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WO2015036737A1 - Biomarqueurs pour la stratification de maladies - Google Patents

Biomarqueurs pour la stratification de maladies Download PDF

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Publication number
WO2015036737A1
WO2015036737A1 PCT/GB2014/052683 GB2014052683W WO2015036737A1 WO 2015036737 A1 WO2015036737 A1 WO 2015036737A1 GB 2014052683 W GB2014052683 W GB 2014052683W WO 2015036737 A1 WO2015036737 A1 WO 2015036737A1
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mir
antibody
seq
composition according
pulmonary hypertension
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PCT/GB2014/052683
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English (en)
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Allan LAWRIE
Alex ROTHMAN
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University Of Sheffield
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/10—Production naturally occurring
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • the disclosure relates to a microRNA [miR] used as a diagnostic biomarker for the detection of pulmonary hypertension (PH) and the use of the miR in the treatment, prevention or regression of PH.
  • miR microRNA
  • SURF1 E3 ubiquitin protein ligase
  • Pulmonary Arterial Hypertension is a devastating and debilitating disease. PAH is caused by narrowing of the arteries in the lungs resulting in abnormally high blood pressure. PAH occurs in either its idiopathic (I PAH) or hereditary (hPAH) form and is also associated with other diseases (APAH) [e.g. connective tissue disease].
  • APAH hereditary diseases
  • Pulmonary hypertension can also result from left heart disease, lung diseases (particularly Congestive Obstructive Disease [COPD] and pulmonary fibrosis), thromboembolism as well as many other multifactorial conditions such as portal hypertension, sickle cell disease and HIV.
  • COPD Congestive Obstructive Disease
  • pulmonary fibrosis thromboembolism
  • the prognosis for patients suffering from PH is poor and varies between disease groups.
  • MicroRNAs are small non-coding RNAs that control mRNA function and are involved in post transcriptional gene regulation by binding to target strands and so inhibiting translation or inducing mRNA degradation.
  • miR miRNAs
  • Extracellular miRs are present in most biological fluids and are relatively stable.
  • miR are selectively exported from cells using membrane- derived vesicles (exosomes and microparticles), lipoproteins, and other ribonucleoprotein complexes.
  • WO2012/005572 discloses the use of miR in the diagnosis of diseases or conditions associated with melanoma.
  • WO2013/060894 discloses the use of miR for the diagnosis of atherosclerosis.
  • the miR 140-5p has been extensively studied in zebrafish and was found to regulate the development of structures homologous to the human neurocranium. Mutations in the platelet derived growth factor receptor alpha gene (pdgfra) in zebrafish resulted in hypoplasia of the roof of the mouth and studies confirmed that the pdgfra gene is regulated by the miR 140-5p. Pdgfra '1' mice resembled a cleft face phenotype, and small nucleic polymorphisms in the miRNA gene were found to be associated with non-syndromic cleft palate patients.
  • miR-140-5p for use as a medicament.
  • miR-140-5p comprises the nucleotide sequence CAGUGGUUUUACCCUAUGGUAG [SEQ ID NO: 1], or a polymorphic sequence variant wherein SEQ ID NO: 1 is modified by addition, deletion or substitution of at least one nucleotide base and which has retained or enhanced activity associated with miR-140-5p.
  • Polymorphic variants have high sequence identity to miR-140-5p and retain or have enhanced miR activity and which have at least 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1.
  • miR mir-140-5p is modified.
  • modified describes a nucleic acid molecule in which: i) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide).
  • a synthetic internucleoside linkage i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide.
  • said linkage may be the 5' end of one nucleotide linked to the 5' end of another nucleotide or the 3' end of one nucleotide with the 3' end of another nucleotide; and/or ii) a chemical group, such as cholesterol, not normally associated with nucleic acids has been covalently attached to the nucleic acid.
  • Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, phosphate triesters, acetamidates, peptides, and carboxymethyl esters.
  • modified nucleotides also encompasses nucleotides with a covalently modified base and/or sugar.
  • modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • modified nucleotides may also include 2' substituted sugars such as 2'-0-methyl-; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'- fluoro-; 2'-halo or 2;azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.
  • 2' substituted sugars such as 2'-0-methyl-; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'- fluoro-; 2'-halo or 2;azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses
  • Modified nucleotides include alkylated purines and/or pyrimidines; acylated purines and/or pyrimidines; or other heterocycles. These classes of pyrimidines and purines are known in the art and include, pseudoisocytosine; N4, N4- ethanocytosine; 8-hydroxy-N6-methyladenine; 4-acetylcytosine, 5-
  • miR-140-5p for use in the treatment of pulmonary hypertension or conditions that result in pulmonary hypertension.
  • miR-140-5p comprises the nucleotide sequence set forth in SEQ ID NO: 1 , or a polymorphic sequence variant wherein SEQ ID NO: 1 is modified by addition, deletion or substitution of at least one nucleotide base and which has retained or enhanced activity of miR-140-5p.
