Oligonucleotides for modulating gene expression and uses thereof

Abstract

The present invention regards oligonucleotides for modulating the expression of a gene, in particular for modulating a gene responsible for a pathology of genetic, tumoural or viral origin. Moreover, the present invention relates to the use of said oligonucleotides, possibly chemically modified, for the treatment and/or the diagnosis of said diseases.

Claims

1. A method of treating a tumor in a patient in need thereof with a composition comprising at least one single strand anti-gene oligonucleotide complementary to the anti-sense DNA strand of a target gene said target gene being selected from MYCN and MYC, said oligonucleotide comprising 6-30 nucleotides, and comprising a sequence comprising at least three groups of at least two consecutive guanines, said tumor being caused by an overexpression of said target gene, said method comprising administering to said patient in need an effective amount of said composition; inhibiting said target gene expression; and treating said patient.

2. The method according to claim 1, wherein said composition is in combination with a compound with pharmacological action selected from the group consisting of: NGF, somatostatin, retinoic acid, actinomycin D, asparaginase, bleomycin, busulphan capecitabine, carboplatin, cyclophosphamide, cyclosporine, cisplatin, cytarabine, clorambucil, dacarbazine, daunorubicin, docetaxel, doxorubicin hydrochloride, epirubicin hydrochloride, etoposide, fludarabine phosphate, fluorouracil, gemcitabine, idarubicin hydrochloride, hydroxyurea, ifophosphamide, irinotecan hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, procarbazine, raltitrexed, streptozocin, tegafururacil, temozolomide, thioguanine, thiotepe, topotecan, vinblastine, vincristine sulphate, vindesine and vinorelbine.

3. The method according to claim 1, wherein said tumor is an adult or pediatric tumor selected from the group consisting of: neuroblastoma, retinoblastoma, medulloblastoma, ependymoma, pheochromocytoma, embryonal carcinoma, germ cell tumour, alveolar rabdomyosarcoma, embryonal rabdomiosarcoma, Wilms tumor, clear cell sarcoma of the kidney, synovial sarcoma, hepatoblastoma, acute lymphoid leukaemia, chronic lymphoid leukaemia, acute lymphoblastic leukaemia, chronic lymphoblastic leukaemia, Burkitt's lymphoma, acute myeloid leukaemia, chronic myeloid leukaemia, acute megakaryoblastic leukaemia, B chronic lymphoid leukaemia, T-cell leukaemia, lymphomas, small cell lung cancer (microcytoma), lung adenocarcinoma, squamous cell lung carcinoma, typical and atypical primary lung cancer, large cell lung carcinoma, large-cell neuroendocrine lung carcinoma, glioblastoma, hepatocarcinoma, basal cell carcinoma, ovarian tumour, breast tumor and colon cancer.

4. The method according to claim 1, wherein the groups of at least two consecutive guanines are continuous with each other.

5. The method according to claim 1, wherein the groups of at least two consecutive guanines are spaced apart by at least one nucleotide, said nucleotide not being a guanine.

6. The method according to claim 1, wherein the groups of at least two consecutive guanines are at least four, five or six in number.

7. The method according to claim 1, wherein said oligonucleotide is conjugated with a carrier sequence at the 3 and/or 5 end of said oligonucleotide, said carrier being selected from the group consisting of: SEQ ID NO: 47-56.

8. The method according to claim 1, wherein said oligonucleotide is at least one molecule of natural nucleic acid, at least one molecule of natural nucleic acid chemically modified, or at least one molecule of synthetic nucleic acid selected from PNA, LNA or morpholino, at least one molecule of synthetic nucleic acid chemically modified or a combination of said natural nucleic acid and said synthetic nucleic acid.

9. The method according to claim 8, wherein said PNA has a modified backbone wherein the alpha carbon has the side chain of arginine or lysine as a substituent.

10. The method according to claim 1, wherein said oligonucleotide is selected among: SEQ ID NO: 2-15, SEQ ID NO: 24, 25.

11. The method according to claim 10, wherein SEQ ID NO: 2-15, 66-69, 24, 25 are directed against the MYCN gene.

12. The method according to claim 10, wherein said oligonucleotide is conjugated at the 5 and/or 3 end with SEQ ID NO: 47.

13. The method according to claim 1, wherein said combination is SEQ ID: NO 1 and carboplatin, or etoposide or cisplatin or vincristine; or SEQ ID: NO 5 and carboplatin, or etoposide or cisplatin or vincristine.

14. The method according to claim 1, wherein the tumor is selected from the group consisting of: neuroblastoma, Wilms' tumor, retinoblastoma and small cell lung cancer.

15. The method according to claim 1, wherein said oligonucleotide is selected from among: SEQ ID NO:70-74 directed against the gene MYC.

Description

EXAMPLE 1

(1) Chemical Synthesis of the Oligonucleotides.

(2) The chemical synthesis of the oligonucleotides is based on the use of DNA nucleoside phosphoroamidites modified with a protecting group, 4,4-dimethoxytrityl (DMTr) on 5-OH and -cyanoethyl on the 3-phosphate group; protecting groups are also used for the primary amines (nucleobase heterocycles), which are otherwise too reactive.

