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WO2014180981A1 - Procédé pour diagnostiquer et traiter la stérilité mâle - Google Patents

Procédé pour diagnostiquer et traiter la stérilité mâle Download PDF

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Publication number
WO2014180981A1
WO2014180981A1 PCT/EP2014/059543 EP2014059543W WO2014180981A1 WO 2014180981 A1 WO2014180981 A1 WO 2014180981A1 EP 2014059543 W EP2014059543 W EP 2014059543W WO 2014180981 A1 WO2014180981 A1 WO 2014180981A1
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Prior art keywords
mir
subject
male infertility
expression level
compound
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Mohamed BENAHMED
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Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Nice Sophia Antipolis UNSA
Centre Hospitalier Universitaire de Nice
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Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Nice Sophia Antipolis UNSA
Centre Hospitalier Universitaire de Nice
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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
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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 invention relates to the diagnosis and treatment of male infertility.
  • male infertility represents an ever-increasingly current and complicated problem in view of the fact that many causes may concur to present this disorder. Only recently have the biological processes at the base of spermatogenesis been studied, contributing significantly to a wider comprehension of male fertility, emphasizing how genetic and molecular causes can be directly responsible for the different clinical aspects of male infertility. Thus, the male infertility can be caused by disorders relating to spermatozoa production, emission or functionality and represents approximately 35% of couple infertility causes.
  • the diagnosis of male infertility mostly relies on microscopic assays of semen quality including sperm concentration, motility and morphology. In the case that infertility is suspected to be related to the male, a few types of tests are used to assess the quality of male sperm. Five parameters that are frequently used to analyze sperm quality, and which are recommended by the World Health Organization (WHO) are: (1) sperm count; (2) sperm motility; (3) sperm viability; (4) white blood cell (WBC) count; (5) sperm head morphology.
  • WHO World Health Organization
  • Such assays are time-consuming and expensive since they require spermogram with microscopy, echography and semen biochemical analysis and even in certain cases surgery. Therefore, there is still a need for identifying biomarkers with expression profiles that can be used in a reliable, non-invasive and quick manner to identify male infertility.
  • miRs microRNAs
  • miRs may be detected in semen and that miRs may be differentially expressed between the semen of infertile men and normal men.
  • Liu at al. 2012 have identified, by analysis of miRNA microarray, that 52 miRs were differentially expressed between the semen of infertile men and normal men (21 miRs were significantly overexpressed in the abnormal semen compared with the normal semen and 31 miRs were significantly underexpressed in the abnormal semen compared with the normal semen (including miR-18a).
  • miR-18a the miR-18a
  • the invention relates to a method for identifying whether a subject has, or is at risk of having or developing male infertility, comprising a step of measuring in a blood sample obtained from said subject the expression level of at least one miR.
  • the invention in a second aspect, relates to a method for stratifying a subject having, or being at risk of having or developing male infertility, comprising a step of measuring in a blood sample obtained from said subject the expression level of at least one miR.
  • the invention relates to a compound that inhibits the expression level or the activity of miR18a for use in the prevention or treatment of male infertility.
  • the invention relates to a screening method of a compound that inhibits the expression level of miR- 18a for use in the prevention or treatment of male infertility comprising the steps consisting of:
  • the invention relates to a method for monitoring the efficacy of a compound useful for the prevention or treatment of male infertility administered to a subject in need thereof, comprising a step of measuring in a blood sample obtained from said subject the expression level of miR- 18a.
  • the invention relates to a method for monitoring the efficacy of a compound useful for the prevention or treatment of male infertility administered to a subject in need thereof, comprising the steps consisting of:
  • th einvention relates to a HSF2 (Heat Shock Transcription Factor 2) polypeptide or a functional equivalent thereof, or a nucleic acid encoding thereof, or a vector encoding thereof for use in preventing or treating male infertility.
  • HSF2 Heat Shock Transcription Factor 2
  • the invention relates to a HSP70 (Heat Shock Protein 70) polypeptide or a functional equivalent thereof, or a nucleic acid encoding thereof, or a vector encoding thereof for use in preventing or treating male infertility.
  • HSP70 Heat Shock Protein 70
  • the invention is based on the discovery that miR-18a is up-regulated in the blood plasma and regulates (i.e. decreases) expression level of HSF2 (Heat Shock Transcription Factor 2) and HSP70 (Heat Shock Protein 70) leading to the apoptosis of testicular germ cells.
  • HSF2 Heat Shock Transcription Factor 2
  • HSP70 Heat Shock Protein 70 leading to the apoptosis of testicular germ cells.
  • Up-regulation of miR-18a expression or function thus results notably in no sperm in the ejaculate (azoospermia) or in poor sperm quantity (oligozoospermia) leading to male infertility.
  • expression level of miR-18a family is directly correlated with the number of spermatozoa in semen (or sperm) assessed until now by spermogram and is thus useful for diagnosing and/or stratifying subjects.
  • miRs such as miR18a
  • expression levels of miRs represent valuable new biomarkers useful for diagnosing and/or stratifying subjects with male infertility, for determining apoptosis in testes of germ cells and also represent relevant therapeutic targets for preventing or treating male infertility.
  • miRNAs also called “miR” has its general meaning in the art and refers to microRNA molecules that are generally 21 to 22 nucleotides in length, even though lengths of 19 and up to 23 nucleotides have been reported. miRNAs are each processed from a longer precursor RNA molecule ("precursor miRNA"). Precursor miRNAs are transcribed from non- protein-encoding genes. The precursor miRNAs have two regions of complementarity that enables them to form a stem-loop- or fold-back-like structure, which is cleaved in animals by a ribonuclease Ill-like nuclease enzyme called Dicer. The processed miRNA is typically a portion of the stem.
  • the processed miRNA (also referred to as "mature miRNA”) become part of a large complex to down-regulate a particular target gene. All the miR As pertaining to the invention are known per se and sequences of them are publicly available from the data base http://microrna.sanger.ac.uk/sequences/.
  • the miRNAs of the invention are listed in Table A:
  • Table A list of the miRNAs according to the invention
  • measuring encompasses detecting or quantifying.
  • detecting means determining if at least one miR (such as miR18a) is present or not in a blood sample and “quantifying” means determining the amount of at least one miR (such as miR18a) in a blood sample.
  • blood sample refers to a body fluid sample derived from the subject that contains nucleic acid materials including (whole blood, plasma and serum). Said blood sample is obtained for the purpose of the in vitro evaluation.
