Acid Probes. Part I. Theory and Nucleic Acid Preparation. Tijssen, ed, Elsevier, N.Y., 1993.
[8066] Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing can be performed on a tRNA-derived microRNA (e.g., CU1276).
[8067] Amplification is based on the formation of specific hybrids between
complementary nucleic acid sequences that serve to initiate nucleic acid reproduction.
Amplification can be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), iigase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based ampiification (NASBA). These techniques can be performed using commercially available reagents and protocols. Useful techniques in the art encompass real-time PCR, allele- specific PCR, or PCR based single- strand conformational polymorphism (SSCP). Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction. In one embodiment, ampiification comprises using forward and reverse PCR primers directed to SEQ ID NO: 1. In certain subjects, the downregulation of a tRNA-derived microRNA (e.g., CU1276) corresponds to a subject with a blood cancer, e.g. a lymphoma. In one embodiment, amplification can comprise using forward and reverse PCR primers comprising at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at feast 18, at feast 19, at feast 20 consecutive nucleotides comprising SEQ ID NO: 3 or 4.
[8068] Non-limiting amplification methods include, e.g., polymerase chain reaction, PCR (PCR Protocols, A Guide To Methods And Applications, ed. Innis, Academic Press, N.Y., 1990 and PCR Strategies, 1995, ed. Innis, Academic Press, Inc., N.Y.); ligase chain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren ( 1988) Science 241 : 1077; Barringer (1990) Gene 89: 1 17); transcription amplification (Kwoh (4989) PNAS 86: 1 173); and, self-sustained sequence replication (Guafelii (4990) PNAS 87: 1874); Q Beta replicase ampiification (Smith (1997) J. Clin. Microbiol, 35: 1477- 1491), automated Q-beta replicase amplification assay (Burg (1996) Mol Cell. Probes 10:257-271 ) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario; see also Berger (1987) Methods Enzymol. 152:307-316; U.S. Pat. os. 4,683,195 and 4,683,202; and Sooknanan (1995) Biotechnology 13:563-564). All the references stated above are incorporated by reference in their entireties,
[8069] The invention provides for a nucleic acid primer, wherein the primer can be complementary to and hybridize specifically to a portion of a tRNA-derived microR A (e.g., CU1276), In one embodiment, the blood cancer comprises a lymphoma, such as a B cell lymphoma. Primers can be specific for a tRNA-derived microRNA (e.g., CU1276). By using such primers, the detection of an amplification product indicates the presence of a tRNA-derived microRN A (e.g., CU1276). Examples of primers of this invention can be single-stranded nucleic acid molecules of about 8 to about 1 5 nucleotides in length. Perfect complementarity is useful to ensure high specificity; however, certain mismatch can be tolerated. For example, a nucleic acid primer or a pair of nucleic acid primers as descr ibed above can be used in a method for detecting the presence of a blood cancer in a subject. In one embodiment, primers can be used to detect the absence of reduced level of a cancer- associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276)). In some embodiments, the primers are directed to SEQ ID NO: 1. In another embodiment, the PGR primers comprise at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 3 or 4.
[8(578] The invention provides for a diagnostic kit comprising products and reagents for detecting in a sample from a subject the presence or absence of a cancer-associated tRNA fragment (such as a tRNA-derived microRN A (e.g., CU1276)). The kit can be useful for determining whether a sample from a subject exhibits decreased or reduced expression of a cancer-associated tRNA fragment. For example, the diagnostic kit according to the present invention comprises any primer, or any pair of primers directed specifically to a cancer- associated tRNA fragment. The diagnostic kit according to the present invention can further comprise reagents and/or protocols for performing a hybridization, or amplification. In one embodiment, the kit can comprise nucleic acid primers that specifically hybridize to and can prime a polymerase reaction from a tRN A-derived microRN A (e.g., CU1276) comprising at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 3 or 4, or a combination of the primers. In one embodiment, primers can be used to detect the absence of reduction of a tRNA-derived microRNA (e.g., GUI 276), such as a primer directed to SEQ ID NO: i . In another embodiment, the PCR primer comprises at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleotides comprising SEQ ID NO: 3 or 4. In some embodiments, the kit comprises a probe for detecting a cancer-associated tRNA fragment (such as a tRNA-derived microRNA (e.g., CU1276). In other embodiments, the probe comprises SEQ ID NO: 2.
[0071] The diagnosis methods can be performed in vitro, ex vivo, or in vivo. These methods utilize a sample from the subject in order to assess the status of a tRNA-derived microRNA (e.g., CU1276). The sample can be any biological sample derived from a subject, which contains nucleic acids or polypeptides. Examples of such samples include, but are not limited to, fluids, tissues, cell samples, organs, and tissue biopsies. Non-limiting examples of samples include blood, li ver, plasma, serum, sali va, urine, or seminal fluid. The sample can be collected according to conventional techniques and used directly for diagnosis or stored. The sample can be treated prior to performing the method, in order to render or improve av ailability of nucleic acids or polypeptides for testing. Treatments include, for instance, lysis (e.g., mechanical, physical, or chemical), centrifugation. The nucleic acids and/or polypeptides can be pre-purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides can also be treated with enzymes or other chemical or physical treatments to produce fragments thereof. In one embodiment, the sample is contacted with reagents, such as probes or primers, in order to assess the absence or presence of a tRNA-derived microRNA (e.g., CU1276). Contacting can be perfonned in any suitable device, such as a plate, tube, well, or glass. In some embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate can be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, or polymers. The substrate can be of various forms and sizes, such as a slide, a membrane, a bead, a column, or a gel. The contacting can be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids or polypeptides of the sample.
Nucleic Acid Delivery Methods
[0072] Delivery of nucleic acids into viable cells can be effected ex vivo, in situ, or in vivo by use of vectors, such as viral vectors (e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). Non-limiting techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, eiectroporation, microinjection, cell fusion, DEAE-dextran, and the calcium phosphate precipitation method (See, for example, Anderson, Nature, (1998) supplement to 392(6679):25. introduction of a nucleic acid can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells can also be cultured ex vivo in the presence of therapeut ic compositions of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
[8073] Nucleic acids can be inserted into vectors and used as gene therapy vectors. A number of viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40 (Madzak et al., (1992) J Gen Virol 73( Pi 6):1533-6); adenovirus (U.S. Patent No. 8,460,932; Berkner (1992) Curr Top Microbiol 7WMW?K>/.158:39-66; Berkner (1988) Biolechniques, 6(7):616-29; Gorziglia and Kapikian (1992) ./ Virol. 66(7):4407-12; Quantin et al., (1992) Proc Nail Acad Sci USA. 89(7):2581-4; Rosenfeld et aL, (1992) Cell.
