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WO2015179909A1 - Biomarqueurs micro-arn de la maladie d'alzheimer - Google Patents

Biomarqueurs micro-arn de la maladie d'alzheimer Download PDF

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WO2015179909A1
WO2015179909A1 PCT/AU2015/050264 AU2015050264W WO2015179909A1 WO 2015179909 A1 WO2015179909 A1 WO 2015179909A1 AU 2015050264 W AU2015050264 W AU 2015050264W WO 2015179909 A1 WO2015179909 A1 WO 2015179909A1
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mirna
assay
expression
seq
nos
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Andrew Francis HILL
James Doecke
Lesley Cheng SIM
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Commonwealth Scientific and Industrial Research Organization CSIRO
University of Melbourne
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Commonwealth Scientific and Industrial Research Organization CSIRO
University of Melbourne
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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
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present disclosure relates to nucleic acid biomarkers, the level of expression of which, is instructive as to Alzheimer's disease (AD) including identifying onset and various stages of AD, its various clinical manifestations and amyloidosis-related conditions.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • AD is a progressive degenerative disorder that causes loss of memory, cognitive disturbances and behavioral changes. It is a debilitating condition leading to significant rates of morbidity and mortality. It is the fourth largest cause of death in the United States and affects five percent of people over age 65 and 20 percent of people over age 80. To date, there has been no established treatment which will prevent the onset or delay the progression of AD although there is substantial research ongoing worldwide towards identifying potential treatments, including a number of drug trials.
  • This form of neurodegeneration is characterized by the formation of intracellular neurofibrillary tangles, neuronal and synaptic loss and the accumulation of beta amyloid ( ⁇ ) into amyloid plaque material (Breteler et al. (1992) Epidemiol Rev 14: 59-82).
  • ⁇ is proteolytically cleaved from the amyloid precursor protein (APP) [Cole and Vassar (2008) J Biol Chem 253:29621-29625].
  • APP amyloid precursor protein
  • the most characteristic neuropathological feature of AD is the deposition of ⁇ into plaques in the brain parenchyma and cerebral blood vessels leading to neuronal loss and cerebral atrophy (Terry et al. (1981) Ann. Neurol 70: 184-192).
  • MicroRNAs are non-coding RNA species of approximately 22 nucleotides in length which are transcribed in all tissues and cells (Krol et al. (2010) Nat Rev Genet 77:597-610) and which bind to complementary sites at the 3' untranslated region (3'UTR) of their corresponding mRNA targets resulting in down-regulation of gene expression (Hannon (2004) Nat Rev Genet 5:522-531). miRNAs can be released into the extracellular environment by binding to RNA-binding proteins or through secretion in cell- derived plasma microvesicles such as exosomes (Valadi et al. (2007) Nat Cell Biol 9:654- 659; Mitchell et al.
  • a non-invasive and high throughput biological fluid-based test is required for improved population-based screening and patient care in order to refer patients for further examination. Furthermore, with numerous drug trials aiming to treat AD, a fluid-based test is needed to enrich cohorts of AD cases followed by monitoring the potential benefits and side-effects of therapeutic drugs.
  • SEQ ID NO Nucleotide sequences are referred to by a sequence identifier number (SEQ ID NO).
  • the SEQ ID NOs correspond numerically to the sequence identifiers ⁇ 400>1 (SEQ ID NO: l), ⁇ 400>2 (SEQ ID NO: 2), etc.
  • a summary of the sequence identifiers is provided in Table 1.
  • a sequence listing is provided after the claims.
  • microRNAs defined by miR nomenclature are listed in Table 1 with reference to a sequence identifier number.
  • the method disclosed herein is also applicable to other amyloidosis-related conditions.
  • the stratification is based on an association between deregulated miRNA expression and AD.
  • Deregulated miRNAs can be detected in a range of biological samples such as samples comprising or enriched for membranous microvesicles, blood, serum, plasma, urine, lymph, cerebrospinal fluid, ascites, saliva, mucus, stool, biopsy specimens, breast milk, gastric juice, pleural fluid, semen, sweat, tears, hair, vaginal secretion and interstitial fluid.
  • An example of a membranous microvesicle is an exosome. Such structures may first require chemical or mechanical disruption to release the miRNAs or the miRNAs may be free circulating miRNA. The association between miRNA expression and AD is validated through unbiased next generation sequencing (NGS) followed by qRT-PCR.