  • miR-140-5p treatment results in the regression of pulmonary hypertension.
  • pulmonary hypertension is pulmonary arterial hypertension.
  • pulmonary hypertension is idiopathic (IPAH) or hereditary pulmonary hypertension (hPAH).
  • Pulmonary hypertension can occur in various forms and is associated with a wide range of other diseases (Associated Pulmonary Arterial Hypertension: APAH) such as connective tissue disease, or is the result of left heart disease, lung diseases (particularly Congestive Obstructive Disease [COPD] and pulmonary fibrosis), thromboembolism as well as may other multifactorial conditions such as portal hypertension, sickle cell disease and HIV.
  • APAH Associated Pulmonary Arterial Hypertension
  • COPD Congestive Obstructive Disease
  • pulmonary fibrosis pulmonary fibrosis
  • thromboembolism as well as may other multifactorial conditions such as portal hypertension, sickle cell disease and HIV.
  • composition comprising miR-140-5p, or polymorphic variant, and a pharmaceutically adjuvant and/or carrier.
  • miR-140-5p comprises the nucleotide sequence set forth in SEQ ID NO: 1 , or a polymorphic sequence variant wherein SEQ ID NO: 1 is modified by addition, deletion or substitution of at least one nucleotide base and which has retained or enhanced activity of miR-140-5p.
  • miR-140-5p is modified as herein disclosed.
  • said pharmaceutical composition includes a carrier adapted to deliver miR-140-5p to a subject in need of treatment.
  • a combined pharmaceutical composition comprising miR-140-5p and at least one additional therapeutic agent used in the treatment of PAH.
  • miR-140-5p comprises a nucleotide sequence set forth in SEQ ID NO: 1 , or a polymorphic sequence variant wherein SEQ ID NO: 1 is modified by addition, deletion or substitution of at least one nucleotide base and which has retained or enhanced activity associated with miR-140-5p.
  • said additional therapeutic agent is selected from the group consisting of: calcium channel blockers, diuretics, enthothelin receptor antagonists, prostacyclins, soluble guanalate cyclase and phosphodiesterase inhibitors.
  • said additional therapeutic agent is an antagonistic antibody, or active binding fragment thereof, that binds and inhibits the activity of Tumour Necrosis Factor Apoptosis-lnducing Ligand [TRAIL].
  • said antibody competes with an antibody that binds to the amino acid sequence as represented in SEQ ID NO: 2, 3 or 4.
  • said antibody or binding fragment binds the extracellular domain of TRAIL.
  • said antibody binds an epitope located between amino acid residues 91-281 of the amino acid sequence presented in SEQ ID NO: 2.
  • said additional therapeutic agent is an antagonistic antibody, or active binding fragment thereof, that binds and inhibits the activity of osteoprotegerin [OPG].
  • said antibody competes with an antibody that binds to the amino acid sequence as represented in SEQ ID NO: 5.
  • said antibody is a polyclonal antibody.
  • said antibody is a monoclonal antibody.
  • Immunoglobulins are protein molecules which have specificity for foreign molecules (antigens).
  • Immunoglobulins are a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain ( ⁇ or ⁇ ), and one pair of heavy (H) chains ( ⁇ , ⁇ , ⁇ , ⁇ and ⁇ ), all four linked together by disulphide bonds.
  • L light
  • H heavy chains
  • Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another.
  • H and L chains contain regions that are non-variable or constant.
  • the L chains consist of two domains.
  • the carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the "constant" (C) region.
  • the amino terminal domain varies from L chain to L chain and contributes to the binding site of the antibody. Because of its variability, it is referred to as the "variable” (V) region.
  • the H chains of Ig molecules are of several classes, ⁇ , ⁇ , ⁇ , a, and ⁇ (of which there are several sub-classes).
  • An assembled Ig molecule consisting of one or more units of two identical H and L chains, derives its name from the H chain that it possesses.
  • Ig isotypes there are five Ig isotypes: IgA, IgM, IgD, IgE and IgG (with four sub-classes based on the differences in the H chains, i.e., lgG1 , lgG2, lgG3 and lgG4). Further detail regarding antibody structure and their various functions can be found in, Using Antibodies: A laboratory manual, Cold Spring Harbour Laboratory Press.
  • said fragment is a single chain antibody fragment.
  • a Fab fragment is a multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, covalently coupled together and capable of specifically binding to an antigen.
  • Fab fragments are generated via proteolytic cleavage (with, for example, papain) of an intact immunoglobulin molecule.
  • a Fab 2 fragment comprises two joined Fab fragments. When these two fragments are joined by the immunoglobulin hinge region, a F(ab') 2 fragment results.
  • An Fv fragment is multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region covalently coupled together and capable of specifically binding to an antigen.