  • the blood sample can be fresh or frozen.
  • treating refers to reversing, alleviating or inhibiting the process of one or more symptoms of such disorder or condition.
  • preventing a disorder or a condition refers to keeping from occurring, or to hinder, defend from, or protect from the occurrence of a disorder or a condition or phenotype, including a symptom.
  • a subject denotes a mammal.
  • a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with male infertility.
  • a subject refers to any subject afflicted with a disorder related to an impaired spermatogenesis, underdevelopment of testes, decrease of testicular weight and excess apoptosis in testes (including germ cells apoptosis, such as adult testicular germ cells).
  • disorders involving to an impairment of spermatogenesis leading to infertility are selected from the group consisting of: - Oligospermia or Oligozoospermia - decreased number of spermatozoa in semen
  • the invention relates to a method for identifying whether a subject has, or is at risk of having or developing male infertility, comprising a step of measuring in a blood sample obtained from said subject the expression level of at least one miR.
  • the invention relates to a method for identifying whether a subject has, or is at risk of having or developing male infertility, comprising a step of measuring in a blood sample obtained from said subject the expression level of at least one miR with the proviso that said at least one miR is not a member of the miR-29 family.
  • said at least one miR is miR- 18a.
  • a second aspect of the invention relates to a method of stratifying (or determining the severity of male infertility in) a subject having, or being at risk of having or developing male infertility, comprising a step of measuring in a sample obtained from said subject the expression level of at least one miR.
  • the invention relates to a method of stratifying (or determining the severity of male infertility in) a subject having, or being at risk of having or developing male infertility, comprising a step of measuring in a sample obtained from said subject the expression level of at least one miR with the proviso that said at least one miR is not a member of the miR-29 family.
  • said at least one miR is miR- 18a.
  • the blood plasma miR18-a expression level may be increased by more than 10-fold in oligo or azoospermia subjects (who have number of spermatozoa lower than 15 millions/ml of seminal liquid, e.g.. no spermatozoa in azoospermia subjects or less than 5 millions/ml of seminal liquid in severe azoospermia subjects) compared to fertile ones.
  • the method of the invention may further comprise a step consisting of comparing the expression level of at least one miRNA (such as miR- 18a) in the blood sample obtained from a subject with a control sample, wherein detecting differential expression level of said at least one miRNA between the blood sample and the control is indicative of having or a risk of having or developing a male infertility.
  • the control may consist in blood sample of a healthy subject not afflicted with male infertility.
  • control or “control sample” may be a single value such as a level or a mean expression level of at least one miRNA (such as miR- 18a) as determined in a blood sample obtained from a healthy subject or a group of subjects who are not afflicted with male infertility or who did not develop male infertility.
  • miRNA such as miR- 18a
  • higher expression level of at least one miRNA such as miR- 18a is indicative of a subject having or at risk of having or developing a male infertility.
  • higher expression level of at least one miRNA such as miR- 18a is indicative of a subject having, or being at risk of having or developing severe male infertility.
  • a level of expression of at least one miRNA (e.g. miR- 18a) higher than a control value indicates that the subject has or is at risk of having or developing infertility.
  • the expression level of miR- 18a measured in the blood sample of the subject may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150% or 200% higher than the control value.
  • the invention thus provides a method for identifying whether a subject has, or is at risk of having or developing male infertility, comprising the following steps of:
  • the invention further provides a method for stratifying (or determining the severity of male infertility in) a subject having, or being at risk of having or developing male infertility, comprising the following steps of: (i) measuring in a blood sample obtained from said subject the expression level of miR-18a;
  • measuring the expression level of at least one miR such as miR-18a in a blood sample obtained from the subject may be performed by a variety of techniques.
  • the nucleic acid contained in the blood sample e.g., whole blood, plasma or serum prepared from the subject
  • the blood sample e.g., whole blood, plasma or serum prepared from the subject
  • the extracted miRNAs is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR).
  • hybridization e. g., Northern blot analysis
  • amplification e.g., RT-PCR
  • RT-PCR e.g., Northern blot analysis
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., Northern blot analysis
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • the determination comprises contacting the sample with selective reagents such as probes or primers and thereby detecting the presence, or measuring the amount of miRNAs originally in the sample.
  • Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth.
  • the contacting is performed on a substrate coated with the reagent, such as a miRNA array.
  • the substrate may be a solid or semi- so lid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like.
  • the substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc.
  • the contacting may be made under any condition suitable for a detectable complex, such as a miRNAs hybrid, to be formed between the reagent and the miRNAs of the sample.
  • Nucleic acids exhibiting sequence complementarity or homology to the miRNAs of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).
  • the probes and primers are "specific" to the miRNAs they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 %> formamide, 5x or 6x SCC.
  • Tm melting temperature
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • miRNA arrays or miRNA probe arrays which are macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of miRNA molecules positioned on a support or support material in a spatially separated organization.
  • Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted.
  • Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.
  • Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes, oligonucleotides, etc., onto substrates or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non- identical nucleic acid molecules per square centimeter or higher, e.g. up to about 100 or even 1000 per square centimeter. Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose-based material of filter arrays. By having an ordered array of miR A-complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample.
  • nucleic acid molecules e.g., genes, oligonucleotides, etc.
  • array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art.
  • Useful substrates for arrays include nylon, glass, metal, plastic, latex, and silicon.
  • Such arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non-covalent, and the like.
  • the population of target nucleic acids is contacted with the array or probes under hybridization conditions, where such conditions can be adjusted, as desired, to provide for an optimum level of specificity in view of the particular assay being performed.
  • Suitable hybridization conditions are well known to those of skill in the art and reviewed in Sambrook et al. (2001). Of particular interest in many embodiments is the use of stringent conditions during hybridization. Stringent conditions are known to those of skill in the art.
  • miRNAs quantification method may be performed by using stem-loop primers for reverse transcription (RT) followed by a real-time TaqMan® probe.
  • said method comprises a first step wherein the stem-loop primers are annealed to miRNA targets and extended in the presence of reverse transcriptase. Then miRNA-specific forward primer, TaqMan® probe, and reverse primer are used for PCR reactions. Quantitation of miRNAs is estimated based on measured CT values. Many miR A quantification assays are commercially available from Qiagen (S. A. Courtaboeuf, France) or Applied Biosystems (Foster City, USA).
  • Expression level of a miRNA may be expressed as absolute expression level or normalized expression level.