(3) The chemical synthesis of DNA oligonucleotides takes place in a 3-5 direction. Use is made of a CPG (acronym of controlled pore glass) resin or a polystyrene support, functionalized with the first nucleotide base. The synthesis begins with a step of deprotecting the 5-dimethoxytrityl group using a solution of 3% trichloroacetic acid (TCA) in dichloromethane (DCM). This is followed by activation, using ethylthiotetrazole (ETT) or benzylthiotetrazole (BTT) 0.3M, of the phosphoroamidite corresponding to the second base to be inserted in sequence, which will then be coupled with the previously deprotected 5OH, thereby forming a phosphodiester bond.

(4) The next step is capping, which serves to acetylate the 5OH groups that have not reacted. Capping is carried out using 2 solutions, one containing tetrahydrofuran (THF)/lutidine/acetic anhydride (8:1:1) and the other containing a 10% solution of methylimidazole in THF. The unstable trivalent bond of the phosphite triesters is stabilized by iodine in a THF/pyridine solution which oxidizes them to pentavalent phosphodiesters.

(5) After oxidization, the cycle is repeated, starting with detritylation of the second unit introduced and so forth. This cycle is repeated for the number of times necessary, depending on how many bases it is desired to insert in sequence. Finally, the final 5-DMTr group is removed by means of a treatment with an acid at room temperature.

(6) Depending on the protecting groups present on the bases (which in turn depends on the chemistry of the bases selected, PTO, 2OMe, etc.) it can be left at 55 C. for 16 hours with ammonium hydroxide or at 55 C. for 35 minutes with an ammonium hydroxide/methylamine (AMA) solution in order to deprotect the phosphors by R-elimination of the cyanoethyl groups and remove the protecting groups on the nucleobase heterocycles.

(7) Alternatively, the 5-DMTr group can be maintained throughout the phase of analysis (HPLC, MS) and preparative chromatography in order to better purify the final product from the by-products and finally removed by means of a treatment with acetic acid

(8) The chemical synthesis of RNA oligonucleotides differs from that of DNA oligonucleotides because of the 2OH group present on the ribose and thus the presence of an additional protecting group for each phosphoroamidite.

(9) Consequently, the synthesis of RNA oligonucleotides requires a longer coupling time and further steps to deprotect that group.

(10) The same protocol as described above is used for the synthesis of oligonucleotides with chemically modified monomers, such as phosphorothioates (PTO), 2O-Methyl (2OMe), 2Fluoro (2-F), arabinoside nucleic acid (ANA), and locked nucleic acid (LNA).

(11) The details of the specific techniques for each modified base are provided by the company the monomer molecules are purchased from (Link Technologies Ltd.).

(12) The morpholinos were purchased from the manufacturer (Gene Tools, LLC).

(13) The synthesis of PNA oligonucleotides was carried out on a 10 micromole scale, and included a purification and characterization step.

(14) The synthesis of the molecule was carried out in the solid phase, using a Rink Amide-Chemmatrix resin and a Syro automatic synthesizer (MultiSynTech). The first monomer of the synthesis is manually bound to the resin. Each automatic synthesis cycle is divided into three steps. The first step is deprotection, carried out using a solution of 20% piperidine in DMF (dimethylformammide).

(15) The second step is the coupling reaction between the entering monomer and the growing chain. This reaction is carried out by adding 5 0.22M equivalents (eq) of monomer (FMOC-PNA-G(Bhoc)-OH, FMOC-PNA-A(Bhoc)-OH, FMOC-PNA-C(Bhoc)-OH, FMOC-PNA-T-OH) in NMP (N-methylpirrolidone) and 4.5 0.32M eq of an activator in DMF (in this case HATU) in an alkaline environment with an 8% solution of 2,6-lutidine and DIPEA (N,N-diisopropylethylamine) in DMF. The coupling reaction is repeated, in duplicate, at the point of attachment of the first and second monomer on the preloaded resin, in the passage from the peptide chain to the PNA one, and on the last monomer.

(16) The third step is the capping reaction, which serves to block, by acetylation, the sites that have not reacted during the coupling step. The reaction is achieved using a solution containing 6% 2,6-lutidine and 5% acetic anhydride in DMF. Upon completion of the synthesis, the molecules are removed from the solid support.

(17) This reaction is obtained with a solution of TFA (trifluoroacetic acid) and meta-cresol in 4:1 ratios.

(18) The molecule thus obtained is collected by precipitation in diethyl ether.

(19) Once recovered in water, it is purified in HPLC. The column used for purification is a C18 300A 5u Jupiter ( Phenomenex, Inc.). Purification is carried out using a linear gradient from 100% A (water 95%; acetronitrile 5%; 0.1% TFA)-0% B (water 60%; acetonitrile 40%; 0.1% TFA) to 60% A-40% B in 30 minutes. The complete gradient used is 0-5 minutes 0% B; 5-35 minutes 40% B; 35-37 100% B; 37-42 100% B; 42-44 0% B.

(20) Finally, the purified product is analyzed by ESI mass spectroscopy (@ Waters).

(21) Antigene Oligonucleotides for Blocking Gene Transcription.

(22) For the purpose demonstrating that the oligonucleotides of the invention, selected and designed according to the parameters described in the present invention, work to selectively block the transcription of a gene, PNA-based oligonucleotides directed against the MYCN gene were designed and synthesized; they are shown in Table 1.

