WO2015188899A1 - Novel ttv mirna sequences as an early marker for the future development of cancer and as a target for cancer treatment and prevention - Google Patents

Novel ttv mirna sequences as an early marker for the future development of cancer and as a target for cancer treatment and prevention Download PDF

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WO2015188899A1
WO2015188899A1 PCT/EP2014/078346 EP2014078346W WO2015188899A1 WO 2015188899 A1 WO2015188899 A1 WO 2015188899A1 EP 2014078346 W EP2014078346 W EP 2014078346W WO 2015188899 A1 WO2015188899 A1 WO 2015188899A1
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ttv
mirna
nucleotide sequence
cancer
polynucleic acid
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PCT/EP2014/078346
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French (fr)
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Harald Zur Hausen
Ethel-Michel DE VILLIERS
Angel Cid-Arregui
Victor SARACHAGA DE BENITO
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Deutsches Krebsforschungszentrum
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Priority claimed from PCT/EP2014/062251 external-priority patent/WO2014198833A1/en
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Priority to US14/966,110 priority Critical patent/US10457938B2/en
Publication of WO2015188899A1 publication Critical patent/WO2015188899A1/en

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    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/10—Applications; Uses in screening processes
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • Novel V miRNA sequences as an early marker for the future development of cancer and as a target for cancer treatment and prevention
  • the present invention relates to novel TTV miRNA as well as probes and primers comprising part of said novel TTV miRNA polynucleic acid. Finally, the present invention relates to the use of said compounds as an early marker for the future development of cancer.
  • TTV Torque Teno Virus
  • ssDNA single stranded DNA genome of 3.7-3.8Kb of length
  • ssDNA single stranded DNA genome of 3.7-3.8Kb of length
  • ssDNA single stranded DNA genome of 3.7-3.8Kb of length
  • ssDNA single stranded DNA genome of 3.7-3.8Kb of length
  • ssDNA single stranded DNA genome of 3.7-3.8Kb of length
  • They were first discovered in 1997 in a patient presenting post-transfusion non A to G hepatitis [1] .
  • a high divergence in the nucleotide sequence among different TTV strains is observed, reaching to more than 70% in some cases.
  • the genomic organization is also variable , all of them contain a non-coding region , spanning 1.2Kb [22] .
  • the non-coding region has been demonstrated to harbour a promoter in its 3' end [4] and a highly conserved region of 70 bp within this 3 ' end is hypothesized to be the origin of replication of the viruses. It is estimated that more than 90% of humans are infected with one or more TTV strains. The number of different isolates (more than 200 ⁇ , their ubiquity and the lack of reliable and simple techniques to differentiate between them, have made it difficult to obtain enough epidemiological evidence in support of a causative relationship between TTV infection and a specific disease [23-28] . TTV viruses are known to infect several human tissues [21] . Limited data are available on the replication cycle, and even less on the function of the proteins encoded by these viruses.
  • MicroRNAs are small RNA molecules ranging between 19 and 29 nt and usually of 22 nt in length. They mediate post- transcriptional gene silencing (PTGS) by inducing cleavage, destabilization or translational inhibition of a target messenger RNA (mRNA) [9,10,11,12] . They do that by guiding the RISC complex to a concrete mRNA, interacting with it by base pairing.
  • PTGS post- transcriptional gene silencing
  • TGS transcriptional gene silencing
  • RNAa transcriptional gene activation
  • miRNAs encoded by both Polyoma and Herpes viruses have been demonstrated to help these viruses to escape the host immune response, by regulating viral [17] or host [18,19] protein expression.
  • Another important finding was made some months ago when it was demonstrated that Epstein-Barr virus-encoded miRNAs are sufficient to transform cells by themselves [20], suggesting that viral miRNAs could be able to mediate an oncogenic process under the adequate conditions .
  • TTV encode for miRNA ' s and the role of one of this miRNA ' s in interferon signalling inhibition was demonstrated [78]
  • APC Adomatous Polyposis coli
  • APC is a very important tumour suppressor, especially in the context of colorectal cancer.
  • Virtually all colorectal cancers carry inactivating APC mutations or epigenetic changes inactivating the transcription of this gene.
  • Its tumour suppressor activity is thought to be mediated by its function in inhibition of wnt signalling, although it has also been implicated in migration and correct mitotic spindle assembly.
  • the technical problem underlying the present invention is to provide means (or markers) for diagnosis of cancer or diagnosis of a disposition to said disease. Another technical problem is to provide means for preventing cancer development and cancer recurrence by inhibiting a specific target.
  • TTV encode miRNAs are provided.
  • the miRNA are transcribed from the non-coding region of the virus, in both sense and antisense orientations.
  • Some miRNAs encoded in both orientations can, directly or indirectly, downreguiate the tumor suppressor Adenomatous Polyposis Coli (APC) at the mRNA level.
  • APC tumor suppressor Adenomatous Polyposis Coli
  • FIG. 1 TTV-HDl 4a genomic organization and pre-miRNA location
  • A TTV-HDl 4 .
  • the numbers over the lines indicate the nucleotide number.
  • B Details of the non- coding region. The numbers indicate the nucleotide number.
  • the hairpins over the line indicate the pre-miRNA encoded in sense orientation.
  • the hairpins under the line indicate the pre-miRNA encoded in antisense orientation.
  • the names over and under the hairpins are the names given to the pre-miRNA.
  • Figure 2 Schematic representation of the plasmids used for trans fection and Northern Blots showing the pre-miRNA and mature miRNA
  • A Schematic representation of the plasmids containing the CMV promoter and the non coding region (NCR) in sense ( + ) or antisense (-) orientation.
  • the constructs are named pCDNA3.1 ⁇ +) -TTV-HDl4a-NCR-Sense and pCDNA3.1 ⁇ + ) -TTV-HDl a-
  • NCR-Anti Sense respectively.
  • the numbers over and under the lines indicate the nucleotide number.
  • the hairpins and the vertical lines indicate the pre-miRNA in sense or antisense orientation.
  • the names of the pre-miRNA are written below the lines.
  • TTV-HD14a-mir-2-5p (1,2 and 3) TTV-HD14a-mir-2-3p (4) with APC mRNA. Positions relative to APC transcript variant 2 (NCBI accession number: NM_001127510.2; SEQ ID NO: 82) .
  • TTV-HDl4a ⁇ ASmir-2-3p TTV-HDl4a ⁇ ASmir-2-3p and APC promoters 1, 2 and 3, respectively, as stored in EPDNew Human.
  • the shown positions relate to the transcription start site (TSS) in reference to EPD New Human (EPDNew Human names: APC_1, APC_2 and APC__3) .
  • HEK293TT were transfected in a 24 well format with 300 ng of TV-H 14a virus or pCDNA-3.1 (-) - TV-HD14a-NCR ( 2820- 3516) Sense (referred in the graphic as "Sense") or pCDNA-
  • the mature TTV miRNA molecule according to the invention (a) is a nucleotide sequence underlined in
  • a "mature TTV miRNA” is a polynucleic acid of an miRNA derived from a TTV .
  • polynucleic acid refers to a single-stranded or double-stranded nucleic acid sequence.
  • a polynucleic acid may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides or may have been adapted for diagnostic or therapeutic purposes .
  • a polynucleic acid may also comprise a double stranded cDNA clone which can be used, for example, for cloning purposes.
  • the TTV polynucleic acid and the mature TTV miRNA of the invention can be prepared according to well-known routine methods, for example, by (a) isolating the entire DNA or, preferably, RNA from a sample, (b) detecting the TTV sequence by hybridization or PGR and (c) cloning of the TTV sequence into a vector (d) by synthesis of the respective nucleotides of the miRNA sequence.
  • sequence variants of the polynucleic acid and the mature TTV miRNA molecules of the invention containing eithe r deletions and/or insertions of one or more nucleotides, especially insertions or deletions of one or more codons, mainly a the extremities of oligonucleotides (either 3 ' or 5 1 ) and which show at least
  • Pol nucleic acid sequences according to the present invention which are similar to the sequence as shown in Table 1, 2a or 2b can be characterized and isolated according to any of the techniques known in the art, such as amplification by means of sequence- specific primers, hybridization with sequence-specific probes under more or less stringent conditions, sequence determination of the genetic information of TTV etc .
  • the " variants and fragments of the TTV polynucleic acid sequences of the present invention are still able to interfere with or inhibit the expression of their target gene, for example APC.
  • CAUCCCC CAUCCCU
  • CAUCCUC CAUCCUU
  • the inventors show how the most conserved seed motif (AUCCUC) has three additional possible interaction sites within APC mRNA in addition to the previously described for TTV-HD14a-mir-2-3 .
  • AUCCUC most conserved seed motif
  • Table 8 the invention relates to variants of the polynucleic acid as described above which comprise the seed motif AUCCCUC and bind to at least one of the interaction sites within APC mRNA shown in Table 8 and, preferably, downregulate APC.
  • Also included in the present invention are analogous miRNAs in other human TT virus types and variants and in similarly structured single-stranded DNA viruses of the human or animal origin.
  • Anelloviruses have been demonstrated in domestic animals in part with similar nucleotide sequences as human TT viruses [77] .
  • the present invention relates to oligonucleotide primer comprising part of the polynucleic acid of the present invention, said primer being capable of acting as primer for specifically sequencing or specifically amplifying a certain TTV miSNA.
  • primer refers to a . single stranded DNA oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied.
  • the length and the sequence of the primer must be such that they allow to specifically prime the synthesis of the extension products.
  • the primer is at least about 10, preferably at least 15 nucleotides.
  • the amplification primers do not have to match exactly with the corresponding template sequence to warrant proper amplification.
  • the amplification method used can be either polymerase chain reaction (PGR) , ligase chain reaction (LCR) , nucleic acid sequence-based amplification (NASBA) , transcription-based amplification system (TAS) , strand displacement amplification ( SDA) or amplification by means of QB replicase or any other suitable method to amplify nucleic acid molecules using primer extension.
  • PGR polymerase chain reaction
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • TAS transcription-based amplification system
  • SDA strand displacement amplification
  • the amplified products can be conveniently labelled either using labelled primers or by incorporating labelled nucleotides. Labels may be isotopic ( 32 P, 3 S , etc.) or non-isotopic (biotin, digoxigenin, etc.) .
  • the present invention also relates to an oligonucleotide probe comprising part of the TTV polynucleic acid of the present invention, said probe being capable of acting as a hybridization probe for specific detection of a certain TTV miRNA in vitro and in vivo.
  • probe refers to single stranded sequence-specific oligonucleotides which have a sequence which is complementary to the target sequence of the TTV polynucleic acid to be detected. Preferably, these probes are about 5 to 50 nucleotides long, more preferably from about 10 to 25 nucleotides.
  • the probe can be fixed to a solid support, i.e., any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low.
  • the solid substrate will be a microtiter plate, a membrane ⁇ e.g. nylon or nitrocellulose) or a microsphere (bead) .
  • nucleic acid probe Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fixation or improve the hybridization efficiency. Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NH Z groups, SH groups, carboxylic groups, or coupling with biotin or haptens .
  • the probe is an ant i-miR oligonucleotide.
  • An anti-miR oligonucleotide is a single- stranded RNA complementary to the miRNA molecule according to the invention. It can be delivered in its RNA form or being expressed from a vector, using a polymerase III promoter. Such an anti-miR oligonucleo ide can be used for inhibiting the miRNA of the present invention. Methods for inhibiting miRNA by anti-miRs are described by Stenvang et al. in [81], which publication is incorporated by reference .
  • a further embodiment of the invention are miRNA sponges.
  • a miRNA sponge is a messenger RNA wi h several, preferably 6-8 , perfect complementary binding sites to the polynucleotide acid, i.e. mature TTV miRNA, of the invention. The binding sites can also include mismatches in the nucleotides from 10 to 13 of the mature TTV miRNA, to avoid direct RNA slicing and degradation which makes them more effective.
  • a further embodiment of the invention are tough decoy inhibitors.
  • a tough decoy inhibitor is a miRNA consisting of a " hairpin comprising a large internal bulge exposing two of the miRNA interaction sites of APC shown in Table 8 with imperfect baise-pairing with the TTV miRNA of the invention .
  • the design of such a tough decoy inhibitor and methods of suppressing miRNA by a tough decoy inhibitor are described in [82] and [83] which are incorporated by reference.
  • the anti-miR, miRNA spounge and tough decoy inhibitor according to the invention are inhibitors of the TTV polynucleic acid as described above, in a preferred embodiment of a mature TTV miRNA shown underlined in Table 2A and 2B, which prevent the interaction between the TTV polynucleic acid and APC such that APC is not downregulated.
  • the present invention also relates to a vector containing a TTV polynucleic acid, oligonucleotide primer, oligonucleotide probe, anti-miR, miRNA sponge or tough decoy inhibitor of the invention allowing, e.g., expression, mutagenesis or a modification of a sequence by recombination of DNA sequences .
  • the vectors are plasmids, cosmids, viruses, bacteriophages and other vectors usually used in the field of genetic engineering.
  • Vectors suitable for use in the present invention include, but are not limited to the
  • the polynucleic acid of the invention or part thereof is operatively linked to the regulatory elements in the recombinant vector of the invention that guarantee the transcription and synthesis of an niRNA in prokaryotic and/or eukaryotic cells that can be translated.
  • the nucleotide sequence to be transcribed can be operably linked to a promoter like a T7 , metallothionein I or polyhedrin promoter.
  • the present invention also relates to recombinant host cells transiently or stably containing the TTV polynucleic acid (or fragments thereof) or vectors of the invention.
  • a host cell is understood to be an organism that is capable to take up in vitro recombinant DNA and, if the case may be, to synthesize the polypeptids encoded by the polynucleotides of the invention.
  • these cells are prokaryotic or eukaryotic cells, for example mammalian cells, bacterial cells, insect cells or yeast cells.
  • the present invention also relates to a diagnostic kit containing a TTV polynucleic acid, an oligonucleotide primer, an oligonucleotide probe, a polypeptide and/or an antibody of the present invention.
  • the probes For hybridization based assays, according to the hybridization solution (SSC, SSPE, etc.), the probes should be stringently hybridized to the target (with or without prior amplification) at their appropriate temperature in order to attain sufficient specificity.
  • SSC hybridization solution
  • SSPE hybridization solution
  • the probes or variants thereof can be caused to hybridize specifically at the same hybridization conditions (i.e. the same temperature and the same hybridization solution) .
  • changing the amount (concentration) of probe used may be beneficial to obtain more specific hybridization results.
