WO2017182580A1 - Procédés de diagnostic et de traitement de troubles liés à la reproduction et procédés de contraception - Google Patents

Procédés de diagnostic et de traitement de troubles liés à la reproduction et procédés de contraception Download PDF

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WO2017182580A1
WO2017182580A1 PCT/EP2017/059416 EP2017059416W WO2017182580A1 WO 2017182580 A1 WO2017182580 A1 WO 2017182580A1 EP 2017059416 W EP2017059416 W EP 2017059416W WO 2017182580 A1 WO2017182580 A1 WO 2017182580A1
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nucleic acid
seq
gnrh
mir200
mir155
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Vincent PREVOT
Andrea MESSINA
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Inserm (Institut National De La Sante Et De La Recherche Medicale)
Chru (Centre Hospitalier Régional Universitaire) De Lille
Université De Lille 1
Université Lille 2 Droit et Santé
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Publication of WO2017182580A1 publication Critical patent/WO2017182580A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to the field of the diagnosis and of the therapeutic treatment of reproduction-related disorders and the field of contraception.
  • HPG hypothalamic-pituitary-gonadal
  • a first group of disorders is encompassing hypogonadotropic hypogonadism and delayed puberty, for which impaired secretion of gonadotropins by the pituitary gland is observed, i.e., the follicle- stimulating hormone (FSH) and luteinizing hormone (LH), which secretion is itself driven by GnRH neurons in the brain.
  • FSH follicle- stimulating hormone
  • LH luteinizing hormone
  • a second group of disorders include (i) hypergonadotropic hypogonadism for which the hypothalamic-pituitary axis is over-activated with no response of the gonads, (ii) Turner syndrome, anovulation, premature ovarian failure and menopause-related disorders, for which the gonads should exert a negative feedback on the hypothalamus and the pituitary gland, and (iii) early puberty, for which premature activation of the HPG axisis observed.
  • individuals having hypogonadotropic hypogonadism or delayed puberty may be treated with human chorionic gonadotropin (hCG), with a pulsatile GnRH pump or with a hormone replacement treatment.
  • hCG human chorionic gonadotropin
  • the hCG treatment may often be ineffective and the treatment involving a GnRH pump is a very expensive.
  • many individuals undergoing a hormone replacement treatment have been reported to suffer from negative effects with respect to healthy development.
  • microRNAs mechanistically, several microRNAs (miRNAs or miRs) have been reported to be involved in several regulatory mechanisms associated with fertility and/or puberty.
  • Hasuwa et al. (Science; 2013, 341 :71-73) reported that female mice lacking the miR200b and the miR429 microRNAs are unable to ovulate and are infertile.
  • PCOS polycystic ovary syndrome
  • the invention relates to a method for diagnosing a reproduction- related disorder in an individual, comprising:
  • step b) comparing level value of a miR200 nucleic acid and/or miR155 nucleic acid measured at step a) to a reference level value of the said miR200 acid nucleic and/or miR155 nucleic acid.
  • the invention also relates to a method for preventing or treating a reproduction-related disorder due to a low expression of GnRH in an individual, comprising a step of administering to the said individual a compound selected in a group comprising a miR200 nucleic acid, a compound mimicking a miR200 nucleic acid, a miR155 nucleic acid and a compound mimicking a miR155 nucleic acid.
  • the invention relates to a compound selected in a group comprising a miR200 nucleic acid, a compound mimicking a miR200 nucleic acid, a miR155 nucleic acid and a compound mimicking a miR155 nucleic acid, for use in the prevention or the treatment of a reproduction-related disorder due to a low expression of GnRH in an individual.
  • the invention further relates to a method for preventing or treating a reproduction-related disorder due to a high expression of GnRH in an individual, comprising a step of administering to the said individual a compound selected in a group comprising a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and a compound preventing the binding of miR155 nucleic acid to a target nucleic acid.
  • the invention relates to a compound selected in a group comprising a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and a compound preventing the binding of miR155 nucleic acid to a target nucleic acid, for use in the prevention or the treatment of a reproduction-related disorder due to a high expression of GnRH in an individual.
  • Figure 1 Schematic diagram illustrating the genetic strategy to invalidate Dicer expression specifically in GnRH-expressing cells in mice.
  • D-Fl, D-Rl, D-Dl, C-Fl and C-Rl are primers used for genotyping.
  • Figure 4 Plot illustrating the RT-PCR analysis of the expression of Gnrh promoter activators in FACS-sorted GnRH-GFP neurons from mice selectively lacking Dicer in GnRH neurons (grey bars) or wild-type for Dicer (white bars) All values are expressed relative to wild-type values, set at 1 (t-test, D cer loxP/loxP versus Gnrhr. creiDice?
  • FIG. 5 Plot illustrating the RT-PCR analysis of the expression of miR-200 family members in FACS-sorted cells from the preoptic region micro-dissected from Gnrhr.
  • Figure 7 Plot illustrating the RT-PCR analysis of the expression of Zebl in GnRH-GFP neurons isolated from Gnrh: :Gfp;Dicer +l+ mice at P7 and P12 (a) or from mice in which Dicer expression is invalidated or not in GnRH neurons (b).
  • Figure 8 Plot illustrating the effect of the intracerebroventricular (i.c.v) injection of TSB-200 or a scrambled sequence on the expression of Zebl, Lpinl and Maf.
  • the grey zone shows data that were obtained from P28 mice. Values are expressed relative to P7, wild-type or control values, as appropriate, set at 1. (*P ⁇ 0.05; **P ⁇ 0.01). Values shown are means ⁇ s.e.m.
  • Figure 9 Plot illustrating the effect of the intracerebroventricular (i.c.v) injection of TSB-200 or a scrambled sequence on the expression of GnRH transcript and Gnrh promoter activity in GnRH-GFP neurons of infantile mice at PI 2.
  • the grey zone shows data that were obtained from P28 mice. Values are expressed relative to P7, wild- type or control values, as appropriate, set at 1. (*P ⁇ 0.05; **P ⁇ 0.01). Values shown are means ⁇ s.e.m.
  • Figure 10 Plot illustrating the RT-PCR analysis of the expression of miR-155 in FACS-isolated GnRH-GFP neurons between P7 and P12 (**P ⁇ 0.01).
  • FIG. 11 Plot illustrating the effect of the i.c.v. injection of TSB-155 or a scrambled sequence (CTRL) on the expression of Cebpb, Lpinl and Maf. ⁇ **P ⁇ 0.01). Values are expressed relative to untreated wild-type values, set at 1. Values shown are means ⁇ s.e.m.
  • Figure 12 Plot illustrating the effect of the i.c.v. injection of TSB-200 (white bar) or a scrambled sequence (CTRL, grey bar) on the expression of on Gnrh promoter activity, and GnRH and Zebl transcripts in GnRH-GFP neurons of infantile mice.
  • Figure 13 Plot illustrating the RT-PCR analysis of expression levels of Gpr54 mRNA in FACS-sorted GnRH-GFP neurons (** P ⁇ 0.01).
  • (b) Plot illustrating a L-NAME treatment partially rescues Gnrh mRNA expression in P12 mice harbouring a Dicer deficiency in GnRH neurons. Gnrh mRNA levels were arbitrarily set at 1 in control P12 D cer loxP/loxP mice (line). (*P ⁇ 0.05; ****p ⁇ 0.0001). Values are expressed relative to untreated wild-type values, set at 1. Values shown are means ⁇ s.e.m.
  • Figure 16 Plot illustrating the knocking down of the ability of miR-200 and/or miR-155 to repress Zebl and Cebpb expression, respectively, during the infantile period markedly accelerates puberty. Grey line and arrow denote peripuberty at P38. (1) is for CTRL; (2) for TSB-200; (3) TSB-155; (4) combination of TSB-200 and TBS-155.
  • Figure 17 Plot illustrating the circulating levels of LH in P38, in which TSB-200, TSB-155, TSB-200+155 or scrambled control sequences were infused into the brain during the infantile period (*P ⁇ 0.05).
  • FIG. 18 Plot illustrating the LH levels in P28 juvenile mice, in which TSB-200 or scrambled control sequences were infused into the brain during the infantile period (*P ⁇ 0.05).
  • FIG 19. Plot illustrating the estrous cyclicity before and after the infusion of TSB-200 into the preoptic region of adult mice (arrows "1"). Upward arrows show the time of blood collection in control (arrows “2") and TSB-200-treated (arrows “3") mice. Di, diestrus; E, estrus; P, proestrus.
  • Figure 20 Plot illustrating the LH levels in blood samples collected during proestrus as indicated by the colored arrows "2" and "3" in Figure 19. Values shown are means ⁇ s.e.m.
  • Figure 21 Schematic diagram illustrating the miRNAs whose expression is specifically enriched in GnRH neurons at P12, which include members of the miR-200 family, which is known to control Zebl expression.
  • Figure 22 Schematic diagram illustrating the distribution of putative Zebl -binding sites in the upstream region of GnRH modulator genes as well as in GnRH and Gpr54 genes.
  • FIG. 23 Diagram showing the distribution of putative Zebl binding sites in the human GNRH gene (upper panel) and their validation using a Zebl chromatin immunoprecipitation assay in an immortalized mouse cell line secreting GnRH and cultured in the presence (fbs) or absence (sfm) of fetal bovine serum. Values are expressed relative to the immunoprecipitation of chromatin containing the GnRH promoter region with irrelevant IgG species, arbitrarily set at 1 (dotted grey line). T-test, serum- free medium (sfm) vs.
  • FIG. 24 Schematic diagram illustrating a target site blocker (TSB) designed to selectively impair the ability of miR-200b/200c/429 to target the Zebl transcript in the infantile hypothalamus.
  • TLB target site blocker
  • Figure 25 Schematic diagram illustrating the putative double negative- feedback loop illustrating the potential contribution of mir200, Zebl and PoufZfl, which are both known to control the expression of the former in the infantile control of GnRH gene expression. Values shown are means ⁇ SEM.
  • Figure 26 Scheme illustrating the sequences corresponding to the immature and mature forms of miR-155.
  • Figure 27 Schematic diagram illustrating the predicted consequential pairing of miR-155 (top) with the target region (bottom).
  • FIG 28 Schematic diagram illustrating the strategy used to selectively block the binding of miR-155, which has many gene targets, to Cebpb.