(23) TABLE-US-00001 mRNA Cell Phoenix NIH-3T3 SEQID % inhibi- prolif. prolif. prolif. NO PNASEQUENCE GC tion(%) (%) (%) (%) SEQID ATGCCGGGCATGATCT 56.3 42 70 100 100 NO:1 SEQID GGGTGGATGCGGGGGG 81.3 75 32 100 100 NO:2 SEQI GATGCGGGGGGCTCCT 75 68 41 100 100 NO:3 SEQID GTCGGCGGGAGGTAAG 68.8 65 48 100 100 NO:4 SEQID GCTGGGTGGATGCGGG 75 62 53 100 100 NO:5 SEQID TGGACGCGCTGGGTGG 75 60 54 100 100 NO:6 SEQID CGCGCTGGGTGGATGC 75 58 57 100 100 NO:7 SEQID GTCTGGACGCGCTGGG 75 54 59 100 100 NO:8 SEQID CCCTGCAGTCGGCGGG 81.3 51 64 100 100 NO:9 SEQID CGGCCGCGGGCCGCCA 93.8 51 65 100 100 NO:10 SEQID GGGAACTGTGTTGGAG 56.3 48 68 100 100 NO:11 SEQID TGTCTGGACGCGCTGG 68.8 47 69 100 100 NO:12 SEQID ACGCTCAGGGACCACG 68.8 48 66 100 100 NO:13 SEQID CCCGGACGAAGATGAC 62.5 40 70 100 100 NO:14 SEQID ACTGTGTTGGAGCCGA 56.3 37 77 100 100 NO:15 SEQID CCTGTCGTAGACAGCT 56.3 14 90 100 100 NO:16 SEQID TGTGACAGTCATCTGT 56.3 10 98 100 100 NO:17 SEQID GTGACAGTCATCTGTC 50 10 98 100 100 NO:18 SEQID GACAGTCATCTGTCTG 50 5 100 100 100 NO:19 SEQID CGTCGATTTCTTCCTC 50 5 100 100 100 NO:20 SEQID CTCGAGTTTGACTCGC 56.3 1 100 100 100 NO:21 SEQID GCGCCTCCCCTGATTT 62.5 2 100 100 100 NO:22 SEQID ATATCCCCCGAGCTTC 56.3 2 100 100 100 NO:23

(24) The PNA oligonucleotides were tested both individually and conjugated with a carrier at the 3 and/or 5 end with the aim of favouring cell membrane permeation. In particular, the oligonucleotides were conjugated to the carboxyl terminus at 3 with the amino acid sequence SEQ ID NO: 43, i.e. proline-lysine-lysine-lysine-arginine-lysine-valine.

(25) The oligonucleotide having SEQ ID NO: 1 is the sequence that was the subject of patent EP 1618195 and represents the control sequence and sequence used for comparison.

(26) The oligonucleotides having SEQ ID NO: 2-15 contain a group of two consecutive guanines (SEQ ID NO: 14, 15), or a group of three consecutive guanines (SEQ ID NO: 13), or two groups of two consecutive guanines (SEQ ID NO: 12), or a group of two guanines and a group of three consecutive guanines (SEQ ID NO: 11, 10, 9, 8 and 7), or two groups of two consecutive guanines and a group of three consecutive guanines (SEQ ID NO: 6 and 4), or a group of two consecutive guanines and two groups of three consecutive guanines (SEQ ID NO: 5), or a group of six consecutive guanines (SEQ ID NO: 3), or a group of six consecutive guanines, a group of three consecutive guanines and a group of two consecutive guanines (SEQ ID NO: 2).

(27) The groups of consecutive guanines are shown underlined in Table 1.

(28) The oligonucleotides having SEQ ID NO: 16-23 do not have groups of consecutive guanines, being negative controls.

(29) Moreover, for the purpose of selectively modulating the transcription of a gene, oligonucleotides having SEQ ID NO: 24-25 were also designed and synthesized; these are shown in Table 2.

(30) TABLE-US-00002 TABLE2 SEQIDNO:24 DNA-LNA25-1 ATGCCGGGCATGATC T SEQIDNO:25 DNA-LNA25-2 ATGCCGGGCATGATC T

(31) In particular, single-stranded chimeric oligonucleotides, comprising DNA monomers and LNA monomers (SEQ ID NO: 24 and 25) were designed and synthesized.

(32) The DNA bases in the oligonucleotide sequence are shown as bases in boldface type, whereas the LNA monomers are underlined. Each chimeric oligonucleotide molecule was designed and synthesized by inserting the LNA monomers spaced apart by 1, 2 or 3 DNA bases in order to avoid rapid degradation by the endogenous nucleases, as has been reported in the literature (Koch T, Biochem J 2001, 354 (Pt 3):481-4; Koji Nagahama, Bioorg Med Chem Lett, 2009, 19(10):2707-9).

(33) Antisense Oligonucleotides for Blocking Gene Translation.

(34) For the purpose demonstrating that the oligonucleotides of the invention, selected and designed according to the parameters described in the present invention, work to selectively block the translation of the target gene, antisense oligonucleotides having SEQ ID NO: 26-36, directed against the MYCN gene, were designed, synthesized and experimentally analyzed in vitro. They are shown in Table 3.