  • expression levels are normalized by correcting the absolute expression level of a miRNA by comparing its expression to the expression of a mRNA that is not a relevant for determining subject having or at risk of having or developing an infertility , e.g., a housekeeping RNAthat is constitutively expressed.
  • Suitable RNA for normalization includes housekeeping RNAs such as the U6, U24, U48 S18 and cel-miR-39. This normalization allows the comparison of the expression level in one sample, e.g., a subject sample, to another sample, or between samples from different sources.
  • Methods of monitoring may be applied for monitoring the treatment (e.g., drug compounds) administered to a subject in need thereof.
  • the efficacy of a compound used for the prevention or treatment of male infertility may be monitored during treatments of subjects receiving male infertility treatments by measuring in a blood sample obtained from said subject the expression level of at least one miR (such as miR18a).
  • the invention relates to a method for monitoring the efficacy of a compound useful for the prevention or treatment of male infertility administered to a subject in need thereof, comprising a step of measuring in a blood sample obtained from said subject the expression level of at least one miR.
  • the invention relates to a method for monitoring the efficacy of a compound useful for the prevention or treatment of male infertility administered to a subject in need thereof, comprising a step of measuring in a blood sample obtained from said subject the expression level of at least one miR with the proviso that said at least one miR is not a member of the miR-29 family.
  • said at least one miR is miR- 18a.
  • male infertility treatment that is referred to any type of male infertility therapy undergone by the male infertile subjects, including hormones such as testosterone or diet food.
  • the invention relates a method for monitoring the efficacy of a compound useful for the prevention or treatment of male infertility administered to a subject in need thereof, comprising the steps consisting of: i) measuring before said administration in a blood sample obtained from said subject the expression level of miR-18a;
  • kits suitable for performing the methods of the invention wherein said kit comprises means for measuring the expression level of at least miR, in particular miR18-a in the blood sample obtained from the subject.
  • the kits may include probes, primers macroarrays or microarrays as described above.
  • the kit may comprise a set of miRNA probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers.
  • the kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
  • kits of the invention may comprise amplification primers (e.g. stem- loop primers) that may be pre-labelled or may contain an affinity purification or attachment moiety.
  • the kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.
  • the kit is suitable for a method of identifying whether a subject has, or is at risk of having or developing male infertility, comprising means for measuring in a blood sample obtained from said subject, the expression level of miR-18a.
  • the kit is suitable for a method of stratifying a subject having, or being at risk of having or developing male infertility, comprising means for measuring, in a sample obtained from said subject, the expression level of miR-18a.
  • the kit is suitable for a method of monitoring the efficacy of a compound useful for the prevention or treatment of male infertility administered to a subject in need thereof comprising means for measuring in a blood sample obtained from said subject, the expression level of miR-18a.
  • the kit further comprise means for comparing the expression level of miR-18a in the blood sample with a control, wherein detecting differential in the expression level of miR18a between the blood sample and the control is indicative of a risk of having or developing male infertility or is indicative of a severe male infertility .
  • the control may consist in sample associated with a healthy subject not afflicted with male infertility or in a sample associated with a subject afflicted with male infertility.
  • the invention also relates to a compound that inhibits the expression level or the activity of miR-18a for use in the prevention or the treatment of male infertility.
  • the invention further relates to a compound that inhibits the expression level or the activity of miR-18a for use in the prevention or inhibition or apoptosis of male germ cells.
  • the male germ cells are selected from the group consisting of spermatogenic cells (e.g. spermatogonia, preleptotene and pachytene spermatocytes, spermatozoa), Sertoli cells and Leydig cells.
  • spermatogenic cells e.g. spermatogonia, preleptotene and pachytene spermatocytes, spermatozoa
  • Sertoli cells e.g. spermatogonia, preleptotene and pachytene spermatocytes, spermatozoa
  • inhibiting the expression level or the activity of a miRNA or “inhibiting miRNA expression or activity” mean that the production of miRNA in a sample (e.g. blood plasma) after treatment is less than the amount produced prior to treatment or that the function of the miRNA of interest has been reduced or inhibited (e.g. the down-regulation of target gene expression (i.e. the protein translation of the target mRNA) by a miRNA is reduced or inhibited).
  • a sample e.g. blood plasma
  • the function of the miRNA of interest e.g. the down-regulation of target gene expression (i.e. the protein translation of the target mRNA) by a miRNA is reduced or inhibited).
  • a "compound that inhibits the expression level of a miRNA” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of said miRNA. Such compounds include but are not limited to nucleic acids or small organic molecules.
  • a "compound that inhibits the activity of a miRNA” refers to a natural or synthetic compound that has a biological effect to reduce or inhibit the function of said miRNA. Such compounds include but are not limited to nucleic acids or small organic molecules.
  • Suitable compounds for inhibiting miRNA expression or activity includes miRNA inhibitory nucleic acids selected from the group consisting of antagomirs, antisense nucleic acids, double-stranded RNA (such as short- or small-interfering RNA or "siRNA”) and enzymatic RNA molecules such as ribozymes.
  • miRNA inhibitory nucleic acids can be designed according to methods known in the art. These miRNA inhibitory nucleic acids encompassing oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages as well as oligonucleotides having non- naturally-occurring portions that function similarly.
  • modified or substituted oligonucleotides are may be used over native forms because of desirable properties such as, for example, enhanced affinity for nucleic acid target, and/or increased stability in the presence of nucleases.
  • the miRNA inhibitory nucleic acids include oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both, or modifications thereof.
  • the miRNA inhibitory nucleic acid can be a single-stranded, double stranded, partially double stranded or hairpin oligonucleotide. It preferably consists of, consists essentially of, or comprises at least 12 or more contiguous nucleotides substantially complementary to an endogenous miRNA or a pre-miRNA.
  • partially double stranded refers to double stranded structures that contain fewer nucleotides on one strand. In general, such partial double stranded agents will have less than 75% double stranded structure, less than 50%), or less than 25%, 20%> or 15% double stranded structure.
  • a miRNA inhibitory nucleic acid comprises a region sufficient complementary to the target nucleic acid (e.g., target miRNA, pre-miRNA), and is of sufficient length, such that the miRNA inhibitory nucleic acid forms a duplex with the target nucleic acid.
  • the miRNA inhibitory nucleic acid can modulate the function of the targeted molecule.
