WO2017005771A1 - Utilisation de micro-arn ciblant la glypicane pour le traitement du cancer du foie - Google Patents

Utilisation de micro-arn ciblant la glypicane pour le traitement du cancer du foie Download PDF

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WO2017005771A1
WO2017005771A1 PCT/EP2016/065933 EP2016065933W WO2017005771A1 WO 2017005771 A1 WO2017005771 A1 WO 2017005771A1 EP 2016065933 W EP2016065933 W EP 2016065933W WO 2017005771 A1 WO2017005771 A1 WO 2017005771A1
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mir
mirna
hsa
liver cancer
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PCT/EP2016/065933
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Christophe GROSSET
Flora CARTIER
Sarah LESJEAN
Francis SAGLIOCCO
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Universite de Bordeaux
Institut National De La Sante Et De La Recherche Medicale
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides

Definitions

  • the present invention relates to the field of oncology.
  • it provides miRNAs useful for detecting and/or treating cancer.
  • Hepatoblastoma Another type of liver cancer is Hepatoblastoma (HBL) which is an uncommon malignant liver neoplasm occurring in infants and children (1% of pediatric cancers, 0.02% of all cancers and around 3,500 new cases by year worldwide) with a 10-year survival of 61%.
  • HBL Hepatoblastoma
  • miR-34a (recently renamed miR-34a-5p in the last version of miRBase) is currently tested in unresectable primary liver cancer or solid cancers with liver metastasis.
  • miR-34a regulates several key oncogenic targets including CTNNB 1, BCL2, E2F3, HDAC1, MET, MAK1, CDK4/6, PDGFR-a, WINT1/3 and NOTCH-1 (Bader, Front. Genet., 120, 1-9).
  • a therapeutic solution remains urgently needed for these two cancers, in particular for patients who are unable to benefit from treatment by surgery or liver transplantation, fail to properly respond to first-line treatments, already have unresectable metastasis or relapse.
  • the inventors identified 5 new miRNAs decreasing the level of the oncoprotein glypican-3 (GPC-3) in liver cancer cells. These miRNAs are down-regulated in liver tumors and are capable of inhibiting HCC cell growth and inducing HCC cell cycle arrest. Some of them are even capable of inducing tumor cell apoptosis. They especially showed that in vitro, miR-4510 is more effective than miR-34a (the miRNA currently tested in clinical trials for the treatment of patients with liver cancer or liver tumor involvement) for blocking the growth of HCC and HBL cells and for inducing their apoptosis.
  • GPC-3 oncoprotein glypican-3
  • the present invention relates to a molecule selected from the group consisting of hsa-miR-4510, hsa- miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or any combination thereof or a DNA or RNA encoding for said miRNA for use for treating a liver cancer.
  • the present also relates to the use of a molecule selected from the group consisting of hsa-miR-4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or any combination thereof or a DNA or RNA encoding for said miRNA for the manufacture of a medicament for treating a liver cancer.
  • a molecule selected from the group consisting of hsa-miR-4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or any combination thereof or a DNA or RNA encoding for said miRNA to said patient.
  • the molecule is to be used in combination with one or more therapeutic agents, preferably another antitumor therapy, and in particular with sorafenib, doxorubicin, cisplatin, 5-Fluoro-uracil, gemcitabine, oxaliplatin, mitomycin C, tamoxifen, MSC2156119J, foretinib, refametinib, cabozantinib, tivantinib, or any combination thereof, preferably with doxorubicin, cisplatin, gemcitabine, oxaliplatin, mitomycin C, tamoxifen, sorafenib or any combination thereof.
  • sorafenib doxorubicin, cisplatin, 5-Fluoro-uracil, gemcitabine, oxaliplatin, mitomycin C, tamoxifen, MSC2156119J, foretinib, refametinib,
  • the molecule is to be used in combination with sorafenib, in particular for use in the treatment of HCC.
  • the molecule is to be used in combination with cisplatin and/or doxorubicin, in particular for use in the treatment of HBL.
  • the molecule is to be used in combination with another antitumor therapy and a drug lowering the toxicity and side effects of the antitumor therapy.
  • drug lowering the toxicity and side effects of the antitumor therapy can be sodium thiosulfate or N-acetyl cysteine.
  • the molecule is to be used in combination with one or more immunotherapeutic agents, and in particular with monoclonal antibodies binding antigens on cancer cells or targeting immune system checkpoints (e.g. immune checkpoint inhibitors) and especially drugs targeting PD-1 or PD-L1 such as for example pembrolizumab, nivolumab, atezolizumab.
  • the molecule is to be used in combination with resection, radiofrequency ablation and/or percutaneous ethanol injection.
  • the molecule is to be used after or before resection, radiofrequency ablation and/or percutaneous ethanol injection.
  • the molecule is for use as neo-adjuvant therapy or adjuvant therapy.
  • the subject has liver metastasis, and/or does not respond to the first line treatment and/or is not suitable for tumor resection or ablation.
  • the subject has a miRNA selected from the group consisting of hsa-miR-4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or combination thereof which is under-expressed in comparison with a healthy or non-tumoral control.
  • the subject has liver tumors or liver metastasis expressing the glypican-3 oncoprotein, called GPC-3.
  • the present invention further relates to a method for selecting a subject suitable for a treatment by miRNA as disclosed herein comprising determining the level of a miRNA selected from the group consisting of hsa-miR-4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or of a combination thereof in a biological sample from the subject, and selecting the subject if at least one of the miRNA is under-expressed in comparison with a healthy or non-tumoral control.
  • the present invention further relates to a method for selecting a subject suitable for a treatment by miRNA as disclosed herein comprising determining the level of GPC3 thereof in a biological sample from the subject, and selecting the subject if GPC3 is upper-expressed or overexpressed in comparison with a healthy or non-tumoral control.
  • the present invention also relates to the use of a miRNA selected from the group consisting of hsa-miR- 4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or of any combination thereof as a marker for detecting a liver cancer or a susceptibility to develop a liver cancer.
  • the liver cancer is a hepatocellular carcinoma or a hepatoblasma.
  • the present invention relates to a method for detecting a liver cancer or a susceptibility to develop a liver cancer in a subject, comprising determining the level of a miRNA selected from the group consisting of hsa-miR-4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or of any combination thereof in a biological sample from the subject, an under-expression of at least one of the miRNA in comparison with a healthy or non-tumoral control being indicative of a liver cancer or a susceptibility to develop a liver cancer.
  • the liver cancer can be a hepatocellular carcinoma.
  • the present invention also relates to the use of a miRNA selected from the group consisting of hsa-miR- 4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or of any combination thereof as a marker for the prognosis in a subject having a liver cancer, preferably a hepatocellular carcinoma.
  • a miRNA selected from the group consisting of hsa-miR- 4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or of any combination thereof as a marker for the prognosis in a subject having a liver cancer, preferably a hepatocellular carcinoma.
  • the present invention also relates to a method for determining the prognosis in a subject having a liver cancer, preferably a hepatocellular carcinoma, comprising determining the level of a miRNA selected from the group consisting of hsa-miR-4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or of a combination thereof in a biological sample from the subject, the level of expression of said at least one of the miRNA being correlated with the clinical prognosis. More preferably, the miRNA is selected from the group consisting of hsa-miR-4510, hsa-miR-548aa, and any combination thereof.
  • the present invention relates to a kit for detecting a liver cancer or a susceptibility to develop a liver cancer in a subject or for selecting a subject suitable for a treatment by a molecule as disclosed herein or for determining the prognosis in a subject having a liver cancer, the kit comprising detection means specific for at least one miRNA selected from the group consisting of hsa-miR-4510, hsa-miR- 548aa, hsa-miR-548v, hsa-miR-376b-3p or for any combination thereof.
  • the liver cancer is a hepatocellular carcinoma.
  • FIG. 1 Ten new miRNAs regulate GPC3 expression.
  • Figure 2 Relative expression of 5 GPC3-regulating miRNAs in 19 NTL (Non-tumoral liver) and 98 HCC samples.
  • NTL Non-tumoral liver
  • Figure 2 Non-parametric Mann-Whitney test for unpaired samples: * p ⁇ 0.05; ** p ⁇ 0.01 ; *** p ⁇ 0.001.
  • Figure 3 Expression ratio of 5 GPC3 -regulating miRNAs in HCC in each 19 pairs of tumor and adjacent non-tumoral liver. Results are presented as HCC/NTL expression ratios. The median is shown as a full line and the reference ratio value "1" is shown as a dotted line. The statistical analyses were done with the Wilcoxon matched-pairs signed rank test: * p ⁇ 0.05; *** p ⁇ 0.001.