(35) TABLE-US-00003 TABLE3 SEQID SENSE ANTISENSE NO SEQUENCE SEQUENCE SEQID siMYCN UGAAGAUGAUGAAGAGGAA SEQID UUCCUCUUCAUCAUC NO:26 (795) NO:57 UUCA SEQID siMYCN GAUGAUGAAGAGGAAGAUG SEQID CAUCUUCCUCUUCAU NO:27 (799) NO:58 CAUC SEQID siMYCN UGAUGAAGAGGAAGAUGAA SEQID UUCAUCUUCCUCUUC NO:28 (801) NO:59 AUCA SEQID siMYCN GAGGAAGAUGAAGAGGAAG SEQID CUUCCUCUUCAUCUU NO:29 (808) NO:60 CCUC SEQID siMYCN GGAAGAUGAAGAGGAAGAA SEQID UUCUUCCUCUUCAUC NO:30 (810) NO:61 UUCC SEQID MYCN- CGTGGAGCAGCTCGG NO:31 PTO CAT (1)as SEQID MYCN- CAGGGTGTCCTCTCC NO:32 PTO GGA (763) as SEQID siRNA- GAGGAAGAUGAAGAGGAAG SEQID CUUCCUCUUCAUCUU NO:33 2'- TT NO:62 CCUCTT OMe- RNA 808 SEQID siRNA- CAGGAAGAUGAAGAGGAAG SEQID GUUCCUCUUCAUCUU NO:34 2'F- UU NO:63 CCUCUU RNA 808 SEQID siRNA- GAGGAAGAUGAAGAGGAAG SEQID CUUCCUCUUCAUCUU NO:35 LNA TT NO:64 CCUCTT 808 SEQID siRNA- CAGGAAGAUGAAGAGGAAG SEQID GUUCCUCUUCAUCUU NO:36 ANA AA NO:65 CCUCAA 808

(36) Complementary double-stranded oligonucleotides based on RNA (small interfering RNA (siRNA) (SEQ ID NO: 26-30) were produced to selectively modulate the translation of the target gene, MYCN.

(37) Moreover, oligonucleotides based on DNA SEQ ID NO: 31 and 32, in which the phosphodiester bond was modified into phosphorothioate, were produced.

(38) Double-stranded chimeric oligonucleotides: based on RNA monomers and monomers 2O-Methyl (SEQ ID NO: 33); based on RNA monomers and 2-Fluoro monomers (SEQ ID NO: 34); based on RNA monomers and LNA monomers (SEQ ID NO: 35) and based on RNA monomers and arabinoside RNA monomers (SEQ ID NO: 36), were also produced. In the oligonucleotide sequences SEQ ID NO: 33-36, the 2O-Methyl, 2-Fluoro, LNA and arabinoside (ANA) monomers are shown in boldface, whereas each group of at least two consecutive guanines is underlined.

(39) As the siRNA of the oligonucleotides is double-stranded, the chimera was designed and synthesized in such a manner as to leave two 2O-Me monomers, or two 2-Fluoro monomers, or two LNA monomers, or two RNA arabinoside monomers, both in 3 and in 5, unpaired to the complementary strand and thereby avoid rapid degradation by the cell enzymes.

(40) Treatments with OligonucleotidesQT-PCR

(41) For the purpose of assessing the ability of the oligonucleotides of the present invention to modulate the expression of the target genes, their ability to reduce the quantity of messenger RNA was analyzed using the Real-Time PCR technique.

(42) For this purpose, use was made of 24-well plates, which were seeded with 5.0104 cells with 0.3 ml of OPTI-MEM (GIBCO BRL) medium, 4% FBS and 2 mM L-glutammine (experiments in triplicate) per well.

(43) The cells were incubated for 24 hours at 37 C., in an atmosphere containing 5% CO.sub.2 to permit adherence to the base of the wells.

(44) Each oligonucleotide analyzed, except for the PNA oligonucleotides, was incubated beforehand with 2 l of Lipofectamine 2000 (Invitrogen), with 0.3 mL of serum-free OPTI-MEM (GIBCO BRL) medium.

(45) For each well, the oligonucleotides were analyzed at the following final concentrations: the antisense oligonucleotides siRNA and siRNA gapmer (i.e. the oligonucleotides containing one or more monomers of chemically modified nucleic acids at the 3 or 5 end, while in the central portion they have monomers of nucleic acids that have not been modified or have been modified at the level of the phosphodiester bond as a phosphorothioate bond) were used at 200 nM; the antisense oligonucleotides containing phosphorothioate DNA monomers, the RNA antigene oligonucleotides (agRNA) and the oligonucleotides containing DNA monomers and LNA monomers were used at 10 M; the morpholino oligonucleotides were used at a concentration of 1 M, and the PNA oligonucleotides were administered at a concentration of 1 M.

(46) The cells were treated with the oligonucleotides, adding FBS up to 4% 6 hours after administration of the same. After 24 hours the total RNA was extracted from each well and purified using the RNeasy Mini Kit (QIAGEN).

(47) Assays were performed on 8 cell lines obtained from 5 different human tumours which are correlated with MYCN expression, i.e.: as a neuroblastoma model use was made of the cell lines Kelly, IMR-32 (where the MYCN gene is amplified and superexpressed) and SKNBE2c and LAN1 (where the MYCN gene is amplified and superexpressed and the p53 gene is mutated); as a rabdomiosarcoma model use was made of the cell line RH30, where the MYCN gene is amplified and superexpressed; as a Wilms' tumour model use was made of the cell line WiT49, where the MYCN gene is amplified and superexpressed; as a retinoblastoma model use was made of the cell line Y79, where the MYCN gene is amplified and superexpressed; and as a model of small cell lung cancer use was made of the cell line H69 where the MYCN gene is amplified and superexpressed.

