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WO2017120285A1 - Procédés d'utilisation de micro-arn provenant de liquides organiques pour le diagnostic et la surveillance de troubles neurodéveloppementaux - Google Patents

Procédés d'utilisation de micro-arn provenant de liquides organiques pour le diagnostic et la surveillance de troubles neurodéveloppementaux Download PDF

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WO2017120285A1
WO2017120285A1 PCT/US2017/012258 US2017012258W WO2017120285A1 WO 2017120285 A1 WO2017120285 A1 WO 2017120285A1 US 2017012258 W US2017012258 W US 2017012258W WO 2017120285 A1 WO2017120285 A1 WO 2017120285A1
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mirna
mir
enriched
brain
neurodevelopmental disorder
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Samuil R. Umansky
Kira S. Sheinerman
Vladimir G. TSIVINSKY
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DiamiR LLC
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DiamiR LLC
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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
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention provides methods for diagnosis and monitoring of Rett syndrome and other neurodevelopmental disorders by quantitative analysis of miRNAs in bodily fluids.
  • NDDs Neurodevelopmental disorders
  • Some of NDDs, such as Rett syndrome, Tourette syndrome and others are rare, but some, for example Autism Spectrum Disorders, are much more common.
  • Early minimally invasive tests for early detection of NDDs are very important for the following reasons. First, although there is no effective treatment for most of NDDs, early symptomatic treatment may significantly improve patient health. Second, due to variability of mutations causing particular NDDs and other factors these disorders are characterized by the wide phenotypical and clinical heterogeneity, and biomarkers capable of predicting disease development and outcome would be very useful. Finally and maybe most importantly, such biomarkers would be extremely helpful on all stages of drug/treatment development, including use of animal models in preclinical studies, patient involvement and stratification for clinical studies and treatment monitoring.
  • Rett syndrome is a monogenic X-linked disorder caused by mutations in MECP2 gene, which encodes the methyl-CpG binding protein 2 (Renieri A et al. Rett syndrome: the complex nature of a monogenic disease. J Mol Med (Berl). 2003; 81(6):346-54). Due to severe encephalopathy caused by a mutation in the single copy of MECP2 male fetuses usually die before birth, so the disease affects females almost exclusively ( ⁇ 1 in 10,000).
  • An advantage of using RTT as an example for developing diagnostic tests for NDDs in general is the existence of several mouse models of RTT (see below). Of course, potential biomarkers should be evolutionary conservative to be applicable for both human and animals.
  • MicroRNAs are a class of non-coding RNAs whose final product is an approximately 22 nt functional RNA molecule. They play important roles in the regulation of target genes by binding to complementary regions of messenger transcripts to repress their translation or regulate degradation (Griffiths-Jones Nucleic Acids Research. 2006; 34, Database issue: D140-D144; Jin and Xiao, Frontiers in Genetics. 2015; 6:328). Frequently, one miRNA can target multiple mRNAs and one mRNA can be regulated by multiple miRNAs targeting different regions of the 3' UTR.
  • miRNA can modulate gene expression and protein production by affecting, e.g., mRNA translation and stability (Baek et al. Nature. 2008; 455:64; Selbach et al. Nature. 2008; 455:58; Ambros. Nature. 2004; 431 : 350-355; Bartel. Cell. 2004; 116: 281-297; Cullen. Virus Research. 2004; 102: 3-9; He et al. Nat. Rev. Genet. 2004; 5: 522-531; and Ying et al. Gene. 2004; 342: 25-28).
  • mRNA translation and stability Boek et al. Nature. 2008; 455:64; Selbach et al. Nature. 2008; 455:58; Ambros. Nature. 2004; 431 : 350-355; Bartel. Cell. 2004; 116: 281-297; Cullen. Virus Research. 2004; 102: 3-9; He et al. Nat. Rev. Genet. 2004; 5: 5
  • miRNAs are specific to or over-expressed in certain organs / tissues / cells (see, e.g., Hua et al. BMC Genomics. 2009; 10:214; Liang et al. BMC Genomics. 2007; 8: 166; Landgraf et al. Cell. 2007; 129: 1401-1414; Lee et al. RNA. 2008; 14:35-42) and in different brain areas, such as hippocampus, midbrain, frontal cortex, pituitary gland, and in different cell types, such as neurons and glial cells (Sempere et al. Genome Biol. 2004; 5: R13; Deo et al. Dev. Din.
  • miRNAs are enriched in certain cellular compartments, particularly in axons, dendrites and synapses (see, e.g., Schratt et al. Nature. 2006; 439:283-289; Lugli et al. J. Neurochem. 2008; 106:650-661; Bicker and Schratt. J. Cell. Mol. Med. 2008; 12: 1466-1476; Smalheiser and Lugli. Neuromolecular Med. 2009; 11 : 133-140; Rajasethupathy. Neuron. 2009; 63 :714-716; Kye. RNA. 2007; 13 : 1224-1234; Yu et al. Exp Cell Res.
  • miRNA secretion varies depending on cellular physiology (Palma et al. Nucleic Acids Res. 2012; 40:9125-9138; Pigati et al. PLoS One. 2010; 5: el3515).
  • miRNA appear in circulation due to blebbing of apoptotic bodies, budding and shedding of microvesicles, active secretion in the form of exosomes and of miRNA complexes with proteins (AG02, NPMl and others) and high density lipoproteins (HDL) (reviews: Sun et al. Clin. Chem.
  • the second approach is based on analysis of disease-specific miRNAs identified by comparison of miRNAs isolated from pathologic and normal tissue, organ or cell type.
  • disease-specific miRNAs e.g., by an array followed by RT-PCR
  • their presence in bodily fluids is analyzed.
  • RT-PCR since a limited number of circulating miRNAs is tested, the use of individual RT-PCR is possible which allows to increase sensitivity and reproducibility of the analysis.
  • no correlation was detected between miRNA concentration and pathology-induced changes in the tissue and in bodily fluids (Boeri et al., Proc. Natl. Acad. Sci. USA.
  • RTT Rett Syndrome
  • the invention provides a method for detecting a neurodevelopmental disorder in a subject, which method comprises:
  • a first brain-enriched miRNA e.g., synapse and/or neurite miRNA
  • a second brain-enriched miRNA is enriched in a brain area(s) which is not affected by the neurodevelopmental disorder, or (ii) is enriched in a brain cell type which is not affected by the neurodevelopmental disorder, or (iii) is enriched in the same brain area as the first miRNA, but its expression and/or secretion change differently than expression and/or secretion of the first miRNA during development of the neurodevelopmental disorder;
  • step (c) comparing the ratio of the levels of the miRNAs calculated in step (c) with a corresponding control ratio
  • step (c) (i) identifying the subject as being afflicted with the neurodevelopmental disorder when the ratio of the levels of the miRNAs calculated in step (c) is higher than the corresponding control ratio or (ii) identifying the subject as not being afflicted with the neurodevelopmental disorder when the ratio of the levels of the miRNA calculated in step (c) is not higher than the corresponding control ratio.
  • the method further comprises the following steps, which steps can be performed simultaneously or sequentially with each other and/or with the steps (d)-(e) of the above method: f) comparing the ratio of the levels of the miRNAs calculated in step (c) with the standard range of ratios of said miRNAs characteristic of another pathology ("a second pathology"), and g) (i) excluding the diagnosis of the second pathology in the subject if the ratio of the levels of the miRNAs calculated in step (c) does not fall within the standard range of ratios of said miRNAs characteristic of the second pathology, or (ii) not excluding the diagnosis of the second pathology in the subject if the ratio of the levels of the miRNAs calculated in step (c) falls within the standard range of ratios of said miRNAs characteristic of the second pathology.