  • Figure 29 Schematic diagram illustrating the fact that the TSB sequence encompasses the miR-155 binding site and contains an additional motif that selectively hybridizes with Cebpb.
  • Figure 30 Schematic representation of a miR A-gene network potentially regulating GnRH expression in the infantile period, and its crosstalk with nitrergic neurotransmission.
  • Figure 33 Plot illustrating the RT-PCR analysis of the expression of Zebl in
  • Figure 34 Schematic diagram illustrating the rebound hypothesis.
  • Figures 35-38 Schematic diagrams illustrating the potential contribution of the miR-155 and miR-200 families and their target genes to the increase in GnRH gene expression during the infantile period of postnatal development, and how these events could be intertwined with the integration of postmigratory GnRH neurons into the neural network responsible for regulating the timely onset of puberty. GnRH neuroglial network maturation ( Figures 35-37).
  • hypothalamus Although the morphological development of the hypothalamus is almost complete at birth, axons of the neurons located in the arcuate nucleus of the hypothalamus (ARH), which are thought to mediate at least part of the effects of gonadal steroid on the HPG axis, first reach the preoptic region during the infantile period, when astrogliogenesis, synaptogenesis, and dendritic pruning are also thought to occur. These maturational events are well positioned to trigger the miRNA-evoked changes in the transcription factor-gene network controlling GnRH promoter activity during the infantile period, which we have uncovered here. Hormonal profiles ( Figure 38). The hormonal profiles illustrated in the schematic are those for a female mouse.
  • the first postnatal activational period of the HPG axis coincides with the arrival of ARH fibers in the preoptic region, which results in an infantile surge of FSH that triggers the growth of the first pool of ovarian follicles, destined to ovulate at puberty, as well as the sporadic elevation of LH levels that contributes to their maturation.
  • This critical time-window for sexual maturation is highly pronounced of "mini-puberty" in humans.
  • the second activational period occurs during the peripubertal period, when a diurnal rhythm of LH release that accelerates the functional development of the ovaries is established.
  • a third and final activational period coincides with the moment when ovarian follicles reach full maturity, i.e. the Graafian stage, and release massive amounts of ovarian steroids, specifically estrogens, which exert a positive-feedback effect on the HPG, coordinating the onset of the first preovulatory GnRH and LH/FSH surge and triggering the first ovulation, thus conferring fertility on the individual.
  • the primary events that initiate the onset of puberty originate within the hypothalamus
  • GnRH gonadotropin-releasing hormone
  • the present inventors have shown that a natural switch in miRNA expression patterns in infantile GnRH neurons is likely to occur, which switch would invert the balance between inductive and repressive signals, triggering increased hypothalamic GnRH expression and controlling the crucial transition from the early infantile phase, when its levels are low, to the GnRH-fuelled run-up to puberty.
  • miR200 and miR155 Two essential components of this switch, miR200 and miR155, respectively regulate Zebl, a repressor of Gnrh transcriptional activators and Gnrh itself, and Cebpb, a nitric oxide-mediated repressor of Gnrh that acts both directly and through Zebl, in GnRH neurons.
  • This alteration in the delicate balance between inductive and repressive signals induces the normal GnRH-fuelled run-up to correct puberty initiation, and interfering with this process disrupts the neuroendocrine control of reproduction.
  • MicroRNAs are short ( ⁇ 22-nucleotide) noncoding RNAs that silence gene expression post-transcriptionally, principally by binding to 3' untranslated regions (3'UTR) of target mRNAs. Mature miRNAs are known to be required for the normal differentiation and function of several cell types (5) and, in particular, to regulate somatic growth and fertility at the level of peripheral organs (6, 7). We therefore asked whether miRNAs could play a similarly critical role in the central neuroendocrine control of reproduction.
  • miR200 nucleic acid and/or miR155 nucleic acids are up-regulated or down-regulated, and therefore may be considered as good biomarkers in the occurrence of certain reproduction-related disorders.
  • MicroRNAs are small, noncoding RNAs that are emerging as crucial regulators of biological processes.
  • miRNA means a non-coding RNA of about 18 to about 25 nucleotides in length. These miRs could originate from multiple origins including: an individual gene encoding for a miRNA, from introns of protein coding gene, or from poly-cistronic transcript that often encode multiple, closely related microRNAs.
  • Ste- loop sequence means a RNA having a hairpin structure and containing a mature microRNA sequence. Pre-miRNA sequences and stem-loop sequences may overlap. Examples of stem-loop sequences are found in the microRNA database known as miRBase.
  • MicroRNA precursor means a transcript that originates from a genomic DNA and that comprises a non-coding, structured RNA comprising one or more microRNA sequences.
  • a microRNA precursor is a pre-miRNA.
  • a microRNA precursor is a pri-miRNA.
  • mir-X refers to the pre-miRNA
  • miR-X refers to the mature form.
  • a -3p or -5p suffix When relative expression levels are known, an asterisk following the name indicates a microRNA expressed at low levels relative to the microRNA in the opposite arm of a hairpin.
  • miR-X refers to the mature miRNA including both forms -3p and -5p, if any.
  • microRNA, miRNA and miR designate the same product.
  • the invention relates to a method for diagnosing a reproduction- related disorder in an individual, comprising:
  • step b) comparing level value of a miR200 nucleic acid and/or miR155 nucleic acid measured at step a) to a reference level value of the said miR200 acid nucleic and/or miR155 nucleic acid.
  • the said method is performed in vitro or ex vivo.
  • the individual is a human or non-human mammal, preferably a human mammal.
  • the method further comprises the step of identifying variants in the sequence of a miR200 nucleic acid and/or miR155 nucleic acid in a biological sample from an individual.
  • a non-human mammal includes a mammal comprising, a pet such as a dog, a cat, a domesticated pig, a rabbit, a ferret, a hamster, a mouse, a rat and the like; a primate such as a chimp, a monkey, and the like; an economically important animal such as cattle, a pig, a rabbit, a horse, a sheep, a goat.
  • nucleic acid sequences presented herein are related to humans. It is needless to mention that paralogs, orthologs, and related nucleic acid sequences, especially in chimp, squirrel, mouse, rat, rabbit, pig, cow, cat and dog are also encompassed by the present invention.
  • the reproduction-related disorder is a disorder resulting from a defect in the regulation of the expression of the gonadotropin releasing hormone (GnRH), in particular in the hypothalamic neurons.
  • GnRH gonadotropin releasing hormone
  • the reproduction-related disorder is selected in a group comprising early puberty, delayed puberty, low fertility, infertility, and the like.
  • puberty may be defined by the physiological and physical changes in an individual resulting in the transition from childhood to adulthood, i.e. from a child's body to a mature adult's body capable of sexual reproduction to enable fertilization.
  • puberty encompasses breast development, apparition of pubic hairs, increase in the size and/or volume of the uterus, ovaries, and the follicles, apparition of menstruation, increase of the percentage of body fat, variation of the levels of hormones, such as LH and FSH.
  • hormones such as LH and FSH.
  • puberty encompasses increase of the size and/or volume of testes and/or penis, apparition of erections, foreskin retraction, apparition of pubic hairs, body and facial hairs, voice change, formation of the Adam's apple, modification of the body shape, variation of the levels of testosterone.
  • the average puberty onset is set in the age of 10-11 years old, whereas, in male individuals, the average puberty onset is set in the age of 11-12 years old.
  • an "early puberty” or “precocious puberty” may be defined by a puberty onset, i.e. the appearance of any one of the above- referenced physiological and physical changes, occurring before the age of 9 years old for female individuals, and before the age of 10 years old for male individuals.
  • a "delayed puberty” may be defined by a puberty onset, i.e. the appearance of any one of the above-referenced physiological and physical changes, occurring after the age of 12 years old for female individuals, and after the age of 13 years old for male individuals.
  • Fertility may be evaluated in male individuals by parameters such as, semen volume, sperm count, sperm motility, and sperm morphology, and in female individuals the quality of menstrual cycles, in particular the quality of the ovulation phase, i.e. the ability of production of ovules.
  • low fertility refers to a fertility that is below the acknowledged mean fertility measured after compiling the suitable parameters from the available public databases.
  • the term "infertility” refers to the complete inability for an individual to reproduce under physiological or medically-assisted conditions.
  • a reference level value of the miR200 acid nucleic and/or miR155 nucleic acid may be measured from one or more biological sample obtained from one healthy individual or from one or more biological sample obtained from a population of healthy individuals, namely individuals not suffering from a reproduction- related disorder.
  • a reference level value of the miR200 acid nucleic and/or miR155 nucleic acid may be measured from one or more biological sample obtained from one individual or from one or more biological sample obtained from a population of individuals, which individual s) are known to suffer from a reproduction- related disorder.
  • a reference level value of the miR200 acid nucleic and/or miR155 nucleic acid may be measured from one or more biological sample obtained from one individual or from one or more biological sample obtained from a population of individuals, which individual(s) are known to suffer from a reproduction- related disorder and being administered with a treatment against said disorder.
  • a reference level value of the miR200 acid nucleic and/or miR155 nucleic acid may be obtained by calculating a mean value resulting from a compilation of data gathered from a database.
  • a reference sequence of the miR200 acid nucleic and/or miR155 nucleic acid may be obtained to determine miR155 variants in the sequence of a miR200 nucleic acid and/or miR155 nucleic acid.
  • the method according to the invention further comprising step c) of concluding of the occurrence of a reproduction-related disorder from the comparison performed at step b).
  • miR200 nucleic acid hybridizes with one or more target nucleic acids within the Zebl mRNA.
  • the target nucleic acids within the Zebl mRNA are selected in a group of nucleic acid comprising the sequences CAGUAUU (SEQ ID NO: A) and/or CAGUGUU (SEQ ID NO: B).
  • hybridization of miR-X with its target nucleic acid is not a 100% hybridization, since one or more nucleotides from the miR-X cannot hybridized to the target nucleic acid, due to the absence of one or more complementary nucleotide.
  • miR200 nucleic acid includes, but is not limited to, miR141, miR200a, miR200b, miR200c and miR429.
  • hybridizes encompasses situations wherein a first nucleic acid hybridizes with a second nucleic acid within a mammal cell, and especially within a human cell, including within the nucleus or within the cytoplasm of a mammal cell, especially a human cell.