  • Figure 4 Expression of miR-4510 and miR-548aa in HCC tumors with a good or poor prognosis (see p- value above the box and whiskers graph).
  • Figure 9 Cycling of Huh7 cells at day 3 following transfection by the corresponding small RNAs. ANOVA test: *** p ⁇ 0.0001 ; Holm-Sidak's multiple comparisons test: * p ⁇ 0.05; ** p ⁇ 0.01 ; *** p ⁇ 0.001.
  • Figure 11 Number of Huh7 cells at day 6 following transfection by the corresponding small RNAs.
  • si- ctl control RNA
  • AM anti-miRNA
  • miR miR-4510.
  • ANOVA test *** p ⁇ 0.0001 ; Holm-Sidak's multiple comparisons test: * p ⁇ 0.05; *** p ⁇ 0.001.
  • FIG. 12 Apoptosis of Huh7 cells was determined by Annexin V/7-ADD staining at day 3 following transfection by the corresponding small RNAs and/or incubation with Sorafenib.
  • siCtl control RNA
  • AM anti-miRNA.
  • ANOVA test *** p ⁇ 0.0001
  • Holm-Sidak's multiple comparisons test * p ⁇ 0.05; *** p ⁇ 0.001.
  • FIG. 14 Apoptosis of HuH6 HBL cells at day 3 following transfection by the corresponding small RNAs.
  • si-ctl control RNA
  • AM anti-miRNA
  • miR miR-4510.
  • ANOVA test *** p ⁇ 0.0001
  • Holm-Sidak's multiple comparisons test *** p ⁇ 0.001.
  • Protein size is shown in brackets on the left of the blot. All cropped blots retained at least 6 bandwidths above and below the bands.
  • Figure 17 Kinetic growth of HBL-derived HuH6 cells following transfection by the corresponding small RNAs. ANOVA test: *** p ⁇ 0.0001; Holm-Sidak's multiple comparisons test: * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
  • Figure 20 Relative expression of miR-4510 in 24 pairs of HBL and adjacent normal liver samples.
  • Results are presented as HBL/NTL expression ratios. The median is shown as a full line and the reference ratio value "1" is shown as a dotted line. Two-tailed Wilcoxon matched-pairs signed ranked test. ***p ⁇ 0.001.
  • FIG. 21 Assessment of miR-4510 regulatory activity following encapsulation in stable nucleic acid lipid particles (SNALPs).
  • SNALPs used is a combination of a KAUDO nucleolipid and a dioleoylphosphatidylethanolamine (DOPE) lipid.
  • DOPE dioleoylphosphatidylethanolamine
  • Huh7 cells expressing the reference Tomato transgene and the test eGFP transgene carrying the wild-type GPC-3 3 '-untranslated region were incubated with miR-4510 or a control RNA encapsulated in SNALPs. Three days later, the red and green fluorescence signals were measured and the ratios were calculated using the Dual Fluorescence-FunREG system.
  • Glypican-3 (GPC-3) is described in Uniprot under ID P51654 and has a Reference Sequence of mRNA NM_001164617 and a Reference Sequence of protein NP_001158089.
  • identity refers to a relationship between the sequences of two or more nucleic acid molecules, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between amino acid or nucleic acid molecule sequences, as the case may be, as determined by the match between strings of nucleotide or amino acid sequences. "Identity” measures the percent of identical matches between two or more sequences with gap alignments addressed by a particular mathematical model or computer programs (i.e., "algorithms").
  • the BLAST X program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul] et al., NCB NLM NIH Bethesda, Md. 20894; Altschul et al., J. Mol. Biol. 215:403-410 [1990]).
  • NCBI National Center for Biotechnology Information
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • the GAP program is also useful with the above parameters.
  • the aforementioned parameters are the default parameters for nucleic acid molecule comparisons.
  • Other exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices, thresholds of similarity, etc. can be used by those of skill in the art, including those set forth in the Program Manual, Wisconsin Package, Version 9, September 1997.
  • the inventors identified miR-4510 as a therapeutic agent against cancer, especially liver cancer.
  • the seed sequence of miR-4510 is encompassed in the sequence shown in bold highlighting.
  • the mature miR-4510 is underlined.
  • the seed sequence of miR-548aa is encompassed in the sequence shown in bold highlighting.
  • the mature miR-548aa is underlined.
  • the inventors identified miR-548v as a therapeutic agent against cancer, especially liver cancer.
  • miR-376b-3p as a therapeutic agent against cancer, especially liver cancer.
  • Mature sequence of miR-376b-3p AUCAUAGAGG AAAAUCC AUGUU (MIM AT0002172) (SEQ ID No 8)
  • the seed sequence of miR-376b-3p is encompassed in the sequence shown in bold highlighting.
  • the mature miR-376b-3p is underlined.
  • miR-203a-3p as a therapeutic agent against cancer, especially liver cancer.
  • Mature sequence of miR-203a-3p GUGAAAUGUUUAGGACC ACUAG (MIMAT0000264) (SEQ ID No 10)
  • the seed sequence of miR-203a-3p is encompassed in the sequence shown in bold highlighting.
  • the mature miR-203a-3p is underlined.
  • microRNAs are well-known in the art and a person skilled in the art would understand that they include the conventional naturally occurring sequences (provided herein) but also any chemically modified versions and sequence homologues thereof. Chemically modified versions and sequence homologues of miRNAs are generally called miRNA mimic, analog or derivative. The miRNA mimic, analog or derivative has retained or enhanced activity of the original miRNA.
  • the miRNA can be mature miRNA, precursor (pre)-miRNA, primary (pri)-miRNA, a miRNA mimic, analog or derivative thereof.
  • prefix “hsa” indicates Homo sapiens or human. Even in its absence, all miRNA of the invention are human.
  • miRNA and microRNA can be identical and are substitutable.
  • the miRNA is a single-stranded nucleic acid molecule, especially a RNA molecule, of no more than 30 nucleotides in length, preferably no more than 25 nucleotides in length, and generally about 21-23 nucleotides in length. It comprises a sequence which is identical or substantially identical to the seed sequence. By “substantially identical” is meant that at most 1 or 2 substitutions or deletions are allowed. In a preferred embodiment, it comprises a sequence identical to the seed sequence.
  • the seed sequence usually corresponds to a sequence located between position 2 and position 9 of the mature miRNA. For instance, the seed sequence may consist in the sequence between position 2 and position 7, 8 or 9 of the mature miRNA.
  • the miRNA comprises, essentially consists in or consists in a sequence which is at least 80%, 85%, 90%, 95% or 99% identical to the respective full length sequence of the mature miRNA.
  • the mature miRNA sequence is selected in the group consisting of SEQ ID Nos 1, 3, 6, 8 and 10, preferably selected in the group consisting of SEQ ID Nos 1, 3, 6, and 8, preferably consisting of SEQ ID No 1.
  • the miRNA comprises, essentially consists in or consists in a sequence which is at least 80%, 85%, 90%, 95% or 99% identical to the respective full length sequence of the mature miRNA and comprises a sequence identical to the seed sequence.
  • the miRNAs as pre-miRNA has a stem-loop sequence and comprises a guide strand comprising the mature miRNA, and more specifically the seed sequence, and a passenger strand which is complementary or substantially complementary to the seed sequence of the guide strand.
  • an alternative miRNA can be a double-stranded molecule comprising two separate strands as defined before instead of the stem-loop structure.
  • the guide strand comprises a sequence which is identical or substantially identical to the seed sequence.
  • substantially identical is meant that at most 1 or 2 substitutions or deletions are allowed.
  • the guide strand comprises a sequence which is at least 80%, 85%, 90%, 95% or 99% identical to the respective full length sequence of the mature miRNA.
  • the mature miRNA sequence is selected in the group consisting of SEQ ID Nos 1, 3, 6, 8 and 10, preferably selected in the group consisting of SEQ ID Nos 1, 3, 6, and 8, preferably consisting of SEQ ID No 1.
  • the guide strand of the miRNA comprises a sequence which is at least 80%, 85%, 90%, 95% or 99% identical to the guide strand of pre-miRNA as disclosed in a sequence selected in the group consisting of SEQ ID Nos 2, 4-5, 7, 9 and 11, preferably selected in the group consisting of SEQ ID Nos 2, 4-5, 7, and 9, preferably consisting of SEQ ID No 2.
