Sirna for silencing a new isoform of the mitochondrial chaperone trap1

Abstract

A siRNA characterised by having a sequence selected from the group comprised of: - SEQ ID NO. 1 : 5 ' CUCUUUCCCUUGAAUAAGC 3 '; - SEQ ID NO. 2 : 5 ' UGAUUCCCAAAGCUCACAG 3 '; and - a sequence including at least 16 contiguous nucleotides differing by no more than 3 nucleotides from SEQ ID No. 1 or SEQ ID No. 2, to silence TRAPl-low SEQ ID N°3. The use of the 5 'UTR SEQ ID N°4 region of the TRAPl-low transcript SEQ ID N°3 or a portion of that 5 'UTR region selected from the group comprised of: - the SEQ ID No. 6 portion of said 5 ' UTR region comprising nucleotide positions 1 through 644; - a portion of said region 5 ' UTR comprising the portion of SEQ ID No. 6 extended by a predetermined number of nucleotide positions beyond nucleotide position 644 along said region 5 'UTR.

Description

TITLEsiRNA FOR SILENCING A NEW ISOFORM OF THE MITOCHONDRIAL CHAPERONE TRAP1DESCRIPTIONSCOPE OF THE INVENTION

[0001] The present invention relates to oligonucleotide siRNA sequences specific for a novel isoform of the mitochondrial chaperone TRAP1.PRIOR ART - TECHNICAL PROBLEMS

[0002] The mitochondrial localisation molecular chaperone TRAP1 is known. It interacts with several client proteins to make them work correctly. More in detail, it is known that TRAPl ' s role includes:- ensuring that the correct conformational state of newly synthesised polypeptide chains is achieved and maintained;- directing the correct assembly of multienzyme complexes; - stabilising proteins damaged by chemical or physical stress, facilitating their renaturation and / or degradation.In this way, TRAP1 regulates a large number of biological functions such as cell metabolism, cell survival, sternness and resistance to common chemotherapeutic agents.

[0003] It is also known that TRAP1 is highly expressed in several solid tumour types, particularly in metastatic colorectal cancer, and its function in the development of carcinogenesis and the acquisition of more aggressive features is still a subject of research.

[0004] A bioinformatic analysis of public datasets of colorectal adenocarcinoma patients revealed the existence of a TRAP1 isoform, not yet described in any scientific work,containing fewer amino acids than the canonical form. For this reason, hereinafter this new isoform is referred to as TRAP1- low. It is believed that the latter is the result of transcription from an alternative promoter within the TRAP1 gene, as described in more detail below.

[0005] It was therefore necessary to precisely identify this new isoform, and to study its intracellular localisation and biological function, as well as whether it plays a role in carcinogenesis. In the context of colorectal cancer, it is also desirable to assess whether the canonical TRAP1 isoform is more highly expressed than the new TRAPl-low isoform, or vice-versa.

[0006] siRNAs are also known, i.e., small interfering RNAs. They are short RNA sequences capable of recognising complementary sequences and binding them on messenger RNAs, causing the latter to degrade via the RISC complex, a RNA-induced silencing complex. This makes it possible to reduce the expression of a target protein without interfering at the gene level, but rather by blocking the translation of its messenger. This process is referred to as transient silencing.

[0007] However, to date, no tools are available, such as specific antibodies or siRNAs that are selective enough to enable assessing the biological function of TRAPl-low, i. e. to make it possible to study its activity selectively, while excluding the biological contribution of the canonical form of this chaperone. Indeed, commercially available antibodies and known siRNAs, which are capable of recognising or silencing canonical TRAP1, respectively, turned out to be able to bind the new isoform as well, therefore they do not allow to isolate the latter from the known protein.

[0008] In view of the above, it is desirable to find a siRNA that is able to selectively silence the translation ofthis new TRAPl-low isoform, i.e. to silence it without silencing the translation of TRAP1.

[0009] Furthermore, it is desirable to find possible diagnostic and / or therapeutic tools for colorectal cancer pathology and other cancer pathologies.SUMMARY OF THE INVENTION

[0010] It is therefore an object of the present invention to provide compatible siRNAs that are capable of silencing the TRAPl-low isoform without silencing canonical TRAP1.

[0011] It is also an object of the present invention to provide such siRNAs that have a low production cost and are therefore suitable for large-scale production.

[0012] It is also an object of the present invention to provide sequences on which siRNAs can be synthesised that are capable of selectively silencing TRAPl-low.

[0013] In a first aspect of the invention, these purposes are achieved by a siRNA as defined in claim 1.

