FR3058062A1 - NEW USE OF A DOUBLE STRANDED OLIGONUCLEOTIDE - Google Patents

Abstract

The present invention provides a double-stranded oligonucleotide for use in the prevention and / or treatment of cancer, in combination with a therapy resulting in DNA breakage. The present invention relates to a double-stranded oligonucleotide for use in the prevention and / or treatment of cancer, with the exception of androgen receptor-dependent prostate cancer.

Description

© Publication no .: 3,058,062 (to be used only for reproduction orders)

©) National registration number: 16 60441 ® FRENCH REPUBLIC

NATIONAL INSTITUTE OF INDUSTRIAL PROPERTY

COURBEVOIE © Int Cl ⁸ : A 61 K 31/7088 (2017.01), A 61 K 31/713, A 61 P 35/00

A1 PATENT APPLICATION

(© Filing date: 27.10.16. ©) Applicant (s): SELEXEL Liability company ©) Priority: limited - FR. @ Inventor (s): CABON FLORENCE, BROOKS HILARY, DELMAS STEPHANIE and CHUSSEAU MAUD. (43) Date of public availability of the request: 04.05.18 Bulletin 18/18. (56) List of documents cited in the report preliminary research: Refer to end of present booklet (© References to other national documents (73) Holder (s): SELEXEL Limited liability company related: tee. ©) Extension request (s): ® Agent (s): GROSSET FOURNIER AND DEMA- CHY Limited liability company.

154) NEW USE OF A DOUBLE-STRANDED OLIGONUCLEOTIDE.

15g) The present invention relates to a double-stranded oligonucleotide for use in the prevention and / or treatment of cancer, in combination with therapy resulting in DNA breakage.

The present invention relates to a double-stranded oligonucleotide for use in the prevention and / or treatment of cancer, with the exception of prostate cancer dependent on the androgen receptor.

FR 3 058 062 - A1

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NEW USE OF A DOUBLE-STRANDED OLIGONUCLEOTIDE

The present invention relates to a new use of a double-stranded oligonucleotide, and more particularly to a new use of an oligonucleotide targeting the androgen receptor.

Our organism is constantly subjected to aggressions coming from our environment like UV rays, physical or chemical agents, which cause more or less important damage on our DNA. Cells have thus developed during evolution several mechanisms for verifying and repairing the genome.

Among the anti-tumor therapies, radiotherapy and / or chemotherapy are widely used. Cytotoxic chemotherapy aims to block the division of cancer cells in order to prevent tumor proliferation. External radiotherapy is today essential in oncology since it is programmed in two thirds of the therapeutic schemes, either alone or associated with surgery and / or chemotherapy. The vast majority of these treatments induce DNA damage, and in particular double strand break (DSB). Each double-strand break is lethal in the event of non-repair, and the number of double-strand breaks determines cell radiosensitivity. DSB repair is mainly based on the mechanism of homologous recombination (HR) or that of NHEJ (Non Homologous End-Joining).

DNA breaks, whether due to the environment or induced by chemotherapy or radiotherapy treatments, occur in a context where DNA is not linear but compacted by nucleosomes made up of histones. Some histones play a structural role, others, like H2AX, are essential in the functioning or repair of the genome.

The detection of DNA breaks leads to a cascade of activation of different repair processes which involves the participation of a multitude of proteins and post-translational events which help maintain the integrity of the genome. The mutation of several of these proteins leads to genetic diseases, to predispositions to develop one or more cancers, to neurodegenerative diseases or to other pathologies.

Several proteins are involved in the detection of DSB. This is particularly the case of KU70 and KU80, which play a key role in initiating the mechanism of NHEJ. KU70 and

KU80 recruit DNA-PK (DNA-dependent protein kinase). This enzyme phosphorylates in particular H2AX on serine 139, H2AX is then noted γΗ2ΑΧ. ATM (mutated in ataxiatelangiectasia) and ATR (ataxia telangiectasia and Rad3-related) which are major and very early players in DNA repair are also capable of phosphorylating H2AX.

The phosphorylation of H2AX is an early process of DNA repair, required to allow the recruitment and assembly of many other proteins needed to repair the lesion. The detection of γΗ2ΑΧ, in particular by specific antibodies, is frequently used as a sensitive and quantitative indicator of the presence of DSB.

H2AX can also be acetylated on lysine 36 by CBP / P300 and on threonine 101. These two post-translational modifications are required to allow the survival of cells to ionizing radiation, and this independently of the phosphorylation of H2AX.