68(1): 143-55; Wilkinson et al., (1992) Nucleic Acids Res. 20(9):2233-9; Stratford-Perricaudet et aL, (1990) Hum Gene Ther. l (3):241 -56); vaccinia virus (Moss (1992) Curr Opin
Biotechnol. 3(5):518-22); adeno-associated virus (U.S. Patent No. 8,529,885; Muzyczka, (1992) Curr Top Microbiol Immunol. 158:97-129; Ohi et aL, (1990) Gene. 89(2):279-82); herpesviruses including HSV and EBV (Margolskee (1992) Curr Top Microbiol Immunol. 158:67-95; Johnson et aL, (1992) Brain Res Mol Brain ¾s. l2(l -3):95-102; Fink et al., (1992) Hum Gene Ther. 3( 1): 1 1-9; Breakefield and Geller (1987) Mol Neurobiol. 1(4):339- 71; Freese et aL, (1990) Biochem Pharmacol. 40(10):2189-99); and retroviruses of avian (Bandyopadhyay and Temin (1984) Mol Cell Biol 4(4):749-54; Petropoulos et aL, (1992) J Virol. 66(6):3391-7), murine (Miller et al. (1992) Mol Cell Biol 12(7):3262-72; Miller et al., (1985) J Virol 55(3):521-6; Sorge et aL, (1984) Mol Cell Biol. 4(9): 1730-7; Mann and Baltimore (1985) J Virol. 54(2):401-7; Miller et aL, (1988) J Virol. 62(l l):4337-45), and human origin (Shimada et aL, (1991) J Clin Invest. 88(3): 1043-7; Helseth et aL, (1990) ,/ Virol 64( 12):6314-8; Page et al., ( 1990) J Virol 64(1 1 ):5270-6; Buchschacher and
Panganiban ( 1992) J Virol 66(5) :2731-9).
[0074] Non-limiting examples of in vivo gene transfer techniques include transfection with viral (e.g., retroviral) vectors (see U.S. Pat. Nos. 5,252,479; 8,460,932; 8,529,885, each of which is incorporated by reference in its entirety) and viral coat protein-liposome mediated transfection (Dzau et al., (1993) Trends in Biotechnology 1 1 :205-210), incorporated entirely by reference). For example, naked DNA vaccines are generally known in the art; see Brower, (1998) Nature Biotechnology, 16: 1304- 1305, which is incorporated by reference in its entirety. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. Nos. 5,328,470; 8,529,885; 8,460,932) or by stereotactic injection (see, e.g., Chen, et al, (1994) Proc. Natl, Acad. Sci. USA 91 :3054- 3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
[0075] For reviews of nucleic acid delivery protocols and methods see Anderson et al. (1992) Science 256:808-813: U.S. Pat. Nos. 5,252,479; 5,328,470; 5,747,469; 6,017,524; 6,143,290; 6,410,010: 6,51 1,847; 8,247,385; 8,420,61 1 ; 8,529,885; 8,460,932; and U.S. Application Publication Nos. 2002/0077313; 2013/0004471 ; 2013/0171 1 15; 2013/0210717; 2013/0172403, which are all hereby incorporated by reference in their entireties. For additional reviews, see Friedmann ( 1989) Science, 244: 1275-1281 ; Verma, Scientific American: 68-84 (1990); Miller (1992) Nature, 357: 455-460; Kikuchi et al. (2008) J Dermatol Sci. 50(2.):87-98; Isaka et al. (2007) Expert Opin Drug Deliv. 4(5):561-71 ; Jager et al.(2007) Curr Gene Ther. 7(4):272-83; Waehler et al.(2007) Nat Rev Genet. 8(8):573-87; Jensen et al. (2007) Ann Med. 39(2): 108-15; Herweijer et al. (2007) Gene Ther. 14(2):99- 107; Eliyahu et al. (2.005) Molecules 10(l ):34-64; and Altaras et al. (2.005) Adv Biochem Eng Biotechnol. 99: 193-260, all of which are hereby incorporated by reference in their entireties.
[0076] A tRNA-derived microR A (e.g., CU1276) can also be delivered in a controlled release system. For example, the molecule can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of
administration. In one embodiment, a pump can be used (see Sefton ( 1987) Biomed. Eng. 14:201 ; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med.
321 :574). In another embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fia. (1974); Controlled Drug Bioayailability, Dri3g Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, (1983) J. Macromol. Sci. Rev. Macromol Chem. 23:61 ; see also Levy et al. ( 1985) Science 228: 190; During et al. (1989) Ann. Neurol. 25:351 ; Howard et al. (1989) J Neurosurg. 71 :105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical
Applications of Controlled Release, supra, vol. 2, pp. 1 15-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science (1990) 249: 1527-1533).
Pharmaceutical Compositions and Administration for Therapy
[8077] A tRNA-derived microRNA (e.g., CU1276) can be incorporated into
pharmaceutical compositions suitable for administration, for example the rniRNA and a pharmaceutically acceptable carrier.
[0078] A. tRNA-derived microRNA. (e.g., CO 1276) can be administered to the subject once (e.g., as a single injection or deposition). Alternatively, a tRNA-derived microRNA (e.g., CU1276) can be administered once or twice daily to a subject in need thereof for a period of from about two to about twenty-eight days, or from about seven to about ten days. A tRN A-derived microRN A (e.g., CU1276) can also be administered once or twice daily to a subject for a period of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 times per year, or a combination thereof. Furthermore, a tRNA-derived microRNA (e.g., CU1276) can be co-administrated with another therapeutic, such as a chemotherapy drug. Where a dosage regimen comprises multiple administrations, the effective amount of the tRNA-derived microRNA (e.g., CU1276) administered to the subject can comprise the total amount of gene product administered over the entire dosage regimen.
[8(579] A tRNA-derived microRNA (e.g., CU1276) can be administered to a subject by any means suitable for delivering the composition to cells of the subject, such as cancer cells, e.g., a blood cancer. For example, a tRNA-derived microRNA (e.g., CU1276) can be administered by methods suitable to trarisfect cells. Transtection methods for eukarvoiic cells are well known in the art, and include direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, biobalHstic or particle acceleration; calcium phosphate precipitation, and transtection mediated by viral vectors.
[8080] The compositions can be formulated and administered to reduce the symptoms associated with a blood cancer, e.g., a lymphoma, by any means that produces contact of the active ingredient with the agent's site of action in the body of a subject, such as a human or animal (e.g., a dog, cat, or horse). They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
[8(581] A therapeutically effective dose of tRNA-derived microRNA (e.g., CU 1276) can depend upon a number of factors known to those or ordinary skill in the art. The dose(s) of the composition can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the tRNA-derived microRNA (e.g., CU1276) to have. These amounts can be readily determined by a skilled artisan. Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.
[8082] The compositions can be formulated and administered to inhibit, reduce, or ameliorate a variety of cancer states by any means that produces contact of the active ingredient with the agent's site of action in the body of the subject (e.g., a mammal). They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
[8(583] Pharmaceutical compositions for use in accordance with the invention can be formulaied in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration.
