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WO2018008017A1 - Micro-arn circulants en tant que biomarqueurs pour une thérapie dans la neuromyélite optique (nmo) et de la sclérose en plaques (sep) - Google Patents

Micro-arn circulants en tant que biomarqueurs pour une thérapie dans la neuromyélite optique (nmo) et de la sclérose en plaques (sep) Download PDF

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WO2018008017A1
WO2018008017A1 PCT/IL2017/050741 IL2017050741W WO2018008017A1 WO 2018008017 A1 WO2018008017 A1 WO 2018008017A1 IL 2017050741 W IL2017050741 W IL 2017050741W WO 2018008017 A1 WO2018008017 A1 WO 2018008017A1
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mir
treatment
mirnas
levels
circulating
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Iris LAVON BEN MOSHE
Adi VAKNIN-DEMBINSKY
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Hadasit Medical Research Services and Development Co
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Hadasit Medical Research Services and Development Co
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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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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 circulating microRNAs as biomarkers in Neuromyelitis Optica (NMO) and Multiple Sclerosis (MS), for prediction of responsiveness to treatment thereof.
  • NMO Neuromyelitis Optica
  • MS Multiple Sclerosis
  • NMO Neuromyelitis optica
  • CNS central nervous system
  • inflammatory processes in NMO are mediated by the humoral immune system and primarily target astrocytes [1 , 2].
  • target astrocytes [1 , 2].
  • the most important evidence of this was the identification of the NMO-IgG antibody, anti-Aquaporin-4; NMO-IgG antibodies identify about 82% of patients.
  • miRNAs function to modify the expression of target genes. miRNA-mediated gene regulation is critical during many biological processes including inflammation and neurodegeneration. Because each miRNA can regulate many target genes, the biological impact of dysregulation of a single miRNA can be considerable. Studies have shown that miRNAs have potential as non-invasive biomarkers for the diagnosis and prognosis of disease as well as monitoring of treatment response [3]; miRNAs also represent promising novel targets for therapy [1]. Extracellular circulating miRNAs are remarkably stable [4]. Their stability is achieved via different mechanisms.
  • miRNAs can be packaged in microparticles (exosomes, microvesicles, and apoptotic bodies)[5-7] or associated with RNA- binding proteins (Argonaute2 [Ago2]) or lipoprotein complexes (high-density lipoprotein [HDL]) [8], to prevent their degradation.
  • RNA-bind proteins Aronaute2 [Ago2]
  • HDL high-density lipoprotein [HDL]
  • miRNA-92a Altered miRNA expression has been reported in several human autoimmune diseases, and miR-92a was suggested as a circulating biomarker for disease staging in MS.
  • miR-15a, miR-19a, miR-22, miR-210 and miR-223 were up- regulated in T-reg cells, plasma, blood cells, PBMCs and brain white matter tissues from MS patients; miR-21, miR-142-3p, miR-146a, miR-146b, miR-155 and miR- 326 were up-regulated and miR-181c and miR-328 were down-regulated in PBMCs and brain lesions; and miR-15a and miR-15b were down-regulated in blood, peripheral T cells and B cells or plasma samples from MS patients.
  • NMO neuropeptide
  • Rituximab is a chimeric anti-CD20 monoclonal antibody that depletes B cells. It is commonly used for treating B cell lymphoma and has been found to be effective in the treatment of autoimmune rheumatological and neurological conditions, including NMO. Rituximab is currently considered the most effective therapy for preventing NMO exacerbations [14, 15]. Data relating to the correlation between AQP4-IgG titers and disease activity in the long-term course of NMO are inconsistent [16].
  • FCGR3A- F fragment c gamma receptor 3 A
  • biomarkers and corresponding methods that can be reliably used for monitoring and efficiently determining treatment response in NMO patients and/or MS patients.
  • Such biomarkers should preferably be found in peripheral blood of patients and can be used as predictor for response to therapy, for assisting in the individualized management of the disease.
  • the methods include identification of miRNA biomarkers in the peripheral blood of patients with NMO.
  • the method include identification of miRNA biomarkers in the peripheral blood of patients with MS.
  • the therapy includes administration of a suitable treatment.
  • the treatment may be selected from: Rituximab, B cell depletion treatment, treatment with anti CD20, treatment with anti-CD19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, azathioprine, Cladribine, Lemtrada, Teriflunomide, and the like, or combinations thereof.
  • analysis of global miRNA expression in the blood of patients with NMO before and after suitable treatment identified a distinct miRNA expression signature (profile) associated with the treatment.
  • analysis of global miRNA expression in the blood of patients with MS before and after treatment results in a distinct miRNA expression signature associated with the treatment.
  • a set of miRNAs with elevated expression in the blood of MS patients revert to the levels of matched healthy controls following effective therapy (for example, with rituximab).
  • a set of miRNAs with elevated expression in the blood of NMO patients revert to the levels of matched healthy controls following effective therapy (for example, with rituximab).
  • at least some of the miRNAs are brain-enriched miRNAs.
  • a method for determining efficacy of treatment for neuromyelitis optica (NMO) in a subject in need thereof comprising:
  • modulation in the levels of the at least circulating miRNAs from the first to the second serum sample is indicative of said treatment efficacy.
  • step (a) may include determining the levels of at least three circulating miRNAs, selected from the group consisting of miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180.