  • Suitable assay methods for purposes of the present invention to detect hybrids formed between the oligonucleotide probes and a TTV polynucleic acid in a sample may comprise any of the assay formats known in the art, such as the conventional dot-blot format, sandwich hybridization or reverse hybridization.
  • the detection can be accomplished using a dot blot format, the unlabelled amplified sample being " bound to a membrane, the membrane being incorporated with at least one labelled probe under suitable hybridization and wash conditions , and the presence of bound probe being monitored.
  • An alternative and preferred method is a "reverse" dot-blot format, in which the amplified sequence contains a label.
  • the unlabelled oligonucleotide probes are bound to a solid support and exposed to the labelled sample under appropriate stringent hybridization and subsequent washing conditions. It is to be understood that also any other assay method which relies on the formation of a hybrid between the nucleic acids of the sample and the oligonucleotide probes according to the present invention may be used.
  • the present invention also relates to the use of a TTV polynucleic acid, an oligonucleotide primer, or an oligonucleotide probe of the present invention as an early marker for the future development of cancer, preferably colorectal or colon cancer.
  • an embodiment of the present invention relates to a method of detecting or diagnosing of colon cancer, comprising the steps of:
  • step (b) sequencing the miRNA isolated in step (a) ;
  • oligonucleotides For determining miRNA labelled oligonucleotides may be used.
  • the method may comprise a further step (d) of quantifying the miRNA level in sample to distinguish between patients with colon cancer from healthy controls.
  • step (a) the miRNA is isolated from plasma or serum and the miRNA is quantified by using TaqMan miRNA qRT- PCR-assays as described in [86] which is incorporated by reference .
  • the miRNA may be isolated directly from the tumor and a miRNA sequencing may be performed to detect the miRNA or sections of any kind (e.g. cryo-sect i ons , sections from paraffin embedded tissue) may be made directly on the tumor and an hybridization for the miRNA as described above may be performed in-situ.
  • a miRNA sequencing may be performed to detect the miRNA or sections of any kind (e.g. cryo-sect i ons , sections from paraffin embedded tissue) may be made directly on the tumor and an hybridization for the miRNA as described above may be performed in-situ.
  • the present invention also relates to the use of a TTV polynucleic acid o f the present invention as a lead component for the development of a medicament for prevention or treatment of cancer, preferably colorectal or colon cancer.
  • These medicaments may be inhibitors of any interaction between miRNAs and tumour suppressor genes to avoid cancer development or recurrence and cancer treatment.
  • the specific TT virus miRNA o r o f its derivatives or of related miRNAs are useful for diagnostic, prevention or therapeutic applications in the cancer field.
  • Such inhibitor of an interaction between miRNA and tumor suppressor genes for example ARC, can be an anti-miR, A miRNA sponge or a tough decoy inhibitor as described above .
  • the inhibitor can be delivered to the tumor site by using an adeno associated virus (AAV) in order to deliver the inhibitor to counter effect the TTV miRNA according to the invention .
  • AAV adeno associated virus
  • An AAV gene therapy suitable for delivering one o f the above miRNA inhibitors to the tumor is described in [84] which is incorporated by reference.
  • a further example of a suitable method for delivering the above inhibitors against TTV miRNA to a tumor is known as low pH-induced transmembrane structure (pHILP) [85] .
  • This phILP construct consists of a peptide that crosses the plasma membrane only under acidic ' conditions which are typical of the tumor microenvironment .
  • a peptide nucleic acid of an TTV miRNA inhibitor can be attached to this pHILP in order to be delivered specifically to cells in the tumor microenvironment.
  • this peptide is an anti-miR, which will cause the inhibition of the TTV miRNA according to the invention.
  • a suitable method for delivering a TTV miRNA inhibitor with a pHILP construct is described in [85] which is incorporated by reference.
  • a further embodiment of the invention is a method of delivering a lead, component for the development of a medicament for prevention or treatment of cancer, preferably colorectal cancer comprising the step of administrating to a patient suffering from a cancer, in particular colorectal cancer (a) a pharmaceutical composition comprising an adeno- associated virus expressing an inhibitor of the miRNA of the invention selected from the group consisting of anti-miR, miRNA sponge and tough decoy inhibitor of a miRNA interacting with a tumor suppressor gene and a pharmaceutically acceptable carrier or (b) a pharmaceutical composition comprising an inhibitor of the miRNA of the invention attached to a pHILP construct and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising an adeno- associated virus expressing an inhibitor of the miRNA of the invention selected from the group consisting of anti-miR, miRNA sponge and tough decoy inhibitor of a miRNA interacting with a tumor suppressor gene and a pharmaceutically acceptable carrier
  • a pharmaceutical composition comprising an inhibitor of the miRNA of
  • a further embodiment of the Invention is (a) an adeno- associated virus for the use of delivering an inhibitor of
  • TTV miRNA to tumor cells or (b) a pHILP construct for the use of delivering an inhibitor of a miRNA of the invention to tumor cells .
  • the inhibitor to be delivered is selected from the group consisting of anti-miR, miRNA sponge and tough decoy i nh ibitor and is an inhibitor of a mature TTV miRNA as shown underlined in Table 2B which interacts with APC.
  • Table 2B which interacts with APC.
  • HEK293TT cells [76] cultured in Dulbeco' s Eagle Modified
  • the TTV NCR is PGR amplified using suitable primers.
  • the TTV-HD14a NCR is PGR amplified using primers TT-ON9 5' gattatggtacctttccaactacgactgggtgt (SEQ ID NO: 83) and TT-ON10 5' gattatggtacctctaccattcgtcaccgctgtt (SEQ ID NO: 84) using pCDNA3.1 (+ ) -TTV-HD1 a as template ⁇ a plasmid containing full-length TTV-HD14a linearized and cloned into Xbal site) .
  • PGR product is run on a 1% agarose gel and DNA stained using ethidium bromide. Bands corresponding to the expected size (-1200 bp) are cut and subsequently extracted from agarose using QIAEXII gel extraction kit (QIAGEN) . 4 g of pCDNA3.1 (+ ) (Life technologies) are cut using Kpnl and dephosphorylated using Fas AP (Thermo scientific) . PGR product is cut using the same procedure, but not dephosphorylated. Cut plasmid and PGR products are cleaned up by using QIAEXII gel extraction kit.
  • Ligation of the plasmid and the amplified fragment corresponding to the TTV NCR, for example TTV-HD1 a NCR, is performed using T4DNA ligase (Thermo Scientific) Ligation product is transformed into NovaBlue Singles competent cells (Merck Millipore) according to the manufacturer instructions, and seeded in LB agar plates supplemented with ampicillin as selection marker. Plates are incubated 20 hours at 37°C. Single colonies are picked and seeded in LB medium supplemented with ampicillin. These cultures are incubated 20 hours. Plasmid is extracted using PureLink Quick Plasmid Miniprep Kit (Life technologies j . I g of each plasmid are double cut with SacI and Nhel (Thermo Scientific) .
  • Cut products are run in 1% agarose gels.
  • the restriction strategy allows us to distinguish between inserts clones in the sense and antisense orientation.
  • Two positive plasmids one containing the sense and the other one the antisense insert, are chosen and sent for sequencing. After confirming the sequence, plasmids are prepared for transfection by using Plasmid Maxi Kit (Qiagen) .
  • Cells are harvested 48-72h post-trans feci, i on .
  • Cells are homogenized using QiaShreder (Qiagen) according to manufacturer instructions . Lysates are then subjected to RNA extraction using miRNAeasy mini kit or RNAeasy mini kit (Qiagen) depending on the purpose of the RNA (for miRNA Northern blot or for RT-qPCR) , according to manufacturer instructions .
  • RNA samples are treated with RQ1 Dnase ( Promega ) according to manufacturer instructions, with the addition of RNasin
  • Custom DNA oligos are ordered to Sigma (Table 4 ) . Probes are 3' biotin labeled .
  • Terminal Deoxynucleotidyl transferase ⁇ TdT Terminal Deoxynucleotidyl transferase ⁇ TdT
  • 2,5 nanomoles of Biotin- 11-dUTP Thermo Scientific
  • Probes are subjected to Isoamyl alcohol-Chloroform extraction and the total volume is used for subsequent hybridization .
  • RNA per sample 30-50 ⁇ g of total RNA per sample are separated by electrophoresis using 15% polyacry1 amide (29:1) gels cast in 7M urea and buffered with lxTBE using a Mini Protean cell (Bio-Rad) .
  • the electrophoresis buffer is 0.5x TBE . Gels are stained with EtBr.
  • RNA is cross linked to the membrane by ultraviolet exposure using Stratalinker ( Stratagene ) .
  • Membranes are cut as needed and hybridized with the appropriated biotin labeled probe (Table 4) o/n in
  • Hybridization signals are detected using BrightStar BioDetect Kit (Life technologies) according to the manufacturer instructions. Film used: (Fiji) .
  • RNA 1 ⁇ g of RNA is used to make cD A with superscript III and RnaseOUT (Life technologies) according to manufacturer instructions.
  • cDNA is diluted 1:10.
  • qPCR is performed using Taqman fast master mix and Taqman expression assays, in a qPCR machine StepOne plus (Applied Biosystems).
  • V-mir is set to default configuration, changing the sequence type to circular.
  • CID-miRNA is run on the web- based tool, using the default run configuration for Homo sapiens.
  • Mature Bayes is run on the web-based tool.
  • DIANA microT -3.0 is run on the web-based tool (no options are given for this program) .
  • RNA hybrid is run using constraint nucleotide configuration, from nucleotide
  • Sense ⁇ TTV- HDl6a-mir-3 GGCCGCCATTTTAAGTAA—GGCGGAAGCAACTCCACTTTCTCRCAAAATGGCGGCGGAGCACTTCCGGCTTGCCCAAAATGGCCGCC
  • Sense3 TTV- HD14a-mir-2 GCTGTGACGTCAACG-TCACGTGGG-GAGGACGGCGTGTAACCCGGAAGTCATCCCCA-TCACGTGACCTGACGTCACGGC—
  • TTV- Sanban-mir-2 ACGTCACAAGTCACGTGGGGAGGGTTGGCGTATAGCCCGGAAGTCAATCCT-CCCACGTGGCCTGTCACGT
  • TTV- sle2057 ⁇ mir-2 ACGTCACAAGTCACCTGACTGGGGAGGAGTCACAACCCGGAAGTCCTCTTCGGTCACGTGACTAGTCACGT
  • TTV- sle2057-ASmir- 1 ACGTGAC TA—GTCACGTGACCGAAGAGGACTTCCGGGTTGTGACTCCTCCCCAGTCAGGTGACTTGTGACGT
  • TTV- ⁇ Sanban ⁇ ASmir-2 GCCTTCATTTTGGGTAAG—CCG—GAAGTGCTCCGCCGACATTTTGT—ATATGCCGAGCTGCTTCCGCCTTACTTAAAATGGCGGC
  • TTV- -tth8-ASmir-2 CCATTTTGAGTAGGTGTGGCTGATGGTGACCTTTGAACTCACGCCACCGTCCG
  • TCTCAAC T AGATGG
  • TTV- -CT30F-ASmir-2 CCGCCGCTACTGTCATACTTCCTCTTTTTTTTTTTGAAAAAGCGGAAGGAAGTCACAAGATGGCGG
  • TTV- -Sanban-mir-3 GCCGGGGGGCTGCCGCCCCCCCCGGGGAAAGGGGGGGGCCCCCCCCGGGGGGGGGTTTGCCCCCCGGC
  • TTV mature miRNA present in the TCGA small RNA sequencing datasets of colon adenocarcinoma with similarity in the nucleotides from 1 to 7 (comprising the seed (nt 2 to 7) ) to T?V-HD1 a-mir-2-3p .
  • the TTV miRNA are shown in the context of the pre-rniRNA sequence.
  • identical conserved nucleotides from 1 to 7 (comprising the seed (nt 2 to 7) ) are boxed. Positions containing identical nucleotides are marked by a (*) and positions containing nucleotides originated by a transition are marked by ( 0 ) . They are classified in groups according to their pre-miRNA sequence. In all cases, the 3p mature miRNA is underlined. The seed is written in italicized letters. The box 3 contains the consensus sequence for the nucleotides from 1 to 7. A - adenine, T - thymine, C - cytosine, G-guanine, Y - C or T.
  • TTV-HDl 4 a Genes predicted to be down-regulated by the TTV-HDl 4 a and at 5 least two other TTV strains miRNA. Notice that some strains have more than one putative miRNA that is predicted to down-regulate some of the genes .
  • NCR non-coding region
  • pre- miRNA structure As a candidate, they used the criterion that it should be predicted by both programs, with a cut-off value over 125 for the V-mir program and that it had to be located in the NCR of the virus. After filtering, only 4 pre-miRNA candidates (Table 1 and Figure IB) , two in sense orientation and two in antisense orientation, were considered as putative pre- miRNA and were further evaluated.
  • TTV-HDl 6a (FR751476, version FR751476.1 GI : 339511352 , 07.07.2011)
  • TTV- C3T0F (AB064597, version AB064597.1 GI : 17827196, 25.06.2008)
  • TTV-HD23a (FR751500, version FR751500.1 GI : 339511376, 07.07.2011)
  • TTV-YonKc ' i 97 (AB038624, version AB038624.1 GI : 7415899, 20.09.2000)
  • TTV-SANBAN (AB025946, version AB025946.2 GI : 5572683, 03.11.2009)
  • TTV-Sle2057 (AM712030, version AM712030.1 GI : 156104055, 19.02.2008)
  • 7TV- tth8 (AJ620231, version AJ 20231, version AJ 20
  • TTV-HD14a can transcribe four precursor miRNA encoded in its NCR
  • the inventors could clearly detect bands that match the expected size for a pre-miRNA with the probes directed against the 3 ' and 5 ' arm of TTV-HD14a-mir-2 and TTV-HDl 4a-ASmi r-2. Moreover, the inventors were able to detect a transcript matching the expected size for a mature miRNA within the 5 'arm of TTV- HD14a-mir-2. On the other hand, the inventors were able to detect transcripts matching the expected sizes with the probes directed against the 3 ' arm only of TTV-HD14a-mir-l and TTV-HD14a- ASmir-1.
  • TTV-HD14a encodes for several precursor miRNA in both orientations ; and at least one of them can be processed into a mature miRN .
  • Target prediction It is well known that the major feature of miRNA is down- regulating gene expression in a pes t-1 ra soriptiona 1 manner. It is - also known that this effect is caused by the mature form of the miRNAs , and not by their precursors. Although the inventors were not able to see any mature miRNA for three of the pre-miRNA, they think that low expression levels of these miRNAs rather than their absence might be the reason of this. In any case, it is necessary to identify the sequence of the mature miRNA to perform accurate predictions, and this is hard to determine by experimental methods different from mi R A-aeq . To overcome this problem, the inventors decided to use an in-silico mature miRNA predictor, Mature Bayes [37] .