Many mechanisms for verifying and repairing the genome therefore exist, and the body naturally tries to repair DNA damage. This therefore decreases the effectiveness of therapies aimed at inducing DNA damage such as chemotherapy and radiotherapy.

There is therefore a real need to improve the effectiveness of therapies that induce DNA damage.

The present invention thus aims to provide a new use of a double-stranded oligonucleotide targeting the androgen receptor to improve the effectiveness of therapies inducing DNA damage.

The term “double-stranded oligonucleotide targeting the androgen receptor” refers to the fact that one of the two strands of this oligonucleotide hybridizes with the mRNA encoding the androgen receptor and thus inhibits its protein synthesis.

The present invention is indeed based on the unexpected results of the Inventors according to which the double-stranded oligonucleotide (SEQ ID NO: 1 / SEQ ID NO: 2) targeting the androgen receptor can also target Thistone H2AX. Indeed, the oligonucleotide which hybridizes on the mRNA coding for the androgen receptor also hybridizes on the mRNA coding for the histone H2AX, as illustrated in FIG. 1, and inhibits the protein synthesis of this histone .

The use of a double-stranded oligonucleotide targeting H2AX, in combination with therapies that cause DNA breakage, makes these therapies more effective, since the histone H2AX which is essential in the functioning or repair of the genome is inhibited.

* In a first aspect, the present invention thus relates to a double-stranded oligonucleotide chosen from pairs, as boxed in Table 2 below:

-siAR-1 consisting of: SEQ ID NO: 1, or a sequence having at least 80% identity with said SEQ ID NO: 1 and SEQ ID NO: 2, or a sequence having at least 80% identity with said SEQ ID NO: 2;

-siAR-lb consisting of: SEQ ID NO: 3, or a sequence having at least 80% identity with said SEQ ID NO: 3 and SEQ ID NO: 4, or a sequence having at least 80% identity with said SEQ ID NO: 4;

-siAR-2 consisting of: SEQ ID NO: 5, or a sequence having at least 80% identity with said SEQ ID NO: 5 and SEQ ID NO: 6, or a sequence having at least 80% identity with said SEQ ID NO: 6;

-siAR-2b consisting of: SEQ ID NO: 7, or a sequence having at least 80% identity with said SEQ ID NO: 7 and SEQ ID NO: 8, or a sequence having at least 80% identity with said SEQ ID NO: 8;

-siAR-3 consisting of: SEQ ID NO: 9, or a sequence having at least 80% identity with said SEQ ID NO: 9 and SEQ ID NO: 10, or a sequence having at least 80% identity with said SEQ ID NO: 10;

-siAR-3b consisting of: SEQ ID NO: 11, or a sequence having at least 80% identity with said SEQ ID NO: 11 and SEQ ID NO: 12, or a sequence having at least 80% identity with said SEQ ID NO: 12, for its use in the prevention and / or treatment of cancer, said oligonucleotide being used in combination with a therapy causing DNA breakage.

The oligonucleotides of the present invention are indicated in Table 1 below:

NO: sequence 5 '- 3' sequence Name of the oligonucleotide Name of siRNA SEQ ID NO: 1 GACUCAGCUGCCCCAUCCA [dT] [dT] siAR-1 Strand 1 siAR-1 SEQ ID NO: 2 UGGAUGGGGCAGCUGAGUC [dT] [dT] siAR-1 Strand 2

NO: sequence 5 '- 3' sequence Name of the oligonucleotide Name of siRNA SEQ ID NO: 3 GACUCAGCUGCCCCAUCCA siAR-1 b Strand 1 siAR-1 b SEQ ID NO: 4 UGGAUGGGGCAGCUGAGUC siAR-1 b Strand 2 SEQ ID NO: 5 GACUCAGCUGCCCCAUCCACG [dT] [dT] siAR-2 Strand 1 siAR-2 SEQ ID NO: 6 CGUGGAUGGGGCAGCUGAGUC [dT] [dT] siAR-2 Strand 2 SEQ ID NO: 7 GACUCAGCUGCCCCAUCCACG siAR-2b Strand 1 siAR-2b SEQ ID NO: 8 CGUGGAUGGGGCAGCUGAGUC siAR-2b Strand 2 SEQ ID NO: 9 GACUCAGCUGCCCCAUCCAC [dT] [dT] siAR-3 Strand 1 siAR-3 SEQIDNO: 10 GUGGAUGGGGCAGCUGAGUC [dT] [dT] siAR-3 Strand 2 SEQ ID NO: 11 GACUCAGCUGCCCCAUCCAC siAR-3b Strand 1 siAR-3b SEQ ID NO: 12 GUGGAUGGGGCAGCUGAGUC siAR-3b Strand 2

For each of the sequences numbered from 1 to 12 (first column):

- The second column indicates the sequence in the 5 'to 3' orientation. The notation [dT] [dT] is used to indicate the presence of two overflowing deoxythymidines.