  • the said miR200 nucleic acid hybridizes with one or more target nucleic acids selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
  • the said miR200 nucleic acid hybridizes with 2, 3, 4 or 5 target nucleic acids selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
  • the said miR200 nucleic acid has at least 70 % nucleic acid identity with a polynucleotide complementary to any of the target nucleic acids selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
  • the "percentage identity" between two sequences of nucleic acids means the percentage of identical nucleotides residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly along their length.
  • the comparison of two nucleic acid sequences is traditionally carried out by comparing the sequences after having optimally aligned them, said comparison being able to be conducted by segment or by using an "alignment window".
  • Optimal alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970, by means of the similarity search method of Pearson and Lipman (1988) or by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by the comparison software BLAST NR or BLAST P).
  • the percentage identity between two nucleic acid sequences is determined by comparing the two optimally- aligned sequences in which the nucleic acid sequence to compare can have additions or deletions compared to the reference sequence for optimal alignment between the two sequences. Percentage identity is calculated by determining the number of positions at which the nucleotide residue is identical between the two sequences, preferably between the two complete sequences, dividing the number of identical positions by the total number of positions in the alignment window and multiplying the result by 100 to obtain the percentage identity between the two sequences.
  • nucleotide sequences having at least 70% nucleotide identity with a reference sequence encompass those having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference sequence.
  • the term "complementary" is intended to mean that a first nucleic acid is complementary to a second nucleic acid when these nucleic acids have the base on each position which is the complementary (i.e. A to T, C to G) and in the reverse order.
  • the complementary sequence to TTAC is GTAA. If one strand of the double-stranded DNA is considered the sense strand, then the other strand, considered the antisense strand, will have the complementary sequence to the sense strand.
  • the said miR200 nucleic acid is selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
  • the miR200 nucleic acid comprising the nucleic acid having the sequence SEQ ID NO: 11 hybridizes with any nucleic acid selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
  • the miR200 nucleic acid comprising the nucleic acid having the sequence SEQ ID NO: 11 hybridizes with any nucleic acid selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.
  • the miR200 nucleic acid comprising the nucleic acid having the sequence SEQ ID NO: 12 hybridizes with any nucleic acid selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
  • the miR200 nucleic acid comprising the nucleic acid having the sequence SEQ ID NO: 12 hybridizes with any nucleic acid selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.
  • the miR200 nucleic acid comprising the nucleic acid having the sequence SEQ ID NO: 13 hybridizes with any nucleic acid selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
  • the miR200 nucleic acid comprising the nucleic acid having the sequence SEQ ID NO: 13 hybridizes with any nucleic acid selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.
  • miR155 nucleic acid hybridizes with one or more target nucleic acids within the Cebpb mRNA.
  • the target nucleic acid within the Cebpb mRNA comprises the sequence AGCAUUAA (SEQ ID NO: C).
  • the said miR155 nucleic acid hybridizes with one or more target nucleic acids selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 19 and SEQ ID NO: 20.
  • the said miR155 nucleic acid hybridizes with 2, 3, 4 or
  • 5 target nucleic acids selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 19 and SEQ ID NO: 20.
  • the said miR155 nucleic acid has at least 70 % nucleic acid identity with a polynucleotide complementary to any of the target nucleic acids selected in a group comprising a sequence SEQ ID NO: 19 and SEQ ID NO: 20.
  • the said miR155 nucleic acid comprises a nucleic acid having a sequence SEQ ID NO: 21.
  • miR200 nucleic acid and/or miR155 nucleic acid level values may be measured in biological samples obtained from an individual.
  • sequence of miR200 nucleic acid and/or miR155 nucleic acids may be determined from a biological sample from an individual.
  • suitable biological samples include, but are not limited to, a blood-derived sample, including whole blood, plasma, serum; a urine sample; a saliva sample; a tissue or an organ biopsy, i.e. a sample comprising cells, in particular neuronal cells.
  • the biological sample includes induced pluripotent stem cells (iPSCs), in particular patient-derived iPSCs.
  • iPSCs induced pluripotent stem cells
  • the said sample is selected in a group comprising a blood-derived sample and a cell-derived sample.
  • the levels of miR200 nucleic acid and/or miR155 nucleic acid are measured from a biological sample by using techniques commonly used in the state of the art.
  • the whole DNA and RNA content comprised in the biological sample is often harvested and/or isolated in a first step.
  • Harvest and isolation of total RNAs from a sample may be performed by known techniques disclosed in the art and reference may be made to standard DNA and RNA isolation protocols (e.g., Maniatis' Handbook of Molecular Biology.)
  • the method does not require that miR200 nucleic acid and/or miR155 nucleic acid be enriched from a standard RNA preparation.
  • miR200 nucleic acid and/or miR155 nucleic acid can be enriched using, for example, an appropriate separating mean.
  • miR200 nucleic acid and/or miR155 nucleic acid levels may be detected using any appropriate assay known in the art.
  • assays include, but are not limited to, miRNA arrays (including the commercially available sources from AGILENT® and ILLUMINA®), reverse transcriptase polymerase chain reaction (RT- PCR), quantitative real-time reverse transcriptase PCR (qPCR) using TaqMan microRNA assays (e.g.
  • measuring the levels of miR200 nucleic acid and/or miR155 nucleic acid may be performed by amplifying all or part of miRNA nucleic acid sequences such as mature miRNAs, precursor miRNAs, and primary miRNAs.
  • suitable nucleic acid polymerization and amplification methods include, but are not limited to, reverse transcription (RT), polymerase chain reaction (PCR), real-time PCR (quantitative PCR (q- PCR)), nucleic acid sequence-base amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, invader technology (Third Wave), rolling circle amplification, in vitro transcription (IVT), strand displacement amplification, transcription- mediated amplification (TMA), RNA (Eberwine) amplification, and the like.
  • RT reverse transcription
  • PCR polymerase chain reaction
  • q- PCR quantitative PCR
  • NASBA nucleic acid sequence-base amplification
  • ligase chain reaction multiplex ligatable probe amplification
  • IVT in vitro transcription
  • TMA transcription- mediated amplification
  • RNA Erberwine amplification
  • One or more amplification methods may be used, such as reverse transcription followed by real time PCR.
  • suitable DNA and RNA sequencing techniques methods include, but are not limited to, Illumina HiSeq 2500 and mapping of the 450 million 50bp single reads onto the mouse genome (GRCm38) using a splice junction aligner, Tophat v2.0.11.
  • the inventors observed for the first time a correlation between the expression level of GnRH, originating from GnRH neurons, and the expression of miR200 nucleic acid and/or miR155 nucleic acid. This correlation may benefit the therapeutic strategies to treat reproduction-related disorder.
  • the invention also relates to a method for preventing or treating a reproduction-related disorder due to a low expression of GnRH in an individual, comprising a step of administering to the said individual a compound selected in a group comprising a miR200 nucleic acid, a compound mimicking a miR200 nucleic acid, a miR155 nucleic acid and a compound mimicking a miR155 nucleic acid, or a mixture thereof.
  • the invention relates to a compound selected in a group comprising a miR200 nucleic acid, a compound mimicking a miR200 nucleic acid, a miR155 nucleic acid and a compound mimicking a miR155 nucleic acid, or a mixture thereof, for use in the prevention or the treatment of a reproduction-related disorder due to a low expression of GnRH in an individual.
  • Suitable miR200 nucleic acids, compounds mimicking a miR200 nucleic acid, miR155 nucleic acids and compounds mimicking a miR155 nucleic acid are abundantly described elsewhere in the present description, in particular in the above section relating to the diagnosis methods.
  • the individual is an individual in need of such a treatment for treating said reproduction-related disorder due to a low expression of GnRH.
  • a reproduction-related disorder due to a low expression of GnRH may be selected in a group comprising delayed puberty and hypogonadotropic hypogonadism.
  • an individual experiencing a reproduction-related disorder due to a low expression of GnRH may be an adolescent individual having a delayed puberty or an adult individual having a hypogonadotropic hypogonadism.
  • a compound mimicking a miR-X nucleic acid refers to the properties of said compound to exert the naturally occurring functions of said miR-X, namely bind to its site specific target nucleic acid and upregulate or downregulate the expression said target nucleic acid.
  • the said individual has a low expression of a miR200 nucleic acid and/or of a miR155 nucleic acid.
  • the expression "low expression” encompasses no detectable expression or expression below a reference value reflecting the average expression level measured in a biological sample from a healthy individual or a population of healthy individuals or sequence variants (e.g., mutation) with deficient biological activity, or from healthy individual at a comparable stage of development.
  • a low expression refers to expression levels which are at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% lower than a reference value.
  • a low expression refers to expression levels which are at least 5.0 fold, 4.0 fold, 3.0 fold, 2.0 fold, 1.8 fold, 1.6 fold, 1.4 fold, 1.2 fold lower than a reference value.
  • a low expression refers to variants in miR 200 nucleic acid or of a miR155 nucleic acid sequences (e.g., mutation) resulting in deficient biological activity.
  • the inventors show that miRNAs from the miR-200 and miR- 155 families are selectively enriched in GnRH neurons as compared to non GnRH cells (see sections 2.3/ and 2.4/ of the example section).
  • GnRH normal levels of GnRH may be restored through administration of a miR200 nucleic acid, a compound mimicking a miR200 nucleic acid, a miR155 nucleic acid and a compound mimicking a miR155 nucleic acid, or a mixture thereof, would benefit individuals with reproduction-related disorder due to a low expression of GnRH.
  • the invention further relates to a method for preventing or treating a reproduction-related disorder due to a high expression of GnRH in an individual, comprising a step of administering to the said individual a compound selected in a group comprising a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and a compound preventing the binding of miR155 nucleic acid to a target nucleic acid, or a mixture thereof.
  • Block-miR-200 provides a mean for altering the estrous cycle, in order to reduce or inhibit the ovulatory event. Therefore, contraception may be efficiently achieved by an administration of a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and a compound preventing the binding of miR155 nucleic acid to a target nucleic acid, or a mixture thereof.
  • the invention also relates to a contraceptive method comprising a step of administering to the said individual a compound selected in a group comprising a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and a compound preventing the binding of miR155 nucleic acid to a target nucleic acid, or a mixture thereof.