  • NGS next generation sequencing
  • An miRNA signature is selected from an NGS training set in the form of a panel which is used to predict future or potential cognitive decline, early onset AD, the various stages of AD as well as other amyloidosis-related conditions.
  • the miRNA expression profile is the level of expression of one or more miRNAs or 2 or more miRNA comprising a nucleotide sequence selected from the listing consisting of SEQ ID NOs: 1 through 16 or a precursor form of each of SEQ ID NOs: l through 16 compared to a first knowledge base generated from training data.
  • the training data represent the correlation of expression levels of the miRNAs with subjects of known status with respect to AD.
  • the first knowledge base for example, may be miRNA expression levels in healthy individuals (healthy or normal controls). Alternatively, the first knowledge base may comprise levels of expression of the miRNAs in unhealthy controls. Hence, elevation or reduction in expression is dependent on the type of control.
  • the first knowledge base enables determination of ACt levels of a particular miRNAs obtained using qPCR and then ACt cut-off values established which correlate with the AD condition.
  • Expression fold changes and levels of expression can also be measured of individual miRNAs and/or ratios determined of ACt levels, fold levels of expression between any 2 or more of SEQ ID NOs: l through 17
  • the assay may or may not require control sample to be run side-by-side.
  • a diagnostic rule based on the application of a comparison of levels of miRNA expression in a control sample or based on predetermined ACt cut-off values.
  • the diagnostic rule is based on application of statistical and machine learning algorithms.
  • Such an algorithm uses the relationships between miRNA expression and disease status observed in training data (with known disease status) to infer relationships which are then used to predict the status of patients with unknown status (test data).
  • Practitioners skilled in the art of data analysis recognize that many different forms of inferring relationships in the training data may be used without materially changing the scope of the present invention.
  • an assay to stratify a human subject with respect to Alzheimer's disease (AD) or a symptom associated with AD or other amyloidosis-related condition comprising screening a biological sample from the subject for an miRNA comprising a nucleotide sequence selected from the list consisting of SEQ ID NOs: l through 17 or a precursor form of each of SEQ ID NOs: l through 17 or their corresponding cDNA forms or their corresponding cDNA forms wherein the level of expression of a selected miRNA compared to a control is indicative of a stage in the development of AD.
  • control includes a ACt cut-off value based on known ACt levels of particular miRNAs obtained using qPCR. Ratios of expression or fold changes or ACt levels between 2 or more of SEQ ID NOs: 1 through 17 may also be determined.
  • SEQ ID NO: 17 is excluded. Accordingly, the level of expression in the first knowledge base is based on levels in healthy individuals.
  • the expression of one or more miRNAs comprising nucleotide sequences selected from SEQ ID NOs: l through 13 is elevated relative to the healthy control and the expression of one or more miRNAs comprising nucleotide sequences selected from SEQ ID NOs: 14 through 16 is reduced relative to a healthy control in a subject stratified as having a stage of AD or is at risk of developing AD.
  • Reference to any of SEQ ID NOs: l through 16 as well as SEQ ID NO: 17 includes precursor forms thereof.
  • the biological sample comprises membranous microvesicles or a sample enriched for membranous microvesicles, blood, serum, plasma, urine, lymph, cerebrospinal fluid, ascites, saliva, mucus, stool, biopsy specimens, breast milk, gastric juice, pleural fluid, semen, sweat, tears, hair, vaginal secretion or interstitial fluid. Free circulating miRNAs may also be measured.
  • An example of a membranous microvesicle is an exosome.
  • Microvesicles and exosomes may first require chemical or mechanical disruption to release the miRNAs.
  • the miRNAs may also be captured on a solid support such as a bead or the side of a reaction vessel.
  • the sample may comprise or be enriched for membrane microvesicles or miRNAs released from exosomes or membrane vesicles.
  • a non-microvesicle sample of free miRNA is also contemplated herein.
  • the miRNA profile enables diagnosis of AD in subjects who present as healthy individuals yet have amyloid burden and/or other risk factors.
  • the present invention extends to the detection of precursor forms of the miRNAs which comprise the sequences of any one of SEQ ID NOs: 1 through 17.
  • Table 2
  • Figure 1 is a diagrammatic representation showing hierarchical clustering of differentially expressed exosomal miRNA biomarkers obtained from healthy controls (HC) and patients with Alzheimer's disease (AD).
  • HC healthy controls
  • AD Alzheimer's disease
  • Hierarchical clustering is performed using Partek Genomics Suite on significantly differentially expressed miRNA using Euclidean average H05, p (AD, MCI and HC) ⁇ 0 05 and ⁇ 1 -2 fold change); There are two major nodes of the dendrogram.