- The third column indicates the name of the oligonucleotide.

- The fourth column indicates the name of the siRNA constituted by the association of a strand-type oligonucleotide 1 or of an oligonucleotide whose sequence has at least 80% identity with this strand-type oligonucleotide, and of a strand 2 type oligonucleotide or an oligonucleotide whose sequence has at least 80% identity with this strand 2 type oligonucleotide.

When the siRNAs indicated in Table 1 consist of two single-stranded oligonucleotides whose sequence is 100% identical to that listed in Table 1, the target sequence of all these siRNAs (sequence of the mRNA to which they hybridize ) in the mRNA encoding the androgen receptor is identical in many species including man, rat, mouse, monkey, dog. The conservation of the target sequence of an siRNA between humans and other animal species is advantageous since it greatly increases the probability that the results of preclinical studies, especially those of toxicology, are predictive of effects in humans.

The target sequence of all the siRNAs indicated in Table 1 is transcribed from the exonl of the androgen receptor. Thus, all these siRNAs inhibit the expression of all forms of splicing of the androgen receptor, including those partially or completely deleted from the ligand binding domain, such as for example the AR-V7 variants of the receptor.

A “double-stranded oligonucleotide” according to the present invention is understood more particularly to be a siRNA (Small Interfering RNA) which comprises a so-called “guide” strand and a so-called “passenger” strand. More specifically, when the double-stranded oligonucleotide is a siRNA, the guide strand which is loaded into the RISC complex hybridizes with a region of an mRNA of which it is complementary. The mRNA is then cut by RISC and then degraded, or its translation is prevented. The protein corresponding to this mRNA can therefore no longer be synthesized.

The expression "at least 80% identity" means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%, especially 90%, 95% and 99%.

In a preferred aspect of the invention, said double-stranded oligonucleotide is the siAR-1 pair.

In one aspect of the invention, said oligonucleotide is used in a patient undergoing therapy resulting in DNA breakage.

According to the invention, said therapy results in a direct or indirect break in single-stranded or double-stranded DNA.

In one particular aspect, said therapy causing DNA breakage is chosen from radiotherapy and / or chemotherapy.

Radiation therapy refers to a method of locoregional cancer treatment, using radiation to destroy cancer cells by blocking their ability to multiply. The technique and modalities of radiotherapeutic treatment, in particular the doses of radiation delivered, are adapted according to the cancer and the patient. There are three main radiotherapy techniques: external radiotherapy, brachytherapy and vector metabolic radiotherapy. Each of them has its indications according to the type of tumor and its location.

- External radiotherapy is the best known and most used. The radiation source, outside the patient, produces high energy X-ray beams and electron beams.

- Brachytherapy, also called brachytherapy, is a local radiotherapy by implantation of grains or radioactive needles. Such therapy is used in particular in the treatment of prostate cancer.

- Vector metabolic radiotherapy is a radiotherapy technique based on the administration by the oral route or by injection of a radiopharmaceutical agent marked with a radioelement. The radioactive isotope will preferentially bind to the sick target cells. For example, iodine 131 attaches to the thyroid. Bone metastatic pain can thus be treated for example by injection of samarium 153, liquid strontium chloride marked with strontium 894, or radium chloride 223.

Chemotherapy refers to drug therapy (cytostatic and antineoplastic chemotherapeutic agents) against cancer.

In a particular aspect, said chemotherapy causing DNA breakage is carried out using at least one chemotherapeutic agent chosen from: alkylating agents, anti-metabolite agents, cytotoxic antibiotics, topoisomerase I inhibitors, topoisomerase II inhibitors, anti-tumor antibiotics, genotoxic agents, PARP (poly (ADP-ribose) polymerase) inhibitors.