  • a fragment could also be a single chain polypeptide containing only one light chain variable region, or a fragment thereof that contains the three CDRs of the light chain variable region, without an associated heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multi specific antibodies formed from antibody fragments, this has for example been described in US patent 6,248,516.
  • Fv fragments or single region (domain) fragments are typically generated by expression in host cell lines of the relevant identified regions.
  • immunoglobulin or antibody fragments are within the scope of the invention and are described in standard immunology textbooks such as Paul, Fundamental Immunology or Janeway's Immunobiology, Murphy, K., Travers, P. & Walport P. Molecular biology now allows direct synthesis (via expression in cells or chemically) of these fragments, as well as synthesis of combinations thereof.
  • a fragment of an antibody or immunoglobulin can also have bispecific function as described above.
  • said antibody is a chimeric antibody.
  • said antibody is a humanized or human antibody.
  • Chimeric antibodies are recombinant antibodies in which all of the V-regions of a mouse or rat antibody are combined with human antibody C-regions.
  • Humanised antibodies are recombinant hybrid antibodies which fuse the complementarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V- regions. The C-regions from the human antibody are also used.
  • the complementarity determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V- region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen.
  • Antibodies from non-human animals provoke an immune response to the foreign antibody and its removal from the circulation.
  • Both chimeric and humanised antibodies have reduced antigenicity when injected to a human subject because there is a reduced amount of rodent (i.e. foreign) antibody within the recombinant hybrid antibody, while the human antibody regions do not elicit an immune response. This results in a weaker immune response and a decrease in the clearance of the antibody. This is clearly desirable when using therapeutic antibodies in the treatment of human diseases.
  • Humanised antibodies are designed to have less "foreign" antibody regions and are therefore thought to be less immunogenic than chimeric antibodies.
  • compositions of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, transdermal or trans-epithelial.
  • compositions of the invention are administered in effective amounts.
  • An "effective amount” is that amount of a composition that alone, or together with further doses, produces the desired response.
  • the desired response is inhibiting or reversing the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently and ideally reversing disease phenotype. This can be monitored by routine methods.
  • Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • compositions used in the foregoing methods preferably are sterile and contain an effective amount of miR according to the invention for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • doses of the miR according to the invention administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
  • doses of miR of between 1 nM - 1 ⁇ generally will be formulated and administered according to standard procedures. Preferably doses can range from 1 nM- 500nM, 5nM-200nM, and 10nM-100nM.
  • compositions to mammals other than humans, (e.g. for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above.
  • a subject as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.
  • the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents' used in the treatment of PAH. When used in medicine, the salts should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • Compositions may be combined, if desired, with a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
  • carrier in this context denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application, (e.g. liposome or immuno-liposome).
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • the pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable preservatives such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as syrup, elixir or an emulsion or as a gel.
  • Compositions may be administered as aerosols and inhaled.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of agent, which is preferably isotonic with the blood of the recipient.
  • This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, for example, as a solution in 1 , 3-butane diol.
  • the acceptable solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • the pharmaceutical composition is adapted to be delivered as an aerosol.
  • an inhaler comprising a pharmaceutical composition according to the invention.
  • a diagnostic method for determining if a subject has or is predisposed to pulmonary hypertension comprising: i) providing an isolated biological sample to be tested;
  • a preparation comprising said nucleic acid to be detected in said sample, an oligonucleotide probe or probes adapted to anneal to a nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 1 ; a thermostable DNA polymerase, deoxynucleotide triphosphates and co-factors;
  • the levels of miR-140-5p are decreased by at least 2 fold when compared to a normal matched control. More particularly the levels are decreased by 3, 4, 5, 6, 7, 8, 9 or at least 10-fold compared to a normal control level of miR-140-5p.
  • a diagnostic method for determining if a subject has or is predisposed to pulmonary hypertension comprising: i) providing an isolated biological sample to be tested;
  • a preparation comprising said nucleic acid to be detected in said sample, an oligonucleotide probe or probes adapted to anneal to a nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 6; a thermostable DNA polymerase, deoxynucleotide triphosphates and co-factors;
  • said method is a real time PCR method for the detection and quantification of a nucleic acid encoding all or part of the nucleotide sequence set forth in SEQ ID NO: 1 or 6.
  • a diagnostic method for determining if a subject has or is predisposed to pulmonary hypertension comprising the steps of:
  • the level of SMURF1 mRNA [SEQ ID NO: 6] or protein [SEQ ID NO: 7] is increased by at least 2 fold when compared to a normal matched control. More particularly the levels are increased by 3, 4, 5, 6, 7, 8, 9 or at least 10-fold compared to a normal control level of SMURF mRNA or protein.
  • said method further includes the detection of the expression of one or more genes associated with the BMP and/or PDGF pathway.