  • Node 1 contains 15 miRNA which are found to be up-regulated (hsa-miR-361-5p, hsa- miR-30e-5p, hsa-miR-93-5p, hsa-miR-15a-5p, hsa-miR-143-3p, hsa-miR-335-5p, hsa- miR-106b-5p, hsa-miR-101-3p, hsa-miR-424-5p, hsa-miR-106a-5p, hsa-miR-18b-5p, hsa- miR-3065-5p, hsa-miR-20a-5p, hsa-miR-3065-5p and hsa-miR-582-5p).
  • Node 2 contains 3 miRNA which are found to be down-regulated (hsa-miR-1306-5p, hsa-miR-342-3p, and 15b-3p, Table 3).
  • Patient samples are arranged by attributes. HC, healthy control; MCI, mild cognitive impairment; and AD, Alzheimer's disease.
  • the miRNAs are defined in Table 1 with reference to the sequence listing.
  • Figures 2A through D are graphical representations of clustering and random forest testing of correlated miRNA identified in healthy controls and patients with Alzheimer's disease within the discovery set.
  • Figure 3 is a graphical representation of box plots showing validated miRNA differentially expressed in healthy controls (HC) and patients with Alzheimer's disease (AD). Mean centred and scaled data are plotted between HC and AD patients. HC patients are represented by blue circular dots while AD patients are represented by red triangular dots.
  • the present disclosure teaches the identification of microRNAs (miRNAs) or precursor forms thereof, the expression of which, is statistically associated with AD, a stage of AD including asymptomatic or presymptomatic AD and/or a risk of developing AD.
  • AD manifests itself as a spectrum of symptoms from asymptomatic features to mild cognitive impairment to severe cognitive decline and other symptoms or outcomes of major neurodegeneration.
  • the ability to identify asymptomatic or presymptomatic or early onset AD subjects enables early clinical and behavioral intervention leading to significant improvement in quality of life for the individual and surrounding social and family networks.
  • the method described herein can also be used to detect other amyloidosis- related conditions.
  • a diagnostic rule based on the application of a comparison of levels of miRNA expression in a control sample or based on a ACt cut-off value selected from a set of predetermined ACt levels obtained using qPCR and which are associated with a healthy individual with or at risk of developing AD or other amyloidosis-related condition.
  • the diagnostic rule is based on application of statistical and machine learning algorithms. Such an algorithm uses the relationships between miRNA expression and disease status observed in training data (with known disease status) to infer relationships which are then used to predict the status of patients with unknown status (test data). Practitioners skilled in the art of data analysis recognize that many different forms of inferring relationships in the training data may be used without materially changing the scope or essence of the present invention.
  • the instant disclosure teaches an assay to stratify a human subject with respect to AD or a symptom associated with AD or other amyloidosis-related condition, the method comprising screening a biological sample from the subject for one or more miRNA comprising a nucleotide sequence selected from the list consisting of SEQ ID NO: l through 16 or precursor forms thereof or their corresponding cDNA forms wherein the level of expression of the one or more miRNAs is indicative of a stage of AD or of the amyloidosis-related condition.
  • reference to AD also includes other amyloidosis-related conditions. Ratios of expression levels or expression folds or ACt levels between 2 or more of SEQ ID NOs: 1 through 17 may also be determined.
  • the sample may comprise a sample comprising or enriched for membranous microvesicles (e.g. exosomes), blood, serum, plasma, urine, lymph, cerebrospinal fluid, ascites, saliva, mucus, stool, biopsy specimens, breast milk, gastric juice, pleural fluid, semen, sweat, tears, hair, vaginal secretion or interstitial fluid.
  • membranous microvesicles e.g. exosomes
  • an assay to stratify a human subject with respect to AD or a symptom associated with AD comprising obtaining a sample comprising or enriched for membranous microvesicles and subjecting the microvesicles to mechanical or chemical disruption to release miRNA and screening for one or more miRNA comprising a nucleotide sequence selected from the list consisting of SEQ ID NO: l through 16 or precursor forms thereof wherein the level of expression of the one or more miRNAs is indicative of a stage of AD.
  • an assay to stratify a human subject with respect to AD or a symptom associated with AD comprising subjecting a sample comprising or enriched for membranous microvesicles to mechanical or chemical disruption to release miRNA and treating one or more miRNA comprising a nucleotide sequence selected from the list consisting of SEQ ID NO: l through 16 or a precursor form thereof or a corresponding cDNA form to chemical processing and then determining the level of expression of the miRNAs wherein the level of expression of the one or more miRNAs is indicative of a stage of AD.