  • This program predicts the mature miRNA from a pre-miRNA sequence. After doing that with all the predicted miRNA precursors (Table 2), they used DI N -micro -3.0 [38-39] to predict possible targets. They reasoned that, despite the variability in their sequences, the putative TTV mature miRNAs should have some targets in common. So, after performing the predictions, the inventors compared the results among the different TTV strains and considered as good candidates the targets that were predicted for some miRNAs belonging to TTV-KDl4a and, at least, two more TTV strains. Candidate targets are listed in Table 3.
  • RNA Hybrid RNA Hybrid [40] .
  • This program allows to directly detecting the complementary sequence of a given miRNA within a gene, independently of the conservation or localization of complementary sequence. This is useful, as most of the other prediction programs do not take into account the CDS or promoter region of the genes, while it has been demonstrated that a seed pairing with the first one can mediate PTGS and with the second one can cause TGS or RNAa [11, 12,29-33].
  • the inventors found seed complementarity between the APC gene and TTV-HD14a-mir-2-5p in three different points within the APC mRNA sequence, two in the CDS and one in the 3 ' UTR ( Figure 3A - 1, 2 and 3) .
  • a possible interaction site between TTV-HD14a-mir-2-3p and APC mRNA was present in the CDS ( Figure 3A - 4) .
  • the inventors also found complementarity between the TTV-HD14a-ASmir-2-3p and three of the four promoters listed for APC in the Eukaryotic Promoter Database New Human (EPD New Human) [59] (accession names APC_1, APC_2 APC_3 and APC_4 ) ( Figure 3B-D) .
  • APC is down-regulated after transfection with pCDNA3.1 (+) -TTVHD14a-
  • TTV-HDl4a miRNA the inventors transiently transfected
  • HEK293TT cells with the constructs encoding the miRNA, with the full length T'i'V-HD] 4a virus or mock transfected them, followed by RT-qPCR ( Figure 3E+F) .
  • APC down-regulation by the iRNA itself as well as by the full length genome (coding for the miRNA) is significant in comparison to the mock transfected.
  • GAPDH (Glyceraldehyde-3-phosphate-dehydrogenase ) is a gene usually used as internal control (housekeeping gene) , at the mRNA and protein levels, because its levels of expression are very constant among very different conditions.
  • GAPDH is up-regulated in the majority of cancers and under hypoxic conditions [72, 73, 74] .
  • the inventors suggest that the TTV miRNA dependent up-regulation of GAPDH is mediated indirectly by APC down- regulation .
  • Table 5 includes all the genes that were consistently deregulated between the transfection with the constructs and with the full-length virus.
  • ARPC2 actin related protein 2/3 complex, subunit 2, 34kDa 7160327 ARPC2 actin related protein 2/3 complex, subunit 2, 34kDa
  • ILVBL ilvB bacterial acetolactate synthase
  • APITD1 apoptosis-inducing, TAF9-like domain 1
  • carboxyl ester lipase (bile salt-stimulated lipase
  • CDKN2D cyclin-dependent kinase inhibitor 2D (pl9, inhibits CDK4)
  • ILVBL ilvB bacterial acetolactate synthase
  • TBP TAF1D TATA box binding protein
  • RNA 1090687 P0LR1D polymerase (RNA) 1 polypeptide D, 16kDa
  • TTV miRNA might be deregulating several pathways important for cancer progression.
  • the TCGA (The Cancer Genome Atlas) is an initiative of the NIH .
  • the data stored within this repository consist of sequencing datasets from cancer and normal tissue extracted from patients.
  • the data extracted by this analysis can be considered as M in vivo", since it comes directly from tumors of patients.
  • the small-R A sequencing data for colon adenocarcinoma, lung adenocarcinoma, breast carcinoma and hepatocellular carcinoma from the TCGA initiative was mapped against all the full-length TTV genomes included in the NCBI database plus several newly identified TTV from the inventors ' s laboratory.
  • miRNA taken into consideration complied to the following: mapping with 2 mismatches or less to TTV genomes and mapping in a region where the RNA is predicted to acquire the characteristic hairpin structure of a pre-miRNA (Table 7) .
  • TTV positive pa were considered when having at least one read mapping to a TTV miRNA.
  • Patients positive for TTV encoding a mature miRNA presenting the "con sequence” where considered when having at least one read mapping to a TTV strain that encodes for a mature miRNA that contains the "consens sequence”.
  • the "consensus sequence” the TTV strains found in the TCGA containing the consensus sequence and the mature miRNA form these strains are listed in Table 2B.+
  • TTV-HD14a-2-3p analogous miRNA meaning, with 80% homology or more in the nucleotides from 1 to 7 of the miRNA, comprising -the seed) ⁇ Table 2B) were found at higher frequency in colon cancer patients than in the other three type of cancer being screened so far.
  • Seed interaction sites are shown in black bold letters.
  • APC exerts its tumor suppressor activity by down-regulating canonical Wnt pathway, although other putative roles for this protein have been suggested. This effect is mediated by its participation in the "destruction complex".
  • the destruction complex is formed by APC, AXIN , and GSK3-beta, among others. This complex phosphorylates beta-catenin, allowing its ubiquitination and degradation by the proteasome. In the absence of any of the proteins of the destruction complex, its function is impaired. The final outcome is the cytoplasmic accumulation of beta-catenin, that can be then translocated into the nucleus , where it activates transcription of its target genes , together with the transcription factor TCF4 or LEF1.
  • HEK293TT ceils were transfected with the plasmids encoding for TTV-HDl a miRNA, with the TTV-HD14a full genome, or mock transfected, together with a plasmid encoding for Firefly luciferas - under the control of a minimum promoter and seven binding sites for the TCF4 /beta catenin complex (TOPFLASH plasmid) .
  • Renilia Luciferase under the control of CMV promoter was used for normalization purposes.
  • An upregulation of wnt pathway resulted in cells with the plasmid encoding for the sense-miRNA or with the TTV-HD14a virus in comparison to mock transfected cells (Figure 5).
  • TTV replicate in several tissues [21], but they only repl icate in peripheral blood mononuclear cells when these cells are activated [42] . It was recently demonstrated that TTV replicate more efficiently when they are co-infecting cells with Epstein Barr virus [41] .
  • TTV-HDl4a and Table 2B can down-regulate APC, an important tumor suppressor.
  • being infected with any of the TTVs encoding for the miRNA' s included in the present invention could represent a risk factor for the development of colon cancer, as well as many other cancer types .
  • TTV miRNA ' s that down-regulate APC in a higher frequency in colon adenocarcinoma patients in comparison to other three types of cancer (lung adenocarcinoma, hepatocellular carcinoma and breast invasive carcinoma). Consequently, TTV miRNA ' s presented here represent a target for the prevention of colon cancer, as well as a putative biomarker for the early detection of a subset of these cancers.
  • CID-miRNA a web serve for prediction of novel miRNA precursors in human genome . Biochemical and biophysical research communications , 372(4), 831- 834.
  • DIANA-microT web server elucidating microRNA functions through target prediction. Nucleic Acids Research 2009 Jul 1; 37 (Web Server issue) : 273-6
  • TT virus replicates in stimulated but not in nonstimulated peripheral blood mononuclear cells .
  • TT viruses oncogenic or tumor-suppressive properties?. In TT Viruses (pp. 109- 116) . Springer Berlin Heidelberg .
  • TTV viral load as a marker for immune reconstitution after initiation of HAART in HIV-infected patients, HIV Clinical Trials, 3(4), 287-295 46. Thorn, K. , & Petrik, J. (2007) . Progression towards AIDS leads to increased Torque teno virus and Torque teno minivirus titers in tissues of HIV infected individuals . Journal of medical virology, 79(1) , 1-7. 47. Van Es , J. H . , Kirkpatrick, C . , Van de Wetering, M . , Molenaar, M . , Miles , A., Kuipers , J., & Clevers , H. (1999) .
  • APC2 a homologue of the adenomatous polyposis coli tumour suppressor .
  • Current biology 9(2), 105-S2.
  • APC promoter methylation and protein expression in colorectal cancer metastasis Carcinogenesis, 26(1), 37-43.
  • APC promoter hypermethylation contributes to the loss of APC expression in colorectal cancers with allelic loss on 5ql . Cancer biology & therapy, 3 ( 10 ) , 960-964.
  • Sex-determining region Y box 4 is a transforming oncogene in human prostate cancer cells . Cancer research, 66(8), 4011-4019.
  • Phenotypic subpopulations of metastatic colon cancer stem cells genomic analysis . Cancer Genomics-Proteomics , 6(1), 19-29.

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Abstract

Described are TTV raiRNAs and probes and primers comprising part of said TTV miRNA polynucleic acid. The use of said compounds for diagnosis of cancer or predisposition of cancer is also described.

Description

Novel V miRNA sequences as an early marker for the future development of cancer and as a target for cancer treatment and prevention
The present invention relates to novel TTV miRNA as well as probes and primers comprising part of said novel TTV miRNA polynucleic acid. Finally, the present invention relates to the use of said compounds as an early marker for the future development of cancer.
Torque Teno Virus (TTV) is a viral species belonging to the family Aneiloviridae , genus Alphatorquevirus . Viruses classified into this specie present a circular, single stranded DNA (ssDNA) genome of 3.7-3.8Kb of length, and are non-enveloped [2,3] . They were first discovered in 1997 in a patient presenting post-transfusion non A to G hepatitis [1] . A high divergence in the nucleotide sequence among different TTV strains is observed, reaching to more than 70% in some cases. Although the genomic organization is also variable , all of them contain a non-coding region , spanning 1.2Kb [22] . The non-coding region has been demonstrated to harbour a promoter in its 3' end [4] and a highly conserved region of 70 bp within this 3' end is hypothesized to be the origin of replication of the viruses. It is estimated that more than 90% of humans are infected with one or more TTV strains. The number of different isolates (more than 200 } , their ubiquity and the lack of reliable and simple techniques to differentiate between them, have made it difficult to obtain enough epidemiological evidence in support of a causative relationship between TTV infection and a specific disease [23-28] . TTV viruses are known to infect several human tissues [21] . Limited data are available on the replication cycle, and even less on the function of the proteins encoded by these viruses.
MicroRNAs (miRNA) are small RNA molecules ranging between 19 and 29 nt and usually of 22 nt in length. They mediate post- transcriptional gene silencing (PTGS) by inducing cleavage, destabilization or translational inhibition of a target messenger RNA (mRNA) [9,10,11,12] . They do that by guiding the RISC complex to a concrete mRNA, interacting with it by base pairing. This interaction is thought to be mediated mainly by a perfect match between the target mRNA 3' untranslated region (UTR) and the miRNA "seed" (nucleotides from 2 to 7) [7,8,80], whereas a perfect match means that each of the "seed" nucleotides hybridizes by a Watson and Crick pairing with respective nucleotides of the target mRNA. In contrast, recent findings suggest that non-perfect matches (no Watson and Crick pairing or seeds containing one mismatch) in this region are more abundant than perfect matches [6] . The same study suggests that miRNA- RNA pairings in coding sequences (CDS) are as abundant as those in 3'UTRs. Moreover, they demonstrate that some miRNAs tend to hybridize with mRNAs in a region totally different from the seed, and they are still able to exert PTGS . To increase even more the complexity of the miRNA-based gene expression regulation, in the last few years some examples of transcriptional gene silencing (TGS ) and transcriptional gene activation (known as RNA activation (RNAa ) ) mediated by miRNA have appeared [29-33] . While the mechanisms mediating these two events are still poorly understood, it cannot be discarded that TGS and RNAa are general features of some miRNA. The number of known endogenous human miRNAs has increased very fast in the last few years. The number of mature miRNA annotated in miRBase is 2042 [13-16] . In addition, a large number of virally encoded miRNA has also been shown to use the cellular miRNA silencing machinery. Since the discovery of the first human viral encoded miRNA [5] its number has increased to 157 [13-16] . The majority of these miRNA are encoded by DNA viruses, especially those belonging to Herpesviridae and Polyomaviridae families. Recently, a bovine oncogenic RNA virus (Bovine Leukemia Virus) was reported to encode 8 mature miRNA, demonstrating that this type of viruses also can express them. Despite the large number of viral miRNA discovered, the function of most of them still remains elusive, although in the last years some reports have shed light over this issue. For instance, miRNAs encoded by both Polyoma and Herpes viruses have been demonstrated to help these viruses to escape the host immune response, by regulating viral [17] or host [18,19] protein expression. Another important finding was made some months ago when it was demonstrated that Epstein-Barr virus-encoded miRNAs are sufficient to transform cells by themselves [20], suggesting that viral miRNAs could be able to mediate an oncogenic process under the adequate conditions . Very recently, it was shown that TTV encode for miRNA's, and the role of one of this miRNA's in interferon signalling inhibition was demonstrated [78]
APC (Adenomatous Polyposis coli) is a very important tumour suppressor, especially in the context of colorectal cancer. Virtually all colorectal cancers carry inactivating APC mutations or epigenetic changes inactivating the transcription of this gene. Its tumour suppressor activity is thought to be mediated by its function in inhibition of wnt signalling, although it has also been implicated in migration and correct mitotic spindle assembly.
The technical problem underlying the present invention is to provide means (or markers) for diagnosis of cancer or diagnosis of a disposition to said disease. Another technical problem is to provide means for preventing cancer development and cancer recurrence by inhibiting a specific target.
The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
Few aspects are known concerning the interaction between TTVs and their host. In the studies resulting in the present invention it was elucidated that TTV encode miRNAs, as well as their significance for the TTV infection and pathogenicity, mainly focusing on their possible role in cancer. Pre-miRNAs expressed by TTV strains are provided. The miRNA are transcribed from the non-coding region of the virus, in both sense and antisense orientations. Some miRNAs encoded in both orientations can, directly or indirectly, downreguiate the tumor suppressor Adenomatous Polyposis Coli (APC) at the mRNA level. Surprisingly, the inventors identified a link between TTV and tumour suppressor regulation and this finding suggests a role of TTV in cancer development. This work represents the first molecular link between TTV and cancer.
Brief description of the drawings
Figure 1 : TTV-HDl 4a genomic organization and pre-miRNA location
(A) TTV-HDl 4 . The numbers over the lines indicate the nucleotide number. NCR - Non- coding region. (B) Details of the non- coding region. The numbers indicate the nucleotide number. The hairpins over the line indicate the pre-miRNA encoded in sense orientation. The hairpins under the line indicate the pre-miRNA encoded in antisense orientation. The names over and under the hairpins are the names given to the pre-miRNA.