In one particular aspect, a chemotherapeutic agent is chosen in particular from: Arabinosylcytosine, Bléomycine, Busulfan, Capecitabine, Carboplatine, Carmustine, Chlorambucil, Cisplatine, Cladribine, Cyclophosphamide, Dacarbazine, Daunorubicine, Doxorubicine, Epiruborine Flourine, Flouridin, Flouridin, Flouridin, Epirubinine 5-Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Ifosfamide, Iniparib, Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine and in particular 6Mercaptopurine, Methotrexate, Mitomycine C, Mitoxantrone HCl, Mustinalararar, Nirapin, Nirapin, Rin , Talazopanib, Thioguanine and in particular 6-Thioguanine, Topotecan, Veliparib, Actinomycin D, Amsacrine, Anthracyclines, Camptotecin, Epipodophyllotoxin, Plicamycin, Temozolomide.

In one particular aspect, the cancer is a primary tumor, or a metastasis of a primary tumor.

In one particular aspect, the cancer is a prostate cancer or a non-prostatic cancer.

In a particular aspect, cancer is cancer of the anus, appendix, mouth, bronchi and / or upper airways, bile duct, nasal and paranasal cavity, brain, heart , cervix, colon, uterine body, stomach, liver, salivary glands, throat, tongue, lips, nasopharynx, esophagus, bone, ovary, pancreas, parathyroid, penis, pleura, lung, prostate, rectum, kidney, breast, adrenal, testes, head and neck, thymus, thyroid, urethra, vagina, gallbladder, bladder, vulva, gastrointestinal cancer, lymphoma, melanoma or non-melanoma skin cancer, myeloma , sarcoma, leukemia, mesothelioma, cholangiocarcinoma, osteosarcoma, glioblastoma, astrocytoma, oligodendroglioma, chondrosarcoma, liposarcoma, rhabdomyosarcoma, a pheochromocytoma, or the metastases of these cancers that develop in other organs.

In one particular aspect, said therapy leading to DNA breakage and the administration of said oligonucleotide are simultaneous or successive. The term "successive" is understood according to the invention of the expression "separated, spread over time".

In a particular aspect, said oligonucleotide is formulated for a systemic administration mode, continuously or as a bolus, in single or repeated doses.

According to the invention, the terms "formulated for" also include terms "administered by".

In a particular aspect, said systemic mode of administration is chosen from the subcutaneous, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, intravaginal, intrarectal, sublingual, intrathecal, intracerebral, oral administration mode, and is in particular sub- skin.

In a preferred aspect, said mode of administration is continuous, subcutaneous.

In a particular aspect, said oligonucleotide is formulated for a mode of administration at a therapeutically effective dose, and in particular at doses from 0.005 mg / kg / day to 30 mg / kg / day, in particular 0.01 mg / kg / daily at 10 mg / kg / day.

From "0.005 mg / kg / day to 30 mg / kg / day" means all doses from 0.005 mg / kg / day to 30 mg / kg / day, for example 0.008; 0.01; 0.05; 0.1; 0.5; 1.0; 1.5; 10.0;

10.5; 14.0; 14.5; 20; 20.5; 25; 25.5; 29.5 mg / kg / day.

According to the invention, said oligonucleotide may present chemical modifications on one and / or the other strand, on one or more nucleotides located at the 3 ′ or 5 ′ terminal ends, and / or on one or more nucleotides constituting the skeleton internal. For example, said chemical modifications are located on ribose and / or base and / or phosphoric acid. Said chemical modifications comprise, for example, at least one substitution of the OH group at 2 'of the ribose with a 2'-O-methyl RNA (2'OMe) or 2'-O-methoxyethyl (2'MOE) or 2'-fluoro group. (2'F) or 2'-fluoro-3-arabinonucleotide (FANA), a 2 'alkylation of oxygen to aminoethyl-, guanidinoethyl-, cyanoethyl- or allyl, a replacement of the phosphodiester group by a phosphorothioate, an alkylation or a thiloation of one or more nucleotides of the oligonucleotide, the replacement of a ribonucleotide by a deoxyribonucleotide, or the replacement of a nucleotide by a Locked Nucleic Acid (LNA).

In a particular aspect, said oligonucleotide is devoid of chemical modification.

In a particular aspect, said oligonucleotide is devoid of chemical modification and comprises two deoxynucleotides protruding at the 3 ′ end, in particular two protruding deoxythymidines.