  • the guide strand of the miRNA comprises, essentially consists in or consists in a sequence of the guide strand of pre-miRNA as disclosed in a sequence selected in the group consisting of SEQ ID Nos 2, 4-5, 7, 9 and 11, preferably selected in the group consisting of SEQ ID Nos 2, 4-5, 7, and 9, preferably consisting of SEQ ID No 2.
  • substantially complementary is intended that at most 1 or 2 mismatches and/or deletions are allowed.
  • the passenger strand comprises a sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to the complement of the respective full length sequence of the mature miRNA.
  • the mature miRNA sequence is selected among the SEQ ID Nos 1, 3, 6, 8 and 10, preferably selected among the SEQ ID Nos 1, 3, 6, and 8, preferably consisting of SEQ ID No 1.
  • the passenger strand of the miRNA comprises a sequence which is at least 80%, 85%, 90%, 95% or 99% identical to the passenger strand of pre-miRNA as disclosed in a sequence selected in the group consisting of SEQ ID Nos 2, 4-5, 7, 9 and 11, preferably selected in the group consisting of SEQ ID Nos 2, 4-5, 7, and 9, preferably consisting of SEQ ID No 2.
  • the passenger strand of the miRNA comprises, essentially consists in or consists in a sequence of the passenger strand of pre-miRNA as disclosed in a sequence selected in the group consisting of SEQ ID Nos 2, 4-5, 7, 9 and 11 , preferably selected in the group consisting of SEQ ID Nos 2, 4-5, 7, and 9, preferably consisting of SEQ ID No 2.
  • the miRNA is between 17 and 30 nucleotides in length, preferably 22-23 nucleotides in length, and comprises (i) a microRNA region having a sequence from 5' to 3' that is at least 80 % identical to at least one of SEQ ID Nos 1, 3, 6, 8 and 10, preferably SEQ ID Nos 1, 3, 6, and 8; and (ii) a complementary region having a sequence from 5' to 3' that is 60-100 % complementary to the microRNA region.
  • the microRNA region has a sequence that is at least 80, 85, 90, 95 or 100 % identical to at least one of SEQ ID Nos 1, 3, 6, 8 and 10, preferably SEQ ID Nos 1, 3, 6, and 8.
  • the miRNA comprises a hairpin structure.
  • the miRNA is between 17 and 30 nucleotides in length, preferably 22-23 nucleotides in length, and comprises (i) a first polynucleotide having a sequence from 5' to 3' that is at least 80 % identical to at least one of SEQ ID Nos 1, 3, 6, 8 and 10, preferably SEQ ID Nos 1, 3, 6, and 8; and (ii) a second separate polynucleotide having a sequence that is 60-100 % complementary to the first polynucleotide.
  • the microRNA region has a sequence that is at least 80, 85, 90, 95 or 100 % identical to at least one of SEQ ID Nos 1, 3, 6, 8 and 10, preferably SEQ ID Nos 1, 3, 6, and 8.
  • the miRNA can include some chemical modifications, in particular for increasing its stability, resistance to degradation and/or its cellular uptake.
  • microRNA molecules may be modified to stabilize the miRNAs against degradation, to enhance half -life, or to otherwise improve efficacy. Desirable modifications are described, for example, in US20070213292, US20060287260, US20060035254, US20060008822, WO2015131115, US2016053264, WO2010144485 and US20050288244, each of which is hereby incorporated by reference in its entirety.
  • the miRNA can include 5' cap, 3' cap, backbone modifications, ribose modifications, mismatch, as well as nucleobase modifications.
  • a 3' may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3' end of the oligonucleotide.
  • Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars can block 3'-5'-exonucleases.
  • Modified bases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2- thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (including 5-brom
  • the guide strand of pre-miRNA or mature miRNA can include 2'-fluorine modifications while the passenger strand can include 2'-0-methyl modifications.
  • the miRNA comprises one or more of the following (i) a replacement group for phosphate or hydroxyl of the nucleotide at the 5' terminus of the complementary strand or passenger strand (5' cap); (ii) one or more sugar modifications in the first or last 1-6 residues of the complementary strand or passenger strand; or (iii) non-complementarity between one or more nucleotides in the last 1 - 5 residues at the 3' end of the complementary strand or passenger strand and the corresponding nucleotides of the microRNA region or guide strand.
  • the miRNA comprises a fully complementary passenger strand comprising (i) modified nucleotides in the first and last two nucleotides of the passenger strand, and/or (ii) a terminal modification of the nucleotide at the 5 'end.
  • the passenger strand comprises modified nucleotides and fewer than half of the total number of nucleotides in the passenger are modified nucleotides. For instance, 2-10, 4-8 or 5-7 nucleotides in the passenger are modified nucleotides. In a particular embodiment, the modified nucleotides are selected from the group consisting of the two-three first and the two last nucleotides of the passenger strand.
  • the guide strand comprise at least one or two modified nucleotides.
  • the guide strand does not comprise modified nucleotides in the first two positions at the 5' end of the guide strand and/or in the last two positions at the 3' end of the guide strand.
  • the microRNA molecules may comprise alternate stretches or portions of nucleotides with 2'-0-methyl modifications and stretches or portions of nucleotides without the modification.
  • alternate stretches or portions it is meant that, when considering the double-stranded RNA molecule, for each pair of nucleotides, at least one nucleotide of the pair, preferably only one, has a 2'-0-methyl modification.
  • the length of the stretches/portions can vary from 1 to 7 consecutive nucleotides. Accordingly, just for illustrating this aspect, the mature miRNA may present one of the following structures: Sens 5' NNNNNNNN NNNNNNNN 3'
  • N refers to a nucleotide having 2' -O-methyl modification.
  • the microRNA molecules may comprise stretches or portions of nucleotides with 2' -O-methyl modifications.
  • both nucleotide of the pair have 2' -O-methyl modifications.
  • the length of the stretches/portions can vary from 1 to 7 consecutive nucleotides.
  • N refers to a nucleotide having 2' -O-methyl modification.
  • the miRNA when considering the specific example of miR-4510, the miRNA may present one of the following structures:
  • the miRNA can present a modification at one or both 3' ends, preferable a modified sugar.
  • modified sugar is Triantennary N-acetyl galactosamine (GalNAC 3 ).
  • the miRNA when considering the specific example of miR-4510, the miRNA may present the following structure: V4
  • the miRNA when a molecule increasing the cellular uptake such as cholesterol or tocopherol is linked to the miRNA, the molecule is linked to the passenger strand of the pre -miRNA.
  • the miRNA may be linked to a moiety allowing the targeting of the liver.
  • the disclosure provides a nucleic acid molecule or any modified molecule derivatives encoding or leading to a miRNA as disclosed above and a recombinant expression vector comprising a recombinant nucleic acid sequence operatively linked to an expression control sequence, wherein expression of the recombinant nucleic acid sequence provides a miRNA sequence, a precursor miRNA sequence, or a primary miRNA sequence as described herein.
  • the resulting sequence (e.g., primary or precursor miRNAs) can optionally be further processed to provide the miRNA sequence.
  • the recombinant expression vector comprises at least one sequence selected from the group consisting of SEQ ID Nos 1-11, preferably of SEQ ID Nos 1-9, more preferably of SEQ ID Nos 1-2.
  • Any suitable expression vector can be used such as, for example, a DNA vector (e.g., viral vector, plasmid, etc.).
  • the expression vector is selected for expression in a eukaryotic cell such as, for example, a mammalian cell.
  • the expression cassette is comprised in a viral vector, or plasmid DNA vector or other therapeutic nucleic acid vector or delivery vehicle, including liposomes and the like. miRNA therapeutic uses.
  • the miRNA miR-4510, miR-548aa, miR-548v, miR-376b-3p, and any combination thereof as disclosed above can be for use for treating a solid cancer in a subject, preferably a liver cancer or a solid cancer with liver metastasis.
  • the present disclosure also relates a pharmaceutical composition comprising miR- 4510, miR-548aa, miR-548v, miR-376b-3p, and any combination thereof.
  • miR-4510 miR-548v, and miR-203a-3p
  • miR-4510 miR-376b-3p
  • miR-4510 miR-548aa, miR-548v and miR-376b-3p
  • miR-4510 miR-548aa, miR-548v and miR-203a-3p
  • miR-4510 miR-548v, miR-376b-3p and miR-203a-3p
  • miR-4510 miR-548aa, miR-
  • the combination can further comprise an additional miRNA, for instance miR-34a.
  • the combination may include at least miR-548aa and 1-4 miRNAs selected among miR-4510, miR-548v, miR-376b-3p, and miR-203a-3p, e.g., miR-548aa and miR-548v; miR- 548aa and miR-376b-3p; miR-548aa and miR-203a-3p; miR-548aa, miR-548v and miR-376b-3p; miR- 548aa, miR-548v and miR-203a-3p; miR-548aa, miR-376b-3p and miR-203a-3p; and miR-548aa, miR- 548v, miR-376b-3p and miR-203a-3p.