  • the target molecule is an miRNA, such as miR-18a
  • the miRNA inhibitory nucleic acid can inhibit the gene silencing activity of the target miRNA, which action will up-regulate expression of the mRNA targeted by the target miRNA.
  • An miRNA inhibitory nucleic acid can be partially or fully complementary to the target miRNA.
  • the miRNA inhibitory nucleic acid can be further stabilized against nucleo lytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • the miRNA inhibitory nucleic acid includes a phosphorothioate at least the first, second, or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence.
  • the miRNA inhibitory nucleic acid includes a 2'-modified nucleotide, e.g., a 2'- deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2 * -0-AP), 2 * -0-dimethylamino ethyl (2 * -0-DMAOE), 2 * -0-dimethylaminopropyl (2 * -0- DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA).
  • a 2'-modified nucleotide e.g., a 2'- deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-amin
  • the miRNA inhibitory nucleic acid includes at least one 2'-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the miRNA inhibitory nucleic acid include a 2'-0-methyl modification.
  • the miRNA inhibitory nucleic acid can be modified so as to be attached to a ligand that is selected to improve stability, distribution or cellular uptake of the agent, e.g., cholesterol.
  • the oligonucleotide miRNA inhibitory nucleic acid can further be in isolated form or can be part of a pharmaceutical composition used for the methods described herein, particularly as a pharmaceutical composition formulated for parental administration.
  • the pharmaceutical compositions can contain one or more oligonucleotide agents.
  • a miRNA inhibitory nucleic acid can be constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. Alternatively, the miRNA inhibitory nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned.
  • the expression vectors can be DNA plasmids or viral vectors.
  • the expression vectors capable of expressing the miRNA inhibitory nucleic acids can be delivered as described herein, and can persist in target cells.
  • viral vectors can be used that provide for transient expression of nucleic acid molecules.
  • Such vectors can be repeatedly administered as necessary.
  • Viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus (lentivirus), adenovirus, or alphavirus.
  • Pol II or Pol Ill-based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886).
  • the miRNA inhibitory nucleic acid interacts with the target RNA (e.g., miRNA or pre-miRNA) and inhibits miRNA activity.
  • the target RNA e.g., miRNA or pre-miRNA
  • liposomes may be used to deliver miRNA inhibitory nucleic acid to a subject. Liposomes can also increase the blood half-life of the gene products or nucleic acids. Liposomes suitable for use in the invention can be formed from standard vesicle- forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream.
  • the miRNA inhibitory nucleic acid is an antagomir, which is a chemically-modified, single-stranded RNA that is antisense to the miRNA sequence.
  • An antagomir is from about 12 to about 33 nucleotides in length, preferably at least about 15 nucleotides in length.
  • a preferred modification is 2'-0-methylation of the ribose. Additional or alternative modifications can include phosphorothioate linkage near 5' and 3' ends or a cholesterol moiety conjugated to the 3' end.
  • Antagomirs form highly stable, sequence-specific duplexes with their corresponding target miRNAs and potently attenuate miRNA activity.
  • antagomirs act as competitive inhibitors of endogenous target mRNA binding to the miRNA, resulting in suppression of miRNA function.
  • Antagomirs can also induce degradation of target miRNAs.
  • Antagomirs bind to their target miRNAs through sequence- specific base pairing.
  • a morpholino is an example of an antagomir.
  • a locked nucleic acid (LNA) antisense oligonucleotide is also an example of antagomir.
  • Antagomirs for use in the present invention preferably inhibit miR-18a.
  • the sequence of the antagomir against miR-18a is shown as follows (SEQ ID NO: 2) and as described in Scherr et al., 2007: CTATCTGCACTAGATGCACCTTA
  • Antagomirs can be designed according to methods known in the art. See Krutzfeldt et al. (2005) and U.S. Publication No. 2009/0092980, incorporated herein by reference. Antagomirs are commercially available, for example, from Ambion, Inc. (Austin, Tex.).
  • miRNA inhibitory nucleic acid is a miRNA sponge (Ebert et al, 2007, incorporated herein by reference). Sponges can be designed to bind effectively to multiple miRNAs that contain the same seed region. See Tang et al. 2008, incorporated herein by reference, for a review of small RNA technologies, including miRNA inhibition.
  • small organic molecules refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • Such small organic molecules may be for instance FDA- approved drugs.
  • the invention also relates to a HSF2 (Heat Shock Transcription
  • Factor 2 polypeptide or a functional equivalent thereof for use in preventing or treating male infertility.
  • the invention relates to a HSF2 polypeptide or a functional equivalent thereof for use in preventing or inhibiting apoptosis of male germ cells.
  • the male germ cells are selected from the group consisting of spermatogenic cells (e.g. preleptotene and pachytene spermatocytes), Sertoli cells and Leydig cells.
  • spermatogenic cells e.g. preleptotene and pachytene spermatocytes
  • Sertoli cells e.g. serotonin cells
  • Leydig cells spermatogenic cells
  • HSF2 Heat shock Transcription Factor 2
  • HSF2 has its general meaning in the art and refers to a main transcriptional regulator of the stress-induced expression of heat shock proteins (hsp) genes. HSF2 is thus able to bind the hsp70 promoter in response to a heat shock response which is a defense reaction activated by proteotoxic damage induced by physiological or environmental stress.
  • HSPs that function as molecular chaperones and maintain the vital homeostasis of protein folds.
  • the naturally occurring human HSF2 gene has a nucleotide sequence as shown in Genbank Accession number NM 001135564 and the naturally occurring human HSF2 protein has an aminoacid sequence of 518 amino acids as shown in GenBank database under accession number NP 001129036 and is shown as follows (SEQ ID NO: 3):
  • the invention further relates to a HSP70 (Heat Shock Protein 70) polypeptide or a functional equivalent thereof for use in preventing or treating male infertility.
  • HSP70 Heat Shock Protein 70
  • the invention relates to a HSP70 polypeptide or a functional equivalent thereof for use in preventing or inhibiting apoptosis of male germ cells.
  • HSP70 Heat Shock Protein 70
  • HSPA1B Heat Shock Protein 70
  • the naturally occurring human HSP70 gene has a nucleotide sequence as shown in Genbank Accession number NM 005346 and the naturally occurring human HSP70 protein has an aminoacid sequence of 641 amino acids as shown in GenBank database under accession number NP 005337 and is shown as follows (SEQ ID NO: 4):
  • polypeptide means herein a polymer of amino acids having no specific length. Thus, peptides, oligopeptides and proteins are included in the definition of “polypeptide” and these terms are used interchangeably throughout the specification, as well as in the claims.