In a particular aspect, said oligonucleotide is devoid of chemical modification and does not comprise two deoxynucleotides projecting at the 3 ′ end, in particular two projecting deoxythymidines.

In a particular aspect, said oligonucleotide is in a solution containing no vectoring agent.

In a particular aspect, said oligonucleotide is in association with a vectoring agent.

According to the invention, a vectoring agent is an agent which facilitates the penetration of the oligonucleotide into the cells of the organism. This agent can be chemically conjugated or noncovalently associated with the oligonucleotide. These are, for example, lipids, polymers, peptides, dendrimers, polyethylenimine derivatives, nanoparticles, magnetic spheres, or inorganic or organic nanostructures.

In all aspects of the present invention, said oligonculeotide can be associated with a vectoring agent, whether it is associated with an addressing molecule or whether it is not associated with an addressing molecule.

According to the invention, an addressing molecule is a molecule addressing the oligonucleotide either passively towards a tumor, such as for example PEG, or actively towards a particular cell type, such as for example endothelial cells or cells cancerous. For example and in a non-exhaustive manner, an addressing molecule can be, an aptamer, an antibody, transferrin, an RGD peptide, a ligand, or a ribozyme, this targeting molecule targeting a molecule expressed on the surface of cells targeted, such as a receptor or PSMA (Prostate Specifies Membrane Antigen).

In addition, and in all aspects of the present invention, said oligonucleotide can be associated with an addressing molecule, whether it is associated with a vectoring agent or whether it is not associated with a vectoring agent.

In a preferred aspect, said oligonucleotide is associated with an addressing molecule and is not associated with a vectoring agent.

In a preferred aspect, said oligonucleotide is not associated with an addressing molecule and is not associated with a vectoring agent.

According to the invention, said prevention and / or said treatment is carried out in a human patient.

In one aspect, the invention also relates to a combination product containing:

- a double-stranded oligonucleotide chosen from pairs: siAR-1, siAR-lb, siAR2, siAR-2b, siAR-3, siAR-3b, and at least one chemotherapeutic agent which causes DNA breakage, for use simultaneous or successive in the prevention and / or treatment of cancer.

In one aspect, the invention also relates to a combination product containing:

a double-stranded oligonucleotide chosen from pairs: siAR-1, siAR-1b, siAR-2, siAR-2b, siAR-3, siAR-3b, and at least one radiopharmaceutical agent which causes DNA breakage, for simultaneous or successive use in the prevention and / or treatment of cancer.

* In a second aspect, the invention also relates to a double-stranded oligonucleotide chosen from pairs: siAR-1, siAR-lb, siAR-2, siAR-2b, siAR-3, siAR-3b for its use in prevention and / or the treatment of cancer, with the exception of prostate cancer dependent on the androgen receptor.

"Androgen receptor-dependent prostate cancer" is defined as prostate cancer that expresses and is dependent on the androgen receptor for signaling activated by this receptor.

In a particular aspect of this second aspect, said cancer is cancer of the liver, breast, kidney, bladder, or the metastases of these cancers developing in other organs.

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In a particular aspect of this second aspect, said cancer is cancer of the anus, appendix, mouth, bronchi and / or upper airways, bile duct, nasal and paranasal cavity, brain, heart, cervix, colon, uterine body, stomach, liver, salivary glands, throat, tongue, lips, nasopharynx, l esophagus, bones, ovary, pancreas, parathyroid, penis, pleura, lung, androgen-independent prostate, rectum, kidney, breast, adrenals, testes, head and neck, thymus, thyroid, urethra, vagina, gallbladder, bladder, vulva, gastrointestinal cancer, lymphoma, melanoma or cancer skin excluding melanoma, myeloma, sarcoma, leukemia, mesothelioma, cholangiocarcinoma, osteosarcoma, glioblastoma, astrocytoma, oligode ndroglioma, chondrosarcoma, liposarcoma, rhabdomyosarcoma, pheochromocytoma, or metastases from these cancers developing in other organs, and more particularly androgen-independent prostate cancer.

In a particular aspect of this second aspect, said oligonucleotide is formulated for a continuous or bolus systemic administration mode, in single or repeated doses.

In a particular aspect of this second aspect, said continuous systemic administration mode is chosen from the subcutaneous, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, intravaginal, intrarectal, sublingual, intrathecal, intracerebral, oral administration mode. and is notably subcutaneous.