  • the method further comprises the following steps, which steps can be performed simultaneously or sequentially with each other and/or with the steps (a)-(e) of the above method:
  • a third brain-enriched miRNA e.g., synapse and/or neurite miRNA
  • step (h) comparing the ratio of the levels of the miRNAs calculated in step (h) with the standard range of ratios of said miRNAs characteristic of the second pathology;
  • step (h) (i) identifying the subject as being afflicted with the second pathology in addition to the neurodevelopmental disorder if the ratio of the levels of the miRNAs calculated in step (h) falls within the standard range of ratios of said miRNAs characteristic of the second pathology, or (ii) excluding the diagnosis of the second pathology in the subject if the ratio of the levels of the miRNAs calculated in step (h) does not fall within the standard range of ratios of said miRNAs characteristic of the second pathology.
  • the invention provides a method for detecting a neurodevelopmental disorder in a subject, which method comprises: a) measuring the level of a first brain-enriched miRNA (e.g., synapse and/or neurite miRNA) in a bodily fluid sample collected from the subject, wherein said first brain-enriched miRNA is enriched in a brain area(s) affected by the neurodevelopmental disorder;
  • a first brain-enriched miRNA e.g., synapse and/or neurite miRNA
  • a second brain-enriched miRNA is enriched in a brain area(s) which is not affected by the neurodevelopmental disorder, or (ii) is enriched in a brain cell type which is not affected by the neurodevelopmental disorder , or (iii) is enriched in the same brain area as the first miRNA, but its expression and/or secretion change differently than expression and/or secretion of the first miRNA during development of the neurodevelopmental disorder, and
  • the invention also provides a computer-implemented method of assigning a subject into a category of being afflicted with a neurodevelopmental disorder, which method comprises: a. measuring the level of a first brain-enriched miRNA (e.g., synapse and/or neurite miRNA) in a bodily fluid sample collected from the subject, wherein said first brain-enriched miRNA is enriched in a brain area(s) affected by the neurodevelopmental disorder;
  • a. measuring the level of a first brain-enriched miRNA e.g., synapse and/or neurite miRNA
  • step (c) calculating, by the processor and based on the ratio determined in step (c), a first probability based on a first predefined probability distribution curve, wherein the first predefined probability distribution curve corresponds to the neurodevelopmental disorder;
  • step (c) e. calculating, by the processor and based on the ratio determined in step (c), a second probability based on a second predefined probability distribution curve, wherein the second predefined probability distribution curve corresponds to a matched control (e.g., matched by gender and/or age and/or race, etc.) or another pathology;
  • a matched control e.g., matched by gender and/or age and/or race, etc.
  • step (d) determining, by the processor, a difference between the first probability calculated in step (d) and the second probability calculated in step (e), and
  • step (f) identifying, by the processor, the subject as being afflicted with the neurodevelopmental disorder when the difference between the first probability and the second probability calculated in step (f) is positive or (ii) identifying the subject as not being afflicted with the neurodevelopmental disorder when the difference between the first probability and the second probability calculated in step (f) is negative.
  • the invention provides a method for treating a neurodevelopmental disorder in a subject in need thereof, which method comprises:
  • a first brain-enriched miRNA e.g., synapse and/or neurite miRNA
  • a second brain-enriched miRNA is enriched in a brain area(s) which is not affected by the neurodevelopmental disorder, or (ii) is enriched in a brain cell type which is not affected by the neurodevelopmental disorder , or (iii) is enriched in the same brain area as the first miRNA, but its expression and/or secretion change differently than expression and/or secretion of the first miRNA during development of the neurodevelopmental disorder;
  • step (c) optionally calculating the ratio of the levels of the miRNAs measured in steps (a) and (b); d) optionally comparing the ratio of the levels of the miRNAs calculated in step (c) with a corresponding control ratio;
  • step (c) optionally (i) identifying the subject as being afflicted with the neurodevelopmental disorder when the ratio of the levels of the miRNAs calculated in step (c) is higher than the corresponding control ratio or (ii) identifying the subject as not being afflicted with the neurodevelopmental disorder when the ratio of the levels of the miRNA calculated in step (c) is not higher than the corresponding control ratio, and
  • the invention provides a method for selecting subjects for enrollment in a clinical trial involving treatment of a neurodevelopmental disorder, which method comprises:
  • a first brain-enriched miRNA e.g., synapse and/or neurite miRNA
  • a second brain-enriched miRNA is enriched in a brain area(s) which is not affected by the neurodevelopmental disorder, or (ii) is enriched in a brain cell type which is not affected by the neurodevelopmental disorder , or (iii) is enriched in the same brain area as the first miRNA, but its expression and/or secretion change differently than expression and/or secretion of the first miRNA during development of the neurodevelopmental disorder;
  • step (c) optionally calculating the ratio of the levels of the miRNAs measured in steps (a) and (b); d) optionally comparing the ratio of the levels of the miRNAs calculated in step (c) with a corresponding control ratio;
  • step (c) optionally (i) identifying the subject as being afflicted with the neurodevelopmental disorder when the ratio of the levels of the miRNAs calculated in step (c) is higher than the corresponding control ratio or (ii) identifying the subject as not being afflicted with the neurodevelopmental disorder when the ratio of the levels of the miRNA calculated in step (c) is not higher than the corresponding control ratio, and
  • the neurodevelopmental disorder is Rett Syndrome (RTT).
  • RTT Rett Syndrome
  • the neurodevelopmental disorder is selected from the group consisting of Landau-Kleffner Syndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Angelman Syndrome, Ataxias and Cerebellar or Spinocerebellar Degeneration, Ataxia Telangiectasia, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorders including Asperger Syndrome, Batten Disease, Canavan Disease, and Tourette Syndrome.
  • control ratio is a predetermined value which represents a statistically validated threshold ratio of the levels of said first and second miRNAs (a single "cut-off value) equal to the highest possible value within the range of corresponding values in matched healthy subjects (e.g., matched by gender and/or age and/or race, etc.).
  • control ratio is the ratio of the levels of said first and second miRNAs in a similarly processed bodily fluid sample from the same subject collected in the past.
  • the standard range of ratios of miRNAs characteristic of the second pathology is a statistically validated predetermined range of values established by determining the ratios of the same miRNAs in a cohort of subjects diagnosed with the second pathology.
  • the cohort of subjects diagnosed with the second pathology represents a full range of development stages of said second pathology.
  • the cohort of subjects diagnosed with the second pathology represents one or more development stages of said second pathology.
  • the neurodevelopmental disorder is Rett Syndrome and the second pathology is selected from the group consisting of Landau-Kleffner Syndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Angelman Syndrome, Ataxias and Cerebellar or Spinocerebellar Degeneration, Ataxia Telangiectasia, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorders including Asperger Syndrome, Batten Disease, Canavan Disease, and Tourette Syndrome.
  • the second pathology is selected from the group consisting of Landau-Kleffner Syndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Angelman Syndrome, Ataxias and Cerebellar or Spinocerebellar Degeneration, Ataxia Telangiectasia, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorders including Asperger Syndrome, Batten Disease, Canavan Disease, and Tourette Syndrome.
  • said neurodevelopmental disorder is Rett Syndrome (RTT) and the method further comprises determining the underlying MECP2 mutation (e.g., for predicting disease severity).