  • chemical processing in this context includes reverse transcription to generate cDNA, bisulfite treatment or chemical coupling to an immobilized support or to a solid phase. Free miRNA in a biological sample may also be assayed.
  • Reference to "membranous microvesicles” includes exosomes.
  • the sample may or may not be a purified sample of microvesicles.
  • a non-microvesicle sample may also be employed.
  • a "stage of AD” means from presymptomatic to early onset to from mild to severe forms of AD. It also encompasses individuals with a likelihood of developing AD.
  • Reference to "presymptomatic AD” includes a subject having no to mild cognitive impairment but having a risk factor for development of AD such as one or more ⁇ 4 alleles and/or a high amyloid burden. This may also be determined by PET imaging.
  • the miRNA expression profile is the level of expression of one or more miRNAs comprising a nucleotide sequence selected from the listing consisting of SEQ ID NOs: 1 through 16 or precursor forms thereof or corresponding cDNA compared to a first knowledge base generated from training data. Ratios of levels of expression of 2 or more of SEQ ID NOs: l through 17 can also form the training data.
  • the training data represent the correlation of expression levels of the miRNAs with subjects of known status with respect to AD.
  • the first knowledge base for example, may be miRNA expression levels in healthy individuals (healthy or normal controls). Alternatively, the first knowledge base may comprise levels of expression of the miRNAs in unhealthy controls. Hence, elevation or reduction in expression is dependent on the type of control. Alternatively, ACt levels in healthy or AD patients are predetermined using qPCR and a ACt cut-off value selected which is associated with AD or with a healthy person.
  • the level of expression in the first knowledge base is based on levels in healthy individuals.
  • the expression of one or more miRNAs comprising nucleotide sequences selected from SEQ ID NOs: l through 13 is elevated relative to the healthy control and the expression of one or more miRNAs comprising nucleotide sequences selected from SEQ ID NOs: 14 through 16 is reduced relative to a healthy control in a subject stratified as having a stage of AD or is at risk of developing AD.
  • the level of deregulation relative to a healthy control of individual miRNA expression correlates to a sensitivity of from about 40% to about 100% and a specificity of from about 30% to 100%.
  • determination of miRNAs defined by SEQ ID NOs: l through 16 results in a sensitivity of about 87% or a specificity of about 77%.
  • another miRNA is hsa-miR-3065-5p (SEQ ID NO: 17) which is not always detected by qRT-PCR. This miRNA is still regarded as part of the present invention.
  • the term "deregulation” includes up-regulation and down-regulation.
  • the miRNA panel can be divided into two clusters based on up or down- regulation.
  • the up-regulation cluster includes SEQ ID NOs: l through 13 and the down- regulation cluster includes SEQ ID NOs: 14 through 16.
  • SEQ ID NOs: l through 8 and 9 through 13 are in clusters 1 and 3, respectively, which exhibit a mean increase in expression relative to a healthy control
  • SEQ ID NO: 14 and 15 and SEQ ID NO: 16 are in clusters 2 and 4, respectively, which exhibit showing a mean decrease in expression relative to a healthy control.
  • SEQ ID NO: 16 (hsa-miRNA 1306-5p) is assayed.
  • the level of miRNA expression correlates with an unhealthy subject having AD or a certain likelihood of developing AD.
  • SEQ ID NOs: l through 16 may be assayed. This includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all 16 miRNAs.
  • SEQ ID NO: 17 may also be assayed.
  • Precursor forms of RNAs comprising a nucleotide sequence selected from SEQ ID NOs: l through 17 may also be screened.
  • Corresponding cDNA forms may also be assayed.
  • an assay to stratify a human subject with respect to AD or a symptom associated with AD comprising screening a biological sample from the subject for miRNA comprising a nucleotide sequence selected from the list consisting of SEQ ID NOs: l through 16 or precursor forms thereof or their corresponding cDNA forms wherein up-regulated levels of SEQ ID NOs: l through 13 or precursor forms thereof and/or down-regulated levels of SEQ ID NOs: 14 through 16 or precursor forms thereof or their corresponding cDNA forms relative to a healthy control are indicative of the subject having AD or is at risk of developing symptoms of AD.
  • the biological sample may comprise or be enriched for a membranous microvesicles such as exosomes or may be a non-membranous fluid sample. The latter may comprise free circulating miRNAs.