In a particular aspect of this second aspect, said oligonucleotide is formulated for a mode of administration at a therapeutically effective dose, and in particular at doses from 0.005 mg / kg / day to 30 mg / kg / day, in particular 0.01 mg / kg / day to 10 mg / kg / day.

In a particular aspect of this second aspect, said oligonucleotide exhibits chemical modifications such as chemical modifications on one and / or the other of the two strands, on one or more nucleotides situated at the 3 ′ or 5 ′ terminal ends, and / or on one or more nucleotides constituting the internal skeleton.

In a particular aspect of this second aspect, said oligonucleotide is devoid of chemical modification.

In a particular aspect of this second aspect, said oligonucleotide is devoid of chemical modification, and comprises two deoxynucleotides protruding at the 3 ′ end, in particular two protruding deoxythymidines.

In a particular aspect of this second aspect, said oligonucleotide is devoid of chemical modification and does not comprise two deoxynucleotides protruding at the 3 ′ end, in particular two protruding deoxythymidines.

In a particular aspect of this second aspect, said oligonucleotide is in solution containing no vectoring agent or is not associated with a vectorizing agent.

In a particular aspect of this second aspect, said oligonucleotide is in solution containing a vectoring agent or is associated with a vectorizing agent.

In a particular aspect of this second aspect, said oligonucleotide is associated or is not associated with an addressing molecule.

In a particular aspect of this second aspect, said prevention and / or said treatment is carried out in a human patient.

The invention will be better illustrated by the following examples and figures. The examples below aim to clarify the subject of the invention and illustrate advantageous embodiments, but in no case aims to limit the scope of the invention.

Legend of figures:

Figure 1: Complementarity of siRNAs sequences with mRNA coding in humans the androgen receptor and with that coding H2AX.

The positions shown correspond respectively to those of the human androgen receptor mRNA (AR, Accession Number: NM_000044.3) and of the H2AX mRNA in humans (Accession Number: NM_002105.2). The underlined bases are those which are perfectly complementary to those of the antisense strand of siRNA, that is to say identical to those of the sense strand shown in the figure. AR is represented by SEQ ID NO: 13; H2AX by SEQ ID NO: 14.

Figure 2: Quantification by RT-qPCR of mRNAs encoding AR, PSA and H2AX 48 hours after transfection of a control siRNA (cont) or of siAR-1. Y-axis: quantity of mRNA indicated (AR, PSA, H2AX), normalized by the quantity of mRNA Cyclophilin A. The values measured in the cells treated with siAR-1 (light gray bars) are compared to those measured in the cells transfected with a control siRNA (dark gray bars). LNCaP and C4-2 correspond to tumor cells which have been transfected with the control siRNA and siAR-1.

Figure 3: Quantification by RT-qPCR of mRNAs coding H2AX 48 hours after transfection of a control siRNA, siAR-1 or siAR-2. Ordinate: quantity of H2AX mRNA, normalized by the quantity of Cyclophilin A mRNA in C4-2 cells treated with siAR-1 or siAR-2, compared to that measured in cells transfected with a control siRNA.

Figure 4: Immuno detection by western blot of the expression of AR and H2AX 48 hours after transfection of the cells by a control siRNA or by siAR-1 noted here SXL01. The molecular weights of markers are shown on the left.

Figure 5: Immuno detection of γΗ2ΑΧ (first column) and 53BP1 (second column) in C4-2 cells treated for 6 h with bleomycin (10μΜ, second and fourth lines) or the vehicle (first and third lines) added 72 h after transfection of the cells with a control siRNA or with siAR-1. DAPI (third column) is used to detect cell nuclei.

Figure 6: Cell viability, measured by an MTT metabolic test, 48 hours after treatment with bleomycin 10 μΜ or by its vehicle added 24 hours after transfection of the cells with a control siRNA or with siAR-1. Abscissa: dose of Bleomycin (first line) and siRNA transfected (second line) in LNCaP cells (light gray bars) or C4-2 (black bars). Ordinate, measurement of MTT activity by optical density. The values of the various samples are compared to the average of the values measured in the cells cultured under control condition.

EXAMPLES

Example 1: siRNAs siAR-1, siAR-1b, siAR-2, siAR-2b, siAR-3, siAR-3b hybridize with mRNA encoding AR, and with that encoding H2AX.