  • the combination can further comprise an additional miRNA, for instance miR-34a
  • the combination may include at least miR-548v and 1-4 miRNAs selected among miR-4510, miR-548aa, miR-376b-3p, and miR-203a-3p, e.g., miR-548v and miR-376b- 3p; miR-548v and miR-203a-3p; and miR-548v, miR-376b-3p and miR-203a-3p.
  • the combination can further comprise an additional miRNA, for instance miR-34a-5p.
  • the combination may include at least miR-376b-3p and 1-4 miRNAs selected among miR-4510, miR-548aa, miR-548v, and miR-203a-3p, e.g., miR-376b-3p and miR-203a- 3p.
  • the combination can further comprise an additional miRNA, for instance miR-34a.
  • the miRNA and any combination thereof can be used for treating liver cancer or a solid cancer with liver involvement (e.g. metastasis).
  • liver cancer e.g. metastasis
  • Their use for the treatment of other specific solid cancers with or without liver involvement can also be contemplated, in particular breast, colorectal, esophageal, lung, melanoma, pancreatic, stomach, ovaries, neuroendocrine, uterus, CNS (central nervous system) and brain cancer.
  • the subject can be an adult or a child.
  • the subject has liver metastasis, and/or does not respond to the first line treatment and/or is not suitable for tumor resection or ablation.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or several miRNA selected from the group consisting of hsa-miR-4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or any combination thereof and another drug, in particular an antitumor drug.
  • a product comprising one or several miRNA selected from the group consisting of hsa-miR-4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or any combination thereof and another drug, in particular an antitumor drug, as a combined preparation for simultaneous, separate or sequential use, preferably for treating a solid cancer, in particular a liver cancer or a solid tumor with liver metastasis.
  • miRNA selected from the group consisting of hsa-miR-4510, hsa-miR-548aa, hsa-miR-548v, hsa-miR-376b-3p or any combination thereof and another drug, in particular an antitumor drug, as a combined preparation for simultaneous, separate or sequential use, preferably for treating a solid cancer, in particular a liver cancer or a solid tumor with liver metastasis.
  • the antitumor drug can be selected from the group consisting of an inhibitor of topoisomerases I or II, a DNA crosslinker, a DNA alkylating agent, an anti-metabolic agent and inhibitors of the mitotic spindles. It can also be an immunotherapy.
  • the antitumor drug is sorafenib, in particular for use in the treatment of hepatocellular carcinoma.
  • the antitumor drug is cisplatin or doxorubicin, in particular for use in the treatment of a hepatoblastoma.
  • Inhibitors of topoisomerases I and/or II include, but are not limited to, etoposide, topotecan, camptothecin, irinotecan, amsacrine, intoplicin, anthracyclines such as doxorubicin, epirubicin, daunorubicin, idarubicin and mitoxantrone.
  • Inhibitors of Topoisomerase I and II include, but are not limited to intoplicin.
  • DNA crosslinkers include, but are not limited to, cisplatin, carboplatin and oxaliplatin. In a preferred embodiment, the DNA crosslinker is cisplatin.
  • the DNA-damaging anti-tumoral agent can be alkylating agents including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, metal salts and triazenes.
  • alkylating agents including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, metal salts and triazenes.
  • Non- exhaustive examples thereof include Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN(R)), Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphor amine, Busulfan, Carmustine, Lomustine, cisplatin, carboplatin, oxaliplatin, thiotepa, Streptozocin, dacarbazine, and Temozolomide.
  • the immunotherapeutic drug may target multiple elements of the immune pathway: a therapy that enhances tumor antigen presentation; a therapy that inhibits negative immune regulation e.g., by inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or depleting or blocking Tregs or other immune suppressing cells; a therapy that stimulates positive immune regulation, e.g., with agonists that stimulate the CD- 137, OX-40, and/or GITR pathway and/or stimulate T cell effector function; a therapy that increases systemically the frequency of anti-tumor T cells; a therapy that depletes or inhibits Tregs, such as Tregs in the tumor, e.g., using an antagonist of CD25 (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; a therapy that impacts the function of suppressor myeloid cells in the tumor; a therapy that enhances immunogenicity of tumor cells (e.g., anthracyclines); adopt
  • the immunotherapeutic drug is a drug targeting PD-1 or PD-L1.
  • the PD- 1/PD-Ll agent is preferably selected from the group consisting of Nivolumab (Opdivo, Bristol-Myers Squibb), Pembrolizumab (Keytruda, MK-3475, Merck), Pidilizumab (CT-011, Cure Tech), BMS 936559 (Bristol Myers Squibb), atezolizumab or MPDL3280A (Roche), and a combination thereof.
  • the combined association of one or several miRNA as disclosed herein with another antitumor drug can allow the use of a lower/decreased amount of the other antitumor drug that could result in a reduction of the adverse effects and toxicity.
  • the amount of the other antitumor drug can be a sub-therapeutic amount. More specifically, the other antitumor drug is used at lower dosage than the conventional dosage used in chemotherapy for the same indication and the same administration route when it is used alone (i.e., an amount equal to or preferably lower than the one used in conventional chemotherapy), also called herein a sub-therapeutic amount.
  • the amount can be for instance 90, 80, 70, 60, 50, 40, 30, 20 or 10 % of the conventional therapeutic dosage (in particular for the same indication and the same administration route).
  • the conventional therapeutic dosages are those acknowledged by the drug approvals agencies (e.g., FDA or EMEA) and can be found in reference Manuals such as Merck Manuals (www.merck.com/mmpe/lexicomp/).
  • the administration frequency of the other antitumor drug or its treatment period can be reduced.
  • the treatment period may be reduced, for instance by 90, 80, 70, 60 or 50%.
  • the interval between treatments with the other antitumor drug can be increased, for instance by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% or by 1.5, 2, 2.5 or 3 fold.
  • the molecule is to be used in combination with another antitumor therapy and a drug lowering/decreasing the toxicity and side effects of the antitumor therapy.
  • the drug lowering the toxicity and side effects of the antitumor therapy can be sodium thiosulfate or N-acetyl cysteine.
  • the molecule is to be used in combination with cisplatin and, sodium thiosulfate or N-acetyl cysteine.
  • miRNAs, pharmaceutical compositions, or products of the invention can be used in humans with existing cancer or tumour, including at early or late stages of progression of the cancer.
  • the miRNAs, pharmaceutical compositions, or products of the invention will not necessarily cure the patient who has the cancer but will delay or slow the progression or prevent further progression of the disease, ameliorating thereby the patients' condition or survival.
  • the miRNAs, pharmaceutical compositions, or products of the invention reduce the development of tumors, reduce tumor burden, produce tumor regression in a mammalian host and/or prevent metastasis occurrence and cancer relapse.
  • the pharmaceutical composition of the invention is administered in a therapeutically effective amount.
  • the delivery system can be selected among the lipid-based delivery system, the PEI (polyethylenimine)-based delivery system, dendrimers, PLGA (poly(lactide-co-glycolide)) particles, WO 15023775, and the like.
  • the pharmaceutically acceptable carriers will protect the miRNA against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
  • cationic lipids that are bound or associated with miRNA.
  • miRNAs are encapsulated or surrounded in cationic lipids, e.g. liposomes, for in vivo delivery.
  • exemplary cationic lipids include, but are not limited to, N-[l-(2,3- dioleyloxy)propylJ-N,N,N-trimethylammonium chloride (DOTMA); l,2-bis(oleoyloxy)-3-3- (trimethylammonium)propane (DOT AP) , l,2-bis(dimyrstoyloxy)-3 -3 -(trimethylammonia)propane (DMTAP); l,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); dimethyldioctadecylammonium bromide (DDAB); 3-(N-(N',N'- dimethyl) DOTMA); l
  • exemplary cationic lipids include, but are not limited to, 1-dialkenoyl-sn-glycero-S- ethylphosphocholines (EPCs), such as 1 - dioleoyl-sn-glycero-S-ethylphosphocholine, l,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2- dipalmitoyl-sn-glycero-3-ethylphosphocholine, pharmaceutically acceptable salts thereof, and mixtures thereof.
  • EPCs 1-dialkenoyl-sn-glycero-S- ethylphosphocholines
  • Exemplary polycationic lipids include, but are not limited to, tetramethyltetrapalmitoyl spermine (TMTPS), tetramethyltetraoleyl spermine (TMTOS), tetramethlytetralauryl spermine (TMTLS), tetramethyltetramyristyl spermine (TMTMS), tetramethyldioleyl spermine (TMDOS), pharmaceutically acceptable salts thereof, and mixtures thereof.