  • the invention also relates to a contraceptive method comprising a step of administering to an individual a compound selected in a group comprising a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and a compound preventing the binding of miR155 nucleic acid to a target nucleic acid, or a mixture thereof.
  • the invention further relates to the use of a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and/or a compound preventing the binding of miR155 nucleic acid to a target nucleic acid in contraception or in gynaecological therapies.
  • the invention further relates to the use of a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and/or a compound preventing the binding of miR155 nucleic acid to a target nucleic acid fir the manufacture of a contraceptive.
  • the invention also relates to a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and/or a compound preventing the binding of miR155 nucleic acid to a target nucleic acid for use as a contraceptive agent.
  • the invention also relates to a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and/or a compound preventing the binding of miR155 nucleic acid to a target nucleic acid for use in contraception.
  • the invention also relates to a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and/or a compound preventing the binding of miR155 nucleic acid to a target nucleic acid for use in gynaecological therapies.
  • a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and/or a compound preventing the binding of miR155 nucleic acid to a target nucleic acid may be used as a contraceptive agent, preferably in the form of a "birth control pill”.
  • the contraception and the contraceptive methods may be of use in a human individual, a non-human animal individual, e.g. a dog, a cat, preferably in a healthy individual.
  • the said individual is a female individual.
  • a reproduction-related disorder due to a high expression of GnRH may be selected in a group comprising early puberty, hypergonadotropic hypogonadism, Turner syndrome, anovulation, premature ovarian failure and menopause- related disorders.
  • an individual experiencing a reproduction-related disorder due to a high expression of GnRH may be an adolescent individual having an early puberty or an adult individual having a hypergonadotropic hypogonadism.
  • the said individual has a high expression of a miR200 nucleic acid and/or of a miR155 nucleic acid.
  • the invention relates to a compound selected in a group comprising a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and a compound preventing the binding of miR155 nucleic acid to a target nucleic acid, or a mixture thereof, for use in the prevention or the treatment of a reproduction-related disorder due to a high expression of GnRH in an individual.
  • the individual is an individual in need of such a treatment for treating said reproduction-related disorder due to a high expression of GnRH.
  • the expression "high expression” encompasses expression above a reference value reflecting the average expression level measured in a biological sample from a healthy individual or a population of healthy individuals, or from a healthy individual at a comparable stage of development.
  • adolescent individuals with early puberty may have a level of GnRH that is high with respect to their development stage, whereas this level might be considered as being "normal" in an individual in a later development stage.
  • a higher expression refers to expression levels which are at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% higher than a reference value.
  • a low expression refers to expression levels which are at least 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold higher than a reference value.
  • Block-miRs are also encompassed by the instant invention.
  • Block-miR refers to a nucleic acid compound that prevents other molecules from binding to a site specific region of a target nucleic acid, and more particularly prevent the binding of a given miRNA nucleic acid to its target nucleic acid.
  • the compound preventing the binding of a miR200 nucleic acid to a target nucleic acid consists of a Block-miR200 nucleic acid.
  • the Block-miR200 nucleic acid is a nucleic acid having at least 18 consecutive nucleotides being 100% complementary to a target nucleic acid selected in a group comprising a sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.
  • the expression "at least 18 consecutive nucleotides” encompasses at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 consecutive nucleotides.
  • Block-miR200 nucleic acid is selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
  • Block-miR200 nucleic acid of sequence SEQ ID NO: 14 hybridizes with any one of the nucleic acid selected in a group comprising a nucleic acid of sequence SEQ ID NO: 1 and SEQ ID NO: 2.
  • Block-miR200 nucleic acid of sequence SEQ ID NO: 15 hybridizes with any one of the nucleic acid selected in a group comprising a nucleic acid of sequence SEQ ID NO: 3 and SEQ ID NO: 4.
  • Block-miR200 nucleic acid of sequence SEQ ID NO: 16 hybridizes with any one of the nucleic acid selected in a group comprising a nucleic acid of sequence SEQ ID NO: 5 and SEQ ID NO: 6.
  • SEQ ID NO: 17 hybridizes with any one of the nucleic acid selected in a group comprising a nucleic acid of sequence SEQ ID NO: 7 and SEQ ID NO: 8.
  • Block-miR200 nucleic acid of sequence SEQ ID NO: 18 hybridizes with any one of the nucleic acid selected in a group comprising a nucleic acid of sequence SEQ ID NO: 9 and SEQ ID NO: 10.
  • the compound preventing the binding of a miR155 nucleic acid to a target nucleic acid consists of a Block-miR155 nucleic acid.
  • the Block-miR155 nucleic acid is a nucleic acid having at least 18 consecutive nucleotides being 100% complementary to a target nucleic acid selected in a group comprising a nucleic acid having a sequence SEQ ID NO: 19 and SEQ ID NO: 20.
  • Block-miR155 nucleic acid comprises a nucleic acid having a sequence SEQ ID NO: 22.
  • Block-miR155 results in a strong inhibition of both the GnRH promoter activity and the GnRH mRNA levels (see section 2.4/ of the example section). Then, blocking the functioning of miR-200 nucleic acid and/or miR-155 nucleic acid, through the use of suitable Block-miRs, may restore lower levels of GnRH in individuals having a reproduction-related disorder due to a high expression of GnRH, said lower levels of GnRH being compatible with the average physiological conditions.
  • miR200 nucleic acid may be used in combination with one or more other therapies aimed at alleviating a reproduction-related disorder due to a low expression of GnRH in an individual.
  • a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid may be used in combination with one or more other therapies aimed at alleviating a reproduction-related disorder due to a high expression of GnRH in an individual.
  • a miR200 nucleic acid, a compound mimicking a miR200 nucleic acid, a miR155 nucleic acid and a compound mimicking a miR155 nucleic acid according to the invention may comprise naturally occurring or non-naturally occurring nucleic acid sequences.
  • miR200 nucleic acids encompass nucleic acids comprising the mature or precursor forms of miRNA-200, which optionally further comprise additional flanking nucleotides 5' or 3' to the miRNA-200.
  • miR155 nucleic acids encompass miRNA nucleic acids comprising the mature or precursor forms of miRNA-155, which optionally further comprise additional flanking nucleotides 5' or 3' to the miRNA-155.
  • the length of the said miRNA nucleic acids may vary provided that they still achieve binding to the target nucleic acid and exert its regulatory function (up-regulation or down-regulation).
  • the miRNA nucleic acids may range from about 15 to about 100 nucleotides in length, which encompasses 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 and 99 nucleotides in length.
  • the said miRNA nucleic acids may consist in sequences having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identities with respect to the mature or precursor miRNA-200 sequence.
  • the said miRNA nucleic acids may consist in sequences having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identities with respect to the mature or precursor miRNA- 155 sequence.
  • a naturally occurring miRNA-200 sequence is RNA in nature and comprises a phosphodiester backbone.
  • non-naturally occurring miRNA-200 sequence may comprise RNA elements, such as naturally occurring ribonucleotides, and non- naturally occurring elements, such as non-naturally occurring ribonucleotides or other nucleotide- like residues or backbone linkages other than phosphodiester linkages including but not limited to phosphorothioate linkages.
  • naturally occurring or non-naturally occurring nucleic acids may be synthesized in vivo, in vitro or ex vivo.
  • suitable vectors such as plasmid or viral vectors may be employed following the acknowledged indications and protocols available in the state of the art.
  • the compounds according to the instant invention may be used as an active agent, for therapeutic purposes, in the form of a pharmaceutic composition.
  • the present invention relates to a pharmaceutic composition
  • a pharmaceutic composition comprising (1) a compound selected in a group comprising a miR200 nucleic acid, a compound mimicking a miR200 nucleic acid, a miR155 nucleic acid and a compound mimicking a miR155 nucleic acid and (2) a pharmaceutically acceptable carrier.
  • the present invention relates to a pharmaceutic composition
  • a pharmaceutic composition comprising (1) compound selected in a group comprising a compound preventing the binding of a miR200 nucleic acid to a target nucleic acid, and a compound preventing the binding of miR155 nucleic acid to a target nucleic acid and (2) a pharmaceutically acceptable carrier.
  • the pharmaceutic composition may further comprise one or more salts, one or more buffering agents, one or more preservatives, one or more secondary therapeutic agents.
  • a “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a prophylactically or therapeutically active agent.
  • a suitable pharmaceutically acceptable carrier may be selected in a group including sugars, such as lactose, glucose and sucrose; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; buffering agents, such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and the like.
  • sugars such as lactose, glucose and sucrose
  • glycols such as propylene glycol
  • polyols such as glycerin, sorbitol, mannitol and polyethylene glycol
  • esters such as ethyl oleate and ethyl laurate
  • buffering agents such as magnesium hydroxide and aluminum hydroxide
  • a pharmaceutical composition according to the invention may be in any suitable form encompassed by the state in the art, e.g. in the form of an injectable solution or suspension, a tablet, a coated tablet, a capsule, a syrup, a suppository, a cream, an ointment, a lotion, and the like.
  • an effective amount of said compound is administered to said individual in need thereof.
  • an "effective amount” refers to the amount of said compound that alone stimulates the desired outcome, i.e. alleviates or eradicates the symptoms of the reproduction-related disorder.
  • the effective amount of the compound to be administered may be determined by a physician or an authorized person skilled in the art and can be suitably adapted within the time course of the treatment.
  • the effective amount to be administered may depend upon a variety of parameters, including the material selected for administration, whether the administration is in single or multiple doses, and the individual's parameters including age, physical condition, size, weight, and the severity of the disorder.
  • an effective amount of the active agent may comprise from about 0.001 mg to about 3000 mg, per dosage unit, preferably from about 0.05 mg to about 100 mg, per dosage unit.
  • from about 0.001 mg to about 3000 mg includes, from about 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg,
  • the active agent may be at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day.
  • each dosage unit may be administered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the therapeutic treatment encompasses an administration of a plurality of dosage units, including two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations.