(48) As a control, use was made of the same cell lines as listed above treated with sterile water instead of oligonucleotides. Each RNA sample was quantified (in duplicate) using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). The first strand of cDNA was produced using the cDNA Synthesis Kit for RT-PCR (Roche). For the cDNA synthesis reaction a total of 1 g of RNA was used. For the real-time PCR, 10 ng of cDNA in a final volume of 20 l was used with SYBR Green Master Mix 2 (Applied Biosystems) (3 identical experiments were conducted in triplicate). The sequences and the concentrations of the primer used to carry out the Real-Time PCRs are shown in Table 4. Two housekeeping genes were used as positive controls: GAPDH and beta-actin (ACTB).

(49) The conditions of the QT-PCR reaction were: 10 min at 95 C., 20 sec at 95 C., and 30 sec at 60 C., for 50 cycles.

(50) TABLE-US-00004 TABLE4 Concen- SEQ Primer Sequence tration IDNO MYCN CGACCACAAGGCCCTCAGT 300nM SEQIDNO: sense 37 MYCN TGACCACGTCGATTTCTTCCT 300nM SEQIDNO: anti- 38 sense ACTB GAGCACAGAGCCTCGCCTTTG 300nM SEQIDNO: sense 39 ACTB ACCATCACGCCCTGGTGCCTG 300nM SEQIDNO: anti- 40 sense GAPDH CCAATATGATTCCACCCATGGC 300nM SEQIDNO: sense 41 GAPDH CTTGATTTTGGAGGGATCTCGC 300nM SEQIDNO: anti- 42 sense
Treatments with OligonucleotidesCellular Proliferation Assay.

(51) For the purpose of assessing the gene modulation ability of the oligonucleotides of the present invention, the effect of their administration on cell viability was determined.

(52) For this purpose 510.sup.3 cells per well were seeded into 96-well cell culture plates (experiments conducted in triplicate) with 100 l of OPTI-MEM (GIBCO BRL) medium containing 4% FBS and 2 mM of L-glutammine.

(53) The PNA oligonucleotides were administered at different concentrations (1 M-2.5 M-5 M-10 M) in order to observe a dose-effect relationship.

(54) As regards all the other oligonucleotides, the concentrations at which they administered are listed in the paragraph:

(55) Treatments with OligonucleotidesQT-PCR.

(56) The viability of the treated cells was determined at 48, 72, 96 and 168 hours after treatment.

(57) Cell viability was assessed by means of an ATP-Lite assay (Luminescence ATP Detection Assay System, PerkinElmer) and is reported as the ratio between the average signal of the treated wells compared to the average value of the wells of untreated control cells. The cells were processed following the instructions provided with the kit.

(58) The assays were performed on the same cell lines used to determine the levels of the MYCN gene messenger and listed in the paragraph: Treatments with OligonucleotidesQT-PCR.

(59) Results

(60) As far as the PNA oligonucleotides are concerned, the results regarding their ability to inhibit the transcription of the MYCN gene and proliferation of Kelly cells are shown in Table 1. Table 6 shows the values (in percentage form) of the proliferating Kelly cells at the different concentrations of PNA analyzed.

(61) TABLE-US-00005 TABLE5 Cell Cell Cell Cell Prolif Prolif Prolif. Prolif. SEQID (%)1M (%)2,5M (%)5M (%)10M NO Sequence 72h 72h 72h 72h SEQID ATGCCGGGCATGAT 89 66 50 24 NO:1 CT SEQID GGGTGGATGCGGGG 74 32 12 2 NO:2 GG SEQID GATGCGGGGGGCTC 78 41 19 3 NO:3 CT SEQID GTCGGCGGGAGGTA 78 48 21 3 NO:4 AG SEQID GCTGGGTGGATGCG 79 53 27 5 NO:5 GG SEQID TGGACGCGCTGGGT 82 59 35 13 NO:6 GG SEQID CGCGCTGGGTGGAT 80 54 31 8 NO:7 GC SEQID GTCTGGACGCGCTG 80 57 32 11 NO:8 GG SEQID CCCTGCAGTCGGCG 86 64 42 20 NO:9 GG SEQID TGTCTGGACGCGCT 84 60 40 16 NO:12 GG

(62) The results demonstrate that the inhibition effect of these PNAs on the translated protein of the target genes is highly selective and specific. Moreover, it was observed that the arrest of growth of the tumour cells used as a model (characterized by amplification of the MYCN gene) was directly followed by apoptosis following the administration of the antigene PNAs.

(63) In general, the PNA antigene oligonucleotides having SEQ ID: 2-SEQ ID NO: 13 (containing one or more groups of Gs) have greater antigene effectiveness (i.e. inhibition of MYCN mRNA and of the proliferation of tumour cells with MYCN expression) compared to PNA oligonucleotides devoid of groups of Gs (SEQ ID NO: 16-SEQ ID NO: 23).

(64) In particular, the results shown in Table 1 and in Table 5 demonstrate that the sequences SEQ ID NO: 2-SEQ ID NO: 13 have greater antigene effectiveness than the sequence SEQ ID NO: 1, the subject of patent EP 1618195.

(65) Sequences SEQ ID NO: 7-SEQ ID NO: 12 (containing two groups of two or three consecutive Gs) show greater antigene effectiveness than the sequences SEQ ID NO: 1, and SEQ ID NO: 13-SEQ ID NO: 15 (containing only one group of two or three consecutive Gs).

(66) Sequences SEQ ID NO: 4-SEQ ID NO: 6 (containing three groups of two or three consecutive Gs) show greater antigene effectiveness than the sequences SEQ ID NO: 7-SEQ ID NO: 12 (containing two groups of two or three consecutive Gs).