  • TTPS tetramethyltetrapalmitoyl spermine
  • TTOS tetramethyltetraoleyl spermine
  • TTLS tetramethlytetralauryl spermine
  • TTMTMS tetramethyltetramyristyl spermine
  • TMDOS tetramethyldioleyl spermine
  • polycationic lipids include, but are not limited to, 2,5-bis(3-aminopropylamino)-N-(2-(dioctadecylamino)-2- oxoethyl)pentanamide (DOGS); 2,5-bis(3-aminopropylamino)-N-(2-(di(Z)-octadeca-9-dienylamino)-2-oxoethyl) pentanamide (DOGS- 9-en); 2,5-bis(3-aminopropylamino)-N-(2-(di(9Z,12Z)-octadeca-9,12-dienylamino)-2- oxoethyl)pentanamide (DLinGS); 3-beta-(N4-(N 1, Nd-dicarbobenzoxyspermidinearbamoychole-sterol (GL-67); l,3-dioleoy
  • Non-cationic lipids such as neutral, zwitterionic, and anionic lipids.
  • exemplary non-cationic lipids include, but are not limited to, 1,2- Dilauroyl-sn-glycerol (DLG); 1 ,2-Dimyristoyl-snglycerol (DMG); 1,2- Dipalmitoyl-sn-glycerol (DPG); 1 ,2-Distearoyl-sn-glycerol (DSG); l,2-Dilauroyl-sn-glycero-3- phosphatidic acid (sodium salt; DLPA); l,2-Dimyristoyl-snglycero-3-phosphatidic acid (sodium salt; DMPA); l,2-Dipalmitoyl-sn-glycero-3- phosphatidic acid (sodium salt; DPP A); l,2-Distearoyl-sn-glycero-3-phosphatedic acid (sodium
  • non-cationic lipids include, but are not limited to, polymeric compounds and polymer-lipid conjugates or polymeric lipids, such as pegylated lipids, including polyethyleneglycols, N-(Carbonylmethoxypolyethyleneglycol-2000)-l,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DMPE-MPEG-2000); N-(Carbonyl- methoxypolyethyleneglycol-5000)-l,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (sodium salt; DMPE-MPEG-5000) ; NtCarbonyl-methoxypolyethyleneglycol 2000)-l,2-dipalmitoyl-sn-glycero-3 - phosphoethanolamine (sodium salt; DPPE-MPEG-2000); N-(Carbonyl-methoxypolyethyleneglycol 500O)-l,2-dipalmito
  • non-cationic lipids include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), diphytanoylphosphatidylethanolamine (DPhPE), 1,2- Dioleoyl-sn-Glycero-3- Phosphocholine (DOPC), l,2-Diphytanoyl-sn-Glycero-3-Phosphocholine (DPhPC), cholesterol, and mixtures thereof.
  • DOPE dioleoylphosphatidylethanolamine
  • DPhPE diphytanoylphosphatidylethanolamine
  • DOPC 1,2- Dioleoyl-sn-Glycero-3- Phosphocholine
  • DPhPC 1,2- Dioleoyl-sn-Glycero-3-Phosphocholine
  • cholesterol and mixtures thereof.
  • nucleic acid molecules for use herein are described, e.g., in Akhtar, et al., Trends Cell Bio. 2: 139, 1992; Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995; Maurer, et al., Mol. Membr. Biol. 16: 129-140, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 137: 165-192, 1999; and Lee, et al., ACS Symp. Ser. 752: 184-192, 2000. Sullivan, et al., International PCT Publication No. WO 94/02595, further describes general methods for delivery of enzymatic nucleic acid molecules.
  • the miRNA and pharmaceutical composition can be administered by local or systemic routes.
  • the miRNA and pharmaceutical composition can be administered or suitable for being administered by enteral routes, parenteral routes (including subcutaneous, intravenous, intramuscular intratumoral or intraperitoneal), or by rectal, topical, transdermal, or oral routes.
  • the nucleic acid molecules of the present invention may be alternatively delivered into a target cell using a viral vector.
  • the viral vector may be any virus which can serve as a viral vector. Suitable viruses are those which infect the target cells, can be propagated in vitro, and can be modified by recombinant nucleotide technology known in the art.
  • Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus.
  • Adenovirus-associated vectors are an appealing method since they have acceptable toxicity profiles and have been successfully used to restore miRNA expression.
  • the viral vector is a non-replicating viral vector.
  • the viral vector is a non-integrative viral vector, in particular for preventing any oncogenic effect associated with the knock-down of tumor suppressor gene by insertional mutation.
  • the viral vector is a non-replicating non-integrative viral vector.
  • the non-replicating poxvirus vector is selected from: a Modified Vaccinia virus Ankara (MVA) vector, a NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector.
  • the adenovirus vector is a non- replicating adenovirus vector (wherein non-replicating is defined as above).
  • Adenoviruses can be rendered non- replicating by deletion of the El or both the El and E3 gene regions.
  • an adenovirus may be rendered non-replicating by alteration of the El or of the El and E3 gene regions such that said gene regions are rendered non- functional.
  • a non-replicating adenovirus may lack a functional El region or may lack functional El and E3 gene regions.
  • both El and E3 gene region deletions are present in the adenovirus, thus allowing a greater size of transgene to be inserted. This is particularly important to allow larger antigens to be expressed, or when multiple antigens are to be expressed in a single vector, or when a large promoter sequence, such as the CMV promoter, is used. Deletion of the E3 as well as the El region is particularly favored for recombinant Ad5 vectors. Optionally, the E4 region can also be engineered.
  • the adenovirus vector is selected from: a human adenovirus vector, a simian adenovirus vector, a group B adenovirus vector, a group C adenovirus vector, a group E adenovirus vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector, and an Ad5 vector.
  • One or several miRNA selected from the group consisting of miR-4510, miR-548aa, miR-548v, and miR-376b-3p can be used as a biomarker. More specifically, they can be used as a biomarker of a liver cancer, including hepatocellular carcinoma and hepatoblastoma, preferably a hepatocellular carcinoma. In addition, they can be used as a biomarker of the outcome of a liver cancer, in particular of a hepatocellular carcinoma. Their expression can be correlated with the good or bad prognosis.
  • the present invention relates to kits and methods for providing information useful for detecting a liver cancer or a predisposal or susceptibility to develop a liver cancer, or for predicting clinical prognosis or outcome of a liver cancer or for selecting a subject suitable for a treatment by a miRNA as disclosed above.
  • the present invention also relates to a kit for detecting a liver cancer or a susceptibility to develop a liver cancer in a subject or for selecting a subject suitable for a treatment by a miRNA as disclosed above or for determining the prognosis of a subject having a liver cancer, in particular a hepatocellular carcinoma, the kit comprising detection means for at least one miRNA selected from the group consisting of miR-4510, miR-548aa, miR-548v, and miR-376b-3p or for any combination thereof.
  • the kit comprises detection means specific for at least 2, 3 or 4 of miR-4510, miR-548aa, miR-548v, and miR-376b-3p.
  • the kit does not comprises detection means specific for more than 10 miRNAs.
  • Detection means are preferably primers or probes specific for miR-4510, miR-548aa, miR-548v, or miR-376b-3p.
  • the one or several miRNAs are selected from the group consisting of miR- 4510, miR-548aa, miR-548v, and miR-376b-3p.
  • the one or several miRNA are selected from the group consisting of miR-4510 and miR-548aa, more preferably miR-4510.
  • the kit may comprises detection means specific for one of the following combination: miR-548aa, miR-548v, miR-376b-3p, and miR-203a-3p, e.g., miR-4510 and miR-548aa; miR-4510 and miR-548v; miR-4510 and miR-376b- 3p; miR-4510 and miR-203a-3p; miR-4510, miR-548aa, and miR-548v; miR-4510, miR-548aa, and miR-376b-3p; miR-4510, miR-548aa, and miR-203a-3p; miR-4510, miR-548v, and miR-376b-3p; miR- 4510, miR-548v, and miR-203a-3p; miR-4510, miR-376b-3p and miR-203a-3p; miR-4510, miR-548aa, miR-548v and miR-376b-3p; miR-4510,
  • the present invention also relates to the use of the kit for detecting a liver cancer or a susceptibility to develop a liver cancer in a subject or for selecting a subject suitable for a treatment by a miRNA as disclosed above or for determining the prognosis or clinical outcome in a subject having a liver cancer, especially a hepatocellular carcinoma.