[0014] According to the invention, a siRNA for silencing TRAPl-low is characterised in that it includes a sequence selected from the group comprised of:SEQ ID N°1: 5 ’ CUCUUUCCCUUGAAUAAGC 3 ’;SEQ ID N°2: 5 ’ UGAUUCCCAAAGCUCACAG 3 ’;- a sequence including at least 16 contiguous nucleotides that differ by no more than 3 nucleotides from SEQ ID No. 1 or SEQ ID No. 2.

[0015] Such siRNAs are complementary to an uncommon sequence of the canonical TRAP1 transcript (ENST00000246957. 10), but specific to the TRAPl-low transcript (ENST00000575671. 5).

[0016] In particular, in order to identify a siRNA specific for TRAPl-low, the 5 ' UTR region, SEQ ID No. 4, of TRAPl-low transcript SEQ ID No. 3 was taken into account.The latter is shown below in its entirety, and the 5 ' UTR region is underlined:SEQ ID No. 3 (transcript of TRAPl-low):1 GTGCTCCTCAGCTGCACAGGCCACATTGCAGGTGCTCGGCAGGCTGTCGCATTGCACCTG 6061 GCAGAACGTTCCTGTTGTTGGAGAAAGTTCCATCGGACAACGTGGGCCAGGGCAGTGGGC 120121 GTGTGTATTTTACTTGACTTTGGCCTTAGGACCTTTGGCAGTATTATAGTAGAAAAATGA 180181 AGTGCTGTGCGGTGCTTACAGACGCATGCGCGTGATTTCTACTCTTTTTCCCACAAAGTG 240241 TAGAGTCCAGGAGACTTGAATATGGAAGCCAGTTTGGAGCCTCTCCTCACTCCTGGCCTT 300301 CCTATGGAAGGAGCTTATTCAAGGGAAAGAGCTTATTCAAGCTGCTCTTTCCCCACCCCC 360361 AGGTGTGACCCACAGGCCTCCACCTGTCTGGGACAGGCAGGACTGTGAGCTTTGGGAATC 420421 AAATGGAAAACACACTTGTCCCACTGCTGCCAGCTGGTTGTCCTAGGGCTAATGTGTCAC 480481 CTCTCTGTGACTTAGCTCTTCACGTGTAGAATGGGGTGGAAAAGTACTCGGTGTGGCTGT 540541 TTTCAGGATGAAGGGGTCTTAGCGGTGCTCGCAGAAGAGCCACGGTGTTGGGAGTGTCTG 600601 CTCTCTGGAGGGGAGGCTGCCTCACTAACTGGCCTCTGCCCCACAGGCCTTCCTGGATGC 660661 TCTGCAGAACCAGGCTGAGGCCAGCAGCAAGATCATCGGCCAGTTTGGAGTGGGTTTCTA 720721 CTCAGCTTTCATGGTGGCTGACAGAGTGGAGGTCTATTCCCGCTCGGCAGCCCCGGGGAG ..........ATGGTGGCTGACAGAGTGGAGGTCTATTCCCGCTCGGCAGCCCCGGGGAG 50781 CCTGGGTTACCAGTGGCTTTCAGATGGTTCTGGAGTGTTTGAAATCGCCGAAGCTTCGGG 840 51 CCTGGGTTACCAGTGGCTTTCAGATGGTTCTGGAGTGTTTGAAATCGCCGAAGCTTCGGG 110841 AGTTAGAACCGGGACAAAAATCATCATCCACCTGAAATCCGACTGCAAGGAGTTTTCCAG 900 111 AGTTAGAACCGGGACAAAAATCATCATCCACCTGAAATCCGACTGCAAGGAGTTTTCCAG 170901 CGAGGCCCGGGTGCGAGATGTGGTAACGAAGTACAGCAACTTCGTCAGCTTCCCCTTGTA 960 171 CGAGGCCCGGGTGCGAGATGTGGTAACGAAGTACAGCAACTTCGTCAGCTTCCCCTTGTA 230961 CTTGAATGGAAGGCGGATGAACACCTTGCAGGCCATCTGGATGATGGACCCCAAGGATGT 1020 231 CTTGAATGGAAGGCGGATGAACACCTTGCAGGCCATCTGGATGATGGACCCCAAGGATGT 2901021 CCGTGAGTGGCAACATGAGGAGTTCTACCGCTACGTCGCGCAGGCTCACGACAAGCCCCG 1080 291 CCGTGAGTGGCAACATGAGGAGTTCTACCGCTACGTCGCGCAGGCTCACGACAAGCCCCG 3501081 CTACACCCTGCACTATAAGACGGACGCACCGCTCAACATCCGCAGCATCTTCTACGTGCC 1140 351 CTACACCCTGCACTATAAGACGGACGCACCGCTCAACATCCGCAGCATCTTCTACGTGCC 4101141 