  • polypeptide does not exclude post-translational modifications that include but are not limited to phosphorylation, acetylation, glycosylation and the like.
  • a “native sequence” polypeptide refers to a polypeptide having the same amino acid sequence as a polypeptide derived from nature.
  • a native sequence polypeptide can have the amino acid sequence of naturally-occurring polypeptide from any mammal (including human. Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means.
  • the term "native sequence” polypeptide specifically encompasses naturally-occurring truncated or secreted forms of the polypeptide (e. g., an extracellular domain sequence), naturally-occurring variant forms (e. g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide.
  • a polypeptide "variant” refers to a biologically active polypeptide having at least about 80% amino acid sequence identity with the native sequence polypeptide.
  • variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N-or C-terminus of the polypeptide.
  • a variant will have at least about 80% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity with the native sequence polypeptide.
  • polypeptide having an amino acid sequence at least, for example, 95% "identical" to a amino acid query sequence of the invention it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
  • up to 5% (5 of 100) of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid.
  • the percentage of identity is calculated using a global alignment (i.e., the two sequences are compared over their entire length).
  • the "needle” program which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used.
  • the needle program is for example available on the ebi.ac.uk world wide web site.
  • the percentage of identity in accordance with the invention is preferably calculated using the EMBOSS: :needle (global) program with a "Gap Open” parameter equal to 10.0, a "Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.
  • Polypeptides consisting of an amino acid sequence "at least 80%, 85%, 90%, 95%, 96%), 97%), 98%) or 99% identical" to a reference sequence may comprise mutations such as deletions, insertions and/or substitutions compared to the reference sequence.
  • the polypeptide consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to an allelic variant of the reference sequence. It may for example only comprise substitutions compared to the reference sequence. The substitutions preferably correspond to conservative substitutions as indicated in the table below. Conservative substitutions Type of Amino Acid
  • the term "functional equivalent of the HSF2 polypeptide” includes the variants and the fragments of the polypeptide to which it refers (i.e. the HSF2 polypeptide) and that retain the biological activity and the specificity of the parent polypeptide. Therefore, the "functional equivalent of the HSF2 polypeptide” includes variants and fragments of the polypeptide represented by SEQ ID NO: 3.
  • the term "functional equivalent of the HSP70 polypeptide” includes the variants and the fragments of the polypeptide to which it refers (i.e. the HSP70 polypeptide) and that retain the biological activity and the specificity of the parent polypeptide. Therefore, the "functional equivalent of the HSP70 polypeptide” includes variants and fragments of the polypeptide represented by SEQ ID NO: 4.
  • biological activity of a functional equivalent of the HSF2 or HSP70 polypeptide is meant (i) the capacity to induce the expression of HSP70; and/or (ii) the capacity to prevent the death of germ cells.
  • HSF2 or HSP70 polypeptide is biologically active.
  • an apoptosis assay such as a TUNEL assay which is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades may be performed with each polypeptide.
  • a time-course and a dose- response performed in vitro or in vivo e.g. by using a murine model of infertility as described in the Examples section
  • the polypeptides of the invention may comprise a tag.
  • a tag is an epitope-containing sequence which can be useful for the purification of the polypeptides. It is attached to by a variety of techniques such as affinity chromatography, for the localization of said peptide or polypeptide within a cell or a tissue sample using immuno labeling techniques, the detection of said polypeptide by immunoblotting etc.
  • tags commonly employed in the art are the GST (glutathion-S-transferase)-tag, the FLAGTM-tag, the Strep- tagTM, V5 tag, myc tag, His tag (which typically consists of six histidine residues), etc.
  • the polypeptides of the invention may comprise chemical modifications improving their stability and/or their biodisponibility.
  • Such chemical modifications aim at obtaining polypeptides with increased protection of the polypeptides against enzymatic degradation in vivo, and/or increased capacity to cross membrane barriers, thus increasing its half- life and maintaining or improving its biological activity.
  • Any chemical modification known in the art can be employed according to the present invention. Such chemical modifications include but are not limited to:
  • N-terminal and/or C-terminal ends of the peptides such as e.g. N- terminal acylation (preferably acetylation) or desamination, or modification of the C- terminal carboxyl group into an amide or an alcohol group;
  • acylation preferably acetylation
  • alkylation preferably methylation
  • acylation preferably acetylation
  • alkylation preferably methylation
  • water-soluble polymers Another strategy for improving drug viability is the utilization of water-soluble polymers.
  • Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
  • water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
  • PEG Polyethylene glycol
  • Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity.
  • PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids were explored as novel bio materials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
  • PEGylation techniques for the effective modification of drugs.
  • drug delivery polymers that consist of alternating polymers of PEG and tri- functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.).
  • the PEG chains typically 2000 daltons or less
  • Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain.
  • the reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules.
  • These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer.
  • the molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading).
  • increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold- limiting glomular filtration (e.g., less than 60 kDa).
  • linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue.
  • a specific trigger typically enzyme activity in the targeted tissue.
  • tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology.
  • Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
  • polypeptides of the invention may be fused to a heterologous polypeptide (i.e. polypeptide derived from an unrelated protein).
  • the heterologous polypeptide is a peptide capable of being internalized into a cell.
  • Such peptides capable of being internalized into a cell used herein have in their respective primary amino acid sequences (that is, over their entire length) at least 25%, preferably at least 30% positively charged amino acid residues.
  • the term "positively charged amino acids” (herein also referred to as “basic amino acids”), as used herein, denotes the entirety of lysine (K), histidine (H), and arginine (R) residue present in a particular peptide.
  • the peptides used herein comprise in their respective primary amino acid sequences at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%) positively charged amino acid residues.
  • peptides having said ability to pass through cellular membranes are herein referred to as "cell-penetrating peptides” (CPP) also called “protein transduction domains” (PTD), “membrane translocation sequences” (MTS) or “translocating peptides”.
  • CPP cell-penetrating peptides
  • PTD protein transduction domains
  • MTS membrane translocation sequences
  • translocating peptides any possible mechanism of internalization is envisaged including both energy-dependent (i.e. active) transport mechanisms (e.g., endocytosis) and energy-independent (i.e. passive) transport mechanism (e.g., diffusion).
  • the term "internalization” is to be understood as involving the localization of at least a part of the peptides that passed through the plasma cellular membrane into the cytoplasma (in contrast to localization in different cellular compartments such as vesicles, endosomes or in the nucleus).