Figure 2: Schematic representation of the plasmids used for trans fection and Northern Blots showing the pre-miRNA and mature miRNA
(A) Schematic representation of the plasmids containing the CMV promoter and the non coding region (NCR) in sense ( + ) or antisense (-) orientation. The constructs are named pCDNA3.1 {+) -TTV-HDl4a-NCR-Sense and pCDNA3.1 {+ ) -TTV-HDl a-
NCR-Anti Sense, respectively. The numbers over and under the lines indicate the nucleotide number. The hairpins and the vertical lines indicate the pre-miRNA in sense or antisense orientation. The names of the pre-miRNA are written below the lines.
(B-E) Northern blots showing the results with the indicated probes and trans fections (Sense - HEK293TT cells transfected with pCDNA3.1 ( + ) -TTV-HDl 4 a-NCR-Sense . Anti- sense - HEK293TT cells transfected with pCDNA3.1 (+) -TTV- HD14a-NCR-AntiSense . Mock - HEK293TT cells transfected with pCDNA3.!(+)) . Probe sequences are listed in Table 4. Figure 3: Complementarity of TTV-HD14a-mir-2-5p with APC mRNA and TTV-HD14a-ASmir-2-3p with the APC promoters and APC mRNA down-regulation in transfected cells
(A) Complementarity between TTV-HD14a-mir-2-5p (1,2 and 3) TTV-HD14a-mir-2-3p (4) with APC mRNA. Positions relative to APC transcript variant 2 (NCBI accession number: NM_001127510.2; SEQ ID NO: 82) .
(B, C and D) Complementarity between TTV-HDl4a~ASmir-2-3p and APC promoters 1, 2 and 3, respectively, as stored in EPDNew Human. The shown positions relate to the transcription start site (TSS) in reference to EPD New Human (EPDNew Human names: APC_1, APC_2 and APC__3) .
(E) Relative expression levels of APC as measured by qPCR (Mean for Sense = 0.747 and for AntiSense = 0.650). ACt was calculated respect to HPRT . AACt was calculated respect to mock transfected cells. Differential values were normalized to 1. Sense - Relative values for HEK 293TT cells transfected with pCDNA3.1 (+) -TTVHD14a-NCR-Sense . Antisense - Relative values for HEK293TT cells transfected with pCDNA3.1 (+) -TTVHD14a-NCR-AntiSense . Mock - Relative values for HEK293TT cells transfected with pCDNA3.1 (+) . TTV-HD14a - Relative values for cells transfected with the full-length TTV-HD14a virus, +-SD N=6. Statistical significance calculated using unpaired two-tailed Student T-Test .
Figure 4: GAPDH expression (see Example 6 for detai1s )
Relative expression levels of GAPDH as measured by qPCR. ACt was calculated respect to HPRT. AACt was calculated respect to mock transfected cells. Differential values were normalized to 1. Sense - Relative values for HEK 293TT cells transfected with pCDNA3.1 (+} -TTVHD14a-NCR-Sense . Antisense - Relative values for HEK293TT cells transfected with pCDNA3.1 (+) -TTVHDl4a-NCR-AntiSense . Mock - Relative values for HEK293TT cells transfected with pCDNA3.1 (+) . TTV-HDl4a - Relative values for cells transfected with the full-length TTV-HD14a virus. +-SD N=6. Statistical significance calculated using unpaired two-tailed Student T-Test . Figure 5: rit, upregulation by TTV-HDl4a miRNA' s
HEK293TT were transfected in a 24 well format with 300 ng of TV-H 14a virus or pCDNA-3.1 (-) - TV-HD14a-NCR ( 2820- 3516) Sense (referred in the graphic as "Sense") or pCDNA-
3.1 (-) -TTV-HD14a-NCR (3516-2820) -AntiSense (referred in the graphic as "Antisense" ) or pCDNA3.1 (-) (referred in the graphic as "Vector") or Silencer si PC (Life technologies) plus 60ng of TOPFLASH vector (provided by M.Boutros lab) and 5ng of CMV-renilia . Luciferase activity was measured 72h after transfection. (A) Firefly luciferase units normalized to Renilia luciferase (B) Fold change respect to vector.
Accordingly, the present invention relates to a TTV polynucleic acid comprising: (a) a nucleotide sequence depicted in Table 1, 2a or 2b; (b) a nucleotide sequence having at least 60% identity to the nucleotide sequence of (a) and containing the nucleotide sequence CATCCYY (with Y = C or T) ; (c) a fragment of the nucleotide sequence of (a) or (b) and containing the nucleotide sequence CATCCYY (with Y =
C or T) ; or (d) a nucleotide sequence which is complementary to any of said nucleotide sequences.
A further embodiment of the present invention relates to a TTV polynucleic acid, wherein the TTV polynucleic acid is a mature TTV miRNA molecule consisting of 19 to 29 nt, preferably about 22 nt, and comprise the nucleotide sequence CATCCY (with Y = C or T) or CAUCCYY (with Y: C or U) . In a preferred embodiment the mature TTV miRNA molecule according to the invention (a) is a nucleotide sequence underlined in
Table 2a or 2b,· (b) consists of a nucleotide sequence having at least 60%, preferably at least 80%, most preferably at least 90% identity to the nucleotide sequence of (a) underlined in Table 2A or 2B and comprises the nucleotide sequence CATCCYY (with Y = C or T) or CAUCCYY (with Y: C or
U) ; (c) is a fragment of a nucleotide sequence of (a) underlined in Table 2A or 2B and comprises the nucleotide sequence CATCCYY (with Y = C or T) or CAUCCYY (with Y: C or U) or (d) is a nucleotide sequence being complementary to a nucleotide sequence of (a) , (b) or (c) . In the context of the present invention a "mature TTV miRNA" is a polynucleic acid of an miRNA derived from a TTV .
The term "polynucleic acid" refers to a single-stranded or double-stranded nucleic acid sequence. A polynucleic acid may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides or may have been adapted for diagnostic or therapeutic purposes . A polynucleic acid may also comprise a double stranded cDNA clone which can be used, for example, for cloning purposes. In the following statements and findings made on the DNA level apply to the RNA level accordingly and vice versa.
The TTV polynucleic acid and the mature TTV miRNA of the invention can be prepared according to well-known routine methods, for example, by (a) isolating the entire DNA or, preferably, RNA from a sample, (b) detecting the TTV sequence by hybridization or PGR and (c) cloning of the TTV sequence into a vector (d) by synthesis of the respective nucleotides of the miRNA sequence. Also included within the present invention are sequence variants of the polynucleic acid and the mature TTV miRNA molecules of the invention containing eithe r deletions and/or insertions of one or more nucleotides, especially insertions or deletions of one or more codons, mainly a the extremities of oligonucleotides (either 3 ' or 51 ) and which show at least
60%, 70%, 80%, 90%, 95% or 98% identity to said polynucleic acid sequence of the invention and contain the consensus sequence CATCCYY (with Y = C or T) . Pol nucleic acid sequences according to the present invention which are similar to the sequence as shown in Table 1, 2a or 2b can be characterized and isolated according to any of the techniques known in the art, such as amplification by means of sequence- specific primers, hybridization with sequence-specific probes under more or less stringent conditions, sequence determination of the genetic information of TTV etc . The" variants and fragments of the TTV polynucleic acid sequences of the present invention are still able to interfere with or inhibit the expression of their target gene, for example APC.
In a particular preferred embodiment the TTV polynucleic acid sequence (if DNA) contains the consensus sequence CATCCYY (with Y = C or T) , i.e. CATCCCC, CATCCCT, CATCCTC or CATCCTT.
In another particular preferred embodiment the TTV polynucleic acid sequence (if RNA) contains the consensus sequence CAUCCYY (with Y = C or U) , i.e. CAUCCCC, CAUCCCU, CAUCCUC or CAUCCUU. In this regard particular reference is made to Table 2b below.
In a particular preferred embodiment the inventors show how the most conserved seed motif (AUCCUC) has three additional possible interaction sites within APC mRNA in addition to the previously described for TTV-HD14a-mir-2-3 . In this regard particular reference is made to Table 8 below. Therefore, in a further embodiment the invention relates to variants of the polynucleic acid as described above which comprise the seed motif AUCCCUC and bind to at least one of the interaction sites within APC mRNA shown in Table 8 and, preferably, downregulate APC.
Also included in the present invention are analogous miRNAs in other human TT virus types and variants and in similarly structured single-stranded DNA viruses of the human or animal origin. Anelloviruses have been demonstrated in domestic animals in part with similar nucleotide sequences as human TT viruses [77] .
Furthermore, the present invention relates to oligonucleotide primer comprising part of the polynucleic acid of the present invention, said primer being capable of acting as primer for specifically sequencing or specifically amplifying a certain TTV miSNA. The term "primer" refers to a . single stranded DNA oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to specifically prime the synthesis of the extension products. Preferably the primer is at least about 10, preferably at least 15 nucleotides. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature, ionic strength etc. The amplification primers do not have to match exactly with the corresponding template sequence to warrant proper amplification. The amplification method used can be either polymerase chain reaction (PGR) , ligase chain reaction (LCR) , nucleic acid sequence-based amplification ( NASBA) , transcription-based amplification system (TAS) , strand displacement amplification ( SDA) or amplification by means of QB replicase or any other suitable method to amplify nucleic acid molecules using primer extension. During amplification or synthesis, the amplified products can be conveniently labelled either using labelled primers or by incorporating labelled nucleotides. Labels may be isotopic (32P, 3 S , etc.) or non-isotopic (biotin, digoxigenin, etc.) . The present invention also relates to an oligonucleotide probe comprising part of the TTV polynucleic acid of the present invention, said probe being capable of acting as a hybridization probe for specific detection of a certain TTV miRNA in vitro and in vivo.
The term "probe" refers to single stranded sequence-specific oligonucleotides which have a sequence which is complementary to the target sequence of the TTV polynucleic acid to be detected. Preferably, these probes are about 5 to 50 nucleotides long, more preferably from about 10 to 25 nucleotides. The probe can be fixed to a solid support, i.e., any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low. Usually the solid substrate will be a microtiter plate, a membrane {e.g. nylon or nitrocellulose) or a microsphere (bead) . Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fixation or improve the hybridization efficiency. Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NHZ groups, SH groups, carboxylic groups, or coupling with biotin or haptens .
In an embodiment of the invention the probe is an ant i-miR oligonucleotide. An anti-miR oligonucleotide is a single- stranded RNA complementary to the miRNA molecule according to the invention. It can be delivered in its RNA form or being expressed from a vector, using a polymerase III promoter. Such an anti-miR oligonucleo ide can be used for inhibiting the miRNA of the present invention. Methods for inhibiting miRNA by anti-miRs are described by Stenvang et al. in [81], which publication is incorporated by reference .
A further embodiment of the invention are miRNA sponges. A miRNA sponge is a messenger RNA wi h several, preferably 6-8 , perfect complementary binding sites to the polynucleotide acid, i.e. mature TTV miRNA, of the invention. The binding sites can also include mismatches in the nucleotides from 10 to 13 of the mature TTV miRNA, to avoid direct RNA slicing and degradation which makes them more effective. A further embodiment of the invention are tough decoy inhibitors. A tough decoy inhibitor is a miRNA consisting of a "hairpin comprising a large internal bulge exposing two of the miRNA interaction sites of APC shown in Table 8 with imperfect baise-pairing with the TTV miRNA of the invention .
The design of such a tough decoy inhibitor and methods of suppressing miRNA by a tough decoy inhibitor are described in [82] and [83] which are incorporated by reference. The anti-miR, miRNA spounge and tough decoy inhibitor according to the invention are inhibitors of the TTV polynucleic acid as described above, in a preferred embodiment of a mature TTV miRNA shown underlined in Table 2A and 2B, which prevent the interaction between the TTV polynucleic acid and APC such that APC is not downregulated.
The present invention also relates to a vector containing a TTV polynucleic acid, oligonucleotide primer, oligonucleotide probe, anti-miR, miRNA sponge or tough decoy inhibitor of the invention allowing, e.g., expression, mutagenesis or a modification of a sequence by recombination of DNA sequences . Preferably, the vectors are plasmids, cosmids, viruses, bacteriophages and other vectors usually used in the field of genetic engineering. Vectors suitable for use in the present invention include, but are not limited to the
T7-based expression vector for expression in mammalian cells and baculovirus-derived vectors for expression in insect ceils . Preferably, the polynucleic acid of the invention or part thereof is operatively linked to the regulatory elements in the recombinant vector of the invention that guarantee the transcription and synthesis of an niRNA in prokaryotic and/or eukaryotic cells that can be translated. The nucleotide sequence to be transcribed can be operably linked to a promoter like a T7 , metallothionein I or polyhedrin promoter. The present invention also relates to recombinant host cells transiently or stably containing the TTV polynucleic acid (or fragments thereof) or vectors of the invention. A host cell is understood to be an organism that is capable to take up in vitro recombinant DNA and, if the case may be, to synthesize the polypeptids encoded by the polynucleotides of the invention. Preferably, these cells are prokaryotic or eukaryotic cells, for example mammalian cells, bacterial cells, insect cells or yeast cells.
The present invention also relates to a diagnostic kit containing a TTV polynucleic acid, an oligonucleotide primer, an oligonucleotide probe, a polypeptide and/or an antibody of the present invention.
For hybridization based assays, according to the hybridization solution (SSC, SSPE, etc.), the probes should be stringently hybridized to the target (with or without prior amplification) at their appropriate temperature in order to attain sufficient specificity. However, by slightly modifying the polynucleotide, (DNA and/or RNA) probes, either by adding or deleting one or a few nucleotides at their extremities (eithe 3 ' or 5'), or substituting some non- essential nucleotides (i.e. nucleotides not essential to discriminate between types) by others (including modified nucleotides or inosine) these probes or variants thereof can be caused to hybridize specifically at the same hybridization conditions (i.e. the same temperature and the same hybridization solution) . Also changing the amount (concentration) of probe used may be beneficial to obtain more specific hybridization results.