CGACATGAAACCGTCCATGTTTGATGTGAGCCGGGAGCTGGGCTCCAGCGTTGCACTGTA 1200 411 CGACATGAAACCGTCCATGTTTGATGTGAGCCGGGAGCTGGGCTCCAGCGTTGCACTGTA 4701201 CAGCCGCAAAGTCCTCATCCAGACCAAGGCCACGGACATCCTGCCCAAGTGGCTGCGCTT 1260 471 CAGCCGCAAAGTCCTCATCCAGACCAAGGCCACGGACATCCTGCCCAAGTGGCTGCGCTT 5301261 CATCCGAGGTGTGGTGGACAGTGAGGACATTCCCCTGAACCTCAGCCGGGAGCTGCTGCA 1320 531 CATCCGAGGTGTGGTGGACAGTGAGGACATTCCCCTGAACCTCAGCCGGGAGCTGCTGCA 5901321 GGAGAGCGCACTCATCAGGAAACTCCGGGACGTTTTACAGCAGAGGCTGATCAAATTCTT 1380 591 GGAGAGCGCACTCATCAGGAAACTCCGGGACGTTTTACAGCAGAGGCTGATCAAATTCTT 6501381 CATTGACCAGAGTAAAAAAGATGCTGAGAAGTATGCAAAGTTTTTTGAAGATTACGGCCT 1440 651 CATTGACCAGAGTAAAAAAGATGCTGAGAAGTATGCAAAGTTTTTTGAAGATTACGGCCT 7101441 GTTCATGCGGGAGGGCATTGTGACCGCCACCGAGCAGGAGGTCAAGGAGGACATAGCAAA 1500 711 GTTCATGCGGGAGGGCATTGTGACCGCCACCGAGCAGGAGGTCAAGGAGGACATAGCAAA 7701501 GCTGCTGCGCTACGAGTCCTCGGCGCTGCCCTCCGGGCAGCTAACCAGCCTCTCAGAATA 1560 771 GCTGCTGCGCTACGAGTCCTCGGCGCTGCCCTCCGGGCAGCTAACCAGCCTCTCAGAATA 8301561 CGCCAGCCGCATGCGGGCCGGCACCCGCAACATCTACTACCTGTGCGCCCCCAACCGTCA 1620 831 CGCCAGCCGCATGCGGGCCGGCACCCGCAACATCTACTACCTGTGCGCCCCCAACCGTCA 8901621 CCTGGCAGAGCACTCACCCTACTATGAGGCCATGAAGAAGAAAGACACAGAGGTTCTCTT 1680 891 CCTGGCAGAGCACTCACCCTACTATGAGGCCATGAAGAAGAAAGACACAGAGGTTCTCTT 9501681 CTGCTTTGAGCAGTTTGATGAGCTCACCCTGCTGCACCTTCGTGAGTTTGACAAGAAGAA 1740 951 CTGCTTTGAGCAGTTTGATGAGCTCACCCTGCTGCACCTTCGTGAGTTTGACAAGAAGAA 10101741 GCTGATCTCTGTGGAGACGGACATAGTCGTGGATCACTACAAGGAGGAGAAGTTTGAGGA 1800 1011 GCTGATCTCTGTGGAGACGGACATAGTCGTGGATCACTACAAGGAGGAGAAGTTTGAGGA 10701801 CAGGTCCCCAGCCGCCGAGTGCCTATCAGAGAAGGAGACGGAGGAGCTCATGGCCTGGAT 1860 1071 CAGGTCCCCAGCCGCCGAGTGCCTATCAGAGAAGGAGACGGAGGAGCTCATGGCCTGGAT 11301861 GAGAAATGTGCTGGGGTCGCGTGTCACCAACGTGAAGGTGACCCTCCGACTGGACACCCA 1920 1131 GAGAAATGTGCTGGGGTCGCGTGTCACCAACGTGAAGGTGACCCTCCGACTGGACACCCA 11901921 CCCTGCCATGGTCACCGTGCTGGAGATGGGGGCTGCCCGCCACTTCCTGCGCATGCAGCA 1980 1191 CCCTGCCATGGTCACCGTGCTGGAGATGGGGGCTGCCCGCCACTTCCTGCGCATGCAGCA 12501981 GCTGGCCAAGACCCAGGAGGAGCGCGCACAGCTCCTGCAGCCCACGCTGGAGATCAACCC 2040 1251 GCTGGCCAAGACCCAGGAGGAGCGCGCACAGCTCCTGCAGCCCACGCTGGAGATCAACCC 13102041 CAGGCACGCGCTCATCAAGAAGCTGAATCAGCTGCGCGCAAGCGAGCCTGGCCTGGCTCA 2100 1311 CAGGCACGCGCTCATCAAGAAGCTGAATCAGCTGCGCGCAAGCGAGCCTGGCCTGGCTCA 13702101 GCTGCTGGTGGATCAGATATACGAGAACGCCATGATTGCTGCTGGACTTGTTGACGACCC 2160 1371 GCTGCTGGTGGATCAGATATACGAGAACGCCATGATTGCTGCTGGACTTGTTGACGACCC 14302161 TAGGGCCATGGTGGGCCGCTTGAATGAGCTGCTTGTCAAGGCCCTGGAGCGACACTGACA 2220 1431 TAGGGCCATGGTGGGCCGCTTGAATGAGCTGCTTGTCAAGGCCCTGGAGCGACACTGA.. 14882221 GCCAGGGGGCCAGAAGGACTGACACCACAGATGACAGCCCCACCTCCTTGAGCTTTATTT 22802281 ACCTAAATTTAAAGGTATTTCTTAACCCG. 2309