  • RTT Rett Syndrome
  • the invention provides a method for monitoring changes in development of a neurodevelopmental disorder in a subject (e.g., a subject who had been previously diagnosed with said neurodevelopmental disorder), which method comprises: a) measuring the level of a first brain-enriched miRNA (e.g., synapse and/or neurite miRNA) in two or more bodily fluid samples collected from the subject, wherein said first brain- enriched miRNA is enriched in a brain area(s) affected by the neurodevelopmental disorder; b) measuring the level of a second brain-enriched miRNA in the same bodily fluids samples as in step (a), wherein said second brain-enriched miRNA (i) is enriched in a brain area(s) which is not affected by the neurodevelopmental disorder, or (ii) is enriched in a brain cell type which is not affected by the neurodevelopmental disorder, or (iii) is enriched in the same brain area as the first miRNA, but its expression and/or secretion change differently than expression and/or secretion of
  • step (c) comparing the ratios of the levels of the miRNA calculated in step (c) between the earlier collected and later collected bodily fluid sample(s), and
  • step (c) (i) determining that the neurodevelopmental disorder in the subject has progressed if the ratio of the levels of the miRNA calculated in step (c) is increased in the later collected bodily fluid sample(s) as compared to the earlier collected sample(s), or (ii) determining that the neurodevelopmental disorder in the subject has not progressed if the ratio of the levels of the miRNA calculated in step (c) is not changed in the later collected bodily fluid sample(s) as compared to the earlier collected sample(s).
  • the invention provides a method for monitoring the effect of a treatment on development of a neurodevelopmental disorder in a subject (e.g., a subject who had been previously diagnosed with said neurodevelopmental disorder), which method comprises: a) measuring the level of a first brain-enriched miRNA (e.g., synapse and/or neurite miRNA) in a bodily fluid sample collected from the subject prior to initiation of the treatment, wherein said first brain-enriched miRNA is enriched in a brain area(s) affected by the neurodevelopmental disorder;
  • a first brain-enriched miRNA e.g., synapse and/or neurite miRNA
  • a second brain-enriched miRNA is enriched in a brain area(s) which is not affected by the neurodevelopmental disorder, or (ii) is enriched in a brain cell type which is not affected by the neurodevelopmental disorder, or (iii) is enriched in the same brain area as the first miRNA, but its expression and/or secretion change differently than expression and/or secretion of the first miRNA during development of the neurodevelopmental disorder;
  • step (d) measuring the level of the same first miRNA as in step (a) in one or more bodily fluid sample(s) collected from the subject in the course of or following the treatment;
  • step (d) measuring the level of the same second miRNA as in step (b) in the same bodily fluid sample(s) as in step (d);
  • step (f) (i) determining that the treatment is effective for said neurodevelopmental disorder if the ratio of the levels of the miRNA calculated in step (c) is higher than the corresponding ratio(s) calculated in step (f), or (ii) determining that the treatment is not effective for said neurodevelopmental disorder if the ratio of the levels of the miRNA calculated in step (c) is not higher than the corresponding ratio(s) calculated in step (f).
  • the invention provides a method for identifying a compound useful for slowing down the progression or treating a neurodevelopmental disorder in a subject (e.g., a subject who had been previously diagnosed with said neurodevelopmental disorder), which method comprises:
  • a first brain-enriched miRNA e.g., synapse and/or neurite miRNA
  • a second brain-enriched miRNA is enriched in a brain area(s) which is not affected by the neurodevelopmental disorder, or (ii) is enriched in a brain cell type which is not affected by the neurodevelopmental disorder, or (iii) is enriched in the same brain area as the first miRNA, but its expression and/or secretion change differently than expression and/or secretion of the first miRNA during development of the neurodevelopmental disorder;
  • step (d) measuring the level of the same first miRNA as in step (a) in one or more bodily fluid samples collected from the subject following administration of a test compound;
  • step (d) measuring the level of the same second miRNA as in step (b) in the same bodily fluid sample(s) as in step (d);
  • step (c) comparing the ratio of the levels of the miRNA calculated in steps (c) and (f), and h) (i) identifying that the test compound is useful for slowing down the progression or treating the neurodevelopmental disorder if the ratio of the levels of the miRNA calculated in step (f) is lower than the ratio of the levels of the miRNA calculated in step (c); (ii) identifying that the test compound is not useful for slowing down the progression or treating the neurodevelopmental disorder if the ratio of the levels of the miRNA calculated in step (f) is not lower than the ratio of the levels of the miRNAs calculated in step (c).
  • the neurodevelopmental disorder is Rett Syndrome (RTT).
  • RTT Rett Syndrome
  • the neurodevelopmental disorder is selected from the group consisting of Landau-Kleffner Syndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Angelman Syndrome, Ataxias and Cerebellar or Spinocerebellar Degeneration, Ataxia Telangiectasia, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorders including Asperger Syndrome, Batten Disease, Canavan Disease, and Tourette Syndrome.
  • the first brain-enriched miRNA is a synapse and/or neurite miRNA.
  • the second miRNA is a brain-enriched miRNA, which (1) is enriched in a brain area(s) which is not affected by the neurodevelopmental disorder (e.g., RTT) or (2) is enriched in a brain cell type which is not affected by the neurodevelopmental disorder (e.g., RTT).
  • the second miRNA is a brain-enriched miRNA selected from the group consisting of miRNAs which are mainly expressed in brain areas not involved in RTT, miRNAs which are mainly expressed in glial cells, and brain-enriched miRNAs downregulated in RTT.
  • the first brain-enriched miRNA is enriched in neurons and the second brain-enriched miRNA is enriched in glial cells.
  • the pair of the first miRNA and the second miRNA is selected from the group consisting of: miR-107/miR-323-3p, miR-107/miR- 335-5p, miR-491-5p/miR-323-3p, miR-491-5p/miR-335-5p, miR-491-5p/miR-132, miR-491- 5p/miR-411, miR-41 l/miR-335-5p, miR-41 l/miR-132, miR-107/miR-132, miR-323-3p/miR- 335-5p, and miR-323-3p/miR-132.
  • the pair of the first miRNA and the second miRNA is selected from the pairs provided in Tables 3-5, below.
  • the method comprises measuring the level and calculating the ratios of the levels for two or more different pairs of miRNA. In one specific embodiment, the method comprises measuring the level and calculating the ratios of the levels for one or more pair combinations selected from the group consisting of:
  • the method comprises measuring the levels of the miRNAs in two or more bodily fluid samples collected from the subject, wherein the samples have been collected at spaced apart time points.
  • the bodily fluid samples are obtained several months apart (e.g., 3-6 months apart).
  • the method further comprises normalizing the levels of the first and second miRNAs to the level of a normalizer miRNA.
  • the normalizer miRNA is miRNA which is expressed in numerous tissues but is not significantly expressed in brain.
  • the subject does not have clinical symptoms of the neurological disorder.
  • the subject is human.
  • the human subject is an infant or a child.
  • the subject is an experimental animal (e.g., an animal model of a neurodevelopmental disorder such as, e.g., RTT).
  • the bodily fluid is selected from the group consisting of blood plasma, serum, urine, and saliva. Any other bodily fluid can also be used, preferably, those bodily fluids that allow low cost non-invasive or minimally invasive collection and analysis.
  • the method comprises the step of collecting the bodily fluid sample(s) from the subject (e.g., prior to step (a)).