  • said gene is selected from the group consisting of: SMURF1 , WWP2, RUNX2, BMP, PDGFRA, OPG, SP1 , TRAIL, ID1 , TGRBR1 or VEGFA.
  • said isolated biological sample is selected from the group consisting of: blood, blood plasma or serum, lymph fluid, saliva, sputum, lavage, bronchoaveolar lavage, or human tissue biopsy.
  • said biological sample is a blood sample.
  • said biological sample is a peripheral blood sample.
  • said sample is a tissue biopsy.
  • said biopsy is a lung tissue biopsy.
  • said method is combined with an alternative diagnostic method for the detection of pulmonary hypertension in a subject.
  • said method is selected from the group echocardiogram, electrocardiogram, chest X-ray, lung function tests, exercise tests, ventilation-perfusion scan, blood tests or right heart catheterisation.
  • a treatment method for PAH comprising:
  • said medicament comprises miR 140.5p.
  • said subject is administered a second medicament or pharmaceutical composition comprising one or more agents used in the treatment of PAH.
  • said agent[s] are selected form the group calcium channel blockers, diuretics, endothothelin receptor antagonists, prostacyclins, soluble guanalate cyclase and phosphodiesterase inhibitors.
  • said agent[s] is an antagonistic antibody, or active binding fragment thereof, that binds and inhibits the activity of Tumour Necrosis Factor Apoptosis-lnducing Ligand [TRAIL] or osteoprotegerin as herein disclosed.
  • TRAIL Tumour Necrosis Factor Apoptosis-lnducing Ligand
  • said medicaments or compositions are administered simultaneously or sequentially.
  • pulmonary hypertension is pulmonary arterial hypertension.
  • pulmonary hypertension is idiopathic (I PAH) or hereditary pulmonary hypertension (hPAH).
  • FIG. 1 A) Levels of miR-140-5p are reduced in patients with PH (idiopathic PH, PAH associated with connective tissue disease (CTD-PH) and lung disease (Lung-PH), and Chronic Thromboembolic Pulmonary Hypertension (CTEPH)) compared to healthy volunteers (HV) and patients with connective tissue disease without PAH.
  • B) Levels of miR-140-5p reduce with increasing mean pulmonary artery pressure (mPAP) (B), pulmonary vascular resistance (PVR) (C) and the cardiac stress marker N-terminal Pro brain natriuretic peptide (NT-pro-BNP) (D).
  • mPAP mean pulmonary artery pressure
  • PVR pulmonary vascular resistance
  • H Whole blood mRNA levels of ID1 mRNA are higher (G) and its downstream target SMURF1 are lower (H) at diagnosis in patients with I PAH.
  • RVSP Right Ventricular Systolic Pressure
  • I Left Ventricular hypertrophy
  • J Right ventricular hypertrophy
  • K Levels of miR-140-5p are reduced in the monocrotaline (K; lung) and sugen hypoxia (L; whole blood) rat models of PAH during the onset and progression of PAH.
  • FIG. 2 A) Administration of nebulised pre-miR-140-5p prevents disease development in the monocrotaline rat: A) Pulmonary artery acceleration time (PAAT), B) RVSP C) CO. D) Whole lung miR140-5p is increased with administration of pre-miR-140-5p (Pre) compared to the Anti-miR-140-5p (Anti) and scrambled (SCR) molecules. E) Whole lung mRNA levels of SMURF1. Administration of nebulised pre-miR-140-5p reverses established disease in the monocrotaline rat: F) PAAT, G) RVSP H) CO. I) Levels of miR140-5p are increased with administration of pre-miR-140-5p.
  • nebulised pre-miR-140-5p reverses established disease in the Sugen hypoxic rat: K) RVSP and K) CO.
  • L) Levels of miR140-5p are increased with administration of pre-miR- 140-5p.
  • FIG. 3 Suppression of miR-140-5p with an anti-miR-140-5p oligo induces further proliferation of human PASMCs in response to serum (A), and spontaneous proliferation in serum free conditions, which is then inhibited with SMURF1 siRNA (B); C) Western blot measuring protein expression of PDGFA normalised to the endogenous control gene GAPDH following the transfection of human PASMC with either the pre- or anti- miR-140-5p oligo.
  • Cell transfected with the pre-miR-140-5p display a reduced expression of PDGFRA, whereas cells transfected with the anti-miR-140-5p demonstrate increased expression compared to a scrambles molecule (SCR).
  • SCR scrambles molecule
  • FIG. 4 Treatment of established PAH (8 week Paigen diet) with an anti-TRAIL antibody induces reverse remodelling of disease: ApoE " ' " mice with either anti-TRAIL antibody of IgG for 4 weeks. Mice that received the anti-TRAIL antibody displayed a significant reduction in pulmonary vascular remodelling as shown by the reduction in media/CSA of the small pulmonary arteries/arterioles ( Figure 4A). This was associated with decrease in proliferating (PCNA) cells ( Figure 4B) and an increase in apoptotic (TUNEL) cells within remodelled pulmonary arteries ( Figure 4C).