  • the selected treatment may be selected from, but not limited to: Rituximab, B cell depletion treatment, treatment with anti CD20, treatment with anti-CD 19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, azathioprine, Cladribine, Lemtrada, and Teriflunomide.
  • the selected treatment includes Rituximab.
  • step a) may be conducted at a time point prior to step b).
  • steps a) and c) may be conducted at distinct time points during step b).
  • the method may further include repeating step c) for one or more times to determine the levels of the at least two circulating miRNAs in consecutive serum samples, obtained at designated time intervals; and comparing the levels of the at least two circulating miRNAs between said consecutive serum samples, wherein a modulation in the levels of the at least two circulating miRNAs between consecutive serum samples is indicative of said treatment efficacy.
  • modulation comprises decrease in the level of the at least two miRNAs. In some embodiments, modulation comprises an increase.
  • the method may further include a step of isolating RNA from the first serum sample and/or the second serum prior to identification of the at least two miRNAs.
  • the level of the at least two miRNAs in the first serum sample and/or the second serum sample may be determined by a method selected from: amplification reaction, sequencing reaction, microarray, or combinations thereof.
  • the treatment regime with the selected treatment is adjusted based on the determined treatment efficacy.
  • the method may further include a step of comparing the levels of the at least two circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of corresponding miRNAs obtained from healthy control subjects.
  • a method for assessing treatment efficacy of a selected treatment in a subject afflicted with neuromyelitis optica comprising determining the level of at least two circulating miRNA molecules, selected from the group consisting of: miR-138, miR- 660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180, in serum samples of said subjects, wherein said samples are obtained before and after treatment with the selected treatment and comparing the levels of the at least two circulating miRNA molecules between the serum samples, wherein modulation of expression of the miRNA molecules between the serum samples obtained before and after treatment is indicative of increased treatment efficacy.
  • NMO neuromyelitis optica
  • the methods for determining treatment efficacy of NMO patient include evaluating the treatment efficacy by clinical parameters, including determining the level of anti Aquaporin 4 expression levels.
  • kits for determining efficacy of a selected treatment for neuromyelitis optica includes means for determining the levels of at least two circulating miRNAs, selected from: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for the neuromyelitis optica (NMO).
  • miRNAs selected from: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for
  • the means in the kit may include specific nucleic acid molecules for identification of circulating miRNAs in the serum samples.
  • the nucleic acid molecules may include specific primers for identification of miRNAs in an amplification reaction performed on RNA isolated from the serum sample.
  • the nucleic acid molecules include specific probes for identification of circulating miRNAs in a sequencing reaction performed on RNA or DNA isolated from the serum sample.
  • the kit instructions further include comparing the levels of circulating miRNAs in the serum samples of said subject to the circulating levels of miRNAs obtained from healthy control subjects.
  • a subject in need thereof comprising:
  • modulation in the levels of the at least circulating miRNAs from the first to the second serum sample is indicative of said treatment efficacy.
  • the selected treatment is selected from: Rituximab, B cell depletion treatment, treatment with anti CD 20, treatment with anti- CD 19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, azathioprine, Cladribine, Lemtrada, and Terifiunomide.
  • step a) may be conducted at a time point prior to step b).
  • steps a) and c) may be conducted at distinct time points during step b).
  • the method may further include repeating step c) for one or more times to determine the levels of the at least two circulating miRNAs in consecutive serum samples, obtained at designated time intervals; and comparing the levels of the at least two circulating miRNAs between said consecutive serum samples, wherein a modulation in the levels of the at least two circulating miRNAs between consecutive serum samples is indicative of said treatment efficacy.
  • the modulation may include decrease or increase in the level of the at least two miRNAs.
  • the treatment regime using the selected treatment may be adjusted based on the determined treatment efficacy.
  • the method may further include a step of comparing the levels of the at least two circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of corresponding miRNAs obtained from healthy control subjects.
  • a method for assessing treatment efficacy of a selected treatment in a subject afflicted with Multiple Sclerosis (MS) comprising determining the level of at least two circulating miRNA molecules, selected from the group consisting of: miR-138, miR- 660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180, in serum samples of said subjects obtained before and after treatment with the selected treatment, and comparing the levels of the at least two circulating miRNA molecules between the serum samples, wherein modulation of expression of the miRNA molecules between the serum samples obtained before and after treatment is indicative of increased treatment efficacy.
  • the method may further include comparing the at least two circulating miRNA molecules between the consecutive serum samples.
  • the method may further include evaluating the treatment efficacy by clinical parameters.
  • the modulation includes increased levels of the miRNAs or reduced levels of the miRNAs.
  • the method may further include a step of comparing the levels of circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of miRNAs obtained from healthy control subjects.
  • kits for determining efficacy of a selected treatment for multiple sclerosis includes means for determining the levels of at least two circulating miRNAs, selected from: miR- 138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR- 203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for MS.
  • miRNAs selected from: miR- 138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR- 203a, miR-124, miR-7 and miR-1180
  • the means in the kit may include specific nucleic acid molecules for identification of circulating miRNAs in the serum samples.
  • the nucleic acid molecules may include specific primers for identification of miRNAs in an amplification reaction performed on RNA isolated from the serum sample.