  • an under-expression of the miRNAs as disclosed herein is indicative of a cancer, especially a liver cancer or liver metastasis, a predisposition to develop a cancer, especially a liver cancer or liver metastasis or a suitability to be treated with the miRNA as disclosed herein.
  • the under-expression of the miRNAs as disclosed herein is indicative of a hepatocellular carcinoma.
  • the miRNA is selected from the group consisting of miR-4510, miR-548aa, miR-548v and miR-376b.
  • the present invention relates to a method for determining if a subject has or is predisposed to a liver cancer or liver metastasis, comprising determining the level of one or several miRNA selected from the group consisting of miR-4510, miR-548aa, miR-548v, and miR-376b-3p in the biological sample from the subject, and wherein the subject has or is predisposed to a liver cancer or liver metastasis if the level of one of said one or several miRNA is decreased when compared to a non-tumoral control or healthy subject.
  • the method may further determine the level of miR-203a-3p.
  • the liver cancer is a hepatocellular carcinoma.
  • the method may further comprise determining the expression level of GPC-3, an increased level of expression when compared to a healthy or non-tumoral control being indicative of a cancer, especially a liver cancer or liver metastasis, a predisposition to develop a cancer, especially a liver cancer or liver metastasis.
  • the method may comprise an initial step of providing a biological sample from the subject.
  • the miRNA is selected from the group consisting of miR-4510, miR-548aa, miR-548v and miR-376b.
  • the present invention also relates to a method for selecting a subject suitable for a treatment by a miRNA selected from the group consisting of miR-4510, miR-548aa, miR-548v, and miR-376b-3p or any combination thereof, comprising determining the level of one or several miRNA selected from the group consisting of miR-4510, miR-548aa, miR-548v, and miR-376b-3p in the biological sample from the subject, and selecting the subject if at least one of said miRNAs is under-expressed in comparison with a healthy or non-tumoral control.
  • the method may further determine the level of miR-203a- 3p.
  • the liver cancer is a hepatocellular carcinoma.
  • the method may comprise an initial step of providing a biological sample from the subject.
  • the present invention also relates to a method for selecting a subject suitable for a treatment by a miRNA selected from the group consisting of miR- 4510, miR-548aa, miR-548v, and miR-376b-3p or any combination thereof, determining the level of GPC3 thereof in a biological sample from the subject, and selecting the subject if GPC3 is upper- expressed or overexpressed in comparison with a healthy or non-tumoral control.
  • the method may further comprise administering an effective amount of a miRNA selected from the group consisting of miR-4510, miR-548aa, miR-548v, miR-376b-3p and miR-203a-3p or any combination thereof to said subject.
  • a miRNA selected from the group consisting of miR-4510, miR-548aa, miR-548v, miR-376b-3p and miR-203a-3p or any combination thereof to said subject.
  • the present invention further relates to a method for determining a clinical prognosis or outcome in a subject having a liver cancer, especially a hepatocellular carcinoma.
  • the method comprises determining the level of one or several miRNA selected from the group consisting of miR-4510, miR-548aa, miR- 548v, and miR-376b-3p in the biological sample from the subject. More preferably, the miRNA is selected from the group consisting of miR-4510, miR-548aa, and combination thereof.
  • the clinical prognosis is correlated with the level of expression of the miRNA. More particularly, when compared with a non-tumoral control or healthy subject, an under-expression of one of several of these miRNA is associated with a poor prognosis.
  • the liver cancer is a hepatocellular carcinoma.
  • a patient may be considered to have a "poor prognosis” or “bad prognosis” where, for example, the survival rate associated with the cancer subtype is less than the survival rate associated with other related cancer subtypes.
  • the level of miRNA can be determined by any method available to the one skilled in the art such as Northern blot analysis, RT-PCR, quantitative RT-PCR, microarray, in situ hybridization, RNA sequencing. miRNA expression can be quantified in a two-step polymerase chain reaction process of modified RT-PCR followed by quantitative PCR. miRNA expression can be quantified by hybridization on a microarray, RNA sequencing, slides or chips. For instance, probes or primers may be coupled to a support. Such supports are well known to those of ordinary skill in the art and include, but are not limited to glass, plastic, metal, or latex. The support can be planar or in the form of a bead or other geometric shapes or configurations known in the art.
  • the determination of the expression level can be carried out by forming a preparation comprising nucleic acid from said biological samples, an oligonucleotide probe or probes adapted to anneal to one or several miRNAs selected from the group consisting of miR-4510, miR-548aa, miR- 548v, miR-376b-3p or miR-203a-3p, a thermostable DNA polymerase, deoxynucleotide triphosphates and co-factors; providing polymerase chain reaction conditions sufficient to amplify all or part of said nucleic acid molecule; analyzing the amplified products of said polymerase chain reaction for the presence of miRNA; and optionally comparing the amplified product with a normal matched control.
  • miRNAs selected from the group consisting of miR-4510, miR-548aa, miR- 548v, miR-376b-3p or miR-203a-3p, a thermostable DNA polymerase, deoxynucleotide triphosphat
  • the method can further comprise one or more of the steps including: (a) obtaining a sample from the patient, (b) isolating nucleic acids from the sample, (c) labeling the nucleic acids isolated from the sample, and (d) hybridizing the labeled nucleic acids to one or more probes.
  • the levels of miRNA are considered as under-expressed when decreased by at least 1.5 fold when compared to a normal control. More particularly, the levels are decreased by about 2, 3, 4, 5, 6, 7, 8, 9 or at least 10-fold compared to a normal control level.
  • the biological sample from the subject can be a sample from blood, blood plasma or serum, lymph fluid, spinal or cerebrospinal fluid, saliva, sputum, lavage, urine, feces, bronchoaveolar lavage, or human tissue biopsy, especially a tumor sample.
  • the sample is a blood sample, a liver sample or a liver tumor sample.
  • a normal or non-tumoral or healthy control is the miRNA in a sample from a histologically matched sample, for instance a subject which has no cancer or the miRNA in a normal or non-tumoral or healthy tissue taken at a reasonable distance of the tumor in a patient with a cancer.
  • the inventors worked on the regulation of genes by miRNAs and the role of these post-transcriptional regulations in two primary liver cancers: the hepatocellular carcinoma (HCC), and the hepatoblastoma (HBL). They focused their study on Glypican-3 (GPC3), a Wnt signaling pathway-associated gene.
  • GPC3 is an oncogene in liver and actively participates in hepatocarcinogenesis by sustaining tumoral cell proliferation, dedifferentiation and sternness.
  • Their work aimed at identifying miRNAs negatively regulating GPC3 in tumoral hepatic cells and at determining those that are involved in liver carcinogenesis by facilitating GPC3 overexpression and its oncogenic effect.
  • DFSFunREG Dual Fluorescence-FunREG
  • UTRs GPC3 5 '+3 '-untranslated regions
  • Tomato transgene a library of 1712 miRNA mimics (Qiagen, miRBase V17.0). 28 miRNAs modulating the eGFP/Tomato ratio above or below arbitrary threshold values were pre-selected as candidates.
  • MiR-4510, miR-203a-3p, miR-548aa, miR-376b-3p and miR-548v exert antitumor effects
  • the inventors identified 5 miRNAs, namely miR-4510, miR-548aa, miR-548v, miR-376b- 3p and miR-203a-3p, that down-regulate GPC3 expression in Huh7 cells and inhibit HCC cell growth in vitro (Table 1).
  • the down-regulation of miR-4510, miR-548aa, miR-548v, miR-376b-3p and miR-203a-3p constitutes a diagnostic biomarker and the decreased expression of miR-4510 and miR- 458aa also constitutes a prognosis biomarker.
  • miR-4510, miR-548aa, miR- 548v, miR-376b, miR-203a-3p act as tumor suppressors in HCC and could mediate their antitumor effect through the down-regulation of GPC3 oncogene and other target genes that remain to be identified. These 5 miRNAs therefore act as tumor inhibitors in HCC.
  • miR- 4510 and miR-548aa display higher antitumoral properties in vitro than miR-34a. Based on the results summarized in Table 2, the inventors more particularly focused their subsequent work on miR-4510 and its antitumor character in liver cancer.
  • miR-1271 a previously reported GPC3- regulating miRNA (Maurel et al, 2013, Hematology 57, 195-204), was used as a comparison.
  • miR-4510 is one of the most effective miRNAs for inhibiting the growth of HCC Huh7 cells (Figure 6), the most effective for inducing cell apoptosis ( Figure 7) and one of the two most effective for triggering cell cycle arrest through GO phase entering and S phase decreasing ( Figure 9).