Techniques and formulations generally can be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa (1985), the entire disclosure of which is herein incorporated by reference. For systemic administration, injection is useful, including intramuscidar, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, for example, in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use.
[8(584] According to the invention, a pharmaceutically acceptable carrier can comprise any and ail solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions. The compositions can be administered alone or in combination with at least one other agent such as a stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a subject alone, or in combination with other agents and/or drugs,
[0085] The present pharmaceutical formulations comprise a tRN A-derived microRNA
(e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier. The pharmaceutical formulations can also comprise the miRNA, which is encapsulated by liposomes and a pharmaceutically-acceptable carrier. Useful pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid, and the like.
[0086] Pharmaceutical compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTP A or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
- 11 - [8(587] Sterile injectable solutions can be prepared by incorporating the miR A in the required amount in an appropriate sol vent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile po wders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[8088] For solid pharmaceutical compositions of the invention, conventional nontoxic solid pharmaceutically-acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
[8089] For oral administration, the therapeutic compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregeiatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystailine cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch giycoiaie); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with waf er or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
[8090] Preparations for oral administration can be suitably formulated to give controlled release of the active agent. For buccal administration the therapeutic compositions can take the form of tablets or lozenges formulated in a conventional manner. For administration by inhalation, the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellent, e.g., dichlorodifluoromethane,
trichforofluorometliane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflate or can be formulated containing a powder mix of the therapeutic agents and a suitable powder base such as lactose or starch.
[8091] The therapeutic compositions can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.
[8092] "Subcutaneous" administration can refer to administration just beneath the skin (i.e., beneath the dermis). Generally, the subcutaneous tissue is a layer of fat and connective tissue that houses larger blood vessels and nerves. The size of this layer varies throughout the body and from person to person. The interface between the subcutaneous and muscle layers can be encompassed by subcutaneous administration. This mode of administration can be feasible where the subcutaneous layer is sufficiently thin so that the factors present in the compositions can migrate or diffuse from the locus of administration. Thus, where intradermal administration is utilized, the bolus of composition administered is localized proximate to the subcutaneous layer.
[8093] Suitable enteral administration routes for the present methods include oral, rectal, or intranasal delivery. Suitable parenteral administration routes include intravascular administration (e.g. intravenous bolus injection, intravenous infusion, mtra-arterial bolus injection, intra -arterial infusion and catheter instillation into the vasculature); peri- and intra- tissue injection (e.g., peri-tumoral and intra-tumoral injection, intra-retinal injection, or subretinaf injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps): direct application to the tissue of interest, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation. The miR A can be administered by injection or infusion.
[8094] In addition to the formulations described previously, the therapeutic compositions can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneous!}' or intramuscularly) or by intramuscular injection. Thus, for example, the therapeutic compositions can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt,
[0095] Systemic administration can also be by transmucosal or transdermal means. For transniucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives, in addition, detergents can be used to facilitate permeation, Transmucosal administration can be through nasal sprays or using suppositories. For topical administration, the compositions of the invention are formulated into ointments, salves, gels, or creams as generally kno wn in the art, A. wash solution can be used locally to treat an injury or inflammation to accelerate healing. For oral administration, the therapeutic compositions are formulated into conventional oral administration forms such as capsules, tablets, and tonics. In some embodiments, the miRNA can be applied via transdermal delivery systems, which slowly releases the active compound for percutaneous absorption. Permeation enhancers can be used to facilitate transdermal penetration of the active factors in the conditioned media. Transdermal patches are described in for example, U.S. Pat. No. 5,407,713; U.S. Pat, No. 5,352,456; U.S. Pat No. 5,332,213; U.S. Pat, No. 5,336,168; U.S. Pat. No. 5,290,561 ; U.S. Pat. No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No.
5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,1 10; and U.S. Pat. No. 4,921,475.
A miRNA composition can also be formulated as a sustained and/or timed release formulation. Such sustained and/or timed release formulations can be made by sustained release means or delivery devices that are well known, to those of ordinary skill in the art, such as those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809;
3,598, 123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of which are each incorporated herein by reference. The pharmaceutical compositions of the present invention can be used to provide slow or susta ined release of one or more of the active ingredients using, for example, hydropropylm ethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions. Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the invention. Thus, single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gel caps, caplets, powders, and the like, that are adapted for sustained release are encompassed by the present invention.
[8097] According to the present methods, the iR A-derived microRNA (e.g., CU1276) can be administered to the subject either as naked RN A, in conjunction with a delivery reagent, or as a nucleic acid (e.g., a recombinant plasmid or viral vector) comprising sequences which expresses the gene product. Suitable delivery reagents for administration of the miRNA molecule include the Minis Transit TKO lipophilic reagent; lipofectm;
lipofectamine; celifectm; or polycations (e.g., pofylysine), or liposomes.
[0098] The dosage administered will be a therapeutically effective amount of the composition sufficient to result in treatment and/or amelioration of symptoms of a blood cancer (e.g., a ceil lymphoma) and can vary depending upon known factors such as the pharmacodynamic characteristics of the particular active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired.
[0099] Toxicity and therapeutic efficacy of therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (The Dose Lethal To 50% of the Population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapeutic agents that exhibit large therapeutic indices are useful. Therapeutic compositions that exhibit some toxic side effects can be used.
[80180] Appropriate doses depend upon a number of factors known to those or ordinary skill in the art, e.g., a physician. The dose(s) will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the composition to have upon the nucleic acid or polypeptide of the invention. Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight. In some embodiments, the effective amount of the administered tRNA-derived microRNA is at least about 0.0001 ^ig/kg body weight, at least about 0.00025 ug kg body weight, at least about 0.0005 μ2/^ body weight, at least about 0.00075 ug kg body weight, at least about 0.001 μ ¾¾ body weight, at least about 0.0025 g/kg body weight, at least about 0.005 ^ig/kg body weight, at least about 0.0075 ug kg body- weight, at least about 0.01 body weight, at least about 0.025 μg/ g body weight, at least about 0.05 μg kg body weight, at least about 0.075 μg/kg body weight, at least about 0.1 ug/kg body weight, at least about 0.25 μg kg body weight, at least about 0.5 μg/kg body weight, at least about 0.75 μg/kg body weight, at least about 1 μg/kg body weight, at least about 5 μgl |kg body weight, at least about 10 μg kg body weight, at least about 25 g kg body weight, at feast about 50 g/kg body weight, at least about 75 μg kg body weight, at least about 100 ug/kg body weight, at least about 150 jig/kg body weight, at least about 200 g/kg body weight, at least about 250 μg/kg body weight, at least about 300 μg 'kg body weight, at least about 350 μg/'kg body weight, at least about 400 μg kg body weight, at least about 450 ug/kg body weight, at least about 500 pg/kg body weight, at least about 550 ug/kg body weight, at least about 600 ug/kg body weighi, at least about 650 μg/kg body weight, at least about 700 igfkg body weight, at least about 750 μg/kg body weight, at least about 800 ug/kg body weighi, at least about 850 μg kg body weight, at least about 900 ug/kg body weight, at least about 950 μg kg body weight, at least about 1,000 μg kg body weight, at least about 2,000 ug/kg body weight, at least about 3,000 μg/kg body weight, at least about 4,000 μgί'kg body weight, at least about 5,000 ug kg body weight, at least about 6,000 ug/kg body weight, at least about 7,000 ug/kg body weight, at least about 8,000 ug/kg body weight, at least about 9,500 ug/kg body weight, or at least about 10,000 ug''kg body weight,
[00101] These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan.
Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.
?] All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art.
EXAMPLES
[8(5184] A number of Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate the exemplary modes of making and practicing the present invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only , since alternative methods can be utilized to obtain similar results.
[8(5185] Example 1 - A tRNA-derivcd microRNA modulates proliferation and the DNA damage response, and is downregulated in B cell lymphoma
[88186] Sequencing studies from several model systems have suggested that diverse and abundant small RNAs may derive from tRNA, but the function of these molecules remains undefined. Here we demonstrate that one such tR A-derived fragment, cloned from human mature B cells and designated CU1276, in fact possesses the functional characteristics of a microRNA, including a DICERl- dependent biogenesis, physical association with Argonaute proteins, and the ability to repress mR A transcripts in a sequence-specific manner.
Expression of CU 1276 is abundant in normal Germinal Center B cells but absent in Germinal Center-derived lymphomas, suggesting a role in the pathogenesis of this disease.
Furthermore, CU1276 represses endogenous RPAI, an essential gene involved in many aspects of DNA dynamics, and consequently, expression of this tRNA-derived microRNA in a lymphoma cell line suppresses proliferation and modulates the molecular response to DNA damage. These results establish that functionally active microRNAs can derive from tRNA, thus defining a new class of genetic entities with potentially important biological roles.
[88187] In recent decades, sequencing studies have uncovered a diverse menagerie of small RNA molecules expressed in eukaryotic cells, among which microRNAs (miRNAs) are perhaps the single best-understood subclass. miRNAs guide the binding of Argonaute- containing induced silencing complexes (miRISC) to the 3' untranslated region (3'UTR) of genes bearing partially complementary sites, and are typically expressed from within the introns of protein coding genes or as part of long non-coding RNA transcripts (1). However, recent work has demonstrated that miRNAs can also arise from previously unanticipated non- canonical pathways. Specifically, miRNAs can be generated in a DROSHA- (2, 3) or
/CEfli-independent manner (4), and have been demonstrated to arise from cleavage of otherwise functional non-coding RNA molecules, such as small nucleolar RNA (5). [80188] Cloning and sequencing of small RNAs purified from human Naive, Germinal Center (GC), and Memory B cells, as well as from the Burkitt Lymphoma cell line Ramos (6) have been reported. In addition to observing known and new miRNAs, an intriguing class of abundantly expressed small RNAs were noted whose sequences perfectly matched either to mature or to precursor tRNA transcripts. Several other groups have reported similar small RNA species expressed in a variety of human cell types (7- 1 1 ) as well as in other organisms (12-14). However, the functional role of these small RNAs, and in particular the possibility that they may act as miRNAs, has not yet been adequately addressed. Here ai leasi one such sequence, designated CO 1276, is shown to function as a niiRNA.
[8018 ] Results
[8011(5] CU1276 is a DfCERl -dependent tRNA fragment expressed in mature B cells. Taken together, previous reports (8, 9, 1 1) define a minimum of three distinct categories of tRNA fragment: those matching to the 5' end of mature tRNA (tRF-5), those matching to the 3' end of mature tRNA (tRF-3), and those matching to the 3' end of precursor iRNA transcripts (tRF - 1). Further analysis of our published small RNA sequencing data (6) suggested that amongst these classes, tRF-3s are by far the most abundant variety expressed in mature B cells; therefore, in order to investigate the biological function of these molecules, we sought to characterize a representative sequence of the tRF-3 class, designated CU1276.
[80111] CU1276 is a 22nt small R A (5'-TCGATTCCCGGCCAATGCACCA-3'; SEQ D NO: 1) differentially expressed in three stages of mature B cell differentiation and one GC-derived lymphoma cell line, and cloned most frequently in normal GC B cells (FiglA). Despite its roiRNA-like size, CU1276 is a perfect match to the post-transcriptionally modified 3' end of at leasi five annotated human tRNAs (15) (FiglB). In order to clarify (he relationship between CU1276 and tRNA, a CU1276-matching tRN A locus was cloned into an expression vector, and transiently transfected this vector into HEK-293T cells (293T). Northern Biot analy sis of these cells re vealed a clear increase in both the mature tRNA and a 22nt band co-migrating with the endogenous fragment observed in B cells (FiglC), suggesting that CU1276 is indeed tRNA-derived. CU1276 expression was confirmed from a second, independent Gly(GCC) tRNA locus, and the possibility of expression from a candidate precursor genomic locus (6) closely matching the CU 1276 sequence, but not encoding a tRN A (Fig6) was ruled out. [80112] tRNA do not meet the structural criteria of a classical DICERl substrate ( 16): nonetheless, due to the observed similarities in size between CU1276 and DICER /-dependent miRNA, this enzyme could be involved in CUT276 biogenesis. Therefore, a CU.1276- matching tRNA was transiently overexpressed in 293T cell lines stably expressing either a control shRNA or a pool of three DICERl -targe ting shR A. Northern Blot analysis revealed that knockdown of DICER l was sufficient to reduce production of CU1276, regardless of an accumulation of mature tRNA (FiglD). This evidence supports a .D/CH? /-dependent cleavage step in the biogenesis of CU1276, which can be a general feature of fragments derived from mature tRNA.
[8(5113] CU1276 associates with all four human Argonaute proteins, and functions as a miRNA. As a prerequisite for investigating a possible miRNA-like function of CU1276, it was determined whether this small RNA was physically associated with Argonaute proteins. Utilizing a monoclonal antibody with reactivity against all four human Argonaute proteins (17), Argonaute-associated RNAs were purified from the B cell line R1VA; qRT- PCR analysis of the co-precipitated RNA confirms that CU1276 is indeed enriched in the pan-Ago immunoprecipitation (IP) fraction relative to a control IP (Fig2A). In order to dissect CU1276 binding affinity for each individual Argonaute protein, IP of HA-tagged versions of human AGOl , AG02, AG03, and AG04 transiently expressed in 293T cells were performed; the results demonstrate that CU1276 is enriched in the IP fractions of each Argonaute protein relative to that of the HA-EGFP control, indicating that it is specifically incorporated into silencing complexes containing each of the four human Argonautes (Fig2B). The dynamics of this interaction can be influenced by the availability of unoccupied Argonaute complexes, given that the magnitude of CU 1276 enrichment in AGO I complexes increased proportionally with the total levels of this protein, meeting and eventually exceeding the enrichment of the canonical miRNA miR- 16 at high levels of AGO l expression (Fig2C).