  • the nucleic acid molecules include specific probes for identification of circulating miRNAs in a sequencing reaction performed on RNA or DNA isolated from the serum sample.
  • the kit instructions further include comparing the levels of circulating miRNAs in the serum samples of said subject to the circulating levels of miRNAs obtained from healthy control subjects.
  • Fig. 1- Graphs showing differentially expressed miRNAs in circulating blood of NMO patients (responders and non-responders) patients 6 month following Rituximab therapy. RNA was extracted from whole blood of 9 responders and 5 non- responders NMO patients 6 month following Rituximab therapy and miRNAs were quantified using Real time RT PCR. The results of 3 most significant differentially expressed miRNAs are presented in the figure as 2 ⁇ ⁇ ct. Responders are marked in dark black and non-responders in grey.
  • Figs. 2A-B Graphs showing differential expression of the indicated miRNAs between Healthy Controls and NMO patients before and following Rituximab therapy.
  • RNA was extracted from whole blood of 15 healthy controls and 9 NMO patients before and after Rituximab therapy and miRNAs were quantified using deep sequencing. The results of 9 of the 10 miRNAs which revert to normal levels following Rituximab therapy are presented in the figure as normalized counts (logarithmic scale). Healthy controls, NMO patients before Rituximab therapy and NMO patients after therapy are marked as circles, squares and triangles, respectively.
  • the present invention provides methods and kits for determining treatment efficacy of suitable drug in a patient afflicted with Neuromyelitis Optica (NMO) or Multiple Sclerosis (MS), wherein circulating levels of designated miRNAs are used as biomarkers.
  • NMO Neuromyelitis Optica
  • MS Multiple Sclerosis
  • the present invention discloses for the first time the unexpected discovery that the detection of circulating miRNA in serum of NMO or MS patients treated with a suitable drug (such as, for example, rituximab) reflects the treatment efficacy of the treated subject.
  • a suitable drug such as, for example, rituximab
  • the use of detecting circulating levels of specific miRNAs (in particular, one or more of: miR-138, miR-660, miR-125b, miR-760, miR-100, miR- 423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180) as a biomarker of treatment efficacy of suitable treatment, overcomes the drawbacks of currently used methods in providing a more sensitive, reliable, simple and cost effective method for determine efficacy of such treatment.
  • the present invention provides methods, kits and compositions for assessing or determining the effectiveness of an treatment of NMO or MS patients, based on the modulation in the circulating levels of miRNAs, in particular, one or more miRNA's selected from: miR-138, miR-660, miR-125b, miR- 760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7, miR- 1180 and any combination thereof.
  • miRNAs selected from: miR-138, miR-660, miR-125b, miR- 760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7, miR- 1180 and any combination thereof.
  • miRNA's selected from: miR-138, miR-660, miR-125b, miR- 760, miR-100, miR-423, miR-135a, miR-135
  • polynucleotide molecules As referred to herein, the terms “polynucleotide molecules”, “oligonucleotide”, “polynucleotide”, “nucleic acid” and “nucleotide” sequences may interchangeably be used.
  • the terms are directed to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof in the form of a separate fragment or as a component of a larger construct, linear or branched, single stranded (ss), double stranded (ds), triple stranded (ts), or hybrids thereof.
  • ss single stranded
  • ds double stranded
  • ts triple stranded
  • the polynucleotides may be, for example, sense and antisense oligonucleotide or polynucleotide sequences of DNA or RNA.
  • the DNA or RNA molecules may be, for example, but are not limited to: complementary DNA (cDNA), genomic DNA, synthesized DNA, recombinant DNA, or a hybrid thereof or an RNA molecule such as, for example, mRNA, shRNA, siRNA, miRNA, and the like.
  • cDNA complementary DNA
  • RNA molecules such as, for example, mRNA, shRNA, siRNA, miRNA, and the like.
  • the terms "polynucleotide molecules”, “oligonucleotide”, “polynucleotide”, “nucleic acid” and “nucleotide” sequences are meant to refer to both DNA and RNA molecules.
  • the terms further include oligonucleotides composed of naturally occurring bases, sugars, and covalent inter nucleoside linkages, as well as
  • Treating a disease or “treating a condition” is directed to administering a composition, which comprises at least one reagent (which may be, for example, one or more polynucleotide molecules, one or more expression vectors, one or more substance/ingredient, and the like), effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring.
  • a composition which comprises at least one reagent (which may be, for example, one or more polynucleotide molecules, one or more expression vectors, one or more substance/ingredient, and the like)
  • diagnosis refers to the process of identifying/detecting/assessing NMO and/or MS in a subject.
  • organism refers to a mammal. In some embodiments, the organism is human. In some embodiments, the organism is selected from a pet, a rodent, a farm animal, and a lab animal.
  • the term "subject" is interchangeable with an individual or patient. According to some embodiments, the subject is a mammal. According to some embodiments, the subject is a human. According to some embodiments, the subject is symptomatic. According to other embodiments, the subject is asymptomatic. According to some embodiments, the subject may be treated with one or more drugs or other suitable treatment.
  • small interfering RNA and “siRNA” are used interchangeably and refer to a nucleic acid molecule mediating RNA interference or gene silencing.
  • the siRNA inhibits expression of a target gene and provides effective gene knock-down.