  • the level of the miRNAs is determined using a method selected from the group consisting of hybridization, polymerase chain reaction (PCR)-based detection (for example, RT-PCR), sequencing, and microfluidic technologies.
  • PCR polymerase chain reaction
  • useful methods for measuring miRNA level in bodily fluids include hybridization with selective probes (e.g., using Northern blotting, bead-based flow-cytometry, oligonucleotide microchip [microarray], or solution hybridization assays such as Ambion mirVana miRNA Detection Kit), polymerase chain reaction (PCR)-based detection (e.g., stem- loop reverse transcription-polymerase chain reaction [RT-PCR], quantitative RT-PCR based array method [qPCR-array]), direct sequencing by one of the next generation sequencing technologies (e.g., Helicos small RNA sequencing, miRNA BeadArray (Ulumina), Roche 454 (FLX-Titanium), and ABI SOLiD), or various microfluorized.
  • RT-PCR-based techniques allow to achieve good sensitivity and specificity.
  • the miRNA prior to measuring miRNA level, is purified from the bodily fluid sample.
  • miRNAs can be isolated and purified from bodily fluids by various methods, including, without limitation, the use of commercial kits (e.g., miRNeasy kit [Qiagen], MirVana RNA isolation kit [Ambion/ABI], miRACLE [Agilent], High Pure miRNA isolation kit [Roche], and miRNA Purification kit [Norgen Biotek Corp.]), Trizol extraction, concentration and purification on anion-exchangers, magnetic beads covered by RNA-binding substances, or adsorption of certain miRNA on complementary oligonucleotides.
  • commercial kits e.g., miRNeasy kit [Qiagen], MirVana RNA isolation kit [Ambion/ABI], miRACLE [Agilent], High Pure miRNA isolation kit [Roche], and miRNA Purification kit [Norgen Biotek Corp.]
  • Trizol extraction concentration and purification on anion-exchangers, magnetic
  • the method further comprises reducing or eliminating degradation of the miRNAs.
  • Useful methods for reducing or eliminating miRNA degradation include, without limitation, adding RNase inhibitors (e.g., RNasin Plus [Promega], SUPERase-In [ABI], etc.), use of guanidine chloride, guanidine isothiocyanate, N- lauroylsarcosine, sodium dodecyl sulphate (SDS), or a combination thereof. Reducing miRNA degradation in bodily fluid samples is particularly important when sample storage and transportation is required prior to miRNA quantification.
  • RNase inhibitors e.g., RNasin Plus [Promega], SUPERase-In [ABI], etc.
  • SDS sodium dodecyl sulphate
  • QC quality control
  • normalization methods can be used in the present invention:
  • Ubiquitous miRNAs can be used for QC by comparing their concentrations in subject's bodily fluid with pre-established normal values.
  • Synthetic small RNA e.g., non-human miRNA
  • oligonucleotides can be synthesized and used as controls for losses during purification and/or RT-PCR inhibition (e.g., by adding them to bodily fluid samples before RNA purification).
  • miRNA concentration in urine can be normalized on creatinine and/or albumin level.
  • neurodevelopmental disorder detection based on miRNA levels is combined with additional methods of detection.
  • additional methods include, e.g., genetic testing (e.g., MECP2 mutation determination for RTT), hearing test, eye and/or vision exam, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), multiphoton imaging, magnetoencephalography (MEG), and electroencephalography (EEG).
  • genetic testing e.g., MECP2 mutation determination for RTT
  • CT computed tomography
  • MRI magnetic resonance imaging
  • MEG magnetoencephalography
  • EEG electroencephalography
  • the method further comprises administering a therapeutic or preventive treatment to the subject.
  • useful symptomatic and prophylactic treatments include, for example, physical therapies, occupational therapies and/or speech-language therapies, nutritional support, controlling seizures, controlling muscle stiffness, GI treatments, heart treatments, and treatments for breathing problems.
  • the therapeutic or preventative treatment may be administered prior the appearance of one or more clinical symptoms of the neurodevelopmental disorder.
  • effective treatment can mean RTT improvement (decrease of a monitored biomarker miRNA ratio) or prevention/inhibition of further development of RTT (monitored biomarker miRNA ratio stays the same or increases slower).
  • the method further comprises recruiting the subject in a clinical trial.
  • kits of the invention include:
  • a kit for detecting a neurodevelopmental disorder comprising primers and/or probes specific for one or more pairs of miRNAs selected from the group consisting of: miR- 107/miR-323-3p, miR-107/miR-335-5p, miR-491-5p/miR-323-3p, miR-491-5p/miR-335-5p, miR-491-5p/miR-132, miR-491-5p/miR-411, miR-41 l/miR-335-5p, miR-41 l/miR-132; miR- 107/miR-132, miR-323-3p/miR-335-5p, and miR-323-3p/miR-132.
  • a neurodevelopmental disorder e.g., RTT
  • a kit for detecting a neurodevelopmental disorder comprising primers and/or probes specific for one or more pairs of miRNAs selected from the pairs provided in Tables 3-5, below.
  • a kit for detecting a neurodevelopmental disorder comprising primers and/or probes specific for one or more combinations of pairs of miRNAs selected from the group consisting of:
  • kits can further comprise miRNA isolation means and/or miRNA purification means and/or instructions for use.
  • Figures 1A-1E are graphs showing ratios of miRNA levels (biomarker miRNA pairs) in plasma of wild type and Mecp2 tml lJae Null mice. miRNA ratios are presented as log 10 of 2 Average for each pair is indicated.
  • Figures 2A-2B are graphs presenting ROC curves for differentiation between wild type and Mecp2 tml lJae Null mice with miRNA pairs.
  • Figures 3A-3E are graphs showing ratios of miRNA levels (biomarker miRNA pairs identified in Example 3 for differentiating Mecp2 tml lJae Null and wild type mice) in plasma of wild type and Mecp2 tml lBird mice. miRNA ratios are presented as loglO of 2ACt. Average for each pair is indicated.
  • Figures 4A-4H are graphs showing additional biomarker miRNA pairs effectively distinguishing wild type and Mecp2 tml 1Bird Null mice. miRNA ratios are presented as loglO of 2 . Average for each pair is indicated.
  • Figures 5A-5B are graphs presenting ROC curves for differentiation between wild type and Mecp2 tml lBird Null mice with miRNA pairs.
  • Figures 6A-6F are graphs showing ratios of miRNA levels (biomarker miRNA pairs) in plasma of wild type and Mecp2 tml lJae Het mice. miRNA ratios are presented as loglO of 2 Average for each pair is indicated.
  • Figures 7A-7B are graphs presenting ROC curves for differentiation between wild type and Mecp2 tol 1Jae Het mice with miRNA pairs.
  • Figures 8A-8E are graphs showing biomarker miRNA pairs effectively distinguishing wild type and Mecp2 tml lBird Het mice. miRNA ratios are presented as loglO of 2 ACt . Average for each pair is indicated.
  • Figure 9 is a graph presenting ROC curves for differentiation between wild type and Mecp2 tml 1 Bird Het mice with miRNA pairs.
  • DDs differ from neurodegenerative diseases by no or very limited neuronal death [Armstrong et al. Selective Dendritic Alterations in the Cortex of Rett Syndrome. J. Neuropathol. Exp. Neurol. 54, 195 (1995); Katz DM et al. Preclinical research in Rett syndrome: setting the foundation for translational success. Dis. Models and Mechanisms, 5, 733-745 (2012)].
  • NDDs are characterized by synapse and/or neurite dysfunction in particular brain areas.