  • the active agent e.g. in the form of a pharmaceutic composition may be administered by any suitable route, including enteral (e.g. , oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical, mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • enteral e.g. , oral
  • parenteral intravenous, intramuscular, intra-arterial, intramedullary
  • intrathecal subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal
  • topical e.g., mucosal, nasal, buccal, sublingual
  • the active agent is administered by oral administration, systemic intravenous administration. • Other methods
  • the invention relates to a method for screening a drug for treating or preventing a reproduction-related disorder in an individual, comprising:
  • step a) measuring the level of said miR200 nucleic acid and/or miR155 nucleic acid expressed in step a);
  • step b) comparing level value measured at step b) to a reference level value of the said miR200 nucleic acid and/or miR155 nucleic acid obtained in the absence of said drug.
  • the cell type capable of expressing miR200 nucleic acid and/or miR155 nucleic acid is a neuron, preferably a hypothalamic neuron.
  • the variation of the level value of miR200 nucleic acid and/or miR155 nucleic acid in the presence of the drug with respect to the reference level value may indicate the effectiveness of the drug to treat or prevent a reproduction-related disorder in an individual in need thereof.
  • the invention relates to a method for assessing the efficiency of a treatment for treating a reproduction-related disorder in an individual, comprising:
  • the invention relates to a method for assessing the efficiency of a treatment for treating a reproduction-related disorder in an individual, comprising:
  • a parameter associated with reproduction in female individuals may be selected in a group comprising breast development, apparition of pubic hairs, the size and/or volume of the uterus, the size and/or volume of the ovaries, the size and/or volume of the follicles, apparition of menstruation, variation of the percentage of body fat, variation of the levels of hormones, in particular LH and FSH.
  • a parameter associated with reproduction in male individuals may be selected in a group comprising the size and/or volume of testes and/or penis, apparition of erections, foreskin retraction, apparition of pubic hairs, body and facial hairs, voice change, formation of the Adam's apple, modification of the body shape, variation of the levels of testosterone.
  • the method further comprises step d) consisting of adapting the treatment.
  • any variation of the level value measured after the treatment with respect to the level value measured prior to the treatment may indicate the efficiency of said treatment.
  • said method may be of use for helping the physician adjusting the treatment dosage, namely increasing or decreasing the dosage regimen to be administered.
  • a CAGUAUU Nucleic acid (5'-3') consisting of the seed region of the human ZEB 1 nucleic acid targeted by miR-200b-3p, miR-200c-3p and miR-429 nucleic acids
  • mice All mice were group-housed under specific pathogen-free conditions in a temperature-controlled room (21-22 °C) with a 12-h light-dark cycle and ad libitum access to food and water.
  • C57B1/6J Dicer LoxP/LoxP mice were a generous gift of Dr. Catherine Dulac (Howard Hughes Medical Institute, Cambridge MA) (9), Dr. Brian Harfe (University of Florida, FL) (5) and Dr. Daniel J.
  • tdTomato loxP/STOP mice B6.Cg-Gt(ROSA)26Sortm9 (CAG-tdTomato)Hze/J
  • mice were purchased from the Jackson laboratory (Maine, USA).
  • Mice were genotyped by PCR using adapted primers. Animal studies were approved by the Institutional Ethics Committees for the Care and Use of Experimental Animals of the Universities of Lille and Cordoba; all experiments were performed in accordance with the guidelines for animal use specified by the European Union Council Directive of September 22, 2010 (2010/63/EU).
  • Fertility index Male and female fertility indices were calculated from the number of litters per females during a 120-d mating.
  • Hormone level measurements Protocols and doses for in vivo testing of the LH response to GnRH, kisspeptin-10 and NMDA were as described in detail elsewhere (52). Serum LH and FSH levels were measured using radioimmunoassay kits supplied by the National Institutes of Health (Dr. A.F. Parlow, National Hormone and Peptide Program, Torrance, CA). Rat LH-I-10 and FSH-I-9 were labelled with 1251 using lodo-gen tubes, following the instructions of the manufacturer (Pierce, Rockford, IL). Hormone concentrations were determined using reference preparations of LH-RP-3 and FSH-RP-2 as standards.
  • Intra- and inter-assay coefficients of variation were ⁇ 8 and 10% for LH and ⁇ 6 and 9% for FSH, respectively.
  • the sensitivity of the assay was 3.75 pg/tube for LH and 20 pg/tube for FSH.
  • the accuracy of hormone measurements was confirmed by the assessment of rodent serum samples of known concentration (external controls).
  • N-G-nitro-L-arginine methyl ester, HC1 (L-NAME; N5751, Sigma) was used to inhibit the activity of nitric oxide synthase in infantile mice.
  • Gnrh::Gfp;Gnrh::cre;Dicer LoxP/LoxP mice received a single intraperitoneal injection of L-NAME (50 mg/kg, i.p.) or vehicle (saline) on Pl l and were killed 12 h later for GnRH neuron isolation by FACS. 1.4/ Gonadal histology and quantitative analysis.
  • embryos were obtained after cervical dislocation from timed-pregnant Gnrh::cre;Dicer LoxP/+ crossed with Gnrh::cre;Dicer LoxP/LoxP male mice.
  • Embryos were washed thoroughly in cold 0.1 m PBS, fixed in fixative solution (4% paraformaldehyde (PFA), 0.2% picric acid in 0.1 m PBS, pH 7.4) for 6-8 h at 4 °C and cryoprotected in 20% sucrose overnight at 4 °C.
  • fixative solution 4% paraformaldehyde (PFA), 0.2% picric acid in 0.1 m PBS, pH 7.4
  • OCT embedding medium Tissue-Tek
  • Tissues were cryosectioned (Leica cryostat) at 16 ⁇ for embryos and pre- weaning postnatal mice, and at 35 ⁇ (free-floating sections) for post-weaning and adult brains. Immunohistochemistry was performed as previously reported (53, 54), using Alexa-Fluor 488- (1 :400) and Cy3 -conjugated (1 :800) secondary antibodies (Invitrogen, A11008). Fluorescent specimens were mounted using l,4-diazabicyclo[2.2.2]octane (Sigma- Aldrich). The primary antisera used were as follows: rabbit anti-GnRH (1 :3000), a generous gift from Prof. G.
  • Gnrh::Gfp;Gnrh::cre;Dicer LoxP/LoxP mice were micro-dissected and enzymatically dissociated using a Papain Dissociation System (Worthington, Lakewood, NJ) to obtain single-cell suspensions.
  • FACS was performed using an EPICS ALTRA Cell Sorter Cytometer device (BD Bioscience). The sort decision was based on measurements of GFP fluorescence (excitation: 488 nm, 50 mW; detection: GFP bandpass 530/30 nm, auto- fluorescence bandpass 695/40 nm) by comparing cell suspensions from Gnrh::Gfp and wild-type animals. For each animal, 400 to 800 GFP-positive cells were sorted directly into 10 ⁇ of extraction buffer: 0.1% Triton® X-100 (Sigma- Aldrich) and 0.4 unit/ ⁇ R aseOUTTM (Life Technologies).
  • mRNAs obtained from FACS-sorted GnRH neurons were reverse transcribed using Superscript® III Reverse Transcriptase (Life Technologies) and a linear preamplification step was performed using the TaqMan® PreAmp Master Mix Kit protocol (P/N 4366128, Applied Biosystems).
  • Realtime PCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using exon-boundary- specific TaqMan® Gene Expression Assays (Applied Biosystems): Aes (Aes-Mm00507847_ml), Cebpb (Cebpb-Mm00843434_sl), Dicer (Mm00521722_ml), Dlxl(Dlxl-Mm00438424_ml), Dlx5 (Dlx5-Mm00438430_ml), Gfp (Gfp-Mr03989638_mr), Gnrhl (Gnrhl-Mm01315605_ml), Gpr54 (Gpr54- Mm00475046_ml), Lpinl (Lpinl-Mm00550511_ml), Maf (Maf-Mm02581355_sl), Meisl (Meisl-Mm00487664_ml), M
  • MicroRNA expression analyses were performed using stem-loop RT-PCR based TaqMan Rodent MicroRNA Arrays (Applied Biosystems). Briefly, miRNAs obtained from FACS-sorted GnRH neurons were reverse transcribed using the TaqMan miRNA Reverse Transcription Kit (Applied Biosystems) in combination with the stem- loop Megaplex primer pool sets A and B according to the manufacturer's instructions. A linear preamplification step was performed using the TaqMan® PreAmp Master Mix Kit protocol (P/N 4366128, Applied Biosystems) and quantitative real-time PCR were performed using TaqMan Low-Density Arrays (Applied Biosystems) on an Applied Biosystems 7900HT thermocycler using the manufacturer's recommended cycling conditions.
  • Assist 3.0.1 software (Applied Biosystems), with R18S and actin as control housekeeping mRNAs and U6sRNA as control housekeeping miRNA following a standardized procedure (56).
  • Assay-centric heat-maps were generated with Data Assist 3.0.1 by unsupervised hierarchical clustering (complete linkage) using Pearson's correlation as a distance measure. 1.10/ Zebl chromatin immunoprecipitation (ChIP) assays
  • Immortalized GnRH neurons were cultured in DMEM + 10% FBS (Life Technology). Prior to experimental assays, cells were washed twice in PBS and incubated overnight in serum- free medium. The next morning, 10% FBS was added for 2 h. Chromatin isolation was performed using the ChIP-IT Express kit (Active Motif) following the manufacturer's instructions.
  • the preoptic area of the hypothalamus was dissected from each animal using
  • Protein content was determined using the sample buffer (Invitrogen). Samples were boiled for 5 min and stored at -80 °C until use. Samples were reboiled for 5 min after thawing and electrophoresed for 75 min at 150 V in 7% Tris-acetate, or for 50 min at 200 V in precast 4-12%) MES SDS-polyacrylamide gels according to the protocol supplied with the NuPAGE system (Invitrogen). After size fractionation, the proteins were transferred onto 0.2 ⁇ pore-size polyvinylidene difluoride membranes (LC2002; Invitrogen) in the blot module of the NuPAGE system (Invitrogen) for 75 min at room temperature.
  • LC2002 ⁇ pore-size polyvinylidene difluoride membranes
  • Membranes were blocked for 1 h in blocking buffer (TBS with 0.05% Tween 20 (TBST) and 5% nonfat milk) at room temperature, and incubated overnight at 4 °C with the appropriate primary antibody diluted in blocking buffer (rabbit polyclonal anti-RFP, 600-401-379, 1 : 1,000, Rockland Antibodies & Assays; rabbit polyclonal anti-nNOS, sc-8309, 1 :500, Santa Cruz technologies; rabbit polyclonal anti-Serl412 phospho-nNOS, PA1-032; 1 : 1,000, Affinity BioReagents; goat polyclonal anti-actin, sc-1616, 1 : 1,000).