  • PCNA proliferating
  • TUNEL apoptotic
  • FIG. 5 Treatment of established PAH (8 week Paigen Diet) with an anti-OPG antibody induces reverse remodelling of disease: ApoE " ' " mice with anti-OPG antibody of IgG for 4 weeks. Mice that received the anti-OPG antibody displayed a significant reduction in pulmonary vascular remodelling as shown by the reduction in media/CSA of the small pulmonary arteries/arterioles ( Figure 5A). This was associated with decrease in proliferating (PCNA) cells ( Figure 5B) and an increase in apoptotic (TUNEL) cells within remodelled pulmonary arteries ( Figure 5C). The Anti-OPG treatment demonstrates a more pronounced pro-apoptotic response than anti-TRAIL, which was associated with a greater anti-proliferative response.
  • PCNA proliferating
  • TUNEL apoptotic
  • Rats 0.6 ml of Sugen 5416 (20 mg/kg) was administered to each rat (200-250 g) by sub cutaneous injection. Rats were housed in hypoxic chambers at 10% 0 2 and 5% C0 2 for 3 weeks and in normoxia for 5 weeks for experimentation or 11 weeks of normoxia for time course experiments.
  • Left and right ventricular catheterisation was performed using a closed chest method via the right internal carotid artery and right external jugular vein under isoflurane induced anaesthesia.
  • Data was collected using a Millar ultra-miniature pressure-volume SPR-838 (Millar Instruments Inc., Texas, USA) coupled to a Millar MPVS 300 and a PowerLab 8/30 data acquisition system (AD Instruments, Oxfordshire, UK) and recorded using Chart v7 software (AD Instruments).
  • Pressure volume analysis was performed using PVAN v2.3 (Millar Instruments Inc).
  • the right lung was secured tightly at the hilum using 5-0 silk sutures and separated away before being snap frozen in liquid nitrogen for subsequent isolation and determination of whole lung protein and RNA expression.
  • Polyethylene tubing was inserted into the trachea and secured tightly with a suture.
  • the left lung was gently inflated manually with a syringe containing 10% phosphate buffered formalin (0.4% w/v NaH 2 P0 4 2(H 2 0), 0.65% w/v Na 2 HP0 4 2(H 2 0) and 4% v/v formaldehyde in water) and then both heart and left lung were fixed in formalin for 24 hours before transfer into PBS. From the rat prevention study onwards lungs were inflated using 20cm H 2 0 clamp set up to standardise inflation. The left lung was separated from the heart for subsequent histology.
  • RVH right ventricle hypertrophy
  • RVH was defined as the weight of the RV divided by the weight of the left ventricle/septum (RV/LV+S) as first described by Fulton et al. Using a small pair of fine scissors surrounding fat, tissue and great vessels were removed from around the heart. The atria were excised, cleared of any thrombus and weighed. The right ventricle was separated from the left ventricle and septum by the use of anatomical landmarks.
  • RVOT right ventricular outflow tract
  • Paraffin embedded 5 ⁇ lung sections underwent immunohistochemical staining a-SMA for vascular smooth muscle cells, vWF to localise endothelial cells and PCNA for proliferating cells. Immunostaining for OPG was performed to identify any expression within pulmonary vascular lesions. Levels of apoptosis were determined with a colorimetric assay to detect DNA fragmentation (FRAGEL®, Calbiochem, UK) as specified by the manufacturer's instructions. A positive control was generated with DNAse treatment of a control slide.
  • Tissue was then permeabilised by incubation in 0.5% (v/v) tritonXI OO for 10mins at RT (IHC for OPG) b) 0.1 % (w/v) Trypsin/TBS, pH7.8, preheated to 37°C for 10minutes before stopping reaction by immersing in water (IHC for vWF)
  • 3 mg/L (200 M) stock was prepared by re-suspending 250 nmol of HPLC miR/SCR (Invitrogen, UK) in 1.25 ml_ of DNase RNase free water (Ambion, UK).
  • an in vivo transfection mixture (10 doses of 20 nM in 100uL) was prepared by mixing two transfection reagents tubes (Tube 1 : Mix 100 ⁇ _ of 200 ⁇ miR/SCR stock with 100 ⁇ _ of Complexation Buffer (Invivofectamine, Invitrogen, UK); Tube 2: 200 ⁇ _ of Invivofectamine (Invitrogen, UK) at room temperature) then vortexing for 2-3 seconds prior to incubation for 30 min at 50°c.