(67) SEQ ID NO: 3, containing a group of six consecutive Gs, shows greater antigene effectiveness than the sequences SEQ ID NO: 1 and sequences SEQ ID NO: 4-SEQ ID NO: 12 containing one or two or three groups of Gs (in which each group consists of at most two or three consecutive Gs).

(68) Sequence SEQ ID NO: 2, containing three groups of consecutive Gs, which, in addition to two groups of two and three consecutive Gs, also comprises a group of six consecutive Gs, shows greater antigene effectiveness than both SEQ ID NO: 3, containing only one group of six Gs, and sequences SEQ ID NO: 1 and sequences SEQ ID NO: 4-SEQ ID NO: 12 containing one or two or three groups of Gs (in which each group consists of at most two or three consecutive Gs).

(69) Moreover, the oligonucleotides having SEQ ID NO 1-23 were administered in lines of fibroblastoid-type cells (NIH-3T3 and Phoenix) and the results are shown in Table 1 (last two columns).

(70) The results clearly demonstrate that the oligonucleotides analyzed are not specifically effective against and are not toxic for these cells (i.e. cells that do not express the target gene, which in this case is MYCN).

(71) In fact, no changes were observed in cellular proliferation in these two lines of non-tumoural fibroblasts, and this result means that the PNA oligonucleotides act with a specific effect of inhibiting the expression of MYCN, whereas they do not have a non-specific, toxic effect in cells that do not express MYCN.

(72) Therefore, it may be deduced that the PNA oligonucleotides designed on the basis of the parameters described in the present invention act specifically and effectively on the target gene and therefore on the cells which express/overexpress this gene.

(73) The PNAs having SEQ ID NO: 1-12 were also tested on different cell lines which overexpress MYCN (Kelly, SKNBE2c, RH30, WiT49, WERI-Rb1 1 and H69). The results are shown in Table 6 and confirm the selectivity and specificity of the inhibition effect of the PNAs on the product of protein translation of the MYCN gene.

(74) TABLE-US-00006 TABLE 6 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 NO: 6 NO: 7 NO: 8 NO: 9 NO: 10 NO: 11 NO: 12 Kelly 48 75 68 65 62 60 58 54 51 51 50 50 % mRNA Kelly % cell 66 32 41 48 53 54 57 59 60 64 64 65 prol. SKNBE2c % mRNA 41 75 68 60 59 62 60 62 58 41 40 40 SKNBE2c % cell 72 52 53 59 64 57 58 56 64 70 71 71 prol. RH30 39 74 65 57 56 56 55 58 50 29 31 32 % mRNA RH30 76 56 59 62 64 64 63 60 71 80 78 77 % cell prol. WiT49 62 78 75 73 69 68 70 72 66 60 60 62 % mRNA WiT49 74 54 57 60 63 65 60 62 68 70 72 73 % cell prol. WERI-Rb1 31 46 43 41 38 37 38 40 36 30 30 30 % mRNA WERI-Rb1 82 79 78 79 80 80 79 78 79 85 84 84 % cell prol. H69 40 58 55 55 54 55 55 56 50 41 41 40 % mRNA H69 77 61 63 66 68 69 67 66 70 79 78 78 % cell prol.

(75) The antigene PNA oligonucleotide having SEQ ID NO: 5 was analyzed in a more detailed manner.

(76) In particular, modified oligonucleotides, in which the groups of consecutive guanines present in the sequence were modified (the groups of consecutive guanines are underlined, whereas the modified nucleotides are in boldface type) so as to interrupt the consecutiveness of the guanines, were synthesized and analyzed in vitro on Kelly cells (which overexpress MYCN).

(77) The results (summarized in Table 7) clearly demonstrate that the fact of the guanines being consecutive is of fundamental importance for the purposes of the activity of modulating the gene's expression. In fact, the PNAs having SEQ ID NO: 43, in which all the groups of consecutives guanine of the PNAs having SEQ ID NO: 5 were mutated, causes the inhibitory activity of the PNAs to be lost, whereas the mutation of one or two of the three groups of consecutive guanines considerably compromises, but does not completely suppress, the inhibitory activity of the molecule.

(78) TABLE-US-00007 TABLE7 mRNA Inhibition (Kelly)Cell SEQIDNO SEQUENCE (%)(Kelly) Prolif.% SEQIDNO:5 GCTGGGTGGATGCGGG 62 53 SEQIDNO:43 GCTGAGTCGATGCGTG 0 100 SEQIDNO:44 GCTGAGTGGATGCGTG 25 89 SEQIDNO:45 GCTGGGTCGATGCGGG 47 69 SEQIDNO:46 GTTGGGTGGATGTGGG 58 60

(79) Chimeric oligonucleotides comprising DNA monomers and LNA monomers and having the MYCN gene as their target were tested in vitro on alveolar rabdomyosarcoma cells (RH30). The results are summarized in Table 8 and demonstrate these oligonucleotides have an intense, specific antigene activity.