Suitable assay methods for purposes of the present invention to detect hybrids formed between the oligonucleotide probes and a TTV polynucleic acid in a sample may comprise any of the assay formats known in the art, such as the conventional dot-blot format, sandwich hybridization or reverse hybridization. For example, the detection can be accomplished using a dot blot format, the unlabelled amplified sample being" bound to a membrane, the membrane being incorporated with at least one labelled probe under suitable hybridization and wash conditions , and the presence of bound probe being monitored. An alternative and preferred method is a "reverse" dot-blot format, in which the amplified sequence contains a label. In this format, the unlabelled oligonucleotide probes are bound to a solid support and exposed to the labelled sample under appropriate stringent hybridization and subsequent washing conditions. It is to be understood that also any other assay method which relies on the formation of a hybrid between the nucleic acids of the sample and the oligonucleotide probes according to the present invention may be used.
The present invention also relates to the use of a TTV polynucleic acid, an oligonucleotide primer, or an oligonucleotide probe of the present invention as an early marker for the future development of cancer, preferably colorectal or colon cancer.
Accordingly, an embodiment of the present invention relates to a method of detecting or diagnosing of colon cancer, comprising the steps of:
(a) isolating miRNA from a patients sample;
(b) sequencing the miRNA isolated in step (a) ; and
(c) determining, if an miRNA selected from the miRNA shown in Table 2B is present in the sample, whereas the presence of an miRNA shown in Table 2B indicates colon cancer.
For determining miRNA labelled oligonucleotides may be used.
Optionally, the method may comprise a further step (d) of quantifying the miRNA level in sample to distinguish between patients with colon cancer from healthy controls. Preferably, in step (a) the miRNA is isolated from plasma or serum and the miRNA is quantified by using TaqMan miRNA qRT- PCR-assays as described in [86] which is incorporated by reference .
Alternatively, the miRNA may be isolated directly from the tumor and a miRNA sequencing may be performed to detect the miRNA or sections of any kind (e.g. cryo-sect i ons , sections from paraffin embedded tissue) may be made directly on the tumor and an hybridization for the miRNA as described above may be performed in-situ.
Finally, the present invention also relates to the use of a TTV polynucleic acid o f the present invention as a lead component for the development of a medicament for prevention or treatment of cancer, preferably colorectal or colon cancer. These medicaments may be inhibitors of any interaction between miRNAs and tumour suppressor genes to avoid cancer development or recurrence and cancer treatment. Thus , the specific TT virus miRNA o r o f its derivatives or of related miRNAs are useful for diagnostic, prevention or therapeutic applications in the cancer field.
Such inhibitor of an interaction between miRNA and tumor suppressor genes, for example ARC, can be an anti-miR, A miRNA sponge or a tough decoy inhibitor as described above .
The inhibitor can be delivered to the tumor site by using an adeno associated virus (AAV) in order to deliver the inhibitor to counter effect the TTV miRNA according to the invention . An AAV gene therapy suitable for delivering one o f the above miRNA inhibitors to the tumor is described in [84] which is incorporated by reference.
A further example of a suitable method for delivering the above inhibitors against TTV miRNA to a tumor is known as low pH-induced transmembrane structure (pHILP) [85] . This phILP construct consists of a peptide that crosses the plasma membrane only under acidic' conditions which are typical of the tumor microenvironment . A peptide nucleic acid of an TTV miRNA inhibitor can be attached to this pHILP in order to be delivered specifically to cells in the tumor microenvironment. Preferably, this peptide is an anti-miR, which will cause the inhibition of the TTV miRNA according to the invention. A suitable method for delivering a TTV miRNA inhibitor with a pHILP construct is described in [85] which is incorporated by reference.
A further embodiment of the invention is a method of delivering a lead, component for the development of a medicament for prevention or treatment of cancer, preferably colorectal cancer comprising the step of administrating to a patient suffering from a cancer, in particular colorectal cancer (a) a pharmaceutical composition comprising an adeno- associated virus expressing an inhibitor of the miRNA of the invention selected from the group consisting of anti-miR, miRNA sponge and tough decoy inhibitor of a miRNA interacting with a tumor suppressor gene and a pharmaceutically acceptable carrier or (b) a pharmaceutical composition comprising an inhibitor of the miRNA of the invention attached to a pHILP construct and a pharmaceutically acceptable carrier.
A further embodiment of the Invention is (a) an adeno- associated virus for the use of delivering an inhibitor of
TTV miRNA to tumor cells or (b) a pHILP construct for the use of delivering an inhibitor of a miRNA of the invention to tumor cells .
Preferably, the inhibitor to be delivered is selected from the group consisting of anti-miR, miRNA sponge and tough decoy i nh ibitor and is an inhibitor of a mature TTV miRNA as shown underlined in Table 2B which interacts with APC. The following examples illustrate the invention and are not construed as any limitation of the invention. - Example 1
Material and methods
(A) Cell culture and transfections
HEK293TT cells [76] cultured in Dulbeco' s Eagle Modified
Medium (D EM Sigma) supplemented with 10% FBS, l%Glutamax and 1% NGAA. Cells are transfected when 50% confluent, 24h after seeding (7 million for T-75 flask and 800.000 per well for a 6 well plate) . Transfections are performed using Lipofectamine and Plus reagent (Life Technologies, catalog n. 11514 and 18324) according to the manufacturer's instructions.
(B) Plasmid construction
The TTV NCR is PGR amplified using suitable primers. For example, the TTV-HD14a NCR is PGR amplified using primers TT-ON9 5' gattatggtacctttccaactacgactgggtgt (SEQ ID NO: 83) and TT-ON10 5' gattatggtacctctaccattcgtcaccgctgtt (SEQ ID NO: 84) using pCDNA3.1 (+ ) -TTV-HD1 a as template {a plasmid containing full-length TTV-HD14a linearized and cloned into Xbal site) . PGR product is run on a 1% agarose gel and DNA stained using ethidium bromide. Bands corresponding to the expected size (-1200 bp) are cut and subsequently extracted from agarose using QIAEXII gel extraction kit (QIAGEN) . 4 g of pCDNA3.1 (+ ) (Life technologies) are cut using Kpnl and dephosphorylated using Fas AP (Thermo scientific) . PGR product is cut using the same procedure, but not dephosphorylated. Cut plasmid and PGR products are cleaned up by using QIAEXII gel extraction kit. Ligation of the plasmid and the amplified fragment corresponding to the TTV NCR, for example TTV-HD1 a NCR, is performed using T4DNA ligase (Thermo Scientific) Ligation product is transformed into NovaBlue Singles competent cells (Merck Millipore) according to the manufacturer instructions, and seeded in LB agar plates supplemented with ampicillin as selection marker. Plates are incubated 20 hours at 37°C. Single colonies are picked and seeded in LB medium supplemented with ampicillin. These cultures are incubated 20 hours. Plasmid is extracted using PureLink Quick Plasmid Miniprep Kit (Life technologies j . I g of each plasmid are double cut with SacI and Nhel (Thermo Scientific) . Cut products are run in 1% agarose gels. The restriction strategy allows us to distinguish between inserts clones in the sense and antisense orientation. Two positive plasmids , one containing the sense and the other one the antisense insert, are chosen and sent for sequencing. After confirming the sequence, plasmids are prepared for transfection by using Plasmid Maxi Kit (Qiagen) .
(C) RNA extraction and DNAse treatment
Cells are harvested 48-72h post-trans feci, i on . Cells are homogenized using QiaShreder (Qiagen) according to manufacturer instructions . Lysates are then subjected to RNA extraction using miRNAeasy mini kit or RNAeasy mini kit (Qiagen) depending on the purpose of the RNA (for miRNA Northern blot or for RT-qPCR) , according to manufacturer instructions .
After elution, RNA samples are treated with RQ1 Dnase ( Promega ) according to manufacturer instructions, with the addition of RNasin
(Promega) . Phenol-Chloroform extraction followed by ethanol precipitation is performed, and the resulting pellet is resuspended in DEPC water. RNA quality and concentration are tested using NanoDrop 2000c (Thermo Scientific) .
(D) Probes labeling
Custom DNA oligos are ordered to Sigma (Table 4 ) . Probes are 3' biotin labeled .
10 pinoles of each probe are incubated with 4U of
Terminal Deoxynucleotidyl transferase { TdT ) and 2,5 nanomoles of Biotin- 11-dUTP (Thermo Scientific) in IX TdT buffer, overnight. Probes are subjected to Isoamyl alcohol-Chloroform extraction and the total volume is used for subsequent hybridization .
(E) Northern Blot
30-50 μg of total RNA per sample are separated by electrophoresis using 15% polyacry1 amide (29:1) gels cast in 7M urea and buffered with lxTBE using a Mini Protean cell (Bio-Rad) . The electrophoresis buffer is 0.5x TBE . Gels are stained with EtBr.
For blotting, gels are placed over a sheet of nylon hybridization membrane (Hybond-NX®, Amersham/Pharmacia) pre-wetted in 0.5x TBE. This is then sandwiched between pieces of 3MM Whatman filter paper (one layer under the membrane and three over the gel) , also pre-wetted in 0.5x TBE and placed in a Trans-Blot SD semidry transfer cell (Bio-Rad) . Excess liquid and air bubbles are squeezed from the sandwich by rolling the surface with a pipette. Electrophoretic transfer of RNA from the gel to the membrane is carried out at 400W for 60-90"min. After transfer, RNA is cross linked to the membrane by ultraviolet exposure using Stratalinker ( Stratagene ) .
Membranes are cut as needed and hybridized with the appropriated biotin labeled probe (Table 4) o/n in
Ultrahyb Oligo buffer (Life technologies) at 42 ° C . After hybridization, 4 washes are performed; the first one with 2X SSC 30min at 42°C, the second one with
2XSSC 0.5% SDS 30min at 42°C and the last two with 2XSSC
0.5% SDS 30min at 55°C. Hybridization signals are detected using BrightStar BioDetect Kit (Life technologies) according to the manufacturer instructions. Film used: (Fiji) .
(F) RT-qPCR
1 μg of RNA is used to make cD A with superscript III and RnaseOUT (Life technologies) according to manufacturer instructions. cDNA is diluted 1:10. qPCR is performed using Taqman fast master mix and Taqman expression assays, in a qPCR machine StepOne plus (Applied Biosystems).
(G) Pre-miRNA rediction and mature miRNA prediction
V-mir is set to default configuration, changing the sequence type to circular. CID-miRNA is run on the web- based tool, using the default run configuration for Homo sapiens. Mature Bayes is run on the web-based tool.
(H) miRNA target Predictions
DIANA microT -3.0 is run on the web-based tool (no options are given for this program) . RNA hybrid is run using constraint nucleotide configuration, from nucleotide
2 to 8 of the miRNA. G:U pairs are allowed.
Table 1
Predicted pre-rniR A from TTV-HD14a using CID-miR A and V-mir that match three criteria: being predicted by both programs, score over 150 for V-mir and located in the non-coding region of the virus .
Group Orientation Length Starting nucleotide Name
Figure imgf000021_0001
Table 2A. Initial pre-miRNA predicted from different TTV strains grouped according to sequence homology predicted mature miRNA are underlined.
Group Pre-miRNA name Alignment
Senseί TTV- HDl6a-mir-3 GGCCGCCATTTTAAGTAA—GGCGGAAGCAACTCCACTTTCTCRCAAAATGGCGGCGGAGCACTTCCGGCTTGCCCAAAATGGCCGCC
TTV- sle2057-mirl - -CCGCCATTTTAAGTAA—GGCGGAAGCAGCTCCACTTTCTCACAAAATGGCGGCGGAGCACTTCCGGCTTGCCCAAAATGGCGG—
TTV- HD23a-mir-l - -CCGCCATTTTAAGTAA—GGCGGAAGCAGCTCCACCCTCTCACATAATGGCGGCGGAGCACTCCCGGCTTGCCCAAAATGGCGG—
TTV- Sanban-mir-1 - GCCGCCATTTTAAGTA —GGCGGAAGCAGCTCGGCATA—TACAAAATGTCGGCGGAGCACTTCCGGCTTACCCAAAATGAAGGC-
**************** ********** *** * *** **** ************ ******* ********* *
Sense2 TTV- HD14a-mir-l GCCTCGGACCCCCCCTCGACCAGAATCGCTCGCGCGATTCGGACCTG—CGGCCTCGGGGGGGTCGGGGGC
TTV- CT30F-mir-l -CCTCGGACCCCCCCCCGACCCGAATCGCTCGCGCGATTCGGACCTG—CGGCCTCGGGGGGGGTCGGGG-
TTV- HD16a~mir-2- -CCTCGGACCCCCGCTCGTGCTGACGCGCTTGCGCGCGTCAGACCACTTCGGGCTCGCGGGG
************ * * * * * * * * * ***** ** * * * * *** **** ****
Sense3 TTV- HD14a-mir-2 GCTGTGACGTCAACG-TCACGTGGG-GAGGACGGCGTGTAACCCGGAAGTCATCCCCA-TCACGTGACCTGACGTCACGGC—
TTV- Sanban-mir-2 ACGTCACAAGTCACGTGGGGAGGGTTGGCGTATAGCCCGGAAGTCAATCCT-CCCACGTGGCCTGTCACGT
TTV- HD23a-mir-2 GCAGCTACGTCACAAGTCACCTGACTGGGGAGGAGTTACATCCCGGAAGTTCTCCTCGGTCACGTGACTGTACACGTGACTGC
TTV- sle2057~mir-2 ACGTCACAAGTCACCTGACTGGGGAGGAGTCACAACCCGGAAGTCCTCTTCGGTCACGTGACTAGTCACGT
****** **** ** ** * * ********* ****** *
AS1 TTV- HD14a-ASmir-2 GGCCGTGACGT-CAG-GTCACGTGAT-GGGGATGACTTCCGGGTTACACGCCGTCCTCC-CCACGTGACGT-TGACGTCACAGCC r TTV- -CT30F~ASmir-3 CGTGACGT-CAGAGTCACGTGACCAGGGATG-CTTCCGGGTTTAGGCACGCCCCCA-TCACGTGTCTC-AAACGTCACG,
TTV- •HD23a-ASmir-l GCAGTCACGTGTA CA—GTCACGTGACCGAGGAGAACTTCCGGGATGTAACTCCTCCCCAGTCAGGTGACTTGTGACGTAGCTGC-
TTV- sle2057-ASmir- 1 ACGTGAC TA—GTCACGTGACCGAAGAGGACTTCCGGGTTGTGACTCCTCCCCAGTCAGGTGACTTGTGACGT
TTV- HD16a-ASmir-l ACGTGAC CA—GTTACGTGGTTGAGGAT-ACTTCAGTGTTTAAGTACCTCCCCAGTCACGTGACTTATGACGT
**** * * * ***** * **** * * * * * * ** *** * ****
AS2 TTV- -HD16a-ASmir-2 —-GCCATTTTGGGCAAG—CCG--GAAGTGCTCCGCCGCCATTTTGTGAGAAAGTGGAGTTGCTTCCGCCTTACTTAAAATGGC
TTV~ -sle2057-ASmir~ 2 -CCGCCATTTTGGGCAAG--CCG—GAAGTGC CCGCGGCCATTTTGTGAGAAAGTGGAGCTGCTTCCGCCTTACTTAAAATGGCGG-
TTV- -HD23a-ASmir~2 -CCGCCATTTTGGGCftftG—'CCG-—GGAGTGCTCCGCCGCCATTATGTGAGAGGGTGGRGCTGCTT CGCCTTACTTAAAATGGCGG-
TTV- ■Sanban~ASmir-2 GCCTTCATTTTGGGTAAG—CCG—GAAGTGCTCCGCCGACATTTTGT—ATATGCCGAGCTGCTTCCGCCTTACTTAAAATGGCGGC
TTV- -tth8-ASmir-2 CCATTTTGAGTAGGTGTGGCTGATGGTGACCTTTGAACTCACGCCACCGTCCG CCTCAAC—TAC T AGATGG
TTV- -T H~ASmir-3 CCATTTTGTGTAGCTTCCGTCGAGGATGACCTTTAACCTCTA-CGTCAATCCTGA CGTCAGC— ACTTAAAATGG
******* * * * * * ** * * * * * ******* * * * *
AS3 TTV- -HD14a-ASmir-l CCGCCGCTAC-GTCACACTTCCTCTTTTTTTTACAAAAAGCGGAAGGAAGTCACAAGATGGCGG
TTV- -CT30F-ASmir-2 CCGCCGCTACTGTCATACTTCCTCTTTTTTTTTGAAAAAGCGGAAGGAAGTCACAAGATGGCGG
********** ********************* ******************************
TTV- -Sanban-mir-3 GCCGGGGGGCTGCCGCCCCCCCCGGGGAAAGGGGGGGGCCCCCCCCGGGGGGGGGTTTGCCCCCCGGC
Figure imgf000023_0001
Figure imgf000023_0002
Table 2B
TTV mature miRNA present in the TCGA small RNA sequencing datasets of colon adenocarcinoma with similarity in the nucleotides from 1 to 7 (comprising the seed (nt 2 to 7) ) to T?V-HD1 a-mir-2-3p . The TTV miRNA are shown in the context of the pre-rniRNA sequence. The
identical conserved nucleotides from 1 to 7 (comprising the seed (nt 2 to 7) ) are boxed. Positions containing identical nucleotides are marked by a (*) and positions containing nucleotides originated by a transition are marked by ( 0 ) . They are classified in groups according to their pre-miRNA sequence. In all cases, the 3p mature miRNA is underlined. The seed is written in italicized letters. The box 3 contains the consensus sequence for the nucleotides from 1 to 7. A - adenine, T - thymine, C - cytosine, G-guanine, Y - C or T.