  • Float-A- Lyzer dialysis filter (Spectrum Medical Laboratories, UK) was prepared by filling the filter with and submerging in 10% (v/v) isopropranol for 10 min. Alcohol was removed by aspiration and the Float-A-Lyzer flushed, filled and soaked in deionized water for 15-20 min. Deionized water was then aspirated and the Float-A-Lyzer loaded with transfection mixture by pipetting to the bottom of the membrane slowly withdrawing pipet as dispensing. The Float-A-Lyzer (with ring float) was placed vertically in 1 L of PBS and incubated for 2 hours with gentle agitation. The sample was collected by aspirating while moving pipette toward bottom of membrane and marked up to 2 ml_ with PBS in sterile tube.
  • Rodents were anaesthetised with isoflurane (5%) via oxygen (2 L/min) in an induction chamber (Harvard Apparatus, UK) and placed supine on a heated platform at 45° with the head orientated towards the operator. Animals were covered to minimise heat loss and maintenance isoflurane (0.5-1.5%) delivered with oxygen (1 L/min) via a nose cone. Continuous heart rate monitoring was used to monitor depth of anaesthesia. The nose cone was removed to allow delivery of transfection reagent, the vocal cords were visualised with a small animal laryngoscope (Model LS-2, Penn-Century, USA) and the animal intubated with a MicroSprayer Aerosolizer (Model LA-1 B, Penn-Century, USA). 20 ⁇ of miR/SCR was delivered via a 250 ⁇ _ high-pressure syringe (FMJ-250, Penn- Century, USA).
  • polyclonal goat anti-mouse TRAIL Anti-TRAIL
  • polyclonal goat anti- mouse OPG Anti-OPG
  • control goat IgG isotype antibodies R&D systems, UK
  • Interventions were delivered via an Alzet® 1004 micro pump (100 ⁇ reservoir, ⁇ . ⁇ ⁇ /hour for 4 weeks) in mice and via an Alzet® 2002 mini-pump (200 ⁇ reservoir, ⁇ . ⁇ /hr, 85ng/hr for 2 weeks) in rats.
  • Each pump was filled with the appropriate intervention under sterile conditions in a class II laminar flow hood and placed in sterile 0.9% saline at 37°C 24 hours prior to implantation.
  • isoflurane gas anaesthesia (2-3%, IsoFlo® 100% w/w inhalation vapour liquid, Abbot laboratories Ltd, Kent, UK) through 100% oxygen (flow rate 1.5L/min) overlying fur was clipped, the skin cleaned and sterilised prior to making a 1- 1.5cm cutaneous incision over the left posterolateral thoracic wall, inferior to the lower costal margin.
  • pre-filled pumps were implanted into a subcutaneous pocket created with blunt dissection.
  • RNA samples frozen in liquid nitrogen were ground using a pestle and mortar containing liquid nitrogen to a fine powder and weighed. Precautions were taken to minimise contamination by RNAase.
  • Total protein and RNA were isolated using a commercial RNA/Protein purification Kit (#23000, Norgen Biotek, Ontario, Canada) according to the protocol supplied by the manufacturer.
  • the purification kit employed a spin column chromatography technique and allowed elution of proteins and RNA from the same sample within 30 minutes.
  • PA-SMC Human pulmonary artery smooth muscle cells
  • SMURF1 protein levels a putative target of miR-140-5p were assessed by western immunoblotting. Proteins were isolated using RIPA buffer and separated by SDS-Polyacrylamide gel electrophoresis using a commercial electrophoresis kit (NuPAGE® Kit, Invitrogen). All buffers and reagents were part of the NuPAGE range unless otherwise stated. A volume containing 35 ⁇ g of protein purified from control, pre- and anti miR-140p transfected human pulmonary artery smooth muscle cells, sample buffer and a reducing agent made to a final volume of 30 ⁇ (in deionised water) was heated to 70°C for 10 min.
  • Samples and a pre-stained marker ladder were then loaded onto 10 well pre-cast SDS polyacrylamide gels (NuPAGE® 4-12% Bis-Tris Mini gels, Invitrogen).
  • SDS polyacrylamide gels NuPAGE® 4-12% Bis-Tris Mini gels, Invitrogen.
  • a sample of mixed experimental lung tissue was also loaded onto every gel as an additional control to allow for subsequent quantitative analysis.
  • Blots were rinsed three times for 10min. before adding an appropriate, species specific peroxidise labeled secondary antibody diluted in PBS. Following a further rinse step as described enhanced chemoluminescence was performed by adding 1 ml of a commercial assay on to the blots for 5min. in the dark (#34075 West Dura Super Signal, Thermo scientific Fisher). Blots were developed in a dark room using autoradiography film (#28906836, HyperFilmTM GE Amersham, UK) and developer/fixer solutions. Blots were stripped (#2502, Reblot Plus Mild Chemicon solution, Millipore) and reprobed for actin as described above.
  • the developed blots were dried and the ladder marked.