  • microRNA and “miRNA” are directed to a small non-coding RNA molecule that can function in transcriptional and post-transcriptional regulation of target gene expression.
  • circulating with respect to miRNA molecules is directed to the presence of the miRNA in circulating blood (serum) and not localized to an organ or localized to a specific organ (such as the brain).
  • the term "biological sample” refers to any sample obtained from the subject being tested.
  • the biological sample is a biopsy.
  • the biological sample is selected from: cells, tissue and bodily fluid.
  • the biological sample is a fluid sample.
  • the fluid sample is selected from the group consisting of: whole blood, plasma, serum, and the like.
  • the biological sample is obtained or collected from the subject in any method known in the art. The sample may be collected from the subject by noninvasive, invasive or minimal invasive means. Each possibility represents a separate embodiment of the invention.
  • the sample may be treated prior to being subjected to the methods of the present invention.
  • the sample is a fluid sample and is substantially free of residual cells or debris of cells.
  • said cells or debris of cells may be precipitated by centrifugation and the supernatant is taken for determining the specific biomarkers levels.
  • centrifugation of up to 2500 revolutions per minute (rpm) for up to 20 min is performed.
  • a common centrifugation procedure is associated with centrifugation of 1100 rpm for 7 min, or centrifugation of 2000 rpm for 5 min.
  • cells can be removed by filtration.
  • the liquid sample undergoes concentration with a suitable membrane pore cut-off size.
  • a fluid sample treated with such filter is concentrated, namely, any molecule, particle below the size of the cut-off of the filter is removed.
  • the liquid sample is concentrated by up to 100 times the initial concentration.
  • the sample is a fluid sample, such as, serum, and the miRNA molecules within said fluid sample are analysed.
  • the sample is collected, centrifuged, the supernatant is removed and analysed for specific miRNA molecules.
  • the sample is reconstituted (e.g. with fluids, such as, PBS or media).
  • the fluid sample is analyzed together with the cells present therein without prior separation.
  • the sample may conveniently be frozen after being collected from the subject and thawed before use.
  • the biological sample may also optionally comprise a sample that has not been physically removed from the subject.
  • the methods of the invention encompass determining the circulating levels of miRNAs in serum samples of a subject afflicted with NMO or MS, wherein the samples are obtained before and after suitable treatment.
  • the serum samples may be obtained at various time points before commencement of treatment.
  • the time points before commencement of treatment may be any time point of between 1 hour to 6 months.
  • the time points before commencement of treatment may be any time point of between 1-2 months.
  • the serum samples may be obtained at various time points after commencement of treatment.
  • the time points after commencement of treatment may be any time point of between 1 hour to 24 months.
  • the time points after commencement of treatment may be any time point of between 1 month to 24 months. In some embodiments, the time points after commencement of treatment may be any time point of between 2 to 12 weeks. In some embodiments, the time points after commencement of treatment may be any time point of between 1 hour to 12 months.
  • the intervals between time points obtaining of serum samples may be predetermined. In some embodiments, the intervals between time points of obtaining of serum samples may be identical or different. For example, the intervals may include intervals of 1 day, one week, one month (i.e., one month between consecutive samples). For example, the intervals may include intervals of 2-12 weeks. For example, the intervals may include intervals of 1-4 weeks.
  • the intervals may include intervals of 4-10 weeks.
  • the number of samples obtained before and/or after treatment as well as the time intervals between obtaining the samples may be predetermined or may be determined based on the patient condition, treatment regime, and the like.
  • the method of assessing treatment efficacy of NMO or MS patients may encompass, apart for the determining the levels of the indicated circulating miRNA's, any known methodologies used to assess the respective condition and/or treatment efficacy.
  • the term “elevation” or “increase” of circulating levels of miRNAs refers, according to some embodiments, to a statistically significant elevation.
  • the term “decrease” or “reduction” of circulating levels of miRNAs refers, according to some embodiments, to a statistically significant decline.
  • level is amount.
  • levels of miRNAs reflect the expression, secretion and/or presence of these nucleotide molecules in a serum sample of a subject.
  • levels of miRNAs in the serum reflect the expression/presence/secretion of these polynucleotides from within cells or tissues, such as, brain tissue.
  • modulation of circulating levels of miRNAs refers to the change in the levels. In some embodiments, modulation is increase in the levels. In some embodiments, modulation is a decrease in the levels. In some embodiments, the changes may be between various measurements obtained at different time points. In some embodiments, the changes may be between consecutive measurements. In some embodiments, the changes may be between various measurements obtained before and after treatment.
  • the results presented herein demonstrate for the first time that a set of miRNAs with elevated expression in the blood of NMO patients revert to the levels of matched healthy controls following effective therapy with rituximab.
  • the majority of these "normalized" miRNAs are known as brain specific/enriched miRNAs.
  • a diagnostic test to monitor treatment response in NMO is provided herein.
  • the results presented herein demonstrate that specific miRNA signatures in whole blood of patients with NMO can serve as biomarkers for therapy response.
  • monitoring the levels of brain specific/enriched miRNAs in the blood can reflect the degree of disease activity in the CNS of inflammatory demyelinating disorders.
  • the brain enriched miRNAs disclosed herein that revert to healthy control levels after treatment are expressed abundantly or predominantly in the brain.