[80114] Given its demonstrated binding to the functional effectors of miRNA signaling, the effect of CU1276 was tested on a firefly luciferase reporter bearing two antisertse binding sites in its 3 'UTR. Because overexpression of full-length tRNA inevitably produces a complex mixture of RNA molecules, including a previously reported ~-34nt 5' tRN A fragment capable of broadly repressing translation (18), CUI276 was also cloned into a miRNA hairpin in order to investigate its activity in a context free from confounding factors. Expression of either the tRNA or hairpin was indeed sufficient to repress the antisense reporter (Fig2D). Under standard conditions, the potency of tRNA-delivered CU12.76 was less than that of hairpin-delivered CU1276; however, simultaneous expression of exogenous AG02, the Argonaute family member whose slicer activity enables potent repression of mK As bearing a perfect antisense site (19), facilitated tRNA-mediated repression at comparable levels (Fig2D). Without being bound by theory, these data are potentially consistent with a lower affinity for Argonaute proteins or a different distribution among Argonautes as compared to canonical hairpin-derived miRNA; they nonetheless prove that the tRNA-derived CU1276 can repress mRNA targets in an Argonaute-dependent, miRNA- like fashion.
[00115] CU1276 is downregulated m lymphoma cell lines and primary biopsies.
Formation of GC structures is a crucial step in the B cell-mediated adaptive immune response, and GC cells are the cell of origin for the majority of B ceil lymphomas (20). initial small R A sequencing and Northern Blot analysis suggested that CU1276 is abundantly expressed in normal GC B cells, but is low in at least one GC-derived lymphoma cell line (FiglA, C). To expand these findings, GUI 276 was assessed by qRT-PCR in normal GC B ceils and a panel of GC-derived lymphoma cell lines. Strikingly, only normal GC B cells efficiently expressed this small RNA., while the entire pane! of cell lines displayed low CU1276 levels (Fig3A). Additionally, sequencing of small RNAs from a panel of normal GC B cells (n=4) and Diffuse Large B Cell Lymphoma (DLBCL) primary biopsies (n=25) revealed a binary pattern of CU1276 expression, with CU1276 almost completely absent in tumor cells (Fig3B).
[00116] The Gly(GCC) tRN A from which CU1276 is derived can be found in at least five distinct genomic loci (FiglB), making it an unlikely candidate for genetic deletion. Indeed, each ceil line tested expresses roughly equal levels of the mature, post- transcriptionally modified form of the tRNA from which CU1276 is derived (Fig7), indicating that the observed decrease in CU1276 production in lymphomas is probably regulated at the level of tRNA cleavage.
[00117] G UI 276 represses a set of endogenous genes, including RPA1. Genes were identified both significantly downregulated by CU1276 expression and computationally predicted to contain CU 1276 binding sites in their 3'UTR (21). Without being bound by theory, expression of mature Gly(GCC) tRN A can induce changes in gene expression due to CU1276-independent effects on translation; therefore, in order to focus on the most physiologically relevant CU1276-specific targets, expression profiles were compared from 293T cells tratisfected with empty, tRNA-expressing, or GUI 276 hairpin-expressing vectors. This analysis revealed a modest, but statistically significant overlap between the
downregulated probes (Student's i-test, p<0.05) in the two experimental groups (-13% of tRNA-downregulated genes, and ~15% hairpin-downregulated genes; Hypergeometric test, p<lE-40) (Fig8A), confirming that tRNA-delivered and hairpin-delivered CU1276 do exert a similar effect on at least a subset of mRNA. Furthermore, transcripts predicted to contain GUI 2.76 binding sites by the miRN A target prediction algorithm TargetScan (22) were significantly enriched (Hypergeometric test, p=8.6E-8) in the union of genes downregulated by tRNA and/or hairpin expression (FigSB), suggesting that a significant fraction of these genes are repressed based on classical seed-mediated binding.
[80118] To focus on the highest-confidence predictions, attention was focused on those genes a) significantly downregulated in both tRNA- and in hairpin-expressing cells, b) predicted by TargetScan to contain CU1276 binding sites, and c) expressed in the physiological site of CU 1276 expression, GC B cells. Three genes met these criteria, and ail three were tested as targets for CUT 276.
[80119] Two of these genes, WHSCIL1 and STAG2, showed either no response, or an ambiguous response to CU1276 expression (Fig8C-D). In contrast, CUT 276 expression via either tRNA or hairpin was indeed sufficient to significantly (Student's t-test, p=3.9E-5 and p=8.2E-5, respectively) repress a reporter bearing the 3'UTR of the RPAl gene (Fig4A). Mutation of the lone predicted CU1276 binding site rendered the reporter immune to this repression, confirming that the interaction is direct (Fig4A).
[8012(5] Consistent with this result, transient expression of CU1276 by either tRNA- or hairpin-mediated delivery repressed endogenous RPAl protein in 293T cells while leaving another RPA complex member, RPA2, unchanged (Fig4B, Fig.9A). CU1276 expression had minimal effect on RPAl mRNA levels as measured by qRT-PCR (Fig9B), indicating that in this cellular context, CU1276-mediated repression of RPAl is primarily at the transiational level. In order to extend this finding to B cells, a stable B cell lymphoma line was constructed carrying a vector with a doxycycline-mducible bidirectional promoter encoding for GFP alone, or GFP plus GUI 276 hairpin; induction of GUI 276 repressed both endogenous RPAl protein and RPAl mRNA relative to control eelis (Fig4B, Fig9C-D). Thus, RPAl is a bona fide target of the tR A-derived miRNA GUI 276.
[8(5121] Based on the observation of strongly differential CU 1276 expression between normal GC B cells and GC-derived lymphomas (Fig3), without being bound by theory RPAl protein might be de-repressed in cell types lacking CU1276. Consistent with this hypothesis, the majority of tested cell lines express higher levels of RPAl relative to normal GC B ceils (Fig4C). RPAl mRNA levels, as evaluated by gene expression profiling in an independent panel of 5 GC samples and a subset of 8 DLBCL cell lines, were similar between these two groups, consistent with a translatio ai-levei regulatory effect by CU1276 (FiglO). Although sufficient material was not available to directly assess RPAl protein levels in the primary lymphoma biopsies, based on the high levels of expression observed in cell lines, we speculate that loss of GUI 276 expression may also contribute to misregulation of RPAl in the context of primary lymphomas.
[8(5122] CU1276 suppresses proliferation and modulates the molecular response to DNA damage in an HAll-dependent manner. RPAl has a number of well -characterized roles in DNA dynamics, including in replication and DNA. repair (23). Without being bound by theory, through repression of RPAl, CU1276 might influence cellular proliferation and the response to DNA damage.