  • the quantity of TRAIL in the bands was determined by normalising to actin and control samples using the densitometry function on commercial software (Syngene SNAP software, Chemigenius2 bioimaging system, SynGene). Analysis of miRNA Expression in Patients and controls
  • the Sheffield Pulmonary Hypertension Biobank provided access to whole blood samples from patients (at time of diagnosis) and healthy volunteers under the local Sheffield Teaching Hospitals Foundation Trust Observational Cardiovascular Biobank Ethics 08/H 1308/193 and STH 15222).
  • mRNA and miRNAs were extracted from Tempus (Applied Biosystems) whole blood samples according to manufacturer guidelines. Quantitative real time Polymerase Chain Reaction - miR
  • qPCR Quantitative PCR of mature microRNA (miR) was performed according to the manufacturer's instructions using TaqMan MircoRNA Assays. Briefly, total RNA was isolated from the lungs. cDNA was prepared using the TaqMan microRNA Reverse Transcription kit (4366596, Applied Biosystems) and qPCR performed using TaqMan microRNA Assays for U6 and miR-140-5p (Applied Biosystems). The primers used for reverse transcription and qPCR were those supplied with each assay.
  • Each qPCR primer is a stem-loop oligonucleotide containing the sequence of the mature miR (miR- 140-5p: CAGUGGUUUUACCCUAUGGUAG (SEQ ID No 1); U6: GTGCTCGCTTCGGCAGCACATATACTAAAATTGGAACGATACAGAGAAGATTAGCA TGGCCCCTGCGCAAGGATGACACGCAAATTCGTGAAGCGTTCCATATTTT (SEQ ID NO 8)).
  • qPCR was performed in triplicate using a 7900HT Fast Real-Time PCR System (Applied Biosystems). miR-140-5p levels were normalized to mean expression of the control group.
  • This step was performed using components provided in a SuperscriptTM III first strand synthesis system (#18080-051 and #18080-044, InvitrogenTM Life technologies, UK).
  • a volume containing 3 ⁇ g of total RNA isolated from the lungs (and whole blood using PAX- gene tubes) of experimental rodents was made to 10 ⁇ using molecular grade water.
  • 1 ⁇ of random hexamer primers (50ng) and 1 ⁇ of a 10mM dNTP were added to this and heated to 65°C for 5 minutes as a denature step.
  • Amplification of the target lung cDNA derived from the RT step above was then next performed.
  • a volume containing 50ng of each cDNA was diluted to a volume of 4.5 ⁇ using sterile water.
  • 5 ⁇ of a TaqMan® gene expression master mix-2X (#4369016, Applied BiosystemsTM Life Technologies, UK) along with 0.5 ⁇ of the relevant target gene primers (10X) were added to the cDNA in the relevant well of a 384-well plate.
  • Target genes were tested are listed in table 1 (all from Applied BiosystemsTM) 18s and ATP5B were selected as endogenous control genes. Samples (in duplicate) for each gene were loaded on the same plate.
  • the plate was centrifuged at l OOOrpm for 1 min and the reaction was run on a 7900HT fast real time PCR system (Applied BiosystemsTM) using standard manufacturer settings. Relative expression for each gene was quantified by comparing the test gene with the housekeeping control gene and comparing this ratio between an experimental and control subject (delta, delta CT method) for each gene using SDS software (v2.2.1 , Applied BiosystemsTM).
  • Figure 1A shows miR-140-5p levels in whole blood samples taken at the time of diagnosis from patients with different forms of pulmonary hypertension and age and sex matched healthy volunteers. Levels of miR-140-5p are reduced in patients with multiple forms of pulmonary hypertension. Figures 1 B-D show that levels of miR-140-5p are further reduced in patients with more severe haemodynamic markers of disease compared to those with mild disease. Figure 1 E shows that there is a significant survival effect of high verses low levels of miR-140-5p.
  • Figure 1 F shows a western blot of SMURF1 , a putative target of miR-140-5p, 72 hours post transfection of human pulmonary artery smooth muscle cells with 50 nM control miR (SCR), pre miR-140-5p and anti-miR-140-5p.
  • Figures 1 G show that ID1 , a downstream transcription factor from BMP signalling, and therefore influenced by SMURF1 is decreased in patients with IPAH.
  • Figure 1 H shows that SMURF1 is upregulated in patients with IPAH.
  • Figures 11-K show the time course of development of PAH in the monocrotaline rat.
  • Figure 1 L shows miR-140-5p expression from a time course in the Sugen+Hypoxia model of PAH and also demonstrates an early reduction in levels of miR-140-5p compared to normoxia controls.
  • Figure 2A shows a reduction in the pulmonary artery acceleration time (PAAT) in monocrotaline treated rats with a scrambled (SCR) miRNA at 21 days compared to baseline.
  • PAAT pulmonary artery acceleration time
  • SCR scrambled
  • FIGs 2B&C show the protective effects of pre-miR-140-5p prophylactic treatment in the monocrotaline rat model as demonstrated by reduced RVSP (B) increased cardiac output (C).