[0017] For the sake of clarity, the 5 ' UTR region is shown below:SEQ ID No. 4 (5 ' UTR region of the TRAPl-low transcript): GTGCTCCTCAGCTGCACAGGCCACATTGCAGGTGCTCGGCAGGCTGTCGCATTGCACC TGGCAGAACGTTCCTGTTGTTGGAGAAAGTTCCATCGGACAACGTGGGCCAGGGCAGT GGGCGTGTGTATTTTACTTGACTTTGGCCTTAGGACCTTTGGCAGTATTATAGTAGAA AAATGAAGTGCTGTGCGGTGCTTACAGACGCATGCGCGTGATTTCTACTCTTTTTCCC ACAAAGTGTAGAGTCCAGGAGACTTGAATATGGAAGCCAGTTTGGAGCCTCTCCTCAC TCCTGGCCTTCCTATGGAAGGAGCTTATTCAAGGGAAAGAGCTTATTCAAGCTGCTCT TTCCCCACCCCCAGGTGTGACCCACAGGCCTCCACCTGTCTGGGACAGGCAGGA ^G GAGCTTTGGGAATCAAATGGAAAACACACTTGTCCCACTGCTGCCAGCTGGTTGTCCT AGGGCTAATGTGTCACCTCTCTGTGACTTAGCTCTTCACGTGTAGAATGGGGTGGAAA AGTACTCGGTGTGGCTGTTTTCAGGATGAAGGGGTCTTAGCGGTGCTCGCAGAAGAGC CACGGTGTTGGGAGTGTCTGCTCTCTGGAGGGGAGGCTGCCTCACTAACTGGCCTCTG CCCCACAGGCCTTCCTGGATGCTCTGCAGAACCAGGCTGAGGCCAGCAGCAAGATCAT CGGCCAGTTTGGAGTGGGTTTCTACTCAGCTTTC.

[0018] The part of messenger RNA corresponding to the 5 ' UTR region of TRAP1-LOW is also shown as SEQ ID N° 5:SEQ ID N° 5 (part of messenger RNA corresponding to the 5 ' UTR region of TRAP1-LOW):CACGAGGAGUCGACGUGUCCGGUGUAACGUCCACGAGCCGUCCGACAGCGUAACGUGG ACCGUCUUGCAAGGACAACAACCUCUUUCAAGGUAGCCUGUUGCACCCGGUCCCGUCA CCCGCACACAUAAAAUGAACUGAAACCGGAAUCCUGGAAACCGUCAUAAUAUCAUCUU UUUACUUCACGACACGCCACGAAUGUCUGCGUACGCGCACUAAAGAUGAGAAAAAGGG UGUUUCACAUCUCAGGUCCUCUGAACUUAUACCUUCGGUCAAACCUCGGAGAGGAGUG AGGACCGGAAGGAUACCUUCCUCGAAUAAGUUCCCUUUCUCGAAUAAGUUCGACGAGA AAGGGGUGGGGGUCCACACUGGGUGUCCGGAGGUGGACAGACCCUGUCCGUCCUGACA CUCGAAACCCUUAGUUUACCUUUUGUGUGAACAGGGUGACGACGGUCGACCAACAGGA UCCCGAUUACACAGUGGAGAGACACUGAAUCGAGAAGUGCACAUCUUACCCCACCUUU UCAUGAGCCACACCGACAAAAGUCCUACUUCCCCAGAAUCGCCACGAGCGUCUUCUCG GUGCCACAACCCUCACAGACGAGAGACCUCCCCUCCGACGGAGUGAUUGACCGGAGAC GGGGUGUCCGGAAGGACCUACGAGACGUCUUGGUCCGACUCCGGUCGUCGUUCUAGUA GCCGGUCAAACCUCACCCAAAGAUGAGUCGAAAG.