  • the higher quantity of these miRNAs in the circulation of patients with NMO may imply that they were shed to the circulation by degenerative brain cells, with a corresponding decrease in the amount of these miRNAs in the circulation observed following treatment, when the destructive inflammatory process diminishes. Therefore, monitoring the levels of brain specific/enriched miRNAs in the blood reflects the degree of disease activity in the CNS of demyelinating disorders.
  • a method for determining response to therapy in a subject afflicted with Neuromyelitis optica include identification of miRNA biomarkers in the peripheral blood of patients with NMO.
  • NMO neuromyelitis optica
  • step (a) may include determining the levels of at least two circulating miRNAs. In some embodiments, step (a) may include determining the levels of at least three circulating miRNAs.
  • step a) may be conducted at a time point prior to step b).
  • steps a) and c) may be conducted at distinct time points during step b).
  • the method may further include repeating step c) for one or more times to determine the levels of the circulating miRNAs in consecutive serum samples, obtained at designated time intervals; and comparing the levels of the circulating miRNAs between said consecutive serum samples, wherein a modulation in the levels of the at circulating miRNAs between consecutive serum samples is indicative of said treatment efficacy.
  • the method may further include a step of isolating RNA from the first serum sample and/or the second serum prior to identification of the at miRNAs.
  • the method may further include a step of comparing the levels of the circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of corresponding miRNAs obtained from healthy control subjects.
  • a method for determining response to therapy in a subject afflicted with Multiple Sclerosis include identification of miRNA biomarkers in the peripheral blood of patients with MS.
  • a method for determining efficacy of treatment for multiple sclerosis (MS) in a subject in need thereof comprising one or more of the steps of:
  • modulation in the levels of the circulating miRNAs from the first to the second serum sample is indicative of said treatment efficacy.
  • step (a) may include determining the levels of at least two circulating miRNAs. In some embodiments, step (a) may include determining the levels of at least three circulating miRNAs.
  • step a) may be conducted at a time point prior to step b).
  • steps a) and c) may be conducted at distinct time points during step b).
  • the method may further include repeating step c) for one or more times to determine the levels of the circulating miRNAs in consecutive serum samples, obtained at designated time intervals; and comparing the levels of the circulating miRNAs between said consecutive serum samples, wherein a modulation in the levels of the at circulating miRNAs between consecutive serum samples is indicative of said treatment efficacy.
  • the method may further include a step of isolating
  • RNA from the first serum sample and/or the second serum prior to identification of the at miRNAs is derived from the first serum sample and/or the second serum prior to identification of the at miRNAs.
  • the method may further include a step of comparing the levels of the circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of corresponding miRNAs obtained from healthy control subjects.
  • the therapy includes administration of a suitable treatment.
  • the treatment may be selected from: Rituximab, B cell depletion treatment, treatment with anti CD20, treatment with anti-CD19, ocrelizumab, ofatumumab (HuMax-CD20), Natalizumab, azathioprine, Cladribine, Lemtrada, Teriflunomide, and the like, or combinations thereof.
  • analysis of global miRNA expression in the blood of patients with NMO before and after suitable treatment identified a distinct miRNA expression signature (profile) associated with the treatment.
  • analysis of global miRNA expression in the blood of patients with MS before and after treatment results in a distinct miRNA expression signature associated with the treatment.
  • a set of miRNAs with elevated expression in the blood of MS patients revert to the levels of matched healthy controls following effective therapy (for example, with rituximab).
  • a set of miRNAs with elevated expression in the blood of NMO patients revert to the levels of matched healthy controls following effective therapy (for example, with rituximab).
  • the miRNAs are brain enriched miRNAs.
  • the brain enriched miRNAs may be selected from: miR-135a, miR-135b, miR-125b, miR-134, miR-138 and/or miR-76.
  • the methods and kits disclosed herein include determining the levels of one or more circulating miRNAs selected from: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180. Each possibility is a separate embodiment.
  • the methods and kits disclosed herein include determining the levels of one or more circulating miRNAs selected from: miR-138, miR-660, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124 and miR-7. Each possibility is a separate embodiment.
  • the methods and kits disclosed herein include determining the levels of at least two miRNAs, selected from the group consisting of: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR- 135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180. Each possibility is a separate embodiment.
  • the methods and kits disclosed herein include determining the levels of at least three miRNAs, selected from the group consisting of: miR-138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR- 135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180. Each possibility is a separate embodiment.
  • the methods and kits disclosed herein include determining the levels of miR-1180 and miR-125b and at least one additional miR selected from the group consisting of: miR-203, miR-138, miR-660, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124 and miR-7. Each possibility is a separate embodiment.
  • the circulating miRNAs may be selected from one or more of the miRNAs listed in Table 1A and/or Table IB and/or Fig. 1 and/or Figs. 2A-2B.
  • a miRNA signature in whole blood of patients with NMO was identified. Following rituximab therapy, the expression levels of the majority of miRNAs that have higher expression in patients with NMO versus healthy controls (HCs) revert to normal levels following therapy (such as, rituximab). In some embodiments, this miRNA signature can be used as a biomarker for therapy response. In some embodiments, at least some of the identified miRNAs are brain-enriched miRNAs. In some embodiments, monitoring the levels of brain specific miRNAs in the blood of patients with inflammatory demyelinating disorders can reflect the degree of disease activity in the CNS.