  • the cell-penetrating peptide is the HIV transactivator of transcription (TAT) peptide represented by SEQ ID NO: 5 (Vives et ah, J. Biol. Chem., 272, 16010-16017, 1997) :
  • nucleic acid encoding a polypeptide of the invention (such as the HSF2 polypeptide as shown in SEQ ID NO: 3 or the HSP70 polypeptide as shown in SEQ ID NO: 4) or a vector comprising such nucleic acid or a host cell comprising such expression vector may be used in preventing or treating male infertility.
  • a nucleic acid encoding a polypeptide of the invention (HSF2 polypeptide as shown in SEQ ID NO: 3 or the HSP70 polypeptide as shown in SEQ ID NO: 4) or a vector comprising such nucleic acid or a host cell comprising such expression vector may also be used in preventing or inhibition male germ cells.
  • said nucleic acid encoding an amino acid sequence consisting on SEQ ID NO: 3 or SEQ ID NO: 4.
  • Nucleic acids of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination(s).
  • heat shock protein inducers in particular compounds inducing the expression of HSP70, such as geranylgeranylacetone (GGA) or 6, 10, 14, 18-tetramethyl-5, 9, 13, 17-nonadecatetraen-2-one (Teprenone sold under the brand name Selbex) may also be used for the prevention or the treatment of male infertility.
  • GGA geranylgeranylacetone
  • Teprenone sold under the brand name Selbex may also be used for the prevention or the treatment of male infertility.
  • Selbex geranylgeranylacetone
  • the invention relates to a method for the prevention or treatment of male infertility in a subject in need thereof comprising administering to said subject a therapeutically effective amount of at a compound that inhibits the expression level or the activity of miR- 18a.
  • the invention also provides to a method for the prevention or inhibition of apoptosis of male germ cells in a subject in need thereof comprising administering to said subject a therapeutically effective amount of at a compound that inhibits the expression level or the activity of miR- 18a.
  • the invention also relates to a method for the prevention or the treatment of male infertility in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a HSF2 polypeptide or a functional equivalent thereof.
  • the invention further relates to a method for the prevention or the treatment of male infertility in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a HSP70 polypeptide or a functional equivalent thereof.
  • a "therapeutically effective amount" of a compound of the invention is an amount sufficient to prevent or to treat infertility in a subject at a reasonable benefit/risk ratio applicable to any medical treatment.
  • a therapeutically effective amount of said compound to be administered to a given subject can be determined by taking into account factors such as the size and weight of the subject; the extent of disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic.
  • a therapeutically effective amount of said compound can be based on the approximate or estimated body weight of a subject to be treated. Preferably, such effective amounts are administered parenterally or enterally, as described herein.
  • a therapeutically effective amount of the compound is administered to a subject can range from about 5-3000 micrograms/kg of body weight, and is preferably between about 700-1000 micrograms/kg of body weight, and is more preferably greater than about 1000 micrograms/kg of body weight.
  • the compound can be administered to the subject once (e.g., as a single injection or deposition).
  • said compound can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, more preferably from about seven to about ten days.
  • the compound is administered once a day for seven days.
  • a dosage regimen comprises multiple administrations, it is understood that the effective amount of the compound administered to the subject can comprise the total amount of gene product administered over the entire dosage regimen.
  • a culture medium of male germ cells A culture medium of male germ cells
  • the invention in another aspect, relates to a method for improving survival of a population of male germ cells in vitro or ex vivo, comprising a step of contacting said population with a culture medium comprising an effective amount of a compound that inhibits the expression level or the activity of miR-18a.
  • improving cell survival refers to an increase in the number of cells that survive a given condition, as compared to a control, e.g., the number of cells that would survive the same conditions in the absence of treatment. Improved cell survival can be expressed as a comparative value, e.g., twice as many cells survive if cell survival is improved two-fold. Improved cell survival can result from a reduction in apoptosis, an increase in the life-span of the cell, or an improvement of cellular function and condition. In some embodiments, cell survival is improved by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, as compared to control levels.
  • cell survival is by two-, three-, four-, five-, or ten-fold of control levels.
  • improved cell survival can be expressed as a percentage decrease in apoptosis.
  • apoptosis is reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or up to 100%, as compared to a control sample.
  • the term "culture medium" refers to a liquid medium suitable for the in vitro or ex vivo culture of mammalian male germ cells, and preferably human male germ cells.
  • male germ cells refers to a population of spermatogenic cells (spermatogonia, preleptotene and pachytene spermatocytes, spermatozoa), Sertoli cells and Leydig cells.
  • the culture medium used by the invention may be a water-based medium that includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors and hormones, all of which are needed for cell survival.
  • a culture medium according to the invention may be a synthetic tissue culture medium such as the RPMI (Roswell Park Memorial Institute medium) or the CMRL- 1066 (Connaught Medical Research Laboratory) for human use, supplemented with the necessary additives as is further described below (Section Examples).
  • RPMI Roswell Park Memorial Institute medium
  • CMRL- 1066 Connaught Medical Research Laboratory
  • the culture medium of the invention is free of animal- derived substances.
  • the culture medium of the invention consists essentially of synthetic compounds, compounds of human origin and water.
  • said culture medium can be used for culturing cells according to good manufacturing practices (under "GMP" conditions). Screening methods:
  • a further aspect of the invention thus relates to a method for screening a compound that inhibits the expression level of miR-18a family for use in the prevention or the treatment of male infertility.
  • the screening method may involve measuring or, qualitatively or quantitatively, ability of said candidate compound to inhibit, reduce or suppress the expression level of miR-18a in miR-18a-expressing cells, and efficiently protects and treats male infertility by inhibiting, reducing or suppressing apoptosis of testicular germ cells.
  • the screening method of a compound that inhibits the expression level of at least one member of the miR-18a for use in the prevention or the treatment of male infertility comprises the steps consisting of: i) providing a plurality of cells expressing miR- 18a;
  • screening methods involve providing appropriate cells which express at least one member of the miR- 18a.
  • said cells may be selected from the group consisting of the mammal cells reported yet to express miR- 18a (e.g. spermatocytes and round spermatids).
  • miR- 18a e.g. spermatocytes and round spermatids.
  • RNA suspected to induce cell apoptosis based for instance on the use of GC1 cell line (a spermatogonia cell line).