  • Figure 2D demonstrates that the pre-miR-140- 5p delivery did significantly rescue miR-140-5p levels in lung tissue, and this was associated with reduced levels of its target SMURF1 (E).
  • Figure 2F shows a reduction in the pulmonary artery acceleration time (PAAT) in monocrotaline treated rats with a scrambled (SCR) miRNA at day 21 in the Pre-Rx group, and at day 35 in treated groups, compared to baseline.
  • PAAT pulmonary artery acceleration time
  • SCR scrambled miRNA
  • Figures 2G&H show the therapeutic effects of pre-miR-140-5p treatment in the monocrotaline rat model as demonstrated by reduced RVSP (G) increased cardiac output (H).
  • Figure 2I demonstrates that the pre-miR-140-5p delivery did significantly rescue miR-140-5p levels in lung tissue.
  • Figures 2G&H show the therapeutic effects of pre-miR-140-5p treatment in the Sugen+Hypoxia rat model as demonstrated by reduced RVSP (J) increased cardiac output (K).
  • Figure 2L demonstrates that the pre-miR-140-5p delivery did significantly rescue miR-140-5p levels in lung tissue and this was associated with reduced levels of its target SMURF1 (M).
  • Figure 3B shows proliferation of human pulmonary artery smooth muscle cells in 0.2% FBS with 10 ng/mL BMP4 at 72 hours post transfection with 30 nM control miR (SCR), pre miR-140-5p, anti miR-140-5p and anti miR-140-5p with SMURF1 siRNA. Proliferation is increased with anti-mR-140-5p, an effect which is negated by the addition of SMURF1 siRNA.
  • Figure 3C illustrates miR-140-5p targeting of PDGFRA expression.
  • Cells transfected with the pre-miR-140-5p oligo display reduced levels of PDGFA therefore attenuating a PDGF response.
  • Cells transfected with the anti-miR-140-5p, thereby mimicking the disease state show increased expression of PDGFRA thereby increasing PDGF signalling.
  • FIG. 4 shows that treatment of established PAH with an anti-TRAIL antibody induces reverse remodelling of disease: ApoE " ' " mice after 8 weeks of feeding on the Paigen diet were implanted with osmotic pumps delivering either anti-TRAIL antibody of IgG for 4 weeks. Mice that received the anti-TRAIL antibody displayed a significant reduction in pulmonary vascular remodelling as shown by the reduction in media/CSA of the small pulmonary arteries/arterioles ( Figure 4A). This was associated with decrease in proliferating (PCNA) cells ( Figure 4B) and an increase in apoptotic (TUNEL) cells within remodelled pulmonary arteries ( Figure 4C).
  • PCNA proliferating
  • TUNEL apoptotic
  • FIG. 5 shows that treatment of established PAH with an anti-OPG antibody induces reverse remodelling of disease: ApoE " ' " mice after 8 weeks of feeding on the Paigen diet were implanted with osmotic pumps delivering either anti-OPG antibody of IgG for 4 weeks. Mice that received the anti-OPG antibody displayed a significant reduction in pulmonary vascular remodeling as shown by the reduction in media/CSA of the small pulmonary arteries/arterioles (Figure 5A). This was associated with decrease in proliferating (PCNA) cells ( Figure 5B) and an increase in apoptotic (TUNEL) cells within remodeled pulmonary arteries ( Figure 5C).
  • PCNA proliferating
  • TUNEL apoptotic

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Abstract

L'invention concerne l'utilisation de miR-140-5p comme biomarqueur diagnostique pour la détection d'hypertension pulmonaire (PAH) et l'utilisation du miR pour traiter, prévenir ou faire régresser PH.
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WO2018138297A1 (fr) 2017-01-27 2018-08-02 Kymab Limited Anticorps anti-opg
CN115851828A (zh) * 2021-12-15 2023-03-28 上海海洋大学 miRNA-140的应用及调控斑马鱼颅面骨骼和鳞片发育的方法
WO2024089437A1 (fr) * 2022-10-28 2024-05-02 Imperial College Innovations Limited Panneaux de micro-arn pour l'hypertension pulmonaire

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Publication number Priority date Publication date Assignee Title
WO2018138297A1 (fr) 2017-01-27 2018-08-02 Kymab Limited Anticorps anti-opg
US11680101B2 (en) 2017-01-27 2023-06-20 Kymab Limited Anti-OPG antibodies
CN115851828A (zh) * 2021-12-15 2023-03-28 上海海洋大学 miRNA-140的应用及调控斑马鱼颅面骨骼和鳞片发育的方法
WO2024089437A1 (fr) * 2022-10-28 2024-05-02 Imperial College Innovations Limited Panneaux de micro-arn pour l'hypertension pulmonaire

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