  • the level of deregulation is measured relative to a control amount.
  • the control may be a statistically validated level or range which defines up- regulation or down-regulation or normal regulation or the control may be determined on a subject of known AD status (e.g. an unhealthy or disease control).
  • predetermined ACt levels are obtained by qPCR associated with a healthy or not healthy subject and a ACt cut-off value selected to diagnose AD. This avoids the need for a healthy control to be run each time in the assay.
  • a first knowledge base of levels of expression of miRNAs in subjects with known status with respect to AD is determined from training data following a trial of subjects.
  • the level of expression of one or more miRNAs is determined in a subject with unknown status, this creates a second knowledge base.
  • the clinician can determine an index of probability that any one of tested subject has AD or is at risk of developing AD.
  • the control level may be determined using any suitable method, such as the analysis of test results relative to a standard result which reflects individual or collective results obtained from individuals with known AD status. This form of analysis enables the design of kits which require the collection and analysis of a single membranous microvesicles sample, being a test sample of interest, relative to the control.
  • the control level may be determined from the subjects of a specific cohort and hence there may be different controls to test samples derived from different cohorts. Accordingly, there may be determined a number of control values or ranges which correspond to cohorts which differ in respect of characteristics such as age, gender, ethnicity or health status.
  • the "control level" may be a discrete level or a range of levels.
  • the first knowledge base may comprise data from healthy controls or from patients with known levels of AD development.
  • “deregulated” expression needs to be defined relative to whether the control (i.e. first knowledge base) is a healthy control or a disease control (or unhealthy control).
  • a "disease control” is data from individuals with a known stage of AD.
  • the first knowledge base may also comprise ACt levels based on qPCR from which a ACt cut-off value is selected based on correlation to AD. Expression levels or expression fold changes or ACt levels of individual miRNAs or ratios thereof from 2 or more of SEQ ID NOs: 1 through 17 can also be used.
  • the sample may be directly tested or else all or some of the nucleic acid material present in the sample may be isolated or enriched prior to testing.
  • the assay may be conducted on the sample directly or molecules derived therefrom pretreated prior to testing, for example, inactivation of live virus or being run on a gel.
  • the sample may be freshly harvested or it may have been stored (for example by freezing) prior to testing or otherwise treated prior to testing.
  • the sample is a sample comprising or enriched for membranous microvesicles (e.g. exosomes), blood, serum, plasma, urine, lymph, cerebrospinal fluid, ascites, saliva, mucus, stool, biopsy specimens, breast milk, gastric juice, pleural fluid, semen, sweat, tears, hair, vaginal secretion or interstitial fluid.
  • membranous microvesicles e.g. exosomes
  • the subject assay is directed to AD
  • symptoms of AD can arise in other conditions associated with aberrant, unwanted or otherwise inappropriate amyloidosis.
  • the miRNA expression profile may be used to screen for AD as well as Down's syndrome, hereditary cerebral hemorrhage with amyloidosis, amyloidosis associated with chronic inflammation, various forms of malignancy, familial Mediterranean fever, amyloidosis associated with multiple myeloma and other B-cell dyscrasias, amyloidosis associated with type II diabetes, prion disease, long term hemodialysis, carpal tunnel syndrome, amyloidosis associated with senile cardiac amyloid, familial amyloidotic polyneuropathy and amyloidosis associated with endocrine tumors.
  • the condition is AD or is a stage of AD.
  • a stage of AD includes asymptomatic or presymptomatic AD.
  • the subject being assayed may undergo additional testing after being stratified as having or being at risk of developing AD.
  • the additional testing includes a range of behavioral or psychological evaluations such as level of cognitive ability as well as genetic testing for missense mutations in the chromosome 21 gene coding for ⁇ -amyloid precursor protein, a chromosome 14q gene coding for a 467 amino acid protein with 7 putative membrane-spanning domains and inheritance of the ⁇ 4 allele of the gene encoding apolipoprotein E.
  • the presence of one or 2 alleles of ⁇ 4 together with the miRNA signature of the present invention enables the clinician to diagnose for the presence or absence of AD or to assess the likelihood of development of AD or to assess the stage of AD.
  • Physiological testing such as PET imaging may also be conducted.
  • a miRNA is typically approximately 22 nucleotides in length although the present assay extends to miRNAs of from 15 to 30 nucleotides in length or to any fragment or part of an miRNA.
  • a precursor form may also be screened.