  • the present inventors have hypothesized that such synapse and/or neurite dysfunction can be accompanied by changes in miRNA expression and secretion, leading to increased or decreased levels of particular miRNAs in plasma/serum or other bodily fluids, such as, e.g., urine or saliva.
  • the present inventors have hypothesized that, since miRNAs are evolutionary conserved, one can expect that same miRNAs may be used as biomarkers of RTT in human, mouse and other animals.
  • Data on miRNA expression in brain of RTT subjects are limited and obtained mainly in various mouse models with mutant MECP2 [Wu et al. Genome-wide analysis reveals methyl- CpG-binding protein 2-dependent regulation of microRNAs in a mouse model of Rett syndrome. Proc.
  • Mecp2 Disrupted microRNA expression caused by Mecp2 loss in a mouse model of RTT. Epigenetics, 5, 656-663 (2010)].
  • Mecp2 affects synthesis of some miRNAs [Leon- Guerrero et al. In sickness and in health: the role of methyl-CpG binding protein 2 in the central nervous system. Europ. J. Neurosci. 33, 1563-1574, (2011)] and its inactivation changes transcription of those miRNAs.
  • the present invention is based on the following ideas and findings made by the present inventors: (1) changes in concentrations of circulating miRNAs enriched in the brain, and more specifically in brain areas involved in a particular pathology, are more likely to reflect associated pathologic processes in the brain than ubiquitous or other brain-enriched miRNAs;
  • miRNAs present in neurites and synapses should be analyzed, because dysfunction and destruction of neurites and synapses is characteristic of NDDs, and therefore, can affect expression and secretion of these miRNAs;
  • the present inventors used the "biomarker miRNA pair" approach normalizing miRNAs enriched in neurons of damaged brain area(s) by other brain-enriched miRNAs, such as, e.g., (i) miRNAs enriched in a brain area(s) which is not affected by the NDD which is being diagnosed, or (ii) miRNAs enriched in a brain cell type which is not affected by the NDD which is being diagnosed, or (iii) miRNAs enriched in the same brain area as the biomarker miRNA, but its expression and/or secretion change differently than expression and/or secretion of the biomarker miRNA during development of the NDD which is being diagnosed;
  • the present invention is based on analysis of the ratios of the levels for pairs of circulating cell-free miRNA in bodily fluids, wherein both miRNA in the pair are brain-enriched, and either (i) are enriched in certain brain areas, which are (for one miRNA in the pair) or are not (for the other miRNA in the pair) affected by the NDD (e.g., by being involved in NDD development), or (ii) are enriched in different cell types (e.g., neurons and glial cells), or (iii) are enriched in the same brain area but whose expression and/or secretion change differently due to NDD development.
  • both miRNA in the pair are brain-enriched, and either (i) are enriched in certain brain areas, which are (for one miRNA in the pair) or are not (for the other miRNA in the pair) affected by the NDD (e.g., by being involved in NDD development), or (ii) are enriched in different cell types (e.g., neurons and glial cells), or (i
  • Brain-enriched miRNAs which are particularly useful as numerators in the biomarker miRNA pairs of the invention include neuronal miRNAs present in neurites and synapses (i.e., synapse and/or neurite miRNAs), whose normal functioning suffers in RTT or other NDDs. Since various NDDs are characterized by neuronal pathology in different brain areas such biomarker miRNA pairs can be used for differentiating those pathologies from each other independent of their clinical symptoms, if any. In addition, discovered biomarker miRNA pairs, reflecting important events in pathology development, could be used for patient selection and stratification for clinical trials, early patient treatment, disease and treatment monitoring as well as for drug screening. Due to evolutionary conservation of miRNAs the same biomarker miRNA pairs may be also used in animal models for preclinical phase of drug development.
  • brain-enriched miRNA in the methods of the invention significantly increases chances that changes of their levels in bodily fluids are caused by brain pathology, and changes in bodily fluid concentration of miRNA enriched in a particular brain area should be indicative of pathology in that brain part. For example, changes in levels of midbrain- or cortex-enriched miRNA would be associated with RTT, reflecting synapse and neuronal dysfunctions in these brain areas. In addition, concentrations of brain-enriched miRNA in blood cells are low, which decreases contamination of plasma and serum by miRNA leakage during purification of these bodily fluids.
  • the concentrations of miRNAs detected in bodily fluids depend on many biological and technical factors.
  • Biological factors include miRNA levels in various tissues, intensity of secretion and excretion into extracellular space, forms of circulating miRNAs (exosomes and other vesicles, complexes with proteins and lipids) affecting their ability to cross various barriers, e.g. blood-brain, placental, and kidney barriers, and miRNA stability and half-life in the bloodstream.
  • Technical factors include variability in methods of bodily fluid collection and storage, methods used for miRNA extraction, and presence in bodily fluids of various factors affecting miRNA purification and quantitation.
  • miRNA normalization is broadly recognized (Meyer et al., Biotechnol. Lett. 2010; 32: 1777-1788). At the same time, no single normalization method is commonly accepted.
  • the present invention is based on the use of brain-enriched biomarker miRNA pairs instead of (or in addition to) normalization per ubiquitous RNA or an average of numerous miRNAs.
  • the use of brain-enriched biomarker miRNA pairs (one as a numerator and another one as a denominator in a ratio) has several advantages. First, any pathology is usually associated with up-regulation of some miRNAs and down-regulation of other miRNAs, thus considering miRNA pairs of up- and down-regulated miRNAs may increase test sensitivity and specificity. Second, the use of a pair of brain-enriched miRNAs, rather than one brain-enriched miRNA, decreases potential overlap with pathologies of other organs.
  • Another innovative aspect of the present invention is the use of probabilistic approach in addition to or instead of calculating ratios of miRNA concentrations in plasma.
  • the use of integral distribution curves, which characterize probabilities of a subject belonging to control or having a pathology has such advantages as better definition of diagnostic uncertainty zone, simplicity of combining biomarkers of different nature (e.g., protein levels, imaging techniques and miRNA levels) and others.
  • Non-limiting examples of such groups for RTT detection include:
  • brain-enriched means that miRNA concentration in brain is at least 4-5 times higher than in other organs.
  • the term "enriched" means that miRNA concentration in said area of the brain is higher (preferably, at least 2-fold higher, more preferably at least 5-fold higher, most preferably at least 10-fold higher) than in brain in general.
  • the term refers to the difference in concentrations within the brain areas (e.g., as measured using qRT-PCR).
  • synapse and/or neurite miRNA refers to miRNA which (i) is "brain-enriched” and (ii) is present in a synapse and/or neurite (i.e., axon and/or dendrite and/or spine).
  • synapse and/or neurite miRNAs should be detectable in bodily fluids as a result of their release from neurons (e.g., due to secretion, neurite/synapse destruction or neuronal death).
  • neuron refers to any projection from the cell body of a neuron. This projection can be an axon, a dendrite, or a spine.
  • Axon refers to a long, slender projection of a neuron that conducts electrical impulses away from the neuron's cell body or soma.
  • Axons are distinguished from dendrites by several features, including shape (dendrites often taper while axons usually maintain a constant radius), length (dendrites are restricted to a small region around the cell body while axons can be much longer), and function (dendrites usually receive signals while axons usually transmit them).
  • Axons and dendrites make contact with other cells (usually other neurons but sometimes muscle or gland cells) at junctions called synapses.
  • dendrite refers to a branched projection of a neuron that acts to conduct the electrochemical stimulation received from other neural cells to the cell body of the neuron from which the dendrites project.