(80) TABLE-US-00008 TABLE8 mRNA Inhibi- Cell SEQID tion Prolif. NO NAME SEQUENCE (%) (%) SEQID DNA-LNA ATGCCGGGCATGA 23 75 NO:24 25-1 TCT SEQID DNA-LNA ATGCCGGGCATGA 24 78 NO:25 25-2 TCT

(81) The antisense oligonucleotides designed and synthesized by the Applicant according to the parameters described in the present invention were analyzed in vitro on rabdomyosarcoma cells (RH30). The results are summarized in the Table 9 and show that the oligonucleotides are capable of inhibiting the MYCN transcript in a specific and effective manner; moreover, they are also capable of selectively inhibiting tumour cell proliferation in a more effective manner than the standard antisense oligonucleotides presently available for the MYCN gene (Chung D H, Bioch Bioph Res Commun, 2006, 351(1):192-7.

(82) In particular, the siRNA identified by the Applicant and directed against MYCN mRNA, described in Table 11, exert an antisense activity with an inhibition of MYCN mRNA ranging from a minimum of 70% to a maximum of 85%.

(83) TABLE-US-00009 TABLE9 SEQ mRNA Cell ID SEQUENCE SEQUENCE Inhibition Prolif. NO SENSE ANTISENSE (%) (%) SEQID siMYCN UGAAGAUGAU UUCCUCUUCA 82 28 NO:26 (795) GAAGAGGAA UCAUCUUCA SEQID siMYCN GAUGAUGAAG CAUCUUCCUC 70 43 NO:27 (799) AGGAAGAUG UUCAUCAUC SEQID siMYCN UGAUGAAGAG UUCAUCUUCC 78 35 NO:28 (801) GAAGAUGAA UCUUCAUCA SEQID siMYCN GAGGAAGAUG CUUCCUCUUC 85 28 NO:29 (808) AAGAGGAAG AUCUUCCUC SEQID siMYCN GGAAGAUGAA UUCUUCCUCU 81 30 NO:30 (810) GAGGAAGAA UCAUCUUCC SEQID MYCN- CGTGGAGCAG 34 73 NO:31 PTO CTCGGCAT (1)as SEQID MYCN- CAGGGTGTCC 45 65 NO:32 PTO TCTCCGGA (763) as SEQID siRNA- GAGGAAGAUG CUUCCUCUUC 13 85 NO:33 2- AAGAGGAAGT AUCUUCCUCT OMe- T T RNA 808 SEQID siRNA- CAGGAAGAUG GUUCCUCUUC 59 52 NO:34 2F- AAGAGGAAGU AUCUUCCUCU RNA U U 808 SEQID siRNA- GAGGAAGAUG CUUCCUCUUC 34 68 NO:35 LNA AAGAGGAAGT AUCUUCCUCT 808 T T SEQID siRNA- CAGGAAGAUG GUUCCUCUUC 69 35 NO:36 ANA AAGAGGAAGA AUCUUCCUCA 808 A A

(84) For the purpose of verifying whether the oligonucleotides of the present invention are capable of lowering the concentrations of the chemotherapeutic agents presently used in the therapeutic protocols against cancer, the same were administered in concomitance with chemotherapy drugs.

(85) Studies were conducted on the effect of associations between PNA and chemotherapy drugs (carboplatin, etoposide (VP16), cisplatin and vincristine) on different human and mouse neuroblastoma tumour cell lines (SMS-KAN, LAN 1, IMR-32, SMS-KCN, Kelly, NHO2A, SKNBE2c).

(86) The therapeutic schedule used in the treatments carried out provided for the cells to be treated with PNA and then afterwards, at a pre-established time (it could be 6 or 12 hours) the chemotherapeutic agent was administered.

(87) The results demonstrate that the association of these compounds with the oligonucleotides of the invention determines, at specific concentration ranges, a greater therapeutic effect than individual treatments with the same compounds.

(88) In particular, it may be observed that the treatmentbe it concomitant and simultaneous or at different time intervals (3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, etc.)with a combination consisting of the oligonucleotides of the invention and other compounds with a pharmacological effect serves to enhance the desired therapeutic effect. Therefore, combining one or more of the oligonucleotides identified by the Applicant with chemotherapy drugs makes it possible to reduce the chemotherapy drug concentration by as much as 10 times compared to the present standard therapeutic concentration while still obtaining the same effect.

(89) In particular, the results of this study demonstrate that SEQ ID NO: 1 has a synergic effect, in terms of inhibiting cell proliferation, when it is administered in combination with vincristine, or etoposide, or carboplatin, or cisplatin, compared to the effects obtained in treatments with the oligonucleotide or with the aforesaid chemotherapeutic agents on their own.

EXAMPLE 2

(90) For the purpose of supporting the present invention, the oligonucleotides summarized in Table 10 were also synthesized. In particular, Table 10 shows the oligonucleotide sequences, the SEQ ID NO, the percentage value of the (G+C) content and the gene the synthesized oligonucleotides are directed against. The synthesis of the oligonucleotides was achieved as in example 1.