  • blocking buffer TBS with 0.05% Tween 20 (TBST) and 5% nonfat milk
  • Membranes were washed four times with TBST the following day before being exposed to HRP- conjugated secondary antibodies (Vector, peroxidase-labelled anti rabbit and anti-goat IgGs PI- 1000 and PI9500, respectively) diluted in blocking buffer for 1 h at room temperature. Immunoreactions were visualized using the ECL detection kit (NEL101; PerkinElmer, Boston, MA). Immunoblots were scanned using a desktop scanner (Epson Expression 1680 PRO) and Adobe Photoshop, and band intensities were determined using Image J software (NIH, Bethesda).
  • TLBs Target site blockers
  • TSB sequences are designed with a large arm that covers the miRNA binding site and a short arm outside the miRNA seed to ensure target specificity.
  • One sequence was generated to protect the unique miR-155-binding site in the Cebpb 200b/200c/429-binding sites in the Zebl 3'UTR and mixed at equimolar ratios (TSB-200).
  • Gnrh::Gfp mice were placed in a stereotaxic frame (Kopf® Instruments, California) under anesthesia (isoflurane), and a burr hole was drilled 1 mm lateral to the bregma, according to a mouse brain atlas (Paxinos and Franklin, 2004).
  • mice from at least three different litters for each group were used to study sexual maturation and fertility; n > 3 mice from three different litters were collected to perform quantitative RT-PCR analyses in cells isolated by FACS; and n > 3 mice from three different litters each group were collected for anatomy and immunostaining. No randomization method was used to assign subjects in the experimental groups or to collect and process data.
  • mice To determine whether miRNAs are involved in the crucial postnatal increase in GnRH expression, we generated mice in which Dicer, an RNAse-III endonuclease essential for miRNA biogenesis (8), was selectively inactivated in GnRH neurons. Mice harboring a loxP-flanked Dicer allele (5) were crossed with those expressing Cre recombinase under the control of the endogenous Gnrh promoter (9) (Fig. 1), which drives expression in hypothalamic GnRH neurons but not the gonads.
  • Dicer an RNAse-III endonuclease essential for miRNA biogenesis (8)
  • LH pituitary gonadotropins luteinizing hormone
  • FSH follicle- stimulating hormone
  • hypothalamic GnRH peptide content increases progressively between birth and puberty, quickening markedly during the infantile period (P7-P20) (14, 15).
  • Gnrh::cre;Dicer loxP/loxP ;tdTomato loxP/STOP trigenic mice showed that GnRH neurons did not die in the absence of miRNAs but simply lost GnRH immunoreactivity.
  • tdTomato once triggered by Gnrh transcriptional activation and thus Cre production during development, is expressed constitutively in GnRH neurons even without continued Gnrh promoter activity, the number of tdTomato-expressing GnRH neurons did not significantly decrease. However, around -70% of them had lost GnRH peptide at P21 when compared to Gnrh::cre;Dicer +/+ ;tdTomato loxP/STOP littermates.
  • mice To further pinpoint the stage at which GnRH expression is blocked in Dicer-deficient mice, we constructed Gnrh::Gfp;Gnrh::cre;Dicer loxP/loxP reporter mice, which express GFP under the control of an ectopic Gnrh promoter only when the promoter is currently active, unlike tdTomato in the previous experiment. These mice showed a concomitant loss of GnRH peptide and GFP during postnatal development.
  • P7 signals the beginning of the infantile period, while at P12 hypothalamic GnRH peptide content undergoes a functionally relevant increase in normal mice (14, 15) but begins to decrease in Dicer-deficient mice even though most of their GnRH neurons (-80%) still express detectable GFP.
  • Gnrh promoter activators and repressors suggest that, in the absence of miRNAs, repressors of these activators, in addition to repressors of GnRH itself, might be reciprocally increased.
  • silico analysis of the 3'UTRs of transcripts encoding these promoter modulators revealed the presence of putative binding sites for several miRNAs (P ⁇ 0.01), confirming that miRNAs could regulate their expression.
  • Fig. 21 To verify whether the miR-200-Zebl network (19) (Fig. 21) also controlled hypothalamic GnRH expression during postnatal development we analyzed Zebl expression in mice with or without miRNA production in GnRH neurons.
  • TSBs are small, freely diffusible locked-nucleic-acid oligonucleotides with two notable characteristics: (i) they are highly stable (with a reported half- life in vivo on the order of weeks) and resistant to nuclease-mediated degradation in vitro and in vivo, thanks to their modified phosphorothioate backbone (21), and (ii) they are complementary to both the miRNA and the binding site being targeted, making them extremely specific (see, for example, Fig. 26-29). As shown by fluorescence-tagged sequences, TSBs readily targeted the preoptic region and GnRH neurons 72 h after infusion into the lateral ventricle of Gnrh::Gfp;Dicer +/+ pups.
  • 2.4/ Hampering miR-155 and Cebpb binding lowers Gnrh mRNA
  • ChlPBase an integrated resource and platform for decoding transcription factor binding maps on the basis of chromatin immunoprecipitation and sequencing (ChlP-seq) data (28)
  • nNOS neuronal NO synthase
  • TSB-200 and/or TSB-155 were detectable in the brain up to 2 weeks after injection, in keeping with previous reports showing that they remained active for at least a week in vivo (37). While growth was similar in TSB- and scrambled- sequence-injected mice, TSB-injected mice exhibited a precocious onset of puberty when compared to control mice (Fig. 16).
  • TSB-200-infused mice showed significantly elevated peripubertal afternoon surges of plasma LH at P38, while changes in LH levels induced by TSB-155 treatment did not reach statistical significance when compared to control mice, in which plasma LH levels were at the limit of detection (Fig. 17).
  • mice To determine whether the miR-200-Zebl network could also control GnRH function outside of this critical infantile time-window leading up to puberty, we next infused TSB-200 into the hypothalamic preoptic region of cycling adult female mice and followed their estrous cycles (Fig. 19). While scrambled-sequence-infused control mice did not exhibit any marked alteration of their estrous cycles, most mice treated with TSB- 200 (5 out of 6) showed a slight prolongation in the number of days spent in diestrus (a basal stage when LH is released at nadir levels) and eventually reached proestrus (when the preovulatory surge of LH occurs) but displayed an incomplete cycle at least once during the first 2 weeks after injection (Fig. 19).
  • This phenomenon is the first of three activational periods that primes the HPG axis for puberty and adult fertility, setting in motion, for example, the growth of the first wave of ovarian follicles that will ovulate at puberty in females and the development of the testes in males (see ref. 15 for review).
  • a switch in miRNA expression flips a switch in a multi-layered array of GnRH gene activators and repressors, permitting the sustained increase of the neurohormone required for subsequent sexual maturation.
  • miRNA species act as the linchpins of this process: the miR- 200/429 family, which is not only upregulated during this critical period but selectively enhanced in GnRH neurons, and miR-155, which appears to act on other hypothalamic cell types as well, and mediates, for example, the effects of a concomitant release of NO upstream of GnRH neurons. Interfering with the binding and function of these two key species blunts the infantile increase in GnRH expression, and the in vivo alteration of the miR-200/429-transcription factor micro network leads to the disruption of normal puberty onset as well as normal estrous cyclicity in adulthood.
  • GnRH neurons form an extremely small population (-800 neurons in a mouse brain) and their scattered distribution in a continuum from the olfactory bulb to the hypothalamus, albeit with a strong concentration in the hypothalamic preoptic area, have made genetic and epigenetic studies in these neurons a technical challenge until now.
  • Our successful separation of GnRH from non-GnRH cells in the preoptic region of GnRH::Gfp mice has allowed us to determine miRNA expression profiles specific to this limited population of neurons in the postnatal brain (i.e., once differentiation and the establishment of connectivity, including their projection over several millimeters to the hypothalamic median eminence, are complete).
  • GnRH expression appears normal during the perinatal and early infantile periods, and the process of sexual maturation commences normally and leads to vaginal opening in 80% of our mice. However, these mice never achieve puberty because of the progressive decline in Gnrh promoter activity from the second week of postnatal life (P7 to PI 2) and its complete extinction during the juvenile period.
  • miR A species whose expression is upregulated in GnRH neurons during this critical period include miR-155 and members of the miR-200 family. Notably, the 100-fold enrichment of miR-200 family members is restricted to GnRH neurons, indicating a highly specific function for these miRNA species in the regulation of infantile GnRH expression.
  • the miR-200/429 family and its target Zebl which can directly repress the Gnrh promoter, also seem to form a double-negative loop with the key Gnrh promoter activator Pou2fl, while miR-155 counteracts NO/Cebpb-dependent GnRH repression.
  • some promoter modulators act as both activators and repressors depending on physiological conditions and cellular context, or exert a reciprocal regulatory effect on the miRNAs that control their expression.
  • Cebpb a Gnrh promoter repressor
  • Zebl increases Zebl levels in GnRH neurons but not in non-GnRH hypothalamic cells (which do not express miR-200/429), while Zebl is known to provide negative feedback to miR-200/429 (19).
  • the expression patterns of various known Gnrh promoter modulators at P12 are not the same. While activators that are upregulated specifically in GnRH neurons in wild-type mice at P12 show reduced expression with the blockade of miR-200/429 binding (with the exception of Dlx5, whose transcript has no Zebl binding site and is thus probably regulated by other miRNA-effector pathways), the expression of one known activator, Dlxl (ref. 43), is increased. Apart from a possible indirect effect, Dlxl expression shows a downward trend in wild-type GnRH neurons at PI 2, unlike Pou2fl or Meisl, which could explain this seeming paradox.
  • the subsequent formation of new synaptic contacts with GnRH cell bodies and dendritic pruning (44) could be among the triggers of the miRNA- mediated switch in the control of Gnrh promoter activity during this period (Figs. 35-38).