  • apoptotic process in this cell line transfected with "apoptomir” may be visualized with annexinV approaches (immunofluorescence, flux cytometry).
  • the screening method of the invention may further comprising a step consisting of determining whether the candidate compound inhibiting the expression level of miR- 18a selected at step iv also increases the expression level of HSF2 and/or HSP70 polypeptide.
  • the screening method of the invention may further comprising a step consisting of administering the candidate compound selected at step d) to an animal model of male infertility to validate the therapeutic and/or protective effects of said candidate compound on male infertility.
  • said animal model of male infertility may be the animal model described above (i.e. the early (PND1 to 5) postnatal exposure of male rats to xenoestrogen compounds such as the estradiol benzoate EB or anti-androgens such as Flutamide, Vinclozolin or Prochloraz) as described above in Materials & Methods.
  • xenoestrogen compounds such as the estradiol benzoate EB or anti-androgens such as Flutamide, Vinclozolin or Prochloraz
  • the screening method of a compound that inhibits the expression level or activity of at least one member of the miR-18a for use in the prevention or the treatment of male infertility comprises the steps consisting of: i) providing a plurality of male germ cells;
  • a nucleic acid encoding miR-18a may be employed to transfect cells to thereby express miR-18a.
  • Such a transfection may be accomplished by methods well known in the art.
  • said cells may be the CG61 spg (CG-1) mouse spermatagonia type B-spermatocyte cells lines transfected with miR-18a as described above in Materials & Methods.
  • the candidate compounds may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo or natural compounds.
  • the candidate compound may be selected from the group of (a) proteins or peptides, (b) nucleic acids and (c) small organic molecules (natural or not).
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound that inhibits the expression level or the activity of miR-18a or a vector encoding thereof for use in the prevention or the treatment of male infertility.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a HSF2 (Heat Shock Transcription Factor 2) polypeptide or a functional equivalent thereof, or a nucleic acid encoding thereof, or a vector encoding thereof for use in the prevention or the treatment of male infertility.
  • HSF2 Heat Shock Transcription Factor 2
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a HSp70 (Heat Shock Protein 70) polypeptide or a functional equivalent thereof, or a nucleic acid encoding thereof, or a vector encoding thereof for use in the prevention or the treatment of male infertility.
  • HSp70 Heat Shock Protein 70
  • compositions of the present invention are preferably formulated as pharmaceutical compositions, prior to administering to a subject, according to techniques known in the art.
  • Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen- free.
  • pharmaceutical compositions include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
  • the pharmaceutical compositions comprise at least one compound that inhibits the expression level or the activity of miR-18a (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier.
  • the pharmaceutical compositions of the invention can also comprise at least one compound of the invention which are encapsulated by liposomes and a pharmaceutically-acceptable carrier.
  • Preferred pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • the pharmaceutical compositions of the invention comprise at least one compound of the invention which is resistant to degradation by nucleases.
  • nucleases One skilled in the art can readily synthesize nucleic acids which are nuclease resistant, for example by incorporating one or more ribonucleotides that are modified at the 2'-position into the miRNAs. Suitable 2'-modified ribonucleotides include those modified at the 2'-position with fluoro, amino, alkyl, alkoxy, and O-allyl.
  • compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives.
  • Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include, e.g., physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
  • solid pharmaceutically acceptable carriers for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10- 95%, preferably 25%-75%, of the at least one compound of the invention.
  • a pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20% by weight, preferably 1%-10% by weight, of one compound of the invention encapsulated in a liposome as described above, and a propellant.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Neonatal exposure to estrogenic compounds induces adult testicular hypospermatogenesis.
  • FIG. 2 Upstream, miR-18a down regulates HSF2 expression.
  • Figure 3 MiR-18a as biomarker of hypospermatogenesis in blood from adult rats unexposed (open box) or exposed to EB (black box) (A) and in blood from patients (fertile versus azoospermia, B).
  • EXAMPLE 1 MODEL OF MALE INFERT I LITY: MALE RATS EXPOSED TO XENOESTROGENS
  • mice Pregnant Sprague Davvley rats at gestational day (GD) 1 5 (Janvier, Le Gen est Saint Isle, France) were individually housed in tern perat u re-co nt ro 11 ed rooms with 12h light, 12h dark cycles and given free access to water and feed. At birth, each pu was sexed, weighed, and identi fied.
  • Pups were administered vehicle (corn oil; MP Biomedicals, I llkirch, France) or estradiol benzoate (EB) (Sigma-Aldrich, L " Isle D ' Abeau, France) by daily sc injections from post-natal day (PND) 1 to PND5 at doses of 0, 0.75, 1.25, 2.5, 1 2.5 or 25 g d.
  • PND6 post-natal day
  • PND21 post-natal day
  • PND30 PND90 by C02 inhalation.
  • the posit ion of each testis was carefully noticed, and testes were removed and weighed. Only bilateral descended testes were studied.
  • testes were snap frozen for quantitative molecular approaches, whereas the other was fixed for morphological studies. At least seven different animals from four different litters were used for each treatment group. This study was conducted in accordance with current regulations and standards approved by Inst itut National de la Sante et de la Recherche Medicale Animal Care Committee (protocol no. 2008-43).
  • testis sections were counterstained with 4',6'-diaminido-2- phenyiindoie (DAPI), mounted with cover- slips using mounting medium. The results were expressed as the number of TUNEL-positive cells per 100 random round seminiferous tubules.
  • DAPI 4',6'-diaminido-2- phenyiindoie
  • sections were incubated 10 min in PBS 0.1% Triton X- 100 at room temperature, then in PBS-5% FCS-1 % protease inhibitor cocktail (Sigma, St. Louis, MO) fo 0 min. The section were incubated overnight at 4°C with BAX ant ibody (Cell Signaling) diluted at 1 : 100.
  • Sections were then washed three times in PBS 0.1% Triton X- 100 and incubated 1 h at room temperature with a Texas Red-conjugated secondary antibody donkey antirabbit (Amersham, Piscataway, NJ) diluted at 1 :30. The sections were washed in lx PBS and counterstained as described for TUN EL.
  • Western blot analysis Frozen testicular tissues were ground in l iquid nitrogen to obtain tissue powders. Aliquots of powder were homogenized in ice-cold hypoton ic buffer (25 mM Tris-HCl, 0.1% sodium do- decyl sulfate, and 1% protease inhibitor cocktail) (Sigma-Aldrich).