Seed of a miRNA: nucleotides 2 to 7 of the mature form of the miRNA [80]
Figure imgf000025_0001
Table 26
Table 3
Genes predicted to be down-regulated by the TTV-HDl 4 a and at 5 least two other TTV strains miRNA. Notice that some strains have more than one putative miRNA that is predicted to down-regulate some of the genes .
Figure imgf000026_0002
Table 4 - Probes
Figure imgf000026_0001
Example 2 mxRNA prediction
To address the question about the possible function of the non- coding region (NCR) of TTVs beyond its promoter activity, the inventors had the idea that it also generates non-coding RNAs , such as miRNAs . Therefore, they used available miRNA prediction algorithms, with which they identified several candidate pre-miRNAs in the NCR of some TTVs, The inventors chose to use two of such algorithms: CID-miRNA [34] and Vmir [35- 36] . The first one was chosen because of its high specificity and the second one because of its higher sensitivity. To consider a pre- miRNA structure as a candidate, they used the criterion that it should be predicted by both programs, with a cut-off value over 125 for the V-mir program and that it had to be located in the NCR of the virus. After filtering, only 4 pre-miRNA candidates (Table 1 and Figure IB) , two in sense orientation and two in antisense orientation, were considered as putative pre- miRNA and were further evaluated.
In order to check the conservation of the pre-miRNA sequences among different TTV isolates, the inventors performed the same prediction in seven different strains: TTV-HDl 6a (FR751476, version FR751476.1 GI : 339511352 , 07.07.2011), TTV- C3T0F (AB064597, version AB064597.1 GI : 17827196, 25.06.2008), TTV-HD23a (FR751500, version FR751500.1 GI : 339511376, 07.07.2011), TTV-YonKc'i 97 (AB038624, version AB038624.1 GI : 7415899, 20.09.2000), TTV-SANBAN (AB025946, version AB025946.2 GI : 5572683, 03.11.2009), TTV-Sle2057 (AM712030, version AM712030.1 GI : 156104055, 19.02.2008) and 7TV- tth8 (AJ620231, version AJ 20231.1 GI : 49203022, 03.02.2009 (GenBank accession numbers and versions in brackets) They then grouped the resulting pre-miRNA in different classes (Table 2A) , according to their sequence similarity. As can be observed, the conservation of the sequences is rather poor, being strange the total identity between two pre-miRNA from different strains. Mature™ and pre-miRNAs similar to TTV-HD14a pre-miRNA that contain a mature miRNA with an equal or similar seed to that of TTV-HD14a-3p miRNA which also includes TTV-HD18a-like pre-miRNAs we e found within patients by screening TGCA datasets. These miRNAs are shown i Table 2B . The similarity within the nucleotides 1 to 8 of these miRNAs with that of TTV-HD14a miRNAs indicates, that these miRNAs are downregulating APC as well .
Example 3
TTV-HD14a can transcribe four precursor miRNA encoded in its NCR
To address the question whether the predicted pre-miRNAs could be processed, t he NCR of TTV-HD1 a was cloned downstream of the CMV promoter, in sense or antisense orientation, using the plasmid pCDNA3.1 { + ) -zeo as scaffold ( Figure 2A) . The inventors then transfected HEK293TT cells with these plasmids and performed Northern Blot hybridization with specific probes against the 3 ' or 5 ' arm of each putative pre-miRNA (Table 4) (Figure 2B-E) . The inventors could clearly detect bands that match the expected size for a pre-miRNA with the probes directed against the 3 ' and 5 ' arm of TTV-HD14a-mir-2 and TTV-HDl 4a-ASmi r-2. Moreover, the inventors were able to detect a transcript matching the expected size for a mature miRNA within the 5 'arm of TTV- HD14a-mir-2. On the other hand, the inventors were able to detect transcripts matching the expected sizes with the probes directed against the 3 'arm only of TTV-HD14a-mir-l and TTV-HD14a- ASmir-1.
These results demonstrate that TTV-HD14a encodes for several precursor miRNA in both orientations ; and at least one of them can be processed into a mature miRN .
Example 4
Target prediction It is well known that the major feature of miRNA is down- regulating gene expression in a pes t-1 ra soriptiona 1 manner. It is - also known that this effect is caused by the mature form of the miRNAs , and not by their precursors. Although the inventors were not able to see any mature miRNA for three of the pre-miRNA, they think that low expression levels of these miRNAs rather than their absence might be the reason of this. In any case, it is necessary to identify the sequence of the mature miRNA to perform accurate predictions, and this is hard to determine by experimental methods different from mi R A-aeq . To overcome this problem, the inventors decided to use an in-silico mature miRNA predictor, Mature Bayes [37] . This program predicts the mature miRNA from a pre-miRNA sequence. After doing that with all the predicted miRNA precursors (Table 2), they used DI N -micro -3.0 [38-39] to predict possible targets. They reasoned that, despite the variability in their sequences, the putative TTV mature miRNAs should have some targets in common. So, after performing the predictions, the inventors compared the results among the different TTV strains and considered as good candidates the targets that were predicted for some miRNAs belonging to TTV-KDl4a and, at least, two more TTV strains. Candidate targets are listed in Table 3.
In addition to this approach, the inventors performed a direct comparison of the predicted mature miRNA from TTV-HDl4a with the CDS, 3'UTR and promoter regions of several tumor suppressor genes using RNA Hybrid [40] . This program allows to directly detecting the complementary sequence of a given miRNA within a gene, independently of the conservation or localization of complementary sequence. This is useful, as most of the other prediction programs do not take into account the CDS or promoter region of the genes, while it has been demonstrated that a seed pairing with the first one can mediate PTGS and with the second one can cause TGS or RNAa [11, 12,29-33]. The inventors found seed complementarity between the APC gene and TTV-HD14a-mir-2-5p in three different points within the APC mRNA sequence, two in the CDS and one in the 3'UTR (Figure 3A - 1, 2 and 3) . In addition, a possible interaction site between TTV-HD14a-mir-2-3p and APC mRNA was present in the CDS (Figure 3A - 4) . The inventors also found complementarity between the TTV-HD14a-ASmir-2-3p and three of the four promoters listed for APC in the Eukaryotic Promoter Database New Human (EPD New Human) [59] (accession names APC_1, APC_2 APC_3 and APC_4 ) (Figure 3B-D) .
Example 5
APC is down-regulated after transfection with pCDNA3.1 (+) -TTVHD14a-
NCR-Sense
To check the possible APC down-regulation mediated by the
TTV-HDl4a miRNA the inventors transiently transfected
HEK293TT cells with the constructs encoding the miRNA, with the full length T'i'V-HD] 4a virus or mock transfected them, followed by RT-qPCR (Figure 3E+F) . APC down-regulation by the iRNA itself as well as by the full length genome (coding for the miRNA) is significant in comparison to the mock transfected. Example 6
GAPDH up-regulation by TTV miRNA
After transfection with pCDNA3.1 (+) -TTV-HD14a-NCZ-Sense, which is intended to produce 4 mature miRNAs (ITV- KD1 a-mi r···· 1 - 5p , TTV-HD! a- mir-l-3p, TTV-HD14a-mir-2-5p and TTV-HD14a-mir-2-3p) , the inventors can observe a statistically significant increase of GAPDH transcript:
GAPDH (Glyceraldehyde-3-phosphate-dehydrogenase ) is a gene usually used as internal control (housekeeping gene) , at the mRNA and protein levels, because its levels of expression are very constant among very different conditions.
GAPDH is up-regulated in the majority of cancers and under hypoxic conditions [72, 73, 74] . The inventors suggest that the TTV miRNA dependent up-regulation of GAPDH is mediated indirectly by APC down- regulation .
Example 7 M u « Y i ys s revea s t e an scape o TW-HD s ice
alterations
72h after transfection of cells with the two different constructs, the full-length TTV HD14a genome or an empty plasmid RNA was isolated and microarray analysis was performed. Table 5 includes all the genes that were consistently deregulated between the transfection with the constructs and with the full-length virus.