  • the methods disclosed herein relates to specific miRNA signatures in whole blood of patients with MS that can serve as biomarkers for therapy response. Furthermore, monitoring the levels of brain specific/enriched miRNAs in the blood can reflect the degree of disease activity in the CNS of inflammatory demyelinating disorders.
  • a method for determining efficacy of treatment for neuromyelitis optica (NMO) in a subject in need thereof comprising: determining the levels of circulating miRNAs in a first serum sample of said subject; treating said subject with a selected treatment; determining the levels of circulating miRNAs in a second serum sample of said subject; and comparing the levels of the circulating miRNAs in said first and second serum samples, wherein modulation in the levels of circulating miRNAs from the first to the second serum sample is indicative of said treatment efficacy.
  • NMO neuromyelitis optica
  • the method further comprising a step of isolating RNA from the first serum sample and/or the second serum prior to identification of the miRNA.
  • the level of miRNA in the first serum sample and/or the second serum sample may be determined by a method selected from: amplification reaction, sequencing reaction, microarray, or combinations thereof.
  • the treatment regime using the selected treatment is adjusted based on the determined treatment efficacy.
  • the method further includes a step of comparing the levels of circulating miRNAs in the first and/or second serum sample of said subject to the circulating levels of miRNAs obtained from healthy control subjects.
  • modulating comprises increase in the level of miRNA and/or decrease in the level of miRNA. In some exemplary embodiments, modulating includes increase in the level of miRNA.
  • a method for assessing treatment efficacy of a selected treatment in a subject afflicted with neuromyelitis optica comprising determining the level of circulating miRNA molecules in serum samples of said subjects obtained before and after treatment with the selected treatment, and comparing the levels of the circulating miRNA molecules between the serum samples, wherein modulation of expression of the miRNA molecules between the serum samples obtained before and after treatment is indicative of increased treatment efficacy.
  • kits for determining efficacy of a selected treatment for neuromyelitis optica comprising means for determining the levels of circulating miRNAs in serum samples of a subject obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for the neuromyelitis optica (NMO).
  • kits for determining efficacy of a selected treatment for NMO includes means for determining the levels of one or more circulating miRNAs, selected from: miR-138, miR-660, miR- 125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR-203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for NMO.
  • a method of predicting responsiveness of a subject afflicted with neuromyelitis optica (NMO) to Rituximab treatment comprising determining the level of a plurality of circulating miRNAs, comprising one or more of the miRNAs listed in Table IB, in a serum sample obtained from the subject, wherein a significant difference between the level of said circulating miRNAs in said sample, compared to a control value is indicative of the responsiveness of said subject to Rituximab treatment.
  • NMO neuromyelitis optica
  • a method for predicting responsiveness treatment of a subject afflicted with neuromyelitis optica comprising: determining the levels of circulating miRNAs in a first serum sample of said subject and comparing the levels of the circulating miRNAs in said first serum sample to a control value; wherein a significant difference between the level of said circulating miRNAs in said sample, compared to a control value is indicative of the responsiveness of said subject to said treatment.
  • a method for determining efficacy of treatment for MS in a subject in need thereof comprising: determining the levels of circulating miRNAs in a first serum sample of said subject; treating said subject with a selected treatment; determining the levels of circulating miRNAs in a second serum sample of said subject; and comparing the levels of the circulating miRNAs in said first and second serum samples, wherein modulation in the levels of circulating miRNAs from the first to the second serum sample is indicative of said treatment efficacy.
  • a method for assessing treatment efficacy of a selected treatment in a subject afflicted with MS comprising determining the level of circulating miRNA molecules in serum samples of said subjects obtained before and after treatment with the selected treatment, and comparing the levels of the circulating miRNA molecules between the serum samples, wherein modulation of expression of the miRNA molecules between the serum samples obtained before and after treatment is indicative of increased treatment efficacy.
  • kits for determining efficacy of a selected treatment for MS comprising means for determining the levels of circulating miRNAs in serum samples of a subject obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for the MS.
  • kits for determining efficacy of a selected treatment for multiple sclerosis includes means for determining the levels of one or more circulating miRNAs, selected from: miR- 138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR- 203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for MS.
  • miRNAs selected from: miR- 138, miR-660, miR-125b, miR-760, miR-100, miR-423, miR-135a, miR-135b, miR- 203a, miR-124, miR-7 and miR-1180 in serum samples of a subject, said samples obtained before and after the selected treatment, and instructions for using the kit in the determining efficacy of the selected treatment for MS.
  • a method of predicting responsiveness of a subject afflicted with MS to Rituximab treatment comprising determining the level of a plurality of circulating miRNAs, comprising one or more of the miRNAs listed in Tables 1 A and/or Tables IB, in a serum sample obtained from the subject, wherein a significant difference between the level of said circulating miRNAs in said sample, compared to a control value is indicative of the responsiveness of said subject to Rituximab treatment.
  • a method for predicting responsiveness treatment of a subject afflicted with MS comprising: determining the levels of circulating miRNAs in a first serum sample of said subject and comparing the levels of the circulating miRNAs in said first serum sample to a control value; wherein a significant difference between the level of said circulating miRNAs in said sample, compared to a control value is indicative of the responsiveness of said subject to said treatment.