[88123] Indeed, stable expression of CU1276 in a Burkitt Lymphoma cell line modestly but significantly reduces the proliferation rate of these cells (Student's t-test, p=1.8E-3, FigSA), consistent with defective D A replication efficiency. Restoration of RPAl protein expression to wild type levels through simultaneous co-expression of exogenous RPA 1 significantly rescues the observed growth impairment (FigSA), confirming that RPAl is the primary CU1276 target responsible for this phenotype.
[88124] In addition to its effect on cellular proliferation, CU1276 expression is also capable of modulating the molecular response to DNA. damage. Robust expression of CU1276 is sufficient to sensitize a Burkitt Lymphoma cell line to Etoposide-induced DNA damage, as measured by an increase in H2AFX phosphorylation relative to controls (FigSB). Similar to the CU1276-induced reduction in cellular proliferation, this sensitization can be fully rescued by restoration of RPAl protein to wild-type levels (FigSC), confirming that RPAl is also the critical CUI276 target responsible for this effect. [80125] Discussion
[8(5126] An increasing body of literature supports the existence of highly abundant miRNA-like tRNA fragments in a variety of cell types (7-14), but despite several lines of speculation, no conclusive evidence of their function has yet been shown. The data demonstrates that despite its derivation from the 3' end of a mature tRNA (FiglC), the small RNA CU1276 has ail of the structural and functional characteristics of a miRNA, including a. /J/CEfti-dependent biogenesis (FiglD) and binding to all four human Argonauts proteins (Fig2A, B). GU I 276 is furthermore capable of guiding repression to both artificial anti sense sites (Fig2D) and binding sites identified in endogenous 3' UTRs (Fig4A, B).
[80127] In addition to their classical role in delivering amino acids to nascent peptide chains, tRNAs and tRNA fragments have the capacity to regulate a surprising range of cellular processes, including translational efficiency under stress conditions (18), mitochondrial-mediated apoptosis (24), and oncogenic transformation (25). The data presented further extend the regulatory repertoire of tRNA, and greatly expand the pool of candidate miRNAs, by demonstrating that a tRNA fragment can post-transcriptionally regulate endogenous genes in a sequence-specific, miRNA-like fashion.
[80128] Interestingly, the tRNA-derived microRNA GUI 2.76 is strongly downregulated in GC-derived lymphomas relative to their cell of origin (Fig3), Given that mature
Gly(GCC) tRNA expression is largely constant between samples with highly discordant GUI 276 levels (Fig7 and Fig3A), ihe defect in GUI 276 biogenesis is likeiy to lie at the level ofDiCERl cleavage. However, with only one exception (HBLI), all tested lymphoma cell lines express abundant DiCERl protein (Fig4C), indicating that a more complex mode of regulation, such as differences in post-transcriptional modifications that might protect the tRNA from processing (26), is likely to be responsible for the observed differences. Further investigation of the factors regulating G U I 276 biogenesis can shed light on the upstream cause of this repression in cancer cells, while validation of additional GUI 276 targets will help to clarify its full downstream biological consequences,
[80129] Because of its ability to regulate endogenous target genes, GUI 276 was shown to repress endogenous RPA1 (Fig4A, B). RPA1 is an essential gene for many aspects of DNA dynamics, including genome replication. Consequently, stable CU1276 expression in a Burkitt Lymphoma-derived cell line results in an RPA1 -dependent suppression of their proliferation rate (FigSA). This result, combined with the observed downregulation of CU12.76 levels in lymphoma cell lines and biopsies (Fig3) indicates that loss of CU1276 expression, and a corresponding increase in RPAl protein levels, can confer a growth advantage to malignant ceils.
[8013(5] As part of their maturation, GC B-ceils undergo several physiological processes of somatic mutation and DNA rearrangement (20). RPAl is a required component for some types of DNA repair, and additionally has a OC-specific role in facilitating AICDA-mcdiated mutagenic processes (27, 28). Physiological expression of CU 1276 in the GC can contribute to fine -tuning of RPAl levels in GC B cells, and can thereby indirectly influence the efficiency of DNA repair, somatic hypermutation, and class-switch recombination.
Consistent with such a role, CU1276 expression in a Burkitt Lyraphoraa-derived cell line results in an RPA /-dependent sensitization of the molecular response of these cells to DNA damage, as indicated by phosphorylation of H2AFX upon Etoposide treatment (FigSB, C). Thus, the tRNA-derived miRNA CU1276 can be a new genetic participant in the modulation of DNA damage response pathways in the GC. Furthermore, loss of CU1276 expression in lymphomas may decrease their sensitivity to ongoing DNA damage, thereby helping them to tolerate the accumulation of mutations and genomic aberrations during tumor evolution.
[00131] The validation of CU1276 as a miRNA establishes a clear regulatory potential for a large fraction of the abundant tRNA fragments expressed in many cell types. Further investigation can reveal that many additional tRNA fragments also act as miRNA, with potentially far-reaching biological consequences.
[00132] Materials and Methods
[00133] Cell culture and transfection. 293T cells were maintained in DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin. All B cell lines were maintained in IMDM supplemented with 10% FBS and 1% Penicillin/Streptomycin.
Transfection of 293T cells was achieved by standard calcium phosphate precipitation, or by PEI-based transfection, as previously described (29). 293T-shCtrl and 293T-shDICER stable cell lines were established by transfection with pLKO-based vectors (see Supplementary Methods for details of plasmids and cloning information) followed by selection for 4 days with 2}tg/ml puromycin. P3HR1 stable cells were established by electroporation of exponentially growing cells with 5pmol of pRTS l -GLS VP-based vectors according to standard protocol. After a 48hr recovery in IMDM supplemented with 20% FBS, cells were selected with 0.5fig/nil puromycin for 4 days. Induction of expression from stable P3HR1 cells was achieved by addition of doxycyciine to growth media at a concentration of lOOng/ml. DNA damage response of stable P3HR1 ceil lines was assayed by pre-induction with doxycyciine for 24 hours, followed by treatment with Ομιη, Ι Μ, 2 Μ, or ΙΟμιη concentrations of Etoposide (Sigma) for 3 hours.
[60134] Northern blot and QRTPCR. Total RNA was purified with Trizol Reagent (Invitrogen) according to manufacturer's indications. Northern blot was performed as previously described (6), using 30μg of total RNA from each sample. Prehybridization, hybridization, and washing were performed at 55°C. For CU1276 and tRNA detection, 5'- TOGTGC ATTGGCCGGG-3 ' (SEQ ID NO: 2) probe was [γ-32Ρ]ΑΤΡ labeled by polynucleotide kinase (Fermentas). Images were obtained by exposure to film for 24-48 hours. QRTPCR was also performed as previously described (Basso et al., 2009), starting from 2μg total RNA or a percentage of RNA from IP fraction. QPCR analysis of cDNA was performed with ABsoIute Blue Sybr Green Master Mix (Thermo Scientific) using an AB7300 thermocycler (Applied Biosystems).