  • Inventors showed that miR-4510 down-regulates Glypican-3 through its 3 'UTR.
  • MiR-4510 is a potent tumor suppressor in liver cancer and directly interacts with GPC3 3'-UTR
  • MiR-4510 inhibited the growth of HCC-derived Huh7 and Hep3B cells and of HBL- derived Huh6 and HepG2 cells and it was more effective than miR-34a-5p in Hep3B cells ( Figures 10A and 17). Moreover, it significantly induced apoptosis in three hepatoma cell lines ( Figures 10B, 12, 13 and 14). MiR-4510 specifically induced the apoptosis of Hep3B, another HCC cell line ( Figure 13), and of the HBL-derived HuH6 cell line ( Figure 14). Moreover it blocked the growth of these two cell lines at Day 6 more effectively than miR-34a-5p ( Figures 10A).
  • miR-4510 is a powerful tumor suppressor and a relevant antitumor al agent in both HCC and HBL cells.
  • miR-4510 significantly sensitized Huh7 cells and Huh6 cells to Sorafenib- and Cisplatin- mediated cell death, respectively (Figure IOC, Figure 12).
  • Sorafenib is the current treatment of unresectable and metastatic liver cancer.
  • miR-4510 further decreased Huh7 cell growth inhibition mediated by doxorubicin (Fig. 15).
  • the inventors Using Ingenuity and bioinformatic tools, the inventors also found that most of predicted targets of miR- 4510 are associated with cancerous processes and liver tumorigenesis suggesting its role as a central tumor suppressor in liver.
  • MiR-4510 is a central multifunctional regulator in liver
  • MiRNAs are pleiotropic regulators that target many genes in cells.
  • MiRNA:targets prediction tools showed that miR-4510 could potentially interact with around 700 genes.
  • Ingenuity Pathway Analysis program we found that 82% of these genes are associated with cancer and 34% are related to HCC and liver hyperplasia/hyperproliferation.
  • numerous predicted genes are involved in cell survival and cell cycle progression. Among them is the pro-survival B-cell lymphoma-extra large (BCL-XL) protein which is upregulated in HCC.
  • MiR-4510 was also predicted to interact with upstream and downstream regulators of the transforming growth factor-beta 1 (TGF-pi)/SMAD pathway including ACVR2A, TAB1 and SMAD3. TGF-P-pathway signaling activity has been associated with several features of HCC tumors. The inventors showed here that the levels of TAB 1 and SMAD3 decrease following miR-4510 treatment (Fig. 16B). Finally, miR-4510 was predicted to target TCF4, which is one of the main transcriptional effectors of the Wnt pathway. MiR-4510 decreased TCF4 expression and inhibited Wnt activity without affecting ⁇ -catenin expression in Huh7 cells (Fig. 16C). MiR-4510 inhibits HCC tumor growth and induces HCC cell apoptosis in vivo
  • CAM chick chorioallantoic membrane
  • miR-4510 dramatically inhibited the growth of Huh7 cell-mediated tumors (tumor aggressiveness and active growth being characterized by the presence of bleeding) in vivo further stressing the tumor suppressive properties of this miRNA in liver cancer.
  • Control experiments validated the inhibition of GPC3 by miR-4510 in Huh7 cell before CAM implantation (data not shown).
  • day 3 no obvious difference was observed between the control RNA (Ctrl) and miR-4510 in tumor appearance or size, nor in tissue cross-sections stained with Hematoxylin and Eosin (Fig. 23B and C, upper panels), with the exception of a significantly lower number of tumors with bleeding and bloody areas in presence of miR-4510 (Fig. 23D, left panel).
  • GPC3 protein expression was also decreased in tumors transfected with miR-4510 compared to control (data not shown).
  • the growth of miR-4510 tumors was noticeably impeded compared to Ctrl tumors (Fig. 23B-C, upper panels), as assessed by a disappearance of yellowish, bloody and coagulation areas (Fig. 23B, upper panels) and of blood cells and large vessels in tumoral tissue (Fig. 23C, upper panels).
  • Fig. 23D 80% of Ctrl tumors were characterized by bleeding, while only 30 % of miR-4510 tumors presented this macroscopic feature suggesting a reduction of the aggressiveness of miR-4510-transfected HCC cells during tumor development.
  • miR-4510 tumors seemed to hardly develop, Ki67 and Caspase 3 staining was performed.
  • the decrease of the proliferative marker Ki67 in miR-4510 tumors was visible both at day 3 and day 6 of tumor growth demonstrating the inhibition of HCC cells proliferation by miR-4510. While no Caspase-3 staining was visible at day 3 and at day 6 in Ctrl tumors, miR-4510 tumors were markedly stained at day 6 showing that miR-4510 induces HCC cell apoptosis at later stages of tumor development (Fig. 23C, lower panels). Altogether these results showed that miR-4510 induces HCC cell apoptosis and inhibits the growth and angiogenesis of HCC tumors in vivo.
  • miR-4510 acts as a tumor suppressor in liver and constitutes one of the most relevant candidates (amongst the 5 identified Glypican-3 -targeting miRNAs) for a therapeutic use in HCC and in HBL.
  • miR-4510 The regulatory activity of miR-4510 is maintained when encapsulated in stable nucleic acid lipid particles (SNALPs).
  • SNALPs stable nucleic acid lipid particles
  • MiR-4510 was encapsulated in SNALPs using a combination of KAUDO nucleolipid + dioleoylphosphatidylethanolamine (DOPE) lipid and then, incubated with TGG cells at a final concentration of 15nM for three days. Compared to the control RNA, miR-4510 decreased the eGFP/Tomato ratio and it was as efficient as lipofectamine reagent (Figure 21) demonstrating its ability to interact with its target genes in cellulo when nanoformulated with liposomes. Materials and Methods
  • the lentiviral pTRIP-eGFP-GLO, pTRIP-eGFP-GPC3, pL-GFP, pL-GFP-GPC3 (bearing the GPC3 3'UTR) and pL-Tomato plasmids were as previously described (Laloo, B., et al, MCP 2009; Jalvy- Delvaille, S., et al, 2012; Maurel, M., et al, 2013).
  • the lentiviral pL-5'UTR-GPC3-GFP-3'UTR-GPC3 and pL-GFP-5'UTR-GPC3 constructs were obtained by inserting the GPC3 5'UTR in the pL-GFP- 3'UTR-GPC3 and pL-GFP plasmids, respectively.
  • the pGEM-T-hGPC3 plasmid was constructed as follow.
  • the GPC3 Open Reading Frame was PCR amplified using the following primers: ATTCTCTAGAGAATTCGGATCCATGGCCGGGACCGTGCGC (SEQ ID No 12) on the 5' end and CTCACTCTAGAGCGGCCGCTCAGTGCACCAGGAAG (SEQ ID No 13) on the 3' end.
  • the lentiviral pL-hGPC3 was constructed by subcloning the human GPC3 ORF of the pGEM-T-hGPC3 plasmid in the pL-GFP plasmid using the BamH I-Xba I restriction sites.
  • the hepatocellular carcinoma (HCC)-derived Huh7 and Hep3B and the hepatoblastoma (HB)-derived HepG2 cell lines were grown in DMEM medium (Invitrogen) containing 4.5 g/L of D-glucose supplemented with 10% FCS and penicillin/streptomycin antibiotics.
  • the hepatoblastoma-derived HuH6 cell line was grown in DMEM medium (Invitrogen) containing 1 g/L of D-glucose supplemented with 10% FCS and penicillin streptomycin antibiotics.
  • Lentiviral particles were added to the target cells and incubated for 72 h. Then the cells were washed twice in PBS and grown in the presence of complete medium for a week before use. Cells expressing Tomato and eGFP were washed in PBS, detached with trypsin/EDTA, collected and analyzed by FACS using a BD FACS Canto II (BD Biosciences, San Jose, CA, USA) and the BD FACS Diva software as described previously (Laloo, B., et al, 2009). Cell sorting was performed using the BD FACS Aria cell sorter. Small RNAs, miRNA mimic library, Cell transfection and Sorafenib or Doxorubicin or Cisplatin treatment
  • the miRNA mimics were from Qiagen, Sigma and Exiqon. Hairpin inhibitors were from Thermo- Scientific-Dharmacon Products.
  • the Human miScript miRNA Mimic 96 Set (miRBase V17.0) and the 1027281 negative siRNA control (Ctrl) were from Qiagen.