[0019] In the 5 ' UTR region of the TRAP-low transcript, SEQ ID No. 3, a single target sequence SEQ ID No. 6 was identified that is able to discriminate between the two isoforms, canonical TRAP1 isoform and TRAPl-low isoform. SEQ ID No. 6 comprises positions 1 to 644 of SEQ ID No. 4 and SEQ ID No. 3, and is highlighted in bold font therein. However, for the sake of clarity, SEQ ID N° 6 target sequence is shown below:SEQ ID N° 6 (target sequence for the detection of TRAPl-low silencing siRNAs ):GTGCTCCTCAGCTGCACAGGCCACATTGCAGGTGCTCGGCAGGCTGTCGCATTGCACC TGGCAGAACGTTCCTGTTGTTGGAGAAAGTTCCATCGGACAACGTGGGCCAGGGCAGT GGGCGTGTGTATTTTACTTGACTTTGGCCTTAGGACCTTTGGCAGTATTATAGTAGAA AAATGAAGTGCTGTGCGGTGCTTACAGACGCATGCGCGTGATTTCTACTCTTTTTCCC ACAAAGTGTAGAGTCCAGGAGACTTGAATATGGAAGCCAGTTTGGAGCCTCTCCTCAC TCCTGGCCTTCCTATGGAAGGAGCTTATTCAAGGGAAAGAGCTTATTCAAGCTGCTCTTTCCCCACCCCCAGGTGTGACCCACAGGCCTCCACCTGTCTGGGACAGGCAGGACTGTGAGCTTTGGGAATCAAATGGAAAACACACTTGTCCCACTGCTGCCAGCTGGTTGTCCT AGGGCTAATGTGTCACCTCTCTGTGACTTAGCTCTTCACGTGTAGAATGGGGTGGAAA AGTACTCGGTGTGGCTGTTTTCAGGATGAAGGGGTCTTAGCGGTGCTCGCAGAAGAGC CACGGTGTTGGGAGTGTCTGCTCTCTGGAGGGGAGGCTGCCTCACTAACTGGCCTCTG CCCCAC.

[0020] SEQ ID N°1 siRNA consisting of 19 nucleotides recognises the region of mRNA that starts at the 313th nucleotide, nucleotide positions 313-331, and is indicated by single underlining of the corresponding portions of SEQ ID N°4, SEQ ID N°5 and SEQ ID N°6. siRNA SEQ ID N° 1 forms with this region the Duplex 1 structure by complementary interaction.

[0021] Similarly, SEQ ID N°2 siRNA consisting of 19-nucle-otides recognises the region of mRNA that starts at the 403rd nucleotide, nucleotide positions 403-421, indicated by double underlining of the corresponding portions of SEQ ID N°4, SEQ ID N°5 and SEQ 2D N°6. siRNA SEQ ID N° 2 forms with this region the Duplex 2 structure by complementary interaction.

[0022] In view of the above, the present invention solves the problem of the unavailability of TRAPl-low specific antibodies. In other words, the siRNAs according to the invention are able to transiently silence the expression of TRAPl-low in a specific manner, i. e. without affecting the expression of the canonical isoform of TRAP1. Therefore, the siRNAs according to the invention allow the biological function of TRAPl-low to be studied in a manner independent of the functions of canonical TRAP1.

[0023] Moreover, the two identified siRNAs are characterised by a low production cost, corresponding to the cost of a 21 bp oligonucleotide sequence for each siRNA. These siRNAs are therefore suitable for large-scale manufacturing.

[0024] Based on the results of tests performed to assess specific silencing of TRAPl-low, it was found that this isoform can be constitutively expressed in a chronic myeloidleukaemia cell line (K562), or can be induced under starvation conditions of cells in a glutamine-free culture medium.

[0025] According to another aspect of the invention, the above-mentioned objects are also achieved by the use of a siRNA having a sequence selected from the group comprised of:SEQ ID N°l;SEQ ID N°2;- a sequence including at least 15 contiguous nucleotides differing by no more than 3 nucleotides from SEQ ID N°1 or SEQ ID N°2as a medicament, in particular, in the treatment of:- solid tumours such as colorectal cancers, in particular colorectal adenocarcinoma;- haematological tumours, in particular chronic myeloid leukaemia.