[00135] Western blot and 3'UTR reporter assay. Cell pellets were lysed in RIPA buffer followed by 10 minutes of sonication, cleared of debris by centrifugation, and then quantified with Bradford Reagent (B oRad). 50 g protem lysate was run on pre-cast Tris- Glycine gradient gels (Novex), transferred to Hybond ECL membrane (GE Healthcare), and subjected to standard immunoblotting technique. Antibody information, incubation conditions, and quantification procedures are indicated in SI Materials and Methods. For 3'UTR reporter assays, firefly and renilla luciferase activities were assayed according to standard protocol using the Dual Luciferase Reporter Assay System (Promega) and measured on a Lumat LB 9507 luminometer (Berthold Technologies).
[80136] Argonaute Immunoprecipitation. Argonaute immunoprecipitation was performed as previously described (17), starting from 1X10s exponentially growing RIVA cells or -5X10' 293T cells, with anti-pan- AGO antibody (MABE56, Millipore), or control IgG overnight with rotation at 4°C. Protein G magnetic beads (New England Biosciences) were added to lysates, and the mixture was incubated for 3 hours with rotation at 4°C. HA- tagged proteins were imm tioprecipitated by overnight incubation of lysate with EZ vie lM HA affinity beads (Sigma). Beads were washed and resuspended in Trizol Reagent (lnvitrogen) or lysis buffer for downstream RNA and protein analysis, respectively.
[8(5137] Gene expression profiling and data analysis. Gene expression profiles were generated from total RNA using the HG-U133Plus2.0 platform (Affymetrix) according to the manufacturer's indications. Differential expression was determined by t-test using the geWorkbench software suite (30), with a significance cut-off of p<0.05.
[8(5138] Generation and sequencing of small RNA libraries. GC B cells were isolated from tonsil tissue by magnetic cell sorting as previously reported (31 ). Tonsil tissue was collected at the Columbia-Presbyterian Medical Center following approval by the institutional ethical committee. DLBCL primary biopsies were excess from diagnostic tissue frozen at the time of diagnosis. Total RNA was isolated by Trizol Reagent (lnvitrogen) and the small RNA fraction was enriched by fiashPAGE1 fractionation (Ambion). Small RNA libraries were generated using the SOLiD Small RNA Expression Kit (Applied Biosystems) following the manufacturer's indications. SOLID sequencing was performed on 4 libraries of purified GC B cells and 25 libraries of DLBCL. Following removal of artifacts and rRNA fragments SOLID sequences were subject to a previously developed pipeline to identify candidate miRNAs (6) while tRNA-derived fragments were detected by alignment to the UCSC tRNA database (15 ). Normalized small RNA counts were obtained by correcting for the size of each library.
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Proceedings of the National Academy of Sciences of the United States of America 100(5):2639-2644.
[00140] Supplemental Materials and Methods
[80141] Plasmids and shRNA. Expression vectors for transient transfection of tRNA were generated by PCR amplification of Gly(GCC) chrl .tRNA68 (chrl : 161493501- 161493953), Gly(GCC) chrI7.tRNA5 (chrl 7:8028941-8029251 ), and a CU1276 predicted genomic precursor (chrl 9:3 1 15779-351 16077) followed by insertion into the multiple cloning site of pcDNA.3 expression vector (Invitrogen). CU1276 hairpin-expressing vector was generated by restriction digestion of pcDNA3 vector containing the human miR-26a-l locus (chr3:38()10684-3801 1069) with ael and Btgl to remove the miR-26a-l hairpin; annealed oligos with sequences 5"-
CGTGGCCTCGTCGATTCCCGGCCAATGCACCAAGTGCAGGTCCCAATGTGGTGCA
TTGTCTGGGAATTCACGGGGACGCGGGCCTGGACGCC-3' (SEQ ID NO: 3) and 5 '-
GGCGTCCAGGCCCGCGTCCCCGTGAATTCCCAGACAATGCACCACATTGGGACCT
GCACTTGGTGCATTGGCCGGGAATCGACGAGGCC-3' (SEQ ID NO: 4) were ligated in its place. An inducible CU1276 hairpin-expressing vector was created by restriction digest of the pc'DNA3-CUl 276_hairpin vector and subsequent subcloning into the Sfil sites of pRTSl- GLSVP vector (SI ). Self-ligated pRTSl-GLSVP vector containing no insert was used as an empty vector control. shRNA-expressing plasmids pLKO. I -puro-shCtrl, pLKO. I -puro- shDICER-58, pLKO. i-pui -shDICER-61, and pLKO. l-puro-shDICER-62 were purchased from Open Biosystems. pIRESneo-HA-FLAG- AGO I , -AG02, -AG03, -AG04, and -EGFP were obtained from Addgene (S2). The RPA1, WHSC1L1 , and STAG2 3'UTR reporter constructs were generated by PGR amplification of human genomic DNA, followed by insertion into the multiple cloning site of the pmiRGLO vector (Promega). RPA1 3'UTR- MUT reporter was generated by site-directed mutagenesis with primers 5'- CAAATAGGCATAAlTTCCTATATTTCCTCCCACCTCCG-3' (SEQ ID NO: 5) and 5'- GGAAATATAGGAAATTATGCCTATTTGCAAACTTCTGC-3' (SEQ ID NO: 6); STAG2 3'UTR-MUT reporter was generated by site-directed mutagenesis with primers 5'- GCTGTTAGTTGGCTTTTTCCTATATTATTTCATGCTT-3 ' (SEQ ID NO: 7) and 5'- GAAATAATATAGGAAAAAGCCAACTAACAGCGCATAAATAAAATA-3' (SEQ ID NO: 8).