  • synapses refers to specialized junctions, through which neurons signal to each other and to non-neuronal cells such as those in muscles or glands.
  • a typical neuron gives rise to several thousand synapses.
  • Most synapses connect axons to dendrites, but there are also other types of connections, including axon-to-cell-body, axon-to-axon, and dendrite-to-dendrite.
  • each neuron forms synapses with many others, and, likewise, each receives synaptic inputs from many others.
  • the output of a neuron may depend on the input of many others, each of which may have a different degree of influence, depending on the strength of its synapse with that neuron.
  • synapses There are two major types of synapses, chemical synapses and electrical synapses.
  • electrical synapses cells approach within about 3.5 nm of each other, rather than the 20 to 40 nm distance that separates cells at chemical synapses.
  • chemical synapses the postsynaptic potential is caused by the opening of ion channels by chemical transmitters, while in electrical synapses it is caused by direct electrical coupling between both neurons. Electrical synapses are therefore faster than chemical synapses.
  • normalizer miRNA refers to miRNA which is used for normalization of biomarker miRNA concentration to account for factors that affect appearance and/or stability of miRNA in bodily fluids but are not related to a target pathology.
  • neurodevelopmental disorders refer to pathologies caused by disturbances of the nervous system development such as, e.g., Rett Syndrome, Landau-Kleffner Syndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Angelman Syndrome, Ataxias and Cerebellar or Spinocerebellar Degeneration, Ataxia Telangiectasia, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorders including Asperger Syndrome, Batten Disease, Canavan Disease, and Tourette Syndrome.
  • development of a neurodevelopmental disorder is used herein to refer to any negative change in the extent/severity of a metabolic and/or structural change in individual neurons and/or any increase in the number of neurons affected.
  • improvement of a neurodevelopmental disorder and similar terms refer to any positive change in the extent/severity of a metabolic and/or structural change in individual neurons and/or any decrease in the number of neurons affected.
  • microRNA refers to a class of small approximately 22 nt long non-coding RNA molecules. They play important roles in the regulation of target genes by binding to complementary regions of messenger transcripts (mRNA) to repress their translation or regulate degradation (Griffiths- Jones Nucleic Acids Research, 2006, 34, Database issue: D140-D144). Frequently, one miRNA can target multiple mRNAs and one mRNA can be regulated by multiple miRNAs targeting different regions of the 3' UTR.
  • mRNA messenger transcripts
  • miRNA can modulate gene expression and protein production by affecting, e.g., mRNA translation and stability (Baek et al., Nature 455(7209):64 (2008); Selbach et al., Nature 455(7209):58 (2008); Ambros, 2004, Nature, 431, 350-355; B artel, 2004, Cell, 116, 281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat. Rev. Genet., 5, 522-531; and Ying et al., 2004, Gene, 342, 25-28). Unless otherwise noted, the name of a specific miRNA refers to a mature miRNA sequence.
  • miRNAs are preceded with the prefix "hsa-" (i.e., an abbreviation for Homo sapiens). Throughout the specification and figures the hsa- prefix may be dropped for purposes of abbreviation, thus, for example, "hsa-miR-155" and "miR-155" would represent the same RNA sequence.
  • miRNAs useful in the methods of the present invention include, without limitation, miR-107, miR-323-3p, miR-491-5p, miR-335-5p, miR-132, and miR-411. Information on most currently known miRNAs can be found in the miRNA database miRBase (available at the world wide web at mirbase.org). See also Burside et al., BMC Genomics 9: 185 (2008); Williams et al., BMC Genomics 8: 172 (2007); Landgraf et al., Cell 129: 1401 (2007).
  • miRNA array refers to a multiplex technology used in molecular biology and in medicine. It consists of an arrayed series of multiple (e.g., thousands) microscopic spots of oligonucleotides, each containing a specific sequence (probe) complementary to a particular target miRNA. After probe-target hybridization under high-stringency conditions the resulting hybrids are usually detected and quantified by quantifying fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of miRNA. In the methods of the present invention, both custom-made and commercially available miRNA arrays can be used. Examples of useful commercially available miRNA arrays (based on various methods of target labeling, hybrid detection and analysis) include arrays produced by Agilent, Illumina, Invitrogen, Febit, and LC Sciences.
  • next generation sequencing technologies broadly refers to sequencing methods which generate multiple sequencing reactions in parallel. This allows vastly increased throughput and yield of data.
  • next generation sequencing platforms include Helicos small RNA sequencing, miRNA BeadArray (Illumina), Roche 454 (FLX-Titanium), and ABI SOLiD.
  • an “individual” or “subject” or “animal”, as used herein, refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of neurodevelopmental diseases.
  • the subject is a human.
  • RNA purification refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained.
  • RNA purification includes elimination of proteins, lipids, salts and other unrelated compounds present in bodily fluids.
  • a purified miRNA is preferably substantially free of other RNA oligonucleotides contained in bodily fluid samples (e.g., rRNA and mRNA fragments, ubiquitous miRNAs, which are expressed at high levels in almost all tissues [e.g., miR-16], etc.).
  • the term “substantially free” is used operationally, in the context of analytical testing of the material.
  • purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and still more preferably at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, composition analysis, biological assay, and other methods known in the art.
  • similarly processed refers to samples (e.g., bodily fluid samples or purified miRNAs) which have been obtained using the same protocol.
  • the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition, or to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.
  • the term “treat” also encompasses preventing and/or reducing a positive symptom associated with neurodevelopmental disorders, such as, e.g., seizures, muscle stiffness, GI, heart, and breathing problems.
  • the term "about” or “approximately” means within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range.
  • the allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
  • the present inventors used the following approach: selection of a numerator and a denominator for each pair from those circulating miRNAs, which significantly correlate (Spearman's rank correlation coefficient r>0.8) in a respective bodily fluid of different individuals.
  • Concentrations of miRNAs in plasma depend on numerous factors, including (i) levels of miRNA expression in various organs and tissues; (ii) levels of miRNA secretion from different cell types; (iii) stability of miRNAs in extracellular space and their appearance in plasma in different forms, such as exosomes and other microvesicles, complexes with proteins, lipids and, possibly, other molecules; and (iv) blood-brain barrier permeability for brain-enriched miRNAs. A pathological process may affect some or all of these factors.
  • a nominator and a denominator of an effective biomarker miRNA pair should share some of these basic common factors (e.g., both are brain-enriched and secreted in exosomes) and would change differently in response to a pathology. This does not mean that any correlated miRNA will form a good biomarker pair, since if they are similarly changed by pathology their ratio will mask those changes.
  • the present invention provides a method of "promising" miRNA pair selection, which method comprises the following steps: 1. Concentrations of miRNAs pre-selected on the basis of their enrichment in an organ of interest (e.g., brain) are measured in a bodily fluid (e.g., plasma, serum, saliva, urine) of at least two comparative cohorts (e.g., a disease and control for a diagnostic test, two diseases for a test capable of differentiating two pathologies, a disease at different stages of pathologic process development, or a disease before and after treatment for monitoring tests).
  • a bodily fluid e.g., plasma, serum, saliva, urine
  • a comparative cohorts e.g., a disease and control for a diagnostic test, two diseases for a test capable of differentiating two pathologies, a disease at different stages of pathologic process development, or a disease before and after treatment for monitoring tests.
  • the difference between the means for each miRNA from two comparative cohorts is calculated and miRNAs are divided in two groups: (i) with high difference values; and (ii) with low or with opposite sign difference values.