[0026] According to a further aspect of the invention, the above-mentioned purposes are also achieved by the use of siRNAs having a sequence selected between from the group comprised of:SEQ ID N°l;SEQ ID N°2;- a sequence including at least 15 contiguous nucleotides differing by no more than 3 nucleotides from SEQ ID N°1 or SEQ ID N°2in the diagnosis of:- solid tumours such as colorectal cancers, in particular colorectal adenocarcinoma;- haematological tumours, in particular chronic myeloid leukaemia.

[0027] The scope of the present invention also encompasses a pharmaceutical composition including the above-mentioned siRNA, in addition to pharmacologically acceptable excipients, said pharmaceutical composition being suitable, inparticular, for topical, enteral or parenteral administration. Said pharmaceutical composition can have a liquid form, in particular it can have the form of a suspension, an emulsion, a solution, eye drops or oral spray. As an alternative, the pharmaceutical composition can have the form of a cream or a gel. As a further alternative, the pharmaceutical composition can have a solid form of powder, in particular it can have the form of granules, tablets, capsules or pills.

[0028] According to a further aspect of the present invention, the scope of the invention also encompasses the use of the 5 ' UTR SEQ ID No. 4 region of the SEQ ID No. 3 transcript, or- of a portion of the 5 'UTR region selected between:- SEQ ID No. 6 portion of the 5 'UTR region comprising nucleotide positions 1 to 644;- a portion of the region 5 ' UTR including SEQ ID No.6 portion extended by a predetermined number of nucleotide positions beyond nucleotide position 644, along the 5 'UTR regionto identify a siRNA for silencing TRAPl-low.BRIEF DESCRIPTION OF THE INVENTION

[0029] The invention will be illustrated below with a description of some embodiments, by way of example and not of limitation, with reference to the appended drawings, in which: - figure 1 shows an immunoblotting image related to a TRAPl-low silencing assay in HCT116 colorectal adenocarcinoma cell line;- figure 2 refers to a densitometric analysis to qualify the silencing yield in the case of HCT116 cell line; - figure 3 shows to an immunoblotting image related to TRAPl-low silencing assay in K562 chronic myeloid leukaemia cell line;- figure 4 refers to a densitometric analysis to qualifythe silencing yield in the case of K562 cell line.EXAMPLESDetection of TRAPl-low

[0030] As anticipated, the existence of the new TRAP1 isoform named TRAPl-low was deduced from a bioinformatics analysis performed on public datasets of colorectal adenocarcinoma patients.

[0031] More specifically, through a bioinformatics analysis on four open-access databases of curated, non-redun-dant transcription factor binding sites (TFBS), the consensus ChoRE (carbohydrate response element) sequence of the transcription factor known as MondoA was searched for within the human TRAP1 gene sequence. The CIS-BP and SwissRegulon databases identified a DNA-binding motif with 75% homology to the consensus ChoRE sequence of MondoA. The aforementioned ChoRE sequence is located between nucleotide positions from 4034 to 4052 of the human TRAP1 gene sequence of the fourth intron and less than 2000 base pairs (bp) upstream of a hypothetical new transcription start site (TSS) identified also in the interrogated open-access databases. This TSS encodes for a TRAP1 isoform, referred to as TRAPl-low, which lacks the first 5 exons of the canonical TRAP1 isoform. This results into a shorter amino acid sequence, and thus into a shorter protein. Specifically, the mRNA of the canonical form of TRAP1 consists of 18 exons that translate into an amino acid sequence of 704 amino acids, whereas the mRNA predicted by the new TSS transcript consists of 13 exons and translates into a sequence of 495 amino acids.

[0032] The existence of TRAPl-low was then validated using the ISOexpresso platform (http: / / wiki.tgilab. org / ISOex-presso), a web-based tool that collects large-scale messenger (or transcribed) RNAs expression data to study changesin levels of isoforms and / or alterations in a large collection of data of messengers (or transcribed) RNAs sequences from cancer research. This investigation showed that in colorectal cancer, the expression of the primary transcript of TRAP1- low (uc002cvs.3) is three times higher than the expression of the canonical isoform of TRAP1 (uc002cvt.4), compared to healthy tissue.

[0033] In order to validate the data collected in public databases and to verify that the new, lower molecular weight TRAP1 isoform was really expressed, western blotting (WB) and RT-PCR experiments, using primers specific to the transcript encoding for the TRAP1 isoform, were performed simultaneously on various cancer cell lines. RT-PCR data confirmed the presence of the messenger in the cell lines. For WB assays, a primary monoclonal anti-TRAPl nonspecific antibody was used to discriminate between the two isoforms. In all WB assays, a molecular weight band around 45 kDa was always present, even if the expression levels thereof were much lower than those of the canonical TRAP1 isoform. In particular, the expression was remarkably higher when the cells were allowed to grow in a medium lacking glutamine, which is a nutrient capable of regulating MondoA' s transcriptional activity.