  • miR-155 there might also be other microcircuits and feedback loops, possibly involving the expression of miR-155 in other cell types, as suggested by the fact that simultaneously neutralizing both miR-200/429-Zebl binding and miR-155-Cebpb binding resulted in a considerably weaker effect on peripubertal LH levels than miR-200/429 blockade alone. While blocking miR-200/429-Zebl binding during this infantile-juvenile critical period suppressed Gnrh promoter activity at the cellular level, these animals went on to display precocious, not delayed, puberty.
  • RNAseq repositories for human tissues indicate that miR155 is also widely distributed in the hypothalamus and other brain regions in humans.
  • the expression of miR-200 family members is, as described in mice (6), below the limit of detection in human brain tissues including the hypothalamus, an observation consistent with the restriction of these miRNA species to the sparse GnRH neuronal population.
  • the Allen Human Brain Atlas http://human.brain-map.org/
  • Costinean, S. et al. Src homology 2 domain-containing inosito 1-5 -phosphatase and CCAAT enhancer-binding protein beta are targeted by miR-
  • MicroRNA 135 is essential for chronic stress resiliency, antidepressant efficacy, and intact serotonergic activity. Neuron 83, 344-360 (2014).
  • Giacobini P. et al. Brain endothelial cells control fertility through ovarian- steroid-dependent release of semaphorin 3A. PLoS Biol. 12, el001808 (2014).

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Abstract

La présente invention concerne le diagnostic et le traitement de troubles liés à la reproduction, en particulier dus à des taux modifiés de GnrH, et concerne en outre la contraception. Les traitements à base d'administration d'hormones peuvent présenter certaines limites. Les inventeurs ont montré que miR200, miR155 et les miR de bloc respectifs peuvent restaurer des taux appropriés de GnrH. En particulier, la présente invention concerne la prévention ou le traitement d'un trouble lié à la reproduction dû à une faible expression de GnRH chez un individu, comprenant une étape d'administration d'un composé choisi dans un groupe comprenant un acide nucléique miR200 et/ou miR155, un composé mimant un acide nucléique miR200 et/ou miR155. De manière similaire, l'invention concerne également la prévention ou le traitement d'un trouble lié à la reproduction dû à une expression élevée de GnRH, comprenant une étape d'administration d'un composé choisi dans un groupe comprenant un composé empêchant la liaison d'un acide nucléique miR200 ou miR155 à un acide nucléique cible. En particulier, les inventeurs ont évalué ces composés sur des souris.
PCT/EP2017/059416 2016-04-20 2017-04-20 Procédés de diagnostic et de traitement de troubles liés à la reproduction et procédés de contraception WO2017182580A1 (fr)

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WO2020221821A1 (fr) 2019-04-30 2020-11-05 INSERM (Institut National de la Santé et de la Recherche Médicale) Administration de gnrh pulsée pour le traitement de troubles cognitifs
WO2022136417A1 (fr) 2020-12-22 2022-06-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Administration pulsatile de gnrh pour le traitement de troubles liés à l'ingestion d'aliments
WO2024047115A1 (fr) 2022-09-02 2024-03-07 Leibniz-Institut Für Immuntherapie (Lit) Utilisation thérapeutique du snp mir155 rs377265631

Citations (1)

* Cited by examiner, † Cited by third party
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US20120107825A1 (en) * 2010-11-01 2012-05-03 Winger Edward E Methods and compositions for assessing patients with reproductive failure using immune cell-derived microrna

Patent Citations (1)

* Cited by examiner, † Cited by third party
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Non-Patent Citations (70)

* Cited by examiner, † Cited by third party
Title
ABU-HALIMA ET AL., FERTILITY AND STERILITY, vol. 99, no. 5, 2013, pages 1249 - 1255
ABU-HALIMA MASOOD ET AL: "Altered microRNA expression profiles of human spermatozoa in patients with different spermatogenic impairments", FERTILITY AND STERILITY, ELSEVIER SCIENCE INC, NEW YORK, NY, USA, vol. 99, no. 5, 8 January 2013 (2013-01-08), pages 1249, XP028527192, ISSN: 0015-0282, DOI: 10.1016/J.FERTNSTERT.2012.11.054 *
ALISI, A. ET AL.: "Mirnome analysis reveals novel molecular determinants in the pathogenesis of diet-induced nonalcoholic fatty liver disease", LAB. INVEST., vol. 91, 2011, pages 283 - 293, XP002686522, DOI: doi:10.1038/LABINVEST.2010.166
ANCHATE, N.K. ET AL.: "SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome", PLOS GENET., vol. 8, 2012, pages E1002896
ANDREA MESSINA ET AL: "A microRNA switch regulates the rise in hypothalamic GnRH production before puberty", NATURE NEUROSCIENCE, vol. 19, no. 6, 2 May 2016 (2016-05-02), US, pages 835 - 844, XP055306395, ISSN: 1097-6256, DOI: 10.1038/nn.4298 *
ANJA SØRENSEN ET AL: "MicroRNAs Related to Polycystic Ovary Syndrome (PCOS)", GENES, vol. 5, no. 3, 25 August 2014 (2014-08-25), pages 684 - 708, XP055155868, DOI: 10.3390/genes5030684 *
BEAUVILLAIN, J.C.; TRAMU, G.: "Immunocytochemical demonstration of LH-RH, somatostatin, and ACTH-like peptide in osmium-postfixed, resin-embedded median eminence", J.HISTOCHEM.CYTOCHEM, vol. 28, 1980, pages 1014 - 1017
BELLEFONTAINE, N. ET AL.: "Leptin-dependent neuronal NO signaling in the preoptic hypothalamus facilitates reproduction", J. CLIN. INVEST., vol. 124, 2014, pages 2550 - 2559
BELSHAM, D.D.; MELLON, P.L.: "Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression.", MOL. ENDOCRINOL., vol. 14, 2000, pages 212 - 228
BERNSTEIN, E.; CAUDY, A.A.; HAMMOND, S.M.; HANNON, G.J.: "Role for a bidentate ribonuclease in the initiation step of RNA interference.", NATURE, vol. 409, 2001, pages 363 - 366, XP002183119, DOI: doi:10.1038/35053110
BOEHM, U. ET AL.: "Expert consensus document: European Consensus Statement on congenital hypogonadotropic hypogonadism—pathogenesis, diagnosis and treatment", NAT. REV. ENDOCRINOL., vol. 11, 2015, pages 547 - 564
BOURET, S.G.; DRAPER, S.J.; SIMERLY, R.B.: "Formation of projection pathways from the arcuate nucleus of the hypothalamus to hypothalamic regions implicated in the neural control of feeding behavior in mice", J. NEUROSCI., vol. 24, 2004, pages 2797 - 2805
BURK, U. ET AL.: "A reciprocal repression between ZEB1 and members of the miR-200 family promotes EMT and invasion in cancer cells.", EMBO REP., vol. 9, 2008, pages 582 - 589, XP055166026, DOI: doi:10.1038/embor.2008.74
C. TSATSANIS ET AL: "Serum miR-155 as a potential biomarker of male fertility", HUMAN REPRODUCTION, vol. 30, no. 4, 3 March 2015 (2015-03-03), GB, pages 853 - 860, XP055306393, ISSN: 0268-1161, DOI: 10.1093/humrep/dev031 *
CARON, E.; CIOFI, P.; PREVOT, V.; BOURET, S.G.: "Alteration in neonatal nutrition causes perturbations in hypothalamic neural circuits controlling reproductive function", J. NEUROSCI., vol. 32, 2012, pages 11486 - 11494
CHACHLAKI, K.; PREVOT, V.: "Coexpression profiles reveal hidden gene networks", PROC. NATL. ACAD. SCI. USA, vol. 113, 2016, pages 2563 - 2565
CHOE, H.K. ET AL.: "Real-time GnRH gene transcription in GnRH promoter-driven luciferase-expressing transgenic mice: effect of kisspeptin.", NEUROENDOCRINOLOGY, vol. 102, 2015, pages 194 - 199
COSSENZA, M. ET AL.: "Nitric oxide in the nervous system: biochemical, developmental, and neurobiological aspects.", VITAM. HORM., vol. 96, 2014, pages 79 - 125
COSTINEAN, S. ET AL.: "Src homology 2 domain-containing inositol-5-phosphatase and CCAAT enhancer-binding protein beta are targeted by miR-155 in B cells of Emicro-MiR-155 transgenic mice", BLOOD, vol. 114, 2009, pages 1374 - 1382
COTTRELL, E.C.; CAMPBELL, R.E.; HAN, S.K.; HERBISON, A.E.: "Postnatal remodeling of dendritic structure and spine density in gonadotropin-releasing hormone neurons", ENDOCRINOLOGY, vol. 147, 2006, pages 3652 - 3661
D'ANGLEMONT DE TASSIGNY, X.; ACKROYD, K.J.; CHATZIDAKI, E.E.; COLLEDGE, W.H.: "Kisspeptin signaling is required for peripheral but not central stimulation of gonadotropin-releasing hormone neurons by NMDA", J. NEUROSCI., vol. 30, 2010, pages 8581 - 8590, XP055306197, DOI: doi:10.1523/JNEUROSCI.5486-09.2010
DE ROUX, N. ET AL.: "A family with hypogonadotropic hypogonadism and mutations in the gonadotropin-releasing hormone receptor", N. ENGL. J. MED, vol. 337, 1997, pages 1597 - 1602
DWEEP, H.; STICHT, C.; PANDEY, P.; GRETZ, N.: "miRWalk--database: prediction of possible miRNA binding sites by ''walking'' the genes of three genomes", J. BIOMED. INFORM, vol. 44, 2011, pages 839 - 847, XP028293437, DOI: doi:10.1016/j.jbi.2011.05.002