Table 5
Genes differentially expressed between cells transfected with a plasmid encoding for TTV-HD14a NCR in sense orientation in comparison to mock transfected cells
ILLUMINAJD GENE SYMBOL Description
5550379 CAV1 caveolin 1, caveolae protein, 22kDa
1230465 HNRNPK heterogeneous nuclear ribonucleoprotein K
4230673 GRM2 g!utamate receptor, metabotropic 2
7160327 ARPC2 actin related protein 2/3 complex, subunit 2, 34kDa
4640161 C17orf97 chromosome 17 open reading frame 97
2640142 C17orf97 chromosome 17 open reading frame 97
70403 C14orf45 chromosome 14 open reading frame 45
7510097 CMTM 1 CKLF-like MARVEL transmembrane domain containing 1
4850736 CMTM1 CKLF-like MARVEL transmembrane domain containing 1
6290358 TPX2 TPX2, microtubule-associated, homoiog (Xenopus iaevis)
7150270 LRRC26 leucine rich repeat containing 26
6420441 LOC728416 hypothetical LOC728416
2140541 LRRC26 leucine rich repeat containing 26
6220053 FAM57B family with sequence similarity 57, member B
3520128 TUBG2 tubulin, gamma 2
1410470 FA 71E1 family with sequence similarity 71, member El
2490433 RNF32 ring finger protein 32
650717 C22orf23 chromosome 22 open reading frame 23
4070424 C16orf93 chromosome 16 open reading frame 93
4860068 STX1A syntaxin 1A (brain)
2940739 RASSF1 Ras association (RalGDS/AF-6) domain family member 1
2370538 KRCC1 !ysine-rich coiled-coil 1
6130047 CCDC151 coiled-coil domain containing 151
1240731 RFPL3S RFPL3 antisense RNA (non-protein coding)
650689 CLIP3 CAP-GLY domain containing linker protein 3
7320402 APBB3 amyloid beta (A4) precursor protein-binding, family B, member 3
450204 ALG9 asparagine-linked glycosylation 9, alpha-l,2-mannosyltransferase homolog (S. ce 1240523 RPL41 ribosomal protein L41
1940561 CGRRF1 celi growth regulator with ring finger domain 1
6290609 RPS15 ribosomal protein S15
1090564 T EM175 transmembrane protein 175
5820494 ZNF177 zinc finger protein 177
7560731 SNORA64 small nucleolar RNA, H/ACA box 64
7550021 TTC25 tetratricopeptide repeat domain 25
2350477 LRRC6 leucine rich repeat containing 6
2480364 DPP7 dipeptidyl-peptidase 7
3180678 HNRNPH2 heterogeneous nuclear ribonucleoprotein H2 (H)
3400164 C21orf2 chromosome 21 open reading frame 2
730292 RNFT2 ring finger protein, transmembrane 2
7330474 MOBP myelin-associated oligodendrocyte basic protein
6520241 FAM116B family with sequence similarity 116, member B
270026 RASSF1 Ras association (RalGDS/AF-6) domain family member 1 5560253 N6AMT1 N-6 adenine-specific DNA methyltransferase 1 (putative) 3520092 BAX BCL2-associated X protein
6940181 FAM24B family with sequence similarity 24, member B
7320750 ILVBL ilvB (bacterial acetolactate synthase)-like
3840356 TRIM11 tripartite motif-containing 11
6350121 RASSF1 Ras association (RalGDS/AF-6) domain family member 1 7400619 SPATA5L1 spermatogenesis associated 5-like 1
5270343 IQCC IQ motif containing C
7320192 LOC100129148 hypothetical LOC100129148
3140689 KIAA1407 KIAA1407
1990593 IRX6 iroquois homeobox 6
5340725 DYNLRB2 dynein, iight chain, roadblock-type 2
4120279 APBB3 amyloid beta (A4) precursor protein-binding, family B, member 3 5910397 LIAS lipoic acid synthetase
3130035 FAM149B1 family with sequence similarity 149, member Bl
2570707 N6AMT1 N-6 adenine-specific DNA methyltransferase 1 (putative) 2900725 CYP27B1 cytochrome P450, family 27, subfamily B, polypeptide 1 7400246 BCDIN3D BCDIN3 domain containing
5080010 LOC401431 hypothetical LOC401431
830681 C14orf45 chromosome 14 open reading frame 45
5910041 RPL23AP7 ribosomal protein L23a pseudogene 7
1820739 CDK20 cyclin-dependent kinase 20
6350672 PHF21B PHD finger protein 21B
1170022 C17orf81 chromosome 17 open reading frame 81
4640095 RPL9 ribosomal protein L9
5390497 C7orf53 chromosome 7 open reading frame 53
4490348 C9orfS chromosome 9 open reading frame 6
6040156 C6orf52 chromosome 6 open reading frame 52
2480735 KIAA1731 KIAA1731
4180408 SNORD55 small nucleolar RNA, C/D box 55
6660451 NDUFB4 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 4, 15kDa 4150561 AASDH aminoadipate-semialdehyde dehydrogenase 6840291 FOXN4 forkhead box N4
3850754 KIAA1683 KIAA1683
3800400 LDHAL6B lactate dehydrogenase A-like 6B
540403 LRP5L low density lipoprotein receptor-related protein 5-like
5360139 LOC100128221 similar to hCG2041787
2810674 TRIM4 tripartite motif-containing 4
3780450 BANP BTG3 associated nuclear protein
5690280 FXR2 fragile X mental retardation, autosomal homolog 2
1580750 LOC10O130828 hypothetical LOC100130828
4610433 ANGPTL4 angiopoietin-like 4
3120452 MTMR10 myotubularin related protein 10
2750465 C19orf61 chromosome 19 open reading frame 61
2470270 DUS4L dihydrouridine synthase 4-like (S. cerevisiae)
3370288 CBX6 chromobox homolog 6
7210092 TRIT1 tRNA isopentenyltransferase 1
1580021 TCTEX1D2 Tctexl domain containing 2
540041 CASC1 cancer susceptibility candidate 1
2030152 MOBP myelin-associated oligodendrocyte basic protein
2650678 RSPH3 radial spoke 3 homolog (Chlamydomonas)
620414 HSD17B10 hydroxysteroid (17-beta) dehydrogenase 10
5360326 SIP1 survival of motor neuron protein interacting protein 1
5860709 C9orf9 chromosome 9 open reading frame 9
5960709 APITD1 apoptosis-inducing, TAF9-like domain 1
6100014 MRPL47 mitochondrial ribosomal protein L47
4150059 HNRNPH2 heterogeneous nuclear ribonucleoprotein H2 (H)
5820500 Clorf35 chromosome 1 open reading frame 35
3610148 EVI5L ecotropic viral integration site 5-like
3170494 PPP2R3B protein phosphatase 2, regulatory subunit B, beta
2810112 RRAGC Ras-related GTP binding C
5260692 ZRSR2 zinc finger (CCCH type), RNA-binding motif and serine/arginine rich 2
10543 PNC pregnancy up-regulated non-ubiquitously expressed CaM kinase
940021 PEX11B peroxisomal biogenesis factor 11 beta
5340703 KRCC1 lysine-rich coiled-coil 1
70019 SUGP2 SURP and G patch domain containing 2
7150433 TCTEX1D2 Tctexl domain containing 2
3140634 MECR mitochondrial trans-2-enoyl-CoA reductase
4050040 TUBB3 tubulin, beta 3
940576 ZSCAN21 zinc finger and SCAN domain containing 21
4390687 P0MT1 protein-O-mannosyltransferase 1
6520687 SLC7A9 solute carrier family 7 (cationic amino acid transporter, y+ system), member 9
2750280 CDK5R1 cyclin-dependent kinase 5, regulatory subunit 1 (p35)
4210113 CMTM2 CKLF-like MARVEL transmembrane domain containing 2
940435 TRIM8 tripartite motif-containing 8
4850593 TRIM46 tripartite motif-containing 46
7550110 BAX BCL2-associated X protein
1070541 MYH3 myosin, heavy chain 3, skeletal muscle, embryonic
1740576 LMF2 lipase maturation factor 2 , . ,· .. .. ., .. . . . ..
carboxyl ester lipase (bile salt-stimulated lipase;