[0034] MondoA is a transcription factor that forms a heterodimer with MLX to regulate the expression of several glucose-dependent genes, thereby controlling cellular metabolism. Its transcriptional activity is an adaptive response mechanism of cells to glucose and / or glutamine deficiency. Actually, the presence of glutamine inhibits the transcriptional activity of MondoA, promoting the recruitment of a corepressor to the amino terminus of the transcription factor. This results in blocking the expression of a number of genes downstream of MondoA in favour ofglucose uptake and glycolytic metabolism instead. Considering the bioinf ormatic evidence for the presence of an alternative TSS within the TRAP1 gene with a binding motif for MondoA, an increased expression of TRAPl-low is observed under conditions where no glutamine is present in the culture medium. In K562 cell line, instead, the TRAPl-low isoform is constitutively expressed even under conditions of cell growth in a complete medium, which suggests a central role for TRAPl-low in the pathophysiology of this oncohae-matological disease.

[0035] In order to verify whether the ca. 45 kDa molecular weight band actually corresponded to a new isoform of TRAP1 and not to a non-specific binding of the anti-TRAPl antibody, the sequence of this band was verified by mass spectrometry on protein extracts of cells grown in a glutamine-free growth medium. In these samples, peptides that fall in the C-terminal region of the canonical TRAP1 protein and whose sequence shows high homology to HSP75 were isolated and sequenced. Subsequent ChIP (Chromosome Immunoprecipitation) studies confirmed that the TSS of the novel isoform of TRAP1 is under the control of the transcription factor MondoA.

[0036] The existence of the new TRAP1 isoform predicted by bioinf ormatic analysis was confirmed by mass spectrometry analysis. In this regard, the existence of a 45 kDa low molecular weight protein, as predicted by the bioinformatic analysis, having high sequence homology to the canonical isoform of TRAP1 of molecular weight 75 kDa was demonstrated.

[0037] More specifically, in the intragenic space at the level of the fourth intron of the TRAP1 gene, a DNA-binding motif is present having 75% homology to the consensus ChoRE (carbohydrate response element) sequence of the transcription factor known as MondoA or MLXIP (MLX-Interecting Protein). The aforementioned ChoRE sequence is located in thefourth intron of the TRAP1 gene less than 2000 base pairs (bp) upstream of a putative new transcription start site (TSS).

[0038] The mRNA sequence produced from this TSS is missing the first five exons of the canonical isoform of TRAP1. This results into a shorter amino acid sequence, and thus into a shorter protein, that is defective in the following sequences:- N-terminal signal sequence of 59 amino acids, Mitochondria Targeting Sequence - MTS, which targets TRAP1 to the mitochondrion and which is cut off when the protein is imported into an organelle, in particular into mitochondria, and- sequence that encodes for the ATPase domain required to perform chaperone activity.

[0039] In particular, the mRNA of the canonical form of TRAP1 consists of 18 exons that translate into an amino acid sequence of 704 amino acids, whereas the mRNA predicted by the transcription of the new TSS is 13 exons and translates into a sequence of 495 amino acids.

[0040] TRAPl-low has cytosolic localisation, which is consistent with the absence of the MTS sequence. Its expression is under the control of a glutamine-dependent transcription factor, known as MLXIP, which does not bind the promoter of canonical TRAP1, but binds a promoter within the gene. This result made it possible to hypothesise a biological function of TRAPl-low, independent of the canonical form, which is expressed under conditions of cellular stress such as glutamine deprivation.Biological function of TRAPl-low

[0041] TRAPl-low biological function was also investigated. To this purpose, it was necessary to separate itscontribution to any biological phenomenon from the contribution of the canonical form of TRAP1. Since no specific anti-TRAPl-low antibodies were available, which were also almost impossible to find due to the sequence homology of the two proteins, TRAP1 was transiently silenced, i. e. the protein expression was reduced without interfering at the gene level: instead, the translation of its canonical messenger was blocked by multiple available siRNAs, specific for canonical TRAP-1.