  • Tissue homogenates were further sonicated (10 sec at 80 W). Protein concentration was determined using the bicinchoninic acid assay. The experimental procedures were carried out as described in (Benbrahim-Tallaa et a!., 2008).
  • the antibodies used in this study were Mcl-l (1 : 1000; Rockland, GilbertsviUe, PA), Bax (1 : 1000, Cell Signaling), Bcl2 (1 : 1 .000, Santa Cruz Biotechnology), Bcl-x L (1 : 1 ,000, Santa Cruz Biotechnology), Puma (1 : 1000, Cell Signaling) and Act in (1 : 10,000; Jackson ' s Laboratory).
  • Membranes were scanned using a Luminescent Image Analyzer 3000 CCD Camera ( Fuji film, Dusseldorf, Germany ) and quan- tified using MultiGaugecommun (Fujifilm ).
  • Neonatal exposure to estrogenic compounds induces adult testicular hypospermatogenesis.
  • Neonatal exposure to the estrogenic analog estradiol benzoate (EB) induced severe atrophy of adult (PND 90) testes at the highest dose (12.5 and 25 ⁇ ⁇ , Figure 1 A) with massive germ cell loss (Figure IB).
  • EB estradiol benzoate
  • Figure IB massive germ cell loss
  • testicular phenotype was correlated with the absence of modification in body and testicular weight at 2.5 ⁇ g/d and lower doses of EB.
  • testicular phenotype was correlated with the absence of modification in body and testicular weight at 2.5 ⁇ g/d and lower doses of EB.
  • testicular phenotype was correlated with the absence of modification in body and testicular weight at 2.5 ⁇ g/d and lower doses of EB.
  • testicular phenotype was correlated with the absence of modification in body and testicular weight at 2.5 ⁇ g/d and lower doses of EB.
  • the apoptotic process was assessed by a significant increase in pro-apoptotic protein levels such as BAX (but not PUMA, Figure ID, E) and decrease in anti-apoptotic protein levels such as BCL2, BCL-XL and MCL1 ( Figure ID).
  • pro-apoptotic protein levels such as BAX (but not PUMA, Figure ID, E)
  • anti-apoptotic protein levels such as BCL2, BCL-XL and MCL1 ( Figure ID).
  • Such a long-term germ cell death process was identified in adult (PND90) testes but not in immature (PND6) or juvenile (PND21, 30) testes ( Figure IE).
  • RT-PCR reactions were performed using the stem-loop RT-PCR method which is specific for mature miR A (TaqMan miRNA assays, Applied Biosystems, Foster City, CA, USA). Ten nanograms of total RNA were reverse transcribed in a 7.5 ⁇ reaction using Multiscribe Reverse Transcriptase and a TaqMan miR-18a RT primer (Applied Biosystems). The reaction mixture was incubated at 16°C for 30 min, 42°C for 30 min, 85°C for 5 min, and finally held at 4°C until subsequent analysis or stored at 20 C. Five micro!
  • GC-1 spg mouse spermatogonia type B- spermatocyte cell lines were maintained in DMEM/Glutamax medium ( Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum ( Invitrogen) at 37°C in a humidified, C02-controlied (5%) incubator.
  • miR-18a down regulates HSF2 expression: It is noteworthy that HSF2 protein expression was down regulated whereas its mRNA levels were unchanged. This pattern of expression suggests the involvement of a regulation by microRNAs.
  • target prediction program TargetScan
  • alignment of rat HSF2 with miR-18a revealed a putative target site at position 1 16-122 of the Hsf2 3 'UTR. This site is located in a conserved region of the Hsf2 3 'UTR of several species.
  • the regulation of HSF2 expression by miR18a was validated. Conversely, the possibility that miR-18a would target HSP70 directly was excluded using the target prediction programs.
  • mice receive via intravenous injection specific antagomiR. Analysis of lesions sizes and locations are performed. Antagomir against miR-18a, representing by SEQ ID NO: 2 is used. It should be further noted that for this antagomir all nucleotides are 2'- OMe modified and the two first nucleotides and the three last nucleotides are phosphothioate modified.
  • Plasma were frozen at -80°C until use.
  • RNA from plasma was extracted with TRIzol reagent.
  • RT-PCR reactions were performed using the stem-loop RT-PCR method which is specific for mature miRNA (TaqMan miR A assays, Applied Biosystems, Foster City, CA, USA). Ten nanograms of total R A were reverse transcribed in a 7.5 ⁇ reaction using Muitiscribe Reverse Transcriptase and a TaqMan miR-18a RT primer (Applied Biosystems). The reaction mixture was incubated at 16°C for 30 min, 42°C for 30 min, 85°C for 5 min, and finally held at 4°C until subsequent analysis or stored at 20°C.
  • MiR-18a as biomarkers of hypospermatogenesis Neonatal exposure to EB induced an increase in miR-18a expression in the plasma from adult rats ( Figure 3 A). Since miR-18a is plasma biomarkers of testicular tissues events (hypospermatogenesis induced by EB), we analyzed their levels in plasma from fertile and infertile patients. We observed a significant increase in levels of miR-18a in the plasma from azoospermic patients compared to fertile men ( Figure 3B). ROC analyses were performed to evaluate the capability of miR-18a to discriminate between fertile and azoospermic men. miR-18a showed an area under the ROC curve (AUC) of 0.76.
  • AUC area under the ROC curve

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Abstract

L'invention concerne le diagnostic et le traitement de la stérilité mâle. L'invention concerne, plus particulièrement, des procédés pour diagnostiquer ou stratifier la stérilité mâle chez un sujet, consistant à mesurer le niveau d'au moins un miR (tel qu'un miR18a) dans un échantillon sanguin provenant du sujet. L'invention concerne également des procédés pour prévenir ou traiter la stérilité mâle consistant à administrer un composé qui inhibe le niveau d'expression ou l'activité de la famille miR18a chez un sujet.
PCT/EP2014/059543 2013-05-10 2014-05-09 Procédé pour diagnostiquer et traiter la stérilité mâle Ceased WO2014180981A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3543358A1 (fr) * 2018-03-22 2019-09-25 Institut d'Investigació Biomèdica de Bellvitge (IDIBELL) Procédés et marqueurs pour la caractérisation de l'azoospermie
WO2019180136A1 (fr) * 2018-03-22 2019-09-26 Institut D'investigació Biomedica De Bellvitge (Idibell) Procédés et marqueurs de caractérisation d'azoospermie

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