FORNI, P.E.; WRAY, S.: "GnRH, anosmia and hypogonadotropic hypogonadism--where are we?", FRONT. NEUROENDOCRINOL., vol. 36, 2015, pages 165 - 177
FRANZONI, E. ET AL.: "miR-128 regulates neuronal migration, outgrowth and intrinsic excitability via the intellectual disability gene Phf6", ELIFE, vol. 4, 2015, Retrieved from the Internet <URL:http://dx.doi.org/10.7554/eLife.04263>
GARCIA-GALIANO, D. ET AL.: "Kisspeptin signaling is indispensable for neurokinin B, but not glutamate, stimulation of gonadotropin secretion in mice", ENDOCRINOLOGY, vol. 153, 2012, pages 316 - 328
GARTHWAITE, J.: "From synaptically localized to volume transmission by nitric oxide", J. PHYSIOL. (LOND., vol. 594, 2016, pages 9 - 18
GIACOBINI, P. ET AL.: "Brain endothelial cells control fertility through ovarian-steroid-dependent release of semaphorin 3A", PLOS BIOL., vol. 12, 2014, pages E1001808
GIVENS, M.L. ET AL.: "Developmental regulation of gonadotropin-releasing hormone gene expression by the MSX and DLX homeodomain protein families", J. BIOL. CHEM., vol. 280, 2005, pages 19156 - 19165
GRUETER, C.E. ET AL.: "A cardiac microRNA governs systemic energy homeostasis by regulation of MED13.", CELL, vol. 149, 2012, pages 671 - 683, XP028420459, DOI: doi:10.1016/j.cell.2012.03.029
H. HASUWA ET AL: "MiR-200b and miR-429 Function in Mouse Ovulation and Are Essential for Female Fertility", SCIENCE, vol. 341, no. 6141, 5 July 2013 (2013-07-05), US, pages 71 - 73, XP055306196, ISSN: 0036-8075, DOI: 10.1126/science.1237999 *
HAI-YING LIU ET AL: "Transcription factor-microRNA synergistic regulatory network revealing the mechanism of polycystic ovary syndrome", MOLECULAR MEDICINE REPORTS, 21 March 2016 (2016-03-21), GR, XP055306394, ISSN: 1791-2997, DOI: 10.3892/mmr.2016.5019 *
HANCHATE, N.K. ET AL.: "Kisspeptin-GPR54 signaling in mouse NO-synthesizing neurons participates in the hypothalamic control of ovulation.", J. NEUROSCI., vol. 32, 2012, pages 932 - 945
HARFE, B.D.; MCMANUS, M.T.; MANSFIELD, J.H.; HORNSTEIN, E.; TABIN, C.J.: "The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb", PROC. NATL. ACAD. SCI. USA, vol. 102, 2005, pages 10898 - 10903, XP002627663, DOI: doi:10.1073/pnas.0504834102
HASUWA ET AL., SCIENCE, vol. 341, 2013, pages 71 - 73
HASUWA, H.; UEDA, J.; IKAWA, M.; OKABE, M.: "miR-200b and miR-429 function in mouse ovulation and are essential for female fertility", SCIENCE, vol. 341, 2013, pages 71 - 73, XP055306196, DOI: doi:10.1126/science.1237999
HEINZ, S. ET AL.: "Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities.", MOL. CELL, vol. 38, 2010, pages 576 - 589
ISSLER, O. ET AL.: "MicroRNA 135 is essential for chronic stress resiliency, antidepressant efficacy, and intact serotonergic activity", NEURON, vol. 83, 2014, pages 344 - 360, XP029035640, DOI: doi:10.1016/j.neuron.2014.05.042
KLEIN, D. ET AL.: "MicroRNA expression in alpha and beta cells of human pancreatic islets.", PLOS ONE, vol. 8, 2013, pages E55064
KOCH, M.; MOLLENKOPF, H.J.; KLEMM, U.; MEYER, T.F.: "Induction of microRNA-155 is TLR- and type IV secretion system-dependent in macrophages and inhibits DNA-damage induced apoptosis.", PROC. NATL. ACAD. SCI. USA, vol. 109, 2012, pages E1153 - E1162
KUIRI-HANNINEN, T.; SANKILAMPI, U.; DUNKEL, L.: "Activation of the hypothalamic-pituitary-gonadal axis in infancy: minipuberty", HORM. RES. PAEDIATR., vol. 82, 2014, pages 73 - 80
LE BECHEC, A. ET AL.: "MIR@NT@N: a framework integrating transcription factors, microRNAs and their targets to identify sub-network motifs in a meta-regulation network model.", BMC BIOINFORMATICS, vol. 12, 2011, pages 67, XP021098318, DOI: doi:10.1186/1471-2105-12-67
LEE, V.H.; LEE, L.T.; CHOW, B.K.: "Gonadotropin-releasing hormone: regulation of the GnRH gene", FEBS J., vol. 275, 2008, pages 5458 - 5478
LEFTEROVA, M.I. ET AL.: "Cell-specific determinants of peroxisome proliferator-activated receptor gamma function in adipocytes and macrophages.", MOL. CELL. BIOL, vol. 30, 2010, pages 2078 - 2089
LIU ET AL., MOLECULAR MEDICINE REPORTS, vol. 13, 2016, pages 3920 - 3928
LOMNICZI, A. ET AL.: "Epigenetic control of female puberty", NAT. NEUROSCI., vol. 16, 2013, pages 281 - 289
LOMNICZI, A. ET AL.: "Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression", NAT. COMMUN., vol. 6, 2015, Retrieved from the Internet <URL:http://dx.doi.org/10.1038/ncomms10195>
MASON, A.J. ET AL.: "A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the HPG mouse.", SCIENCE, vol. 234, 1986, pages 1366 - 1371
MESSINA, A. ET AL.: "Dysregulation of Semaphorin7A/D 1-integrin signaling leads to defective GnRH-1 cell migration, abnormal gonadal development and altered fertility", HUM. MOL. GENET, vol. 20, 2011, pages 4759 - 4774
NOVAIRA, H.J.; FADOJU, D.; DIACZOK, D.; RADOVICK, S.: "Genetic mechanisms mediating kisspeptin regulation of GnRH gene expression", J. NEUROSCI., vol. 32, 2012, pages 17391 - 17400
OJEDA, S.R.; LOMNICZI, A.: "Puberty in 2013: unraveling the mystery of puberty", NAT. REV. ENDOCRINOL., vol. 10, 2014, pages 67 - 69
PAPAIOANNOU, M.D.; NEF, S.: "microRNAs in the testis: building up male fertility", J. ANDROL., vol. 31, 2010, pages 26 - 33
PARENT, A.S. ET AL.: "The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration.", ENDOCR. REV., vol. 24, 2003, pages 668 - 693
PLANT, T.M. & ZELEZNIK, J.: "Knobil and Neill's Physiology of Reproduction", 2015, ELSEVIER, article PREVOT, V.: "Puberty in mice and rats", pages: 1395 - 1439
PREVOT, V. ET AL.: "Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes.", J. NEUROSCI., vol. 23, 2003, pages 230 - 239
RAMEAU, G.A. ET AL.: "Biphasic coupling of neuronal nitric oxide synthase phosphorylation to the NMDA receptor regulates AMPA receptor trafficking and neuronal cell death", J. NEUROSCI., vol. 27, 2007, pages 3445 - 3455
REKKER KADRI ET AL., FERTILITY AND STERILITY, vol. 104, no. 4, 2015, pages 938 - 946
REKKER KADRI ET AL: "Circulating miR-200-family micro-RNAs have altered plasma levels in patients with endometriosis and vary with blood collection time", FERTILITY AND STERILITY, vol. 104, no. 4, October 2015 (2015-10-01), pages 938, XP029285533, ISSN: 0015-0282, DOI: 10.1016/J.FERTNSTERT.2015.06.029 *
RODRIGUEZ, A. ET AL.: "Requirement of bic/microRNA-155 for normal immune function", SCIENCE, vol. 316, 2007, pages 608 - 611, XP002490506, DOI: doi:10.1126/science.1139253
SCHMITTGEN, T.D.; LIVAK, K.J.: "Analyzing real-time PCR data by the comparative C(T) method", NAT. PROTOC, vol. 3, 2008, pages 1101 - 1108, XP055137608, DOI: doi:10.1038/nprot.2008.73
SCHWANZEL-FUKUDA, M.; BICK, D.; PFAFF, D.W.: "Luteinizing hormone-releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome.", BRAIN RES. MOL. BRAIN RES., vol. 6, 1989, pages 311 - 326, XP024346564, DOI: doi:10.1016/0169-328X(89)90076-4
SORENSEN ET AL., GENES, vol. 5, 2014, pages 684 - 708
SPERGEL, D.J.; KRUTH, U.; HANLEY, D.F.; SPRENGEL, R.; SEEBURG, P.H.: "GABA- and glutamate-activated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice", J. NEUROSCI., vol. 19, 1999, pages 2037 - 2050
TSATSANIS ET AL., HUMAN REPRODUCTION, vol. 30, no. 4, 2015, pages 853 - 860
URBANSKI, H.F.; OJEDA, S.R.: "A role for N-methyl-D-aspartate (NMDA) receptors in the control of LH secretion and initiation of female puberty", ENDOCRINOLOGY, vol. 126, 1990, pages 1774 - 1776
VAN ROOIJ, E.; PURCELL, A.L.; LEVIN, A.A.: "Developing microRNA therapeutics", CIRC. RES., vol. 110, 2012, pages 496 - 507, XP055144403, DOI: doi:10.1161/CIRCRESAHA.111.247916
WIERMAN, M.E.; KISELJAK-VASSILIADES, K.; TOBET, S.: "Gonadotropin-releasing hormone (GnRH) neuron migration: initiation, maintenance and cessation as critical steps to ensure normal reproductive function", FRONT. NEUROENDOCRINOL., vol. 32, 2011, pages 43 - 52
X. D'ANGLEMONT DE TASSIGNY ET AL: "Kisspeptin Signaling Is Required for Peripheral But Not Central Stimulation of Gonadotropin-Releasing Hormone Neurons by NMDA", JOURNAL OF NEUROSCIENCE, vol. 30, no. 25, 23 June 2010 (2010-06-23), US, pages 8581 - 8590, XP055306197, ISSN: 0270-6474, DOI: 10.1523/JNEUROSCI.5486-09.2010 *
YANG, J.H.; LI, J.H.; JIANG, S.; ZHOU, H.; QU, L.H.: "ChIPBase: a database for decoding the transcriptional regulation of long non-coding RNA and microRNA genes from ChIP-Seq data", NUCLEIC ACIDS RES., vol. 41, 2013, pages D177 - D187
YOON, H.; ENQUIST, L.W.; DULAC, C: "Olfactory inputs to hypothalamic neurons controlling reproduction and fertility", CELL, vol. 123, 2005, pages 669 - 682, XP055306194, DOI: doi:10.1016/j.cell.2005.08.039

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