  • miRNAs from different groups are combined as potential biomarker pairs if parameters determined in step 3 differ at least 1.5 times.
  • One miRNA is used as a numerator and another miRNA is used as a denominator in a potential "promising" miRNA pair.
  • miRNA with high positive correlation are selected as a numerator and a denominator for the biomarker pair. This step significantly decreases the number of potential biomarker miRNA pairs, reduces variance of selected biomarkers caused by factors unrelated to processes differentiating two comparative cohorts and significantly increases test sensitivity and specificity.
  • steps 3-4 and step 5 can be switched as follows:
  • step 1 calculate Spearman's rank correlation coefficient (r) for all possible pairs of individual miRNA measured in step 1 in all bodily fluid samples. Then select as potential biomarker pairs of miRNA with a high positive correlation (r>0.8), compare a ratio of miRNA concentrations in two subject cohorts for each selected miRNA pair and determine a miRNA pair as a suitable biomarker if this pair differentiates two subject cohorts with a statistically significant P-value.
  • the invention provides a method for selecting a biomarker miRNA pair for diagnosis and/or monitoring of a pathology, said method comprising the following steps:
  • step (b) measuring the level of each miRNA selected in step (a) in bodily fluid samples from two subject cohorts;
  • step (c) calculating the mean level of each miRNA measured in step (b);
  • step (d) calculating the difference between the mean miRNA levels calculated in step (c);
  • step (e) comparing the differences between the mean miRNA levels calculated in step (d) between all studied miRNAs and selecting as potential biomarker pairs those miRNA pairs for which the difference calculated in step (d) for one miRNA is at least 1.5 times the difference calculated for the other miRNA;
  • step (g) identifying the miRNA pair as a suitable biomarker pair for diagnosis and/or monitoring of said pathology if its (r) value calculated in step (f) is at least 0.8.
  • the invention provides a method for selecting a biomarker miRNA pair for diagnosis and/or monitoring of a pathology, said method comprising the following steps:
  • step (b) measuring the level of each miRNA selected in step (a) in bodily fluid samples from two subject cohorts;
  • step (c) calculating Spearman's rank correlation coefficient (r) for all possible pairs of individual miRNAs measured in step (b);
  • step (d) selecting as potential biomarker pairs those miRNA pairs which have the (r) value calculated in step (c) of at least 0.8;
  • step (e) calculating the mean level of each miRNA selected in step (d);
  • step (f) calculating the difference between the mean miRNA levels calculated in step (e); (g) identifying a miRNA pair as a suitable biomarker pair for diagnosis and/or monitoring of said pathology if the difference calculated in step (f) for one miRNA is at least 1.5 times the difference calculated for the other miRNA.
  • the invention provides a method for selecting a biomarker miRNA pair for diagnosis and/or monitoring of a pathology, said method comprising the following steps:
  • step (b) measuring the level of each miRNA selected in step (a) in bodily fluid samples from two subject cohorts;
  • step (c) calculating Spearman's rank correlation coefficient (r) for all possible pairs of individual miRNAs measured in step (b);
  • step (d) selecting as potential biomarker pairs those miRNA pairs which have the (r) value calculated in step (c) of at least 0.8;
  • step (e) calculating P-value of two subject cohorts separation for each miRNA pair selected in step (d), and
  • the invention provides a computer-implemented method for selecting a biomarker miRNA pair for diagnosis and/or monitoring of a pathology, said method comprising the following steps:
  • step (b) measuring the level of each miRNA selected in step (a) in bodily fluid samples from two subject cohorts;
  • step (c) electronically calculating the mean level of each miRNA measured in step (b);
  • step (d) electronically calculating a difference between the mean miRNA levels calculated in step (c);
  • step (e) selecting from the group of measured miRNAs a set of potential miRNA pairs each comprising a first miRNA and a second miRNA, wherein the calculated difference in the mean level in step (d) of the first miRNA is at least 1.5 times the calculated difference in the mean level of the second miRNA;
  • step (g) selecting from the set of potential miRNA pairs those miRNA pairs, which are suitable for the diagnosis and/or monitoring of the pathology, wherein the (r) value calculated in step (f) is at least 0.8, and
  • step (h) displaying all or part of the miRNA pairs selected in step (g).
  • the invention provides a computer-implemented method for selecting a biomarker miRNA pair for diagnosis and/or monitoring of a pathology, said method comprising the following steps:
  • step (b) measuring the level of each miRNA selected in step (a) in bodily fluid samples from two subject cohorts;
  • step (c) electronically calculating the Spearman's rank correlation coefficient (r) for all possible pairs of individual miRNAs measured in step (b);
  • step (d) selecting from the group of measured miRNAs a set of potential biomarker miRNA pairs, wherein the (r) value calculated in step (c) is at least 0.8;
  • step (e) electronically calculating the mean level of each miRNA selected in step (d);
  • step (f) electronically calculating the difference between the mean miRNA levels calculated in step (e);
  • step (g) selecting from the group of measured miRNAs a set of suitable miRNA biomarker pairs each comprising a first miRNA and a second miRNA, wherein for each suitable biomarker miRNA pair, the calculated difference in the mean level in step (f) of the first miRNA is at least 1.5 times the calculated difference in the mean level of the second miRNA, and
  • the invention provides a computer-implemented method for selecting a biomarker miRNA pair for diagnosis and/or monitoring of a pathology, said method comprising the following steps: (a) selecting a group of miRNAs known to be enriched in an organ affected by the pathology;
  • step (b) measuring the level of each miRNA selected in step (a) in bodily fluid samples from two subject cohorts;
  • step (c) electronically calculating the Spearman's rank correlation coefficient (r) of the levels measured in step (b) for all possible pairs of individual miRNAs;
  • step (d) selecting from the group of measured miRNAs a set of potential biomarker miRNA pairs, wherein the (r) value calculated in step (c) is at least 0.8;
  • step (e) electronically calculating P-value of two subject cohorts separation for each miRNA pair selected in step (d);
  • step (g) displaying all or part of the suitable biomarker miRNA pairs selected in step (f).
  • Non-limiting examples of the methods which can be used to measure miRNA level in any of the above methods of the invention include, e.g., RT-PCR-based methods, miRNA array- based methods, new generation sequencing, and hybridization.
  • Non-limiting examples of the bodily fluid samples which can be used in any of the above methods of the invention include, e.g., plasma, serum, urine, and saliva.
  • the subjects can be, e.g., humans or experimental animals.
  • any two cohorts can be compared.
  • Non- limiting examples of such cohorts include, e.g., pathology versus control [e.g., age, gender, and/or race/ethni city-matched healthy subjects], one pathology of the organ versus another pathology of the same organ, two age groups, males versus females [e.g., age and/or race/ethnicity-matched], two different ethnic or racial groups [e.g., age and/or gender-matched], etc.).
  • a statistically significant P-value can be calculated using any method known in the art.
  • Non-limiting examples of such methods are Student's t-test (for samples with normal distribution) and Mann-Whitney test (for samples with non-random distribution) (Mann and Whitney, Annals Math Stat. 1947, 18: 50-60).
  • P-value >0.05 is usually accepted as statistically significant. If numerous potential biomarkers are tested Bonferroni correction can be applied.
  • kits comprising one or more primer and/or probe sets specific for the detection of the biomarker miRNA pairs.
  • kits can further include primer and/or probe sets specific for the detection of additional normalizer miRNAs.