  • one or more first samples may be taken at a time point prior to initiation of the treatment and one or more second samples are taken at a time point during or after the treatment.
  • one or more first samples may be taken at a time point during the treatment (step b) and one or more second samples may be taken at a time point during the treatment and subsequent to the time point of the one or more first samples.
  • one or more first samples are taken at a time point during the treatment (step b) and one or more second samples are taken at a time point after the treatment has been discontinued.
  • one or more second samples may be obtained consecutively, at designated time intervals (such as, in the range of 1 -10 weeks).
  • a decrease in the level of the miRNAs in the at least one second sample as compared to that determined for the first sample or compared to a previous consecutive second serum sample is indicative that the treatment is effective.
  • control value is zero (i.e., non detectable level). In some embodiments, the control value is any predetermined value. In some embodiments, the methods are qualitative. In some embodiments, the methods are quantitative.
  • identification and/or quantification of the specific miRNAs in the serum may be performed by use of specific probes or tags capable of specifically interacting with the miRNA molecules.
  • tags or probes may include nucleic acid molecules.
  • such tags or probes may include nucleic acid probe, nucleic acid primers, peptidic-tags, and the like.
  • suitable methods to detect and/or quantify miRNA may include such methods as, but not limited to: amplification reaction, such as, PCR, RT-PCR, real time PCR, and the like; Gene expression microarrays, such as Affymetrix, Agilent, and Illumina microarray platforms; NanoString technology, such as nCounter technology which employs unique fluorescent-tagging of individual miRNA species followed by two-dimensional display and optical scanning and counting of miRNA molecules; Sequencing, such as, next Generation Sequencing (NGS) using specific adaptors and/or probes; and the like, or combinations thereof.
  • amplification reaction such as, PCR, RT-PCR, real time PCR, and the like
  • Gene expression microarrays such as Affymetrix, Agilent, and Illumina microarray platforms
  • NanoString technology such as nCounter technology which employs unique fluorescent-tagging of individual miRNA species followed by two-dimensional display and optical scanning and counting of miRNA molecules
  • Sequencing such as, next Generation Sequencing (
  • the level of the circulating miRNAs may be determined at one or more time points after initiation of treatment. In some embodiments, the level of the circulating miRNAs may be determined at various time points after initiation of treatment. In some embodiments, the levels of circulating miRNAs may be compared between consecutive measurements (i.e., measurements obtained between two samples obtained at consecutive time points) and treatment efficacy is determined based on the comparison.
  • the method may further include evaluating the disease status by clinical parameters. In some embodiments, there is a correlation between circulating levels of the miRNAs and the clinical parameters.
  • the methods of the invention are useful for managing the subject treatment by the clinician or physician subsequent to the determination of treatment efficacy.
  • treatment regime may be adjusted or modulated. Adjustment of treatment regime may include, for example, but not limited to: adjustment of dosage (reducing or increasing dosage), ceasing treatment, replacing or adding a drug, and the like.
  • the results presented herein demonstrate for the first time that a set of miRNAs with elevated expression in the blood of NMO patients revert to the levels of matched healthy controls following effective therapy with rituximab.
  • the majority of these "normalized" miRNAs are known as brain specific/enriched miRNAs.
  • a diagnostic test to monitor treatment response in NMO is provided herein.
  • the results presented herein demonstrate that specific miRNA signatures in whole blood of patients with NMO can serve as biomarkers for therapy response. Furthermore, monitoring the levels of brain specific/enriched miRNAs in the blood can reflect the degree of disease activity in the CNS of inflammatory demyelinating disorders. According to some embodiments, the brain enriched miRNAs disclosed herein, that revert to healthy control levels after treatment are expressed abundantly or predominantly in the brain. Without wishing to be bound to any theory or mechanism, the higher quantity of these miRNAs in the circulation of patients with NMO may imply that they were shed to the circulation by degenerative brain cells, with a corresponding decrease in the amount of these miRNAs in the circulation observed following treatment, when the destructive inflammatory process diminishes.
  • monitoring the levels of brain specific/enriched miRNAs in the blood reflects the degree of disease activity in the CNS of demyelinating disorders.
  • the autoantibodies that are directed against aquaporin 4 (AQP4) are expressed by astrocytes. This may indicate that miRNAs may contribute to pathogenesis of diseases in which astrocytes play a major role, like NMO.
  • RNAs in NMO patients and HCs there are differentially expressed miRNAs in NMO patients and HCs, as well as differential miRNA expression after treatment. Accordingly, a signature of miRNA expression was identified that not only differed between NMO patients and HCs, but also reverted to normal in response to therapy (such as, rituximab therapy).
  • the blood samples taken at the six- month time point were taken following analysis of positive CD 19 cell detection in the blood, indicating that patients were not depleted of B cells.
  • the terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
  • the terms “comprises” and “comprising” are limited in some embodiments to "consists” and “consisting”, respectively.
  • the term “consisting of” means “including and limited to”.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • the term "about” in reference to a numerical value stated herein is to be understood as the stated value +/- 10%.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • NMQ The patient cohort included 16 patients with NMO (14 females, 2 males; age 41+14.3 years; disease duration, 5.3+6.2 years; EDSS [Expanded Disability Status Scale], 4.8+1.8), followed at the Hadassah MS Center.