7050326 CDKN2D cyclin-dependent kinase inhibitor 2D (pl9, inhibits CDK4)
1430673 SDCBP2 syndecan binding protein (syntenin) 2
2600392 CENPA centromere protein A
7380634 C20orf2"0 chromosome 20 open reading frame 20
1740392 CO MD10 COMM domain containing 10
3710746 OXSM 3-oxoacyl-ACP synthase, mitochondrial
7550626 BIRC5 baculoviral IAP repeat-containing 5
6020719 RAB23 RAB23, member RAS oncogene family
6400524 LOC390705 protein phosphatase 2, regulatory subunit B, beta pseudogene
5290148 GPT2 glutamic pyruvate transaminase (alanine aminotransferase) 2
3290296 MRPS14 mitochondrial ribosomal protein S14
770044 FBX015 F-box protein 15
380079 SPATA7 spermatogenesis associated 7
4590154 ZDHHC8 zinc finger, DHHC-type containing 8
6770673 SOCS2 suppressor of cytokine signaling 2
3940309 CDAN1 congenital dyserythropoietic anemia, type 1
1470348 RAGE renal tumor antigen
3990259 TMEM91 transmembrane protein 91
730475 PIN4 protein (peptidylprolyl cis/trans isomerase) NIMA-interacting, 4 (parvuiin)
5670075 PAFAH1B1 platelet-activating factor acetylhydrolase lb, regulatory subunit 1 (45kDa)
4670082 RGS5 regulator of G-protein signaling 5
7210438 ATRIP ATR interacting protein
7000333 ASB6 ankyrin repeat and SOCS box-containing 6
3420180 ZNF202 zinc finger protein 202
2760181 C0Q3 coenzyme Q3 homolog, methyltransferase (S. cerevisiae)
3120093 EFCAB6 EF-hand calcium binding domain 6
3140202 MYPOP Myb-related transcription factor, partner of profilin
7380670 MYB v-myb myeloblastosis viral oncogene homolog (avian)
6100609 UAP1L1 UDP-N-acteylglucosamine pyrophosphorylase 1-like 1
6520059 SNF8 SNF8, ESC T-II complex subunit, homolog (S. cerevisiae)
6350070 SCNM1 sodium channel modifier 1
430402 ABCA3 ATP-binding cassette, sub-family A (ABC1), member 3
6580402 UBE2H ubiquitin-conjugating enzyme E2H (UBC8 homolog, yeast)
6280482 NIP7 nuclear import 7 homolog (S. cerevisiae)
4260609 C2orf74 chromosome 2 open reading frame 74
6100056 HSD17B10 hydroxysteroid (17-beta) dehydrogenase 10
3890561 IFT20 intraflagellar transport 20 homolog (Chlamydomonas)
5900491 ZNF34 zinc finger protein 34
4730204 FCRLB Fc receptor-like B
450309 DDX49 DEAD (Asp-Glu-Ala-Asp) box polypeptide 49
2060274 PREB prolactin regulatory element binding
4890692 LOC285943 hypothetical protein LOC285943
3130326 MSH5 mutS homolog 5 (E. coli)
3440403 DHDDS dehydrodolichyl diphosphate synthase
1170121 MRPL4 mitochondrial ribosomal protein L4
2600470 WDR60 WD repeat domain 60 1690711 SNAPC2 small nuclear RNA activating complex, polypeptide 2, 45kDa
5080367 CKLF chemokine-like factor
730414 APOE apolipoprotein E
3290446 RPL36 ribosomal protein L36
5900286 ZFP90 zinc finger protein 90 homolog (mouse)
7610079 HSF2BP heat shock transcription factor 2 binding protein
4480477 SBSN Suprabasin
540519 NAGLU N-acetylglucosaminidase, alpha
6020209 CYTSA cytospin A
6100072 DENND2A DENN/MADD domain containing 2A
4890382 ILVBL ilvB (bacterial acetolactate synthase)-like
2810082 C20orflll chromosome 20 open reading frame 111
5220309 RILPL1 Rab interacting lysosomal protein-like 1
7040609 SIP1 survival of motor neuron protein interacting protein 1
520446 COMT catechol-O-methyltransferase
4670021 NPEPL1 aminopeptidase-like 1
2850180 NUDT16L1 nudix (nucleoside diphosphate linked moiety X)-type motif 16-like 1
650048 MOBP myelin-associated oligodendrocyte basic protein
3170725 C2orf79 chromosome 2 open reading frame 79
7210767 GAK cyclin G associated kinase
240035 RUNDC3B RUN domain containing 3B
1770519 PDRG1 p53 and DNA-damage regulated 1
2190743 RBM23 RNA binding motif protein 23
6620601 ZBTB40 zinc finger and BTB domain containing 40
3140280 C9orf6 chromosome 9 open reading frame 6
1820711 LOC100288144 hypothetical LOC100288144
6420632 MCCC1 methylcrotonoyl-CoA carboxylase 1 (alpha)
2760519 CKLF chemokine-like factor
2490333 ZNF467 zinc finger protein 467
3890274 DPF2 D4, zinc and double PHD fingers family 2
4010452 SLC38A6 solute carrier family 38, member 6
5720154 ZBTB48 zinc finger and BTB domain containing 48
6960692 ZSCA 10 zinc finger and SCAN domain containing 10
6580075 TAF1D TATA box binding protein (TBP)-associated factor, RNA polymerase 1, D, 41kDa
1580402 SLC35B2 solute carrier family 35, member B2
6200561 CCDC28B coiled-coil domain containing 28B
1780095 RPL26L1 ribosomal protein L26-like 1
2100189 KCTD13 potassium channel tetramerisation domain containing 13
7610538 H1FX HI histone family, member X
6200253 THBS4 thrombospondin 4
3930170 CDK20 cyclin-dependent kinase 20
6560750 UBE3C ubiquitin protein ligase E3C
870376 C9orfl52 chromosome 9 open reading frame 152
4490544 LY6G6D lymphocyte antigen 6 complex, locus G6D
1990674 NUP50 nucleoporin 50kDa
240750 TEL02 TEL2, telomere maintenance 2, homolog (S. cerevisiae)
2630102 CCDC28B coiled-coil domain containing 28B 7040131 DAL D3 DALR anticodon binding domain containing 3
6480209 R AGD Ras-related GTP binding D
6580521 UBAC2 UBA domain containing 2
7570315 M RPL45 mitochondrial ribosomal protein L45
6510397 WDR19 WD repeat domain 19
610735 LRRC43 leucine rich repeat containing 43
2190241 AP1M1 adaptor-related protein complex 1, mu 1 subunit
510370 SYCE 2 synaptonemal complex central element protein 2
2360138 ATP6V0C ATPase, H+ transporting, lysosomal 16kDa, VO subunit c
4610201 SNORA10 small nucleolar RNA, H/ACA box 10
3180445 C14orf79 chromosome 14 open reading frame 79
4890647 Clorf25 chromosome 1 open reading frame 25
2810400 KLHL3 kelch-like 3 (Drosophila)
540221 NOP2 NOP2 nucleolar protein homoiog (yeast)
6020458 NR2F2 nuclear receptor subfamily 2, group F, member 2
10626 TRNAU1AP tRNA selenocysteine 1 associated protein 1
3060092 LAT2 linker for activation of T cells family, member 2
1850612 PARP2 poly (ADP-ribose) polymerase 2
3450521 ECM1 extracellular matrix protein 1
4920537 POLA2 polymerase (DNA directed), alpha 2 (70kD subunit)
4760433 C16orf7 chromosome 16 open reading frame 7
3390373 TIM M22 translocase of inner mitochondrial membrane 22 homoiog (yeast)
3710685 CARD9 caspase recruitment domain family, member 9
7100079 PHF8 PHD finger protein 8
150315 C21orf7 chromosome 21 open reading frame 7
6040703 TRIM39 tripartite motif-containing 39
6650451 MYCBP2 MYC binding protein 2
2750324 PRKCZ protein kinase C, zeta
7400707 CIS complement component 1, s subcomponent
1070474 P0C5 POC5 centriolar protein homoiog (Chlamydomonas)
2350221 TSNAXIP1 translin-associated factor X interacting protein 1
2630561 RPL6 ribosomal protein L6
6330440 MSH5 mutS homoiog 5 (E. coli)
4280048 WFDC3 WAP four-disulfide core domain 3
4860367 ATRIP ATR interacting protein
1990196 DACT3 dapper, antagonist of beta-catenin, homoiog 3 (Xenopus laevis)
4120427 PDX1 pancreatic and duodenal homeobox 1
240333 ETS1 v-ets erythroblastosis virus E26 oncogene homoiog 1 (avian)
4850300 ARHGAP39 Rho GTPase activating protein 39
1710639 RBM4B RNA binding motif protein 4B
6620278 ADI1 acireductone dioxygenase 1
3930577 HMG N2 high-mobility group nucleosomal binding domain 2
2490377 C17orf71 chromosome 17 open reading frame 71
asparagine-linked glycosylation 8, alpha-l,3-glucosyltransferase homoiog
4850632 ALG8 (S. cerevisiae)
1780450 ASB6 ankyrin repeat and SOCS box-containing 6
2710546 C0G4 component of oligomeric golgi complex 4
6620634 U BE2S ubiquitin-conjugating enzyme E2S . , . ,„., . , . ,.
5310358 TIA1 TIA1 cytotoxic granule-associated RNA binding protein
3610735 F12 coagulation factor XII ( Hageman factor)
1500678 TFPT TCF3 (E2A) fusion partner (in childhood Leukemia)
5220152 T EM55B transmembrane protein 55B
4260441 CLEC3B C-type lectin domain family 3, member B
610358 PGS1 phosphatidylglycerophosphate synthase 1
6350181 LOC730183 hypothetical protein LOC730183
2100209 FA 24B family with sequence similarity 24, member B
1070053 SUGP2 SURP and G patch domain containing 2
4590424 PDCD2L programmed cell death 2-like
2470296 ZSCAN16 zinc finger and SCAN domain containing 16
1340411 CA SAP1 calmodulin regulated spectrin-associated protein 1
4210500 PSG4 pregnancy specific beta-l-glycoprotein 4
6100196 ZNF653 zinc finger protein 653
270053 GABPA GA binding protein transcription factor, alpha subunit 60kDa
6770097 UBTD1 ubiquitin domain containing 1
4220184 LOC100289410 hypothetical LOC100289410
7330674 KIFC1 kinesin family member CI
3400202 G PATCH 1 G patch domain containing 1
4900044 CDC7 cell division cycle 7 homolog (S. cerevisiae)
1470379 CCDC116 coiled-coil domain containing 116
650626 C16orf68 chromosome 16 open reading frame 68
1300521 INSM2 insulinoma-associated 2
2340180 TAGLN2 transgelin 2
2370064 ASGR1 asialoglycoprotein receptor 1
1070360 APITD1 apoptosis-inducing, TAF9-like domain 1
10075 ZNF768 zinc finger protein 768
5260639 ZNF330 zinc finger protein 330
5360682 IL17F interieukin 17F
7570500 C0Q5 coenzyme Q5 homolog, methyltransferase (S. cerevisiae)
3190246 CHN2 chimerin (chimaerin) 2
6350626 CCDC120 coiled-coil domain containing 120
6330358 C9orf98 chromosome 9 open reading frame 98
3800068 GTF2E1 general transcription factor HE, polypeptide 1, alpha 56kDa
6180598 NDUFAF1 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, assembly factor 1
4850674 PSAT1 phosphoserine aminotransferase 1
6590520 ZNF839 zinc finger protein 839
1090687 P0LR1D polymerase (RNA) 1 polypeptide D, 16kDa
2680020 DAG LB diacylglyceroi lipase, beta
3440315 PPP2R3B protein phosphatase 2, regulatory subunit B, beta
4260731 STM N1 stathmin 1
1450707 RING1 ring finger protein 1
380373 BA P BTG3 associated nuclear protein
5900112 ZNF830 zinc finger protein 830
7160414 C7orf63 chromosome 7 open reading frame 63
4570500 CPNE1 copine 1
2060427 SBDSP1 Shwachman-Bodian-Diamond syndrome pseudogene 1 4570292 PHF12 PHD finger protein 12
3710068 WARS tryptophanyl-tRNA synthetase
4260044 SQSTM1 sequestosome 1
730687 TCHP trichoplein, keratin filament binding
4900431 STUB1 STIP1 homology and U-box containing protein 1
6290239 ATP6V1B1 ATPase, H+ transporting, lysosomal 56/58kDa, VI subunit Bl
6620669 C3orf23 chromosome 3 open reading frame 23
5080431 NBPF3 neuroblastoma breakpoint family, member 3
510112 PTAFR platelet-activating factor receptor
3520746 MTTP microsomal triglyceride transfer protein
7560328 RAVER1 ribonucleoprotein, PTB-binding 1
3930754 PRR3 proline rich 3
2000100 ABI2 abl-interactor 2
5270239 TUBD1 tubulin, delta 1
460768 LOC285943 hypothetical protein LOC285943
5700722 TSSC1 tumor suppressing subtransferable candidate 1
1190129 SL 02 slowmo homolog 2 (Drosophila)
1400601 T0X2 TOX high mobility group box family member 2
520114 PET112L PET112-like (yeast)
5900020 ClOorfllO chromosome 10 open reading frame 110
5860452 BNIP1 BCL2/adenovirus E1B 19kDa interacting protein 1
6350075 CENPBD1 CENPB DNA-binding domains containing 1
4850296 HCFC1 host cell factor CI (VP16-accessory protein)
5050735 TMEM62 transmembrane protein 62
3310681 LOC391578 MAF1 homolog (S. cerevisiae) pseudogene
7050612 TIAM2 T-cell lymphoma invasion and metastasis 2
10187 CNPY3 canopy 3 homolog (zebrafish)
3890398 WBP2 WW domain binding protein 2
pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of
4860184 PCBD1 hepatocyte nuclear factor 1 alpha
130037 PHF5A PHD finger protein 5A
6350292 ClorfSO chromosome 1 open reading frame 50
6420296 MRPL2 mitochondrial ribosomal protein L2
7510687 SRCAP Snf2-related CREBBP activator protein
5820333 RPUSD2 RNA pseudouridylate synthase domain containing 2
2190010 RACGAP1 Rac GTPase activating protein 1
2570288 SH3YL1 SH3 domain containing, Ysc84-like 1 (S. cerevisiae)
2070201 CCKBR cholecystokinin B receptor
1570129 TRAFD1 TRAF-type zinc finger domain containing 1
610670 ISL2 ISL LIM homeobox 2
6370593 BCL7B B-celi CLL/lymphoma 7B
4860291 HMGXB3 HMG box domain containing 3
2360601 NAA25 N(alpha)-acetyltransferase 25, NatB auxiliary subunit t nes a so ene on o ogy ana yses wer e results are shown in Table 6. As can be seen, TTV miRNA might be deregulating several pathways important for cancer progression.
Table 6
Gene enrichment analysis
Category Term Genes Count % P-
Figure imgf000040_0001
Figure imgf000040_0002
Figure imgf000040_0003
syndrome
Figure imgf000040_0004
Examp e
Screening the TCGA for TTV miR A associated with cancer
The TCGA (The Cancer Genome Atlas) is an initiative of the NIH . The data stored within this repository consist of sequencing datasets from cancer and normal tissue extracted from patients. In this regard, the data extracted by this analysis can be considered as Min vivo", since it comes directly from tumors of patients. In an effort to establish a relationship between TTV miRNA and cancer, the small-R A sequencing data for colon adenocarcinoma, lung adenocarcinoma, breast carcinoma and hepatocellular carcinoma from the TCGA initiative was mapped against all the full-length TTV genomes included in the NCBI database plus several newly identified TTV from the inventors ' s laboratory. To exclude artifacts, miRNA taken into consideration complied to the following: mapping with 2 mismatches or less to TTV genomes and mapping in a region where the RNA is predicted to acquire the characteristic hairpin structure of a pre-miRNA (Table 7) .
Cancer type Total TTV positive patients % of TTV positive Patients positive for TTV encoding number of patients a mature miRNA presenting the
patients "consensus sequence" screened
Colon carcinoma 421 76 18,05225653 53 12,5
Hepatocellular 147 19 12,92517007 9 6,12 carcinoma
Lung 213 25 11,7370892 9 4,22 adenocarcinoma
(Ongoing)
Breast 141 11 7,80141844 1 0,70 carcinoma(ongoing)
Table 7- Small RNA sequencing datasets from patients with different malignancies were screened for the presence of TTV miRNA. TTV positive pa were considered when having at least one read mapping to a TTV miRNA. Patients positive for TTV encoding a mature miRNA presenting the "con sequence" where considered when having at least one read mapping to a TTV strain that encodes for a mature miRNA that contains the "consens sequence". The "consensus sequence" , the TTV strains found in the TCGA containing the consensus sequence and the mature miRNA form these strains are listed in Table 2B.+
TTV-HD14a-2-3p analogous miRNA (meaning, with 80% homology or more in the nucleotides from 1 to 7 of the miRNA, comprising -the seed) {Table 2B) were found at higher frequency in colon cancer patients than in the other three type of cancer being screened so far.
The slight differences in the seed of the miRNA shown in Table 2B in respect to TTV-MDl a-mir-2-3p do not alter the predicted binding sites in APC mRNA. Thus, the miRNA shown in Table 2Bare also able to down- regulate APC {Table 8) .
Table 8
It is shown how, despite the single nucleotide polymorphisms (SNP) found in the seed of diverse TTV miRNA' s respect to the
TTV-HD] 4a-mi r-2-3p seed, the predicted interaction site with APC mRNA shown in figure 3 (A. ) would be conserved. (B) Here the inventors show how the most conserved seed motif (AUCCUC) has three additional possible interaction sites within APC mRNA in addition to the
previously described for TTV-HD14a-mir-2-3p .
Positions are shown in relation to the nucleotide number of APC transcript variant 2 mRNA (NCBI accession number: NM__001127510.2,
SEQ ID NO: 82)
Seed interaction sites are shown in black bold letters.
Sequence corresponding to APC mRNA are shown in italicized letters.
Figure imgf000044_0001
Table 8
This supports a causal role for this type of TTV miRNA of Table 2B in this disease or, at least, an association between them.
A significant increase in TTV load in cancer patients compared to normal controls has been demonstrated [44] . In the case of colon cancer, this increase in viral load would presumably be represented mainly by the TTV strains encoding for miRNA analogous to that of TTV- HD14a.
Example 9
Wnt activation by a TTV miRNA
APC exerts its tumor suppressor activity by down-regulating canonical Wnt pathway, although other putative roles for this protein have been suggested. This effect is mediated by its participation in the "destruction complex". The destruction complex is formed by APC, AXIN , and GSK3-beta, among others. This complex phosphorylates beta-catenin, allowing its ubiquitination and degradation by the proteasome. In the absence of any of the proteins of the destruction complex, its function is impaired. The final outcome is the cytoplasmic accumulation of beta-catenin, that can be then translocated into the nucleus , where it activates transcription of its target genes , together with the transcription factor TCF4 or LEF1. It is well documented that this pathway is upregulated in several malignancies, as well as in other diseases. Consequently, we thought that the APC down-regulation could lead to an activation of Wnt pathway. To check this, a gene reporter approach was used. HEK293TT ceils were transfected with the plasmids encoding for TTV-HDl a miRNA, with the TTV-HD14a full genome, or mock transfected, together with a plasmid encoding for Firefly luciferas - under the control of a minimum promoter and seven binding sites for the TCF4 /beta catenin complex (TOPFLASH plasmid) . Additionally, Renilia Luciferase under the control of CMV promoter was used for normalization purposes. An upregulation of wnt pathway resulted in cells with the plasmid encoding for the sense-miRNA or with the TTV-HD14a virus in comparison to mock transfected cells (Figure 5). Conclusions
The above results highlight the importance of the experimental findings as diagnostic method for TTV infection and identifies TTV miRNA as promising target for cancer prevention, treatment or recurrence .
It is known that TTV replicate in several tissues [21], but they only repl icate in peripheral blood mononuclear cells when these cells are activated [42] . It was recently demonstrated that TTV replicate more efficiently when they are co-infecting cells with Epstein Barr virus [41] .
Very few things are known about the molecular mechanisms mediating infection, replication and virus-host interaction of the TTVs. Here, the inventors provide evidence which supports that several TTVs encode miRNA and that some of them have a biologically relevant role, especially in relation to cancer development.
It has been shown in the present invention that the encoded miRNA of
TTV-HDl4a and Table 2B can down-regulate APC, an important tumor suppressor. Hence, being infected with any of the TTVs encoding for the miRNA' s included in the present invention could represent a risk factor for the development of colon cancer, as well as many other cancer types .
To support these findings , the inventors detected TTV miRNA' s that down-regulate APC in a higher frequency in colon adenocarcinoma patients in comparison to other three types of cancer (lung adenocarcinoma, hepatocellular carcinoma and breast invasive carcinoma). Consequently, TTV miRNA' s presented here represent a target for the prevention of colon cancer, as well as a putative biomarker for the early detection of a subset of these cancers.
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Claims

Claims
1. A TTV polynucleic acid comprising:
nucleotide sequence depicted in Table 2b;
(b) a nucleotide sequence having at least 60% identity to the nucleotide sequence of (a) and containing the nucleotide sequence CATCCYY (with Y: C or T) or CAUCCYY (with Y: C or U) , respectively;
(c) a fragment of the nucleotide sequence of (a) or (b) and containing the nucleotide sequence CATCCYY (with Y: C or T) or CAUCCYY (with Y: C or U) , respectively; or
nucleotide sequence being complementary to a nucleotide sequence of (a) , (b) or (c) .
2. A TTV polynucleic acid, wherein the TTV polynucleic acid is a mature TTV miRNA molecule comprising:
(a) a nucleotide sequence underlined in Table 2a or 2b;
(b) a nucleotide sequence having at least 60% identity to the nucleotide sequence of (a) and containing the nucleotide sequence CATCCYY (with Y: C or T) or CAUCCYY (with Y: C or U) , respectively;
(c) a fragment of the nucleotide sequence of (a) or (b) and containing the nucleotide sequence CATCCYY (with Y: C or T) or CAUCCYY (with Y: C or U) , respectively; or
(d) a nucleotide sequence being complementary to a nucleotide sequence of (a) , (b) or (c) .
3. An oligonucleotide primer comprising part of the TTV polynucleic acid of claim 1 or 2 and containing the nucleotide sequence CATCCYY (with Y: C or T) or CAUCCYY (with Y: C or U) , respectively, said primer being capable of acting as primer for specifically sequencing or specifically amplifying the nucleic acid of a certain TTV isolate containing a nucleotide sequence of claim 1.
4. An oligonucleotide probe comprising a TTV polynucleic acid of claim
1 or 2, said probe being capable of acting as a hybridization probe for specific detection of the nucleic acid of a certain TTV isolate containing a nucleotide sequence of claim 1 or 2.
5. An oligonucleotide primer or oligonucleotide probe according to claim 3 or 4 having a nucleotide sequence as depicted in Table 4.
6. A vector containing a TTV polynucleic acid of claim 1 or 2, a oligonucleotide primer of claim 3 or 5 or a oligonucleotide probe of claim 4 or 5.
7. A host cell transfected with the vector of claim 6.
8. A diagnostic kit containing a TTV polynucleic acid of claim 1 or 2, an oligonucleotide rimer of claim 3 or 4, or an oligonucleotide probe of claim 4 or 5.
9. Use of a TTV polynucleic acid of claim 1 or 2 , an oligonucleotide primer of claim 3 or 4, or an oligonucleotide probe of claim 4 or 5 as an early marker for the future development of cancer.
10. The use of claim 9, wherein the cancer is colorectal cancer or colon cancer.
11. Use of a TTV polynucleic acid o claim 1 or 2 as a lead component for the development of a medicament for prevention or treatment of cancer .
12. An inhibitor of miRNA specific for polynucleic acid shown in Table
2A or 2B selected from the group consisting of anti-miR, miRNA spounge and tough decoy inhibitor.
13. A method of diagnosing colon cancer comprising the steps of:
(a) isolating miRNA from a patients sample;
(b) sequencing the miRNA selected in step (a) ; and
(c) determining if a miRNA selected from the miRNAs shown in Table 2B is present in the sample, whereas the presence of an miRNA shown in Table 2B indicates colon cancer.
14. A method of delievering a lead component for the development of medicament for treatment of colorectal cancer to a patient suffering from colorectal cancer comprising the step of administering
(a) an adeno-associated virus expressing an inhibitor of the polynucleic acid of claim 1 or 2 selected from the group consisting of a anti-miR, a miRNA spounge and a tough deco inhibitor; or
(b) a pHILP cons ruct comprising an inhibitor of a polynucleic acid of claim 1 or 2 attached thereto .
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