The alignments shown in Figure 1 show that 17 bases of H2AX hybridize perfectly with those of siAR-lb, siAR-2b or siAR-3b. Hybridization with the oligonucleotides siAR-1, siAR-2, siAR-3 is identical, except for the two deoxythymidines overflowing in 3 ′ which do not hybridize.

Example 2: siAR-1 inhibits the expression of H2AX.

The LNCaP or C4-2 tumor cells were transfected with a control siRNA (cont) or with siAR-1. The cells were harvested 48 hours after transfection, the mRNAs extracted and quantified by RT-qPCR. Cycophilin A mRNA, which is not regulated by AR, is quantified to normalize the results with respect to the mRNA content involved in the reaction (delta-delta CT method).

The results are presented in FIG. 2. It is observed that in the two lines, siAR-1 inhibits the expression of AR and of PSA which is a gene regulated by AR. In addition, siAR-1 inhibits the expression of H2AX by more than 90% in both lines.

Example 3: siAR-1 and siAR-2 inhibit the expression of H2AX.

The C4-2 tumor cells were transfected with a control siRNA (cont), with siAR-1 or with siAR-2. The cells were harvested 48 hours after transfection, the mRNAs extracted and quantified by RT-qPCR. Cycophilin A mRNA, which is not regulated by AR, is quantified to normalize the results with respect to the mRNA content involved in the reaction (delta-delta CT method).

The results are presented in Figure 3. It is observed that siAR-1 and siAR-2 inhibit the expression of H2AX, siAR-1 more effectively than siAR-2.

Example 4: Inhibition of the protein expression of H2AX by siAR-1.

C4-2 castration-resistant prostate tumor cells were transfected with a control siRNA (cont) or with siAR-1, noted SXL01. 48 hours after transfection, the cellular proteins were analyzed by western blot to quantify the expression of AR and H2AX. Tubulin immuno detection is used to ensure that comparable amounts of protein are analyzed under different conditions. The results are presented in FIG. 4. It is observed that siRNA siAR-1 effectively inhibits the expression of AR but also that of H2AX.

Example 5: The inhibition of H2AX prevents the recruitment of 53BP1 on the DNA lesions induced by bleomycin and decreases cell viability.

Bleomycin is an agent that induces DNA breaks. This treatment very quickly induces the phosphorylation of H2AX (then noted γΗ2ΑΧ) and its recruitment on DNA, which allows the assembly of the DNA repair machinery, which notably includes the protein 53BP1.

C4-2 cells were transfected with a control siRNA or with siAR-1. 72 hours after transfection, the cells were then treated for 6 hours with 10 μΜ of bleomycin or with the corresponding vehicle and then fixed. The proteins γΗ2ΑΧ and 53BP1 were detected by indirect immunofluorescence with specific antibodies. DAPI is used to visualize cell nuclei.

The results are presented in FIG. 5. It is observed that in the cells transfected with the control siRNA in the absence of bleomycin, γΗ2ΑΧ is detectable but weakly expressed, while the expression of 53BP1 is mainly diffuse. Treatment with bleomycin strongly increases the expression of γΗ2ΑΧ while the labeling of 53BP1 becomes punctiform, reflecting its recruitment via γΗ2ΑΧ on DNA lesions. In cells transfected with siAR-1, the expression of H2AX in cells not treated with bleomycin is very reduced compared to that of cells transfected with control siRNA and not treated with bleomycin while the expression of 53BP1 is comparable. After treatment with bleomycin, there is no increase in H2AX in cells transfected with siAR-1, nor recruitment of 53BP1 on focal points for repairing DNA damage. This indicates a deficit in DNA repair.

In another experiment, LNCaP or C4-2 cells were transfected with a control siRNA or with siAR-1. 24 hours after transfection, the cells were treated for 48 hours with ΙΟμΜ bleomycin or by the vehicle then their metabolic activity measured by an MTT test, which makes it possible to assess the viability of the cells. The metabolic activity of the cells was related to that measured in the cells under control condition (transfection with the siRNA control, treatment with the vehicle). The results are represented in FIG. 6. It is observed that in the two lines, the treatment with siAR-1 alone and that with bleomycin alone reduce the cell viability. The combination of these two treatments produces effects approximately twice as additive to inhibit proliferation showing a synergistic effect between these two treatments.