  • Brain magnetic resonance imaging (MRI) was normal or compatible with the diagnosis of NMO in all patients, and spinal MRIs revealed long extensive myelitis in 14/16 patients. None of the 9 responders had relapses in the 2 years following rituximab therapy.
  • the patient cohort includes additional 20 patients with MS that respond to rituximab/ocrelizumab therapy, 20 patients with MS that did not respond to the rituximab/ocrelizumab therapy followed at the Hadassah MS Center.
  • the patient cohort includes additional 20 patients with MS that respond to TYSABRI® (natalizumab) therapy, 20 patients with MS that did not respond to the TYSABRI® (natalizumab) therapy, followed at the Hadassah MS Center.
  • Clinical data is collected from patients' files.
  • MS patients are diagnosed according to the MacDonald's diagnostic criteria.
  • An age- and sex- matched control group include 15 healthy individuals.
  • Library preparation entails ligation of barcoded 3' adapters to 20 different samples, pooling of samples, ligation of a 5' adapter, reverse transcription and polymerase chain reaction (PCR), as previously described [18], with modifications allowing multiplexing of several 20-sample libraries on a single HiSeq lane, namely, 40-100 small RNA libraries per lane (see supplementary methods).
  • Libraries were sequenced on an Illumina HiSeq sequencer, and the information obtained was analyzed by an automated computer pipeline to decode and annotate small RNA reads [19]. Normalization of miRNA reads was performed by dividing each miRNA read frequency by the total number of miRNA sequence reads within the subsample, thereby correcting the variable sequencing depth in each subsample.
  • Mature miRNAs were quantified using Perfecta® micro RNA Assays (Quanta Biosciences), according to the manufacturer's instructions.
  • Real-time polymerase chain reaction (PCR) was performed on a StepOne real-time reverse transcription (RT)-PCR (Life Technologies) in triplicate for each sample.
  • RT real-time reverse transcription
  • the fold changes of miRNAs were normalized to RNU6B, (ACT). The data is presented as 2 "ACT .
  • Primer sequences were designed based on the miRNA sequences obtained from the miRBase database (http://microrna.sanger.ac.uk/). Statistical significance was calculated using 2-tailed t test.
  • Example 1 Identification of differentially expressed microRNAs before and after rituximab treatment and between Healthy Controls (HCs) and patients with NMO.
  • HCs Healthy Controls
  • NMO Healthy Controls
  • RNA samples were collected from Nine (9) NMO patients, classified as rituximab responders, prior to and at 6 months following therapy. Blood samples were also collected from Fifteen (15) age-and sex-matched Healthy subjects (HCs). To further analyze the differential expression between untreated NMOs and HCs, Seven (7) additional untreated AQP4+ seropositive NMO patients were also included in the study. Total RNA was extracted from the whole blood samples and miRNA levels were quantified by deep sequencing analysis (RNAseq).
  • RNAseq results demonstrated that the expression levels of 32 miRNAs were decreased and of 14 miRNAs were increased significantly (p ⁇ 0.05) in the 9 NMO treated patients following treatment with rituximab.
  • Tables 1A-1B Differential expression of miRNAs following rituximab therapy (Table 1A) and between untreated NMO patients and HCs (Table IB). miRNA with increased expression in NMO patients that were normalized following rituximab therapy, indicated by (#); brain specific/enriched miRNAs are marked with ( ⁇ )
  • Example 2 Identification of differentially expressed microRNAs before and after treatment and between Healthy Controls (HCs) and patients with MS.
  • RNAseq RNA-binding protein
  • Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A, 2011. 108(12): p. 5003-8.

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Abstract

L'invention concerne des micro-ARN circulants en tant que biomarqueurs dans la neuromyélite optique (NMO) et/ou la sclérose en plaques (SEP). L'invention concerne en outre un profil de miARN circulants pour la prédiction de la sensibilité à un traitement, tel que le rituximab, de sujets souffrant de NMO et/ou de SEP.
PCT/IL2017/050741 2016-07-03 2017-07-03 Micro-arn circulants en tant que biomarqueurs pour une thérapie dans la neuromyélite optique (nmo) et de la sclérose en plaques (sep) Ceased WO2018008017A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005117978A2 (fr) * 2004-06-04 2005-12-15 Genentech, Inc. Methode de traitement de la sclerose en plaques
WO2011158191A1 (fr) * 2010-06-16 2011-12-22 Istituto Nazionale Di Genetica Molecolare - Ingm Surveillance du système immunitaire à l'aide d'une analyse du profil d'expression de micro-arn de sang périphérique et ses utilisations

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005117978A2 (fr) * 2004-06-04 2005-12-15 Genentech, Inc. Methode de traitement de la sclerose en plaques
WO2011158191A1 (fr) * 2010-06-16 2011-12-22 Istituto Nazionale Di Genetica Molecolare - Ingm Surveillance du système immunitaire à l'aide d'une analyse du profil d'expression de micro-arn de sang périphérique et ses utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KELLER, ANDREAS ET AL.: "Next-generation sequencing identifies altered whole blood microRNAs in neuromyelitis optica spectrum disorder which may permit discrimination from multiple sclerosis", JOURNAL OF NEUROINFLAMMATION, vol. 12, no. 196, 31 October 2015 (2015-10-31), pages 1 - 12, XP055450448, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628234> *

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