  • Small non-coding RNAs or hairpin inhibitors were transferred into the target cells by reverse transfection using Lipofectamine RNAi Max (Invitrogen) according to manufacturer's instructions at a final concentration of 12 nM and cells were grown for 3 days before analysis.
  • sorafenib (Selleckchem) at a final concentration of 10 ⁇ or cisplatin at a final concentration of 3.8 ⁇ or doxorubicin at a final concentration of O. ⁇ g/ml were added to the cells during 48 hr or 72 hr, respectively.
  • Tomato/eGFP Huh7 cells were plated per well of 96-well microplates and reverse transfected by each miRNA mimic of the library. Three days after transfection, cells were washed in PBS and fluorescence signals were measured using an Envision multiplate reader (Perkin Elmer). Then eGFP/Tomato ratios were calculated.
  • Huh7 cells were incubated with small RNA-containing SNALPs at a final concentration of 15nM for three days at 37°C and 5 C02. Then, fluorescence signals were measured as described above.
  • Liver tissues were immediately frozen in liquid nitrogen and stored at -80°C until used for molecular studies. All patients were recruited in accordance with French law and institutional ethical guidelines. Liver samples were clinically, histologically, and genetically characterized as previously described (Maurel, M., et al, 2013).
  • a first set of 133 liver samples (112 HCC and 21 non tumourous liver [NTL] samples) was collected from 118 patients surgically treated at French University Hospitals.
  • a second set of 38 liver paired samples (19 HCC and their corresponding NTL samples) was collected from 19 patients surgically treated at French University Hospitals. miRNA quantification
  • Taqman microRNA assays (Applied Biosystems) were used to quantify the relative expression levels of mature miRNAs in the first set of 133 liver samples.
  • Sybergreen microRNA assays (Qiagen) were used to quantify the absolute expression of mature miRNAs in the second kit of 38 liver paired patients or in cell lines. Quantification of GPC3 protein
  • Fluorescence signals were detected and quantified using the Odyssey infrared imaging system. Blocker and Odyssey infrared imaging system were from LI-COR Biosciences (ScienceTec, Les Ulis, France). Specific protein signal was normalized to the house-keeping protein GAPDH and total proteins (SYPRO Ruby). The rabbit monoclonal anti- GPC3 (EPR5547) antibody was from Abeam and the rabbit polyclonal anti-GAPDH (FL-335) antibody was from Santa Cruz.
  • the rabbit monoclonal anti-ACVR2A (EPR7407, 1:2000), anti-BCL-XL (E18, 1 : 1000), anti-CDKl (E161, 1 :2000), anti-GPC3 (EPR5547, 1 :5000), anti-SMAD3 (EP568Y, 1 :2000) and rabbit polyclonal anti-TABl (1 :200) antibodies were purchased from Abeam.
  • mice monoclonal anti-CDK2 (D-12, 1 :200), rabbit polyclonal anti-CDK6 (C-21, 1 :200), anti-GAPDH (FL-335, 1 :2000) and anti-TCF4 (H-125, 1 :200) antibodies were from Santa Cruz and the mouse monoclonal anti- -Catenin (C-14, 1 :4000) was from BD Biosciences.
  • the anti-human GPC3-Allophycocianin (APC) monoclonal antibody and IgG2a-APC isotype control were from R&D systems. Huh7 cells were washed in PBS, detached with PBS/EDTA, collected and incubated with the fluorescent anti-GPC3 or control antibody. Expression of the membrane GPC3 protein was analyzed by FACS. Cells incubated with the IgG2a-APC isotype control were used as negative control to gate the eGFP-positive cell populations and to measure the basal mean fluorescence intensity of the whole cell population. Flow cytometry
  • Huh7 cells were washed in PBS, detached with PBS/5mM EDTA, collected and stained with a fluorescent anti-GPC3 antibody.
  • Expression of membranous GPC3 protein was measured by FACS using a BD FACS Canto II and the BD FACS Diva software as described previously (Laloo, B., et al, 2009).
  • the anti-human GPC3-Allophycocianin (APC) monoclonal antibody and IgG2a-APC isotype control were from R&D systems.
  • Cell growth was measured using the In vitro Toxicology assay kit (Sigma), which measures the total cellular proteins, according to the manufacturer's instructions. Briefly, 3,500 cells were transfected and seeded into 96-well microplates in a volume of 100 ⁇ . One day, three days and six days later, cell growth was stopped by the addition of cold trichloroacetic acid, then Sulforhodamine B staining was performed and absorbance was measured at 565 nm using the CLARIOstar multiplate reader (BMG labtech). For proliferation assay, 200 000 cells were transfected and seeded into 6-well plates in a volume of 2.5 ml. Three days later, total cells were counted with Malassez cell.
  • Sigma In vitro Toxicology assay kit
  • RNAs Prior to cell apoptosis detection, 200 000 cells were transfected with small RNAs at a concentration of 15 nM and seeded into 6-well plates in a volume of 2.5 ml. Three days later, total cells were collected and cell apoptosis was analysed using the Annexin V-PE/7-Amino-Actinomycin (AAD) apoptosis detection kit (BD Pharmingen). Viable cells with intact membranes exclude 7-ADD and are Annexin V-PE negative. Fluorescence generated by the cell-bound Annexin V-PE, which measure the percentage of early apoptotic cells, and the 7AAD, which measure the percentage of late apoptotic cells, were analyzed by the BD FACS CANTO II. Activities of Caspases 3 and 7 were measured using the Luminescent Caspase-Glo 3/7 assay from Promega, except that luminescence was measured using the CLARIOstar multiplate reader (BMG labtech).
  • Wnt transcriptional activity was assessed using the TOPflash/FOPflash assay.
  • 200 000 cells were transfected with Ctrl or miR-4510 and seeded into 6-well plates in a volume of 2.5 ml. Two days later cells were collected, 10 000 cells were seeded into 96-well plates in a volume of 100 ⁇ ⁇ and transfected with the control plasmid pRL-TK-Renilla (Promega) and either the TOPFLASH or FOPFLASH plasmids kindly provided by Hans Clevers (Korinek V et al, Science 1997).
  • TargetScan miRDB
  • TargetMiner miRanda
  • RNA Hybrid RNA Hybrid
  • PICTAR5 DIANAmt
  • IP A Ingenuity Pathways Analysis

Abstract

La présente invention concerne des miARN ciblant la glypicane-3 destinés à être utilisés dans le traitement du foie, en particulier du carcinome hépatocellulaire et de l'hépatoblastome.
PCT/EP2016/065933 2015-07-07 2016-07-06 Utilisation de micro-arn ciblant la glypicane pour le traitement du cancer du foie WO2017005771A1 (fr)

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WO2019059411A1 (fr) * 2017-09-20 2019-03-28 Chugai Seiyaku Kabushiki Kaisha Posologie pour polythérapie utilisant des antagonistes de liaison d'axe pd-1 et un agent de ciblage gpc3
CN110878353A (zh) * 2019-12-25 2020-03-13 中南大学湘雅二医院 miR-376b的应用及检测试剂盒
WO2023280988A1 (fr) * 2021-07-09 2023-01-12 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés d'amélioration de la relaxation de myocytes striés
US11767362B1 (en) 2016-03-15 2023-09-26 Chugai Seiyaku Kabushiki Kaisha Methods of treating cancers using PD-1 axis binding antagonists and anti-GPC3 antibodies

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11767362B1 (en) 2016-03-15 2023-09-26 Chugai Seiyaku Kabushiki Kaisha Methods of treating cancers using PD-1 axis binding antagonists and anti-GPC3 antibodies
WO2019059411A1 (fr) * 2017-09-20 2019-03-28 Chugai Seiyaku Kabushiki Kaisha Posologie pour polythérapie utilisant des antagonistes de liaison d'axe pd-1 et un agent de ciblage gpc3
JP2020534335A (ja) * 2017-09-20 2020-11-26 中外製薬株式会社 Ｐｄ−１系結合アンタゴニストおよびｇｐｃ３標的化剤を使用する併用療法のための投与レジメン
JP7382922B2 (ja) 2017-09-20 2023-11-17 中外製薬株式会社 Ｐｄ－１系結合アンタゴニストおよびｇｐｃ３標的化剤を使用する併用療法のための投与レジメン
CN110878353A (zh) * 2019-12-25 2020-03-13 中南大学湘雅二医院 miR-376b的应用及检测试剂盒
CN110878353B (zh) * 2019-12-25 2023-04-07 中南大学湘雅二医院 miR-376b的应用及检测试剂盒
WO2023280988A1 (fr) * 2021-07-09 2023-01-12 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés d'amélioration de la relaxation de myocytes striés

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