[0042] After testing two well-known siRNAs specific for canonical TRAP1, noting that both also silence TRAPl-low, and are therefore not useful for studying TRAPl-low, the following two 21-nucleotide sequences were identified:SEQ ID N°l:5' CUCUUUCCCUUGAAUAAGC 3' (Duplex_l antisense)SEQ ID N°2:5' UGAUUCCCAAAGCUCACAG 3' (Duplex_2 antisense)

[0043] These sequences were synthesised and tested to silence TRAPl-low:- on K562 cell line, chronic myeloid leukaemia, an onco- haematological line that grows in suspension;- on HCT116 cell line, colorectal adenocarcinoma, a solid tumour line that grows in adhesion.HiPerfect by Qiagen was used as transfecting agent. Silencing optimisation conditions, including transfectant agent and siRNA amounts, were provided in 6-well multi-wells. Once the silencing conditions were optimised, silencing was performed in both 100-mm and 150-mm dishes.

[0044] Successful silencing was assessed by western blot (WB) testing, in which the expression of both the canonical and low molecular weight form was verified. Figs. 1 and 3 are the related immunoblotting images, while the diagramsof Figs. 2 and 4 show the results of corresponding densi-tometric analysis performed in order to quantify the silencing yield. The latter is expressed as a reduction in the expression of the TRAPl-low isoform, in comparison to housekeeping. Each immunoblotting image relates to a single experiment, whereas the densitometric analyses were performed considering four independent experiments representing respective biological replicates.

[0045] A good silencing yield was obtained with both cell lines, thus demonstrating a high specificity for TRAPl-low.

[0046] Actually, a reduction in TRAP1 expression ranging from 30% to 40% was observed with Duplex_l siRNA and from 50% to 60% with Duplex_2 siRNA. Both siRNAs do not significantly interfere with the expression of the canonical isoform of TRAP1 at 75kDa, as observed in the WB images above.

[0047] The maturity level of the proposed technology is at TRL4 level because the inhibitory activity of the siRNAs has already been validated in laboratory tests.

[0048] The foregoing description of exemplary embodiments is capable of showing the invention from a conceptual point of view in such a way that others, using the known technique, will be able to modify and / or adapt the exemplary embodiments in various applications without further research and without departing from the inventive concept, and, therefore, it is understood that such adaptations and modifications will be considered equivalent to the exemplary embodiments described. The means and materials for realising the various functions may be of various kinds without departing from the scope of the invention. It is understood that the expressions or terminology used are purely descriptive and, therefore, not limiting.

[0049] For example, the SIRNA sequences described herein can be made either with unmodified bases, or with modifiedbases, for example with hydroxyl groups blocked in order not to be degraded by RNAsis.

Claims

CLAIMS1. A siRNA for silencing TRAPl-low characterised by having a sequence selected from the group comprised of:SEQ ID N°l: 5' CUCUUUCCCUUGAAUAAGC 3';SEQ ID N°2: 5' UGAUUCCCAAAGCUCACAG 3'; and - a sequence including at least 16 contiguous nucleotides differing by no more than 3 nucleotides from SEQ ID No. 1 or SEQ ID No. 2.

2. The siRNA according to claim 1 for use as a medicament in the treatment of a tumour selected between a solid tumour and a haematological tumour.

3. The siRNA according to claim 2, wherein said solid tumour is a colorectal tumour.

4. The siRNA according to claim 2, wherein said solid tumour is a colorectal adenocarcinoma.

5. The siRNA according to claim 2, wherein said haematological tumour is chronic myeloid leukaemia.

6. The siRNA according to claim 1 for use in diagnosing a tumour selected between a solid tumour and a haematological tumour.

7. The siRNA according to claim 6, wherein said solid tumour is a colorectal cancer.

8. The siRNA according to claim 6, wherein said solid tumour is colorectal adenocarcinoma.

9. The siRNA according to claim 6, wherein said haematological tumour is chronic myeloid leukaemia.

10. A pharmaceutical composition comprising the siRNA according to claim 2 and pharmacologically acceptable excipients.

11. The pharmaceutical composition according to claim 10, suitable for administration topically, enterally or parenterally.

12. The pharmaceutical composition according to claim 10, wherein said composition is in a form selected from the group comprised of:- a liquid form, in particular a form selected from the group comprised of a suspension, an emulsion, a solution, eye drops or oral spray;- a form of a cream or a gel;- a solid form powder, in particular a form selected from the group comprised of granules, tablets, capsules or pills.

13. The use- of 5 'UTR SEQ ID No. 4 region of TRAPl-low transcript SEQ ID No. 3, or- a portion of said 5 ' UTR region selected between:- SEQ ID No. 6 portion of said 5 ' UTR region comprising nucleotide positions 1 through 644; - a portion of said region 5 ' UTR comprising the SEQ ID portion No. 6 extended by a predetermined number of nucleotide positions beyond nucleotide position 644 along said region 5 ' UTR to identify a siRNA for silencing TRAPl-low.

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