[8(5142] qRT-PCR conditions. RNA samples were reverse-transcribed with the Superscript II First Strand S nthesis Kit (Invitrogen) in the presence of 0.2.uM RTFS primer (5'-TGTCAGGCAACCGTATTCACCGTGAGTGGTTGGTGCATTG-3'; (SEQ ID NO: 9)), random hexamers, or Oligo d(T) primer according to the manufacturer's indications. 1/10™ of the cDNA volume was used as a template for PGR amplification in the presence of 70nM SS primer (5 ' -CGTCAGATGTCCGAGTAGAGGGGG AACGGCGTCGATTCCCGGC-3 ' ; (SEQ ID NO: 10», and 70nM each of MPF (5 ' -TGTCAGGC AACCGTATTC ACC-3 ' ; (SEQ ID NO: 1 1)) and MPR (5'-CGTCAGATGTCCGAGTAGAGG-3'; (SEQ ID NO: 12)) universal primers, or gene specific primers, as appropriate. 5s rRNA and RNU66 were detected from cDNA generated by random hexamer reverse transcription, according to gene- specific primers (5srRNAF: 5'-GCCCGATCTCGTCTGATCT-3' (SEQ ID NO: 13), 5srRNAR: 5'-AGCCTACAGCACCCGGTATT-3' (SEQ ID NO: 14), RNU66F: 5' - GGTGATGGAAATGTGTTAGCC-3 ' (SEQ ID NO: 15), RNU66R: 5'- AGGATAGAAAGAACCACCTCA-3 '(SEQ ID NO: 16)). RPA I, RPA2, and GAPDH were detected from cDNA generated by Oligo d(T) reverse transcription, according to gene- specific primers (RPA1F: 5 '-CTTCACGTCC ATCACAGTGG-3 ' (SEQ ID NO: 17), RP .1R: 5 '-TTTCC AGAATGCCAACTTCC-3 ' (SEQ ID NO: 18), RPA2F: 5'- AGGGAGAGCACCTATCAGCA-3' (SEQ ID NO: 19), RPA2R: 5'- TTCAACCCTTCAGGTCTTGG-3 ' (SEQ ID NO: 20), GAPDHF: 5'- CTGACTTCAACAGCGACACC-3 ' (SEQ ID NO: 21 ), GAPDHR: 5'- CCCTGTTGCTGTAGCCAAAT-3' (SEQ ID NO: 22)).
[80Ϊ43] Western blot conditions and antibody information. Ail primary antibody dilutions were prepared in PBS with 0, 1% Tween20 and 5% BSA, Dilutions and incubation conditions were as follows: ACTB (A5441 ; Sigma), 1 :5000, 1 hour at room temperature; DICER (13D6; Abeam), 1 : 1000, overnight at 4°C; GFP (JL-8; Clontech), 1 :5000, 30 minutes at room temperature; yH2AFX (05-636; Millipore), 1 : 1000, overnight 4°C; H2AFX, total (A300-082A; Bethyl Laboratories), 1 :500, overnight at 4°C; RPA1 (2267; Ceil Signaling), 1 :1000, overnight at 4°C; RPA2 (14692; Santa Cruz), 1 :500, overnight at 4°C. HRP- conjugated secondary antibodies anti-mouse IgG HRP (NA931V; GE Healthcare), anti-goat IgG HRP (2020; Santa Cruz), and anti-rabbit IgG HRP (1238850; Boehringer Mannheim) were diluted at 1 : 10000 in PBS with 0.1% Tween20, 5% milk, and used for 30mins-3hr incubations at room temperature. Blots were visualized with ECL substrate or SuperSignal West Dura ECL substrate (Thermo Scientific). Where applicable, blots were quantified using the ImageJ software suite (S3).
[8(5144] Supplemental references
51. Bornkamm GW, et al. (2005) Stringent doxycycline-dependent control of gene
activities using an episomal one- vector system. Nucleic acids research 33(16) :e 137,
52. Meister G, el al. (2004) Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Molecular cell 15(2): 185- 197.
53. Abramoff M, Magalhaes P, & Ram S (2004) Image Processing with ImageJ.
Biophotonics International 11(7):36-42.
EQUIVALENTS
[80145] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein.

Claims

What is claimed is:
1. A method for treating a blood cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a nucleic acid composition comprising 90% of SEQ ID NO: 1, or a fragment thereof, thereby treating the blood cancer in the subject.
2. The method of claim 1, wherein ihe blood cancer is a lymphoma.
3. The method of claim 1, wherein the blood cancer is a B cell lymphoma.
4. A method for decreasing proliferation of a blood cancer ceil, the method comprising delivering to a ceil an effective amount of a nucleic acid composition comprising 90% of SEQ ID NO: 1 , or a fragment thereof, thereby decreasing proliferation of the cancer cell.
5. The method of claim 4, wherein the proliferation comprises cell invasion, cell migration, or a combination thereof.
6. The method of claim 4, wherein the blood cancer is a lymphoma.
7. The method of claim 4, wherein the blood cancer is a B cell lymphoma.
8. A method for reducing the number of blood cancer cells, the method
comprising delivering to a ceil an effective amount of a nucleic acid composition comprising 90% of SEQ ID NO: I, or a fragment thereof, thereby reduces the number of cancer cells.
9. The method of claim 8, wherein the blood cancer is a lymphoma.
10. The method of claim 8, wherein the blood cancer is a B cell lymphoma.
1 1 . A. method of decreasing growth of a solid tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of a nucleic acid composition comprising 90% of SEQ ID NO: I, wherein the composition decreases the size of the solid tumor.
12. The method of claim 1 1, wherein the solid tumor is a lymphoma.
13. A method for treating a B cell lymphoma in a subject in need thereof, the method comprising administering to the subject an effective amount of a nucleic acid composition comprising 90% of SEQ ID NO: 1 , or a fragment thereof, thereby treating B cell lymphoma in the subject.
14. A diagnostic kit for determining whether a sample from a subject exhibits a presence or absence of a cancer-associated tRNA fragment, the kit comprising at least one oligonucleotide that specifically hybridizes to a nucleic acid comprising 90% of SEQ ID NO: 1 , or a portion thereof.
15. The kit of claim 14, wherein the oligonucleotides comprise a set of nucleic acid primers.
16. The kit of claim 14, wherem the oligonucleotide comprises at least 10
consecutive nucleotides comprising SEQ ID NO: 3 or 4.
17. A method for detecting the presence of a cancer-associated tRNA fragment in a human subject, the method comprising:
(a) obtaining a biological sample from a human subject; and
(b) detecting whether or not a nucleic acid sequence comprising 90% of SEQ ID NO: 1 , or a portion thereof is present in the subject.
18. The method of claim 17, wherein the detecting comprises using hybridization, amplification, or sequencing techniques to detect a cancer-associated tRNA fragment.
19. The method of claim 18, wherem the amplification uses primers comprising at least 10 consecutive nucleotides comprising SEQ ID NO: 3 or 4.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100197772A1 (en) * 2007-07-18 2010-08-05 Andrea Califano Tissue-Specific MicroRNAs and Compositions and Uses Thereof
US20120302626A1 (en) * 2009-12-04 2012-11-29 Sandeep Dave Microrna and use thereof in identification of b cell malignancies

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100197772A1 (en) * 2007-07-18 2010-08-05 Andrea Califano Tissue-Specific MicroRNAs and Compositions and Uses Thereof
US20120302626A1 (en) * 2009-12-04 2012-11-29 Sandeep Dave Microrna and use thereof in identification of b cell malignancies

Non-Patent Citations (2)

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Title
MA ET AL.: "Tumour invasion and metastasis initiated by microRNA-10b in breast cancer.", NATURE, vol. 449, no. 7163, 11 October 2007 (2007-10-11), pages 682 - 688 *
MAUTE ET AL.: "tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma.", PROC NAT ACAD SCI, vol. 110, no. 4, 7 January 2013 (2013-01-07), pages 1404 - 1409 *

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