  • kits can be useful for direct miRNA detection in bodily fluid samples isolated from patients or can be used on purified total RNA or miRNA samples.
  • kits of the invention can also provide reagents for primer extension and amplification reactions.
  • the kit may further include one or more of the following components: a reverse transcriptase enzyme, a DNA polymerase enzyme (such as, e.g., a thermostable DNA polymerase), a polymerase chain reaction buffer, a reverse transcription buffer, and deoxynucleoside triphosphates (dNTPs).
  • a kit can include reagents for performing a hybridization assay.
  • the detecting agents can include nucleotide analogs and/or a labeling moiety, e.g., directly detectable moiety such as a fluorophore (fluorochrome) or a radioactive isotope, or indirectly detectable moiety, such as a member of a binding pair, such as biotin, or an enzyme capable of catalyzing a non-soluble colorimetric or luminometric reaction.
  • the kit may further include at least one container containing reagents for detection of electrophoresed nucleic acids.
  • kits include those which directly detect nucleic acids, such as fluorescent intercalating agent or silver staining reagents, or those reagents directed at detecting labeled nucleic acids, such as, but not limited to, ECL reagents.
  • a kit can further include miRNA isolation or purification means as well as positive and negative controls.
  • a kit can also include a notice associated therewith in a form prescribed by a governmental agency regulating the manufacture, use or sale of diagnostic kits. Detailed instructions for use, storage and troubleshooting may also be provided with the kit.
  • a kit can also be optionally provided in a suitable housing that is preferably useful for robotic handling in a high throughput setting.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent.
  • the solvent may also be provided in another container.
  • the container will generally include at least one vial, test tube, flask, bottle, syringe, and/or other container means, into which the solvent is placed, optionally aliquoted.
  • the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other solvent.
  • the kit also will generally contain a second, third, or other additional container into which the additional components may be separately placed.
  • additional components may be separately placed.
  • various combinations of components may be comprised in a container.
  • kits may also include components that preserve or maintain DNA or RNA, such as reagents that protect against nucleic acid degradation.
  • Such components may be nuclease or RNase-free or protect against RNases, for example. Any of the compositions or reagents described herein may be components in a kit.
  • the methods of the instant invention are based on the use of miRNAs enriched in different brain areas as numerators and denominators, which significantly improves test sensitivity and specificity.
  • Table 1 presents lists of brain-enriched miRNAs, miRNAs enriched in synapses, axons, dendrites and spines ("synapse and/or neurite miRNAs") and miRNAs enriched in different brain areas.
  • Medulla oblongata 10a,b, 34a, 451 (all not brain-enriched), 219, 338
  • Tested miRNAs were initially selected by the present inventors based on literature data on their enrichment in brain compartments and presence in neurites (i.e., axons and/or dendrites and/or spines) and/or synapses (Hua et al. BMC Genomics. 2009; 10: 214; Liang et al. BMC Genomics. 2007; 8: 166; Landgraf et al. Cell. 2007; 129: 1401-1414; Lee et al. RNA. 2008; 14: 35-42; Schratt et al. Nature. 2006; 439: 283-289; Lugli et al. J. Neurochem.
  • Mecp2 tml lJae and Mecp2 tml lBird Two mouse models were used, namely Mecp2 tml lJae and Mecp2 tml lBird . Both models are male mice (Null) with nonfunctional Mecp2 due to deletion of exon 3 in Mecp2 tml lJae (Guy, J., Hendrich, B., Holmes, M., Martin, J. E. and Bird, A. (2001).
  • a mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat. Genet. 27, 322-326) or exon 3-4 in Mecp 2tml lBird (Chen, R. Z., Akbarian, S., Very, M. and Jaenisch, R. (2001).
  • mice Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice. Nat. Genet. 27, 327-331), respectively. Since the total amount of plasma that can be obtained from a mouse is relatively low, 8 miRNAs expressed in different brain regions were tested (see Table 2, above).
  • 0.2 ml plasma samples were obtained from 11 Mecp2 tml lJae mice and 9 wild type controls of the same age. Concentrations of miRNAs in plasma were analyzed using RT-qPCR with primers and probes for each individual miRNA (Life Technologies). The amount of RNA equivalent to 25 ⁇ . of plasma were taken in each RT reaction, and the amount of miRNA (cDNA) equivalent to 2 ⁇ . plasma was taken into final PCR. The results obtained for each miRNA were converted into Relative Concentration (RC) of miRNA according to the ABI protocol (2 " ), normalized per potential normalizer miRNA, and this ratio was compared with respective control values. The biomarker miRNA pairs were selected as described above.
  • miR-323-3p Most likely miR-323-3p is present in different brain regions and its plasma concentration depends on involvement of these areas in pathology and respective changes in miR-323-3p expression and secretion. Although accuracy for individual miRNA pairs is sufficiently high (up to 0.89), their combination gives even higher accuracy (0.95).
  • 0.2 ml plasma samples were obtained from 8 Mecp2 tml lBird mouse and 10 wild type controls of the same age. Same 8 miRNAs were tested and experiments were performed as described above in Example 3. Data obtained are presented in Figures 3-5 and Table 4. Again miR-107 and miR-491-5p are very good numerators, while miR-132 and miR-335-5p, enriched in hippocampus are among the best denominators. miR-323-3p is still a good denominator for miR-491-5p but also behaves as a numerator being combined with miR-132 and miR-335-5p, which again agrees with its expression in various brain areas.
  • Table 5 Data summary for effective miRNA pairs.
  • the table lists miRNA pairs identified in the study oiMecp2 tmL1Bird , Null and Wt (shown in Italic if also identified in female models).
  • miRNA pairs identified in all four studies Mecp2 tmL1Bird , Mecp2 tmL1Ja Null and Wt, Het and Wt are shown in bold Italic.

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L'invention concerne des procédés pour le diagnostic et la surveillance du syndrome de Rett et d'autres troubles neurodéveloppementaux par analyse quantitative de micro-ARN dans des liquides organiques.
PCT/US2017/012258 2016-01-05 2017-01-05 Procédés d'utilisation de micro-arn provenant de liquides organiques pour le diagnostic et la surveillance de troubles neurodéveloppementaux Ceased WO2017120285A1 (fr)

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US10781487B2 (en) 2017-07-24 2020-09-22 Diamir, Llc miRNA-based methods for detecting and monitoring aging
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US11098362B2 (en) 2013-11-18 2021-08-24 Diamir, Llc Methods of using miRNAs from bodily fluids for detection and monitoring of Parkinson's disease (PD)
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10246747B2 (en) 2011-04-18 2019-04-02 Diamir, Llc Methods of using miRNA from bodily fluids for early detection and monitoring of mild cognitive impairment (MCI) and Alzheimer's disease (AD)
US10472681B2 (en) 2011-04-18 2019-11-12 Diamir, Llc miRNA-based universal screening test (UST)
US11098362B2 (en) 2013-11-18 2021-08-24 Diamir, Llc Methods of using miRNAs from bodily fluids for detection and monitoring of Parkinson's disease (PD)
US10975436B2 (en) 2016-01-05 2021-04-13 Diamir, Llc Methods of using miRNA from bodily fluids for diagnosis and monitoring of neurodevelopmental disorders
US11149313B2 (en) 2016-03-21 2021-10-19 Diamir, Llc Methods of using miRNAs from bodily fluids for detection and differentiation of neurodegenerative diseases
US10781487B2 (en) 2017-07-24 2020-09-22 Diamir, Llc miRNA-based methods for detecting and monitoring aging

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