(91) TABLE-US-00010 TABLE10 SEQIDNO %GC MYCN SEQIDNO:66 TCGGGAGCAGTGGGCA 68.8 SEQIDNO:67 GCGGGTCGCGGGCACG 87.5 SEQIDNO:68 TGGAGGTCGGCGCCGG 81.3 SEQIDNO:69 TCGGCGGGAGGTAAGG 68.8 MYC SEQIDNO:70 CTCAGAGGCTTGGCGG 68.8 SEQIDNO:71 GCGGCCGGCTAGGGTG 81.3 SEQIDNO:72 CGGCCGGCTAGGGTGG 81.3 SEQIDNO:73 CGACGGCGGTGGCGGG 87.5 SEQIDNO:74 GGACGGGGGCGGTGGA 81.3 BIRC5 SEQIDNO:75 GCGGCGGCATGGGTGC 81.3 SEQIDNO:76 GGCGGCGGCATGGGTG 81.3 ALK SEQIDNO:77 GCAGGAGAGGACGGTA 62.5 SEQIDNO:78 CAGGAGAGGACGGTAC 62.5 SEQIDNO:79 GGCAGGAGAGGACGGT 68.8 BCL2 SEQIDNO:80 GGATGGCGCACGCTGG 75.0 SEQIDNO:81 GGGAAGGATGGCGCAC 68.8 SEQIDNO:82 CCACGGTGGTGGAGGA 68.8 PLK4 SEQIDNO:83 ACGGCAAGCGGCGGGA 75.0 SEQIDNO:84 GGACGGCAAGCGGCGG 81.3

(92) The following, in particular, were synthesized: SEQ ID NO: 66-69 directed against MYCN, SEQ ID NO: 70-74 directed against MYC, SEQ ID NO: 75, 76 directed against BIRC5, SEQ ID NO: 77-79 directed against ALK, SEQ ID NO: 80-82 directed against BCL2 and SEQ ID NO: 83, 84 directed against PLK4.

(93) The oligonucleotides were tested in vitro (at a concentration of 2.5 M) to determine their ability to inhibit the transcription of the gene they are directed against by measuring the levels of the gene messenger in cellular models in which the gene of interest is overexpressed. The methods used are the ones described in example 1.

(94) In particular, SEQ ID NO: 66-69 (directed against MYCN) were tested in vitro in the Kelly and H69 cell lines; SEQ ID NO: 70-74 (directed against MYC) were tested in vitro in the H82 and RD cell lines, SEQ ID NO: 75, 76 (directed against BIRC5) were tested in vitro in the Kelly cell line, SEQ ID NO: 77-79 (directed against ALK) were tested in vitro in the Kelly cell line, SEQ ID NO: 80-82 (directed against BCL2) were tested in vitro in the Kelly cell line and SEQ ID NO: 83, 84 (directed against PLK4) were tested in vitro in the Kelly cell line.

(95) The ability of the tested oligonucleotides to inhibit the messenger of the gene of interest was measured and the proliferative capacity of the cells following administration of the oligonucleotides was also assessed. The results are summarized in Table 11.

(96) The data show a potent inhibitory activity against the mRNA of the target gene and an inhibition of cell proliferation which rises with increases in the number of groups of Gs present in the sequence of the oligonucleotide.

(97) TABLE-US-00011 TABLE11 mRNA Cell inhibit. Prolif. SEQIDNO (%) (%) MYCN SEQID TCGGGAGCACTGGGC 57 60 Kelly NO:66 A SEQID GCGGGTCGCGGGCAC 60 56 NO:67 G SEQID TGGAGGTCGGCCCCG 74 35 NO:68 G SEQID TCGGCGGGAGGTAAG 76 30 NO:69 G MYCN SEQID TCGGGAGCAGTGGGC 41 73 H69 NO:66 A SEQID GCGGGTCGCGGGCAC 42 71 NO:67 G SEQID TGGAGGTCGGCGCCG 62 49 NO:68 G SEQID TCGGCGGGAGGTAAG 67 45 NO:69 G MYC SEQID CTCAGAGGCTTGGCG 43 79 H82 NO:70 G SEQID GCGGCCGGCTAGGGT 46 75 NO:71 G SEQID CGGCCGGCTAGGGTG 52 64 NO:72 G SEOID CGACGGCGGTGGCGG 56 65 NO:73 G SEQID GGACGGGGGCGGTGG 61 61 NO:74 A MYC SEQID CTCAGAGGCTTGGCG 34 73 RD NO:70 G SEQID GCGGCCGGCTAGGGT 42 65 NO:71 G SEQID CGGCCGGCTAGGGTG 58 53 NO:72 G SEQID CGACGGCGGTGGCGG 59 51 NO:73 G SEQID GGACGGGGGCGGTGG 64 47 NO:74 A BIRC5 SEQID GCGGCGGCATGGGTG 69 43 Kelly NO:75 C SEQID GGCGGCGGCATGGGT 78 35 NO:76 G ALK SEQID GCAGGAGAGGACGGT 57 55 Kelly NO:77 A SEQID CAGGAGAGGACGGTA 68 46 NO:78 C SEQID GGCAGGAGAGGACGG 79 39 NO:79 T BCL2 SEQID GGATGGCGCACGCTG 59 62 Kelly NO:80 G SEQID GGGAAGGATGGCGCA 62 58 NO:81 C SEQID CCACGGTGGTGGAGG 68 55 NO:82 A PLKA4 SEQID ACGGCAAGCGGCGGG 62 59 Kelly NO:83 A SEQID GGACGGCAAGCGGCG 74 49 NO:84 G

(98) Finally, the oligonucleotides having SEQ ID NO 66-84 were administered in cell lines of a fibroblastoid type (Phoenix and NIH-3T3). In these cells the oligonucleotides tested did not show to be toxic.

(99) These results indicate that the PNA oligonucleotides act through a specific inhibition effect on the expression of the gene of interest, whereas they do not have any non-specific, toxic effect in cells which do not express the gene.

(100) It can thus be deduced that the PNA oligonucleotides designed on the basis of the parameters described in the present invention act specifically and effectively on the target gene and therefore on the cells which express/overexpress this gene.