Claims (11)

1. Double-stranded oligonucleotide chosen from the pairs siAR-1, siAR-1b, siAR-2, siAR-2b, siAR-3, siAR-3b, as indicated in Table 2, for its use in the prevention and / or the treatment of cancer, said oligonucleotide being used in combination with a therapy resulting in a break in the single strand or double strand DNA. 2. Double stranded oligonucleotide for use according to claim 1, wherein said therapy causing DNA breakage is chosen from radiotherapy and / or chemotherapy. 3. Double-stranded oligonucleotide for use according to claim 2, wherein said chemotherapy causing DNA breakage is carried out using at least one chemotherapeutic agent chosen from: alkylating agents, antimetabolite agents, antibiotics cytotoxic agents, topoisomerase I inhibitors, topoisomerase II inhibitors, anti-tumor antibiotics, genotoxic agents, PARP inhibitors, and is especially chosen from Arabinosylcytosine, Bleomycin, Busulfan, Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin , Cladribine, Cyclophosphamide, Dacarbazine, Daunorubicin, Doxorubicin, Epirubicin, Etoposide, Floxuridine, Fludarabine, Fluorouracil and in particular 5-Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicine, Ifosfamide, Iniparibine, Irinomine, Irinpine, Minephaine , Methotrexate, Mitomycin C, Mitoxantrone HCl, Mustine, Ni raparib, Olaparib, Oxaliplatin, Procarbazine, Rubitecan, Rucaparib, Streptozocin, Talazopanib, Thioguanine and in particular 6-Thioguanine, Topotecan, Veliparib, Actinomycin D, Amsacrine, Anthracyclines, Camptotecin, Epipodophylline. 4. Double-stranded oligonucleotide for use according to any one of the preceding claims, in which the cancer is cancer of the anus, appendix, mouth, bronchi and / or upper airways, bile duct, nasal and paranasal cavity, brain, heart, cervix, colon, uterine body, stomach, liver, salivary glands, throat, tongue, lips, nasopharynx, esophagus, bones, ovary, pancreas, parathyroid, penis, pleura, lung, prostate, rectum, kidney, breast, adrenal, testes, head and neck, thymus, thyroid, urethra, vagina, gallbladder, bladder, vulva, gastrointestinal cancer, lymphoma, melanoma or non-melanoma skin cancer, myeloma, sarcoma, leukemia, mesothelioma, cholangiocarcinoma, osteo sarcoma, glioblastoma, astrocytoma, oligodendroglioma, chondrosarcoma, liposarcoma, rhabdomyosarcoma, pheochromocytoma, or metastases from these cancers that develop in other organs. 5. Double-stranded oligonucleotide for use according to any one of the preceding claims, in which said oligonucleotide is formulated for a continuous or bolus systemic administration mode, in single or repeated doses, and in particular in which said mode of administration systemic is chosen from the subcutaneous, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, intravaginal, intrarectal, sublingual, intrathecal, intracerebral, oral, and is in particular subcutaneous administration mode continuous and subcutaneous. 6. Double stranded oligonucleotide for use according to any one of the preceding claims, wherein said oligonucleotide is formulated for a mode of administration at a therapeutically effective dose, and in particular at doses of 0.005 mg / kg / day to 30 mg / kg / day, more particularly 0.01 mg / kg / day to 10 mg / kg / day. 7. Double-stranded oligonucleotide chosen from the pairs siAR-1, siAR-1b, siAR-2, siAR-2b, siAR-3, siAR-3b, as indicated in Table 2, for its use in prevention and / or the treatment of cancer, with the exception of prostate cancer dependent on the androgen receptor. 8. Double-stranded oligonucleotide for use according to claim 7, in which said oligonucleotide is formulated for a continuous or bolus systemic administration mode, in single or repeated doses, and in particular in which said continuous systemic administration mode is chosen. among the subcutaneous, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, intravaginal, intrarectal, sublingual, intrathecal, intracerebral, oral, and in particular continuous and subcutaneous mode of administration. 9. Double-stranded oligonucleotide for use according to claim 7 or 8, wherein said oligonucleotide is formulated for a mode of administration at a therapeutically effective dose, and in particular at doses of 0.005 mg / kg / day to 30 mg / kg / day, more particularly 0.01 mg / kg / day to 10 mg / kg / day. 10. Double stranded oligonucleotide for use according to any one of claims 1 to 9, wherein said oligonucleotide is in a solution containing a vectoring agent or not containing a vectorizing agent. 11. Double-stranded oligonucleotide for use according to any one of claims 1 to 10, wherein said oligonucleotide is in a solution, and is associated or not with an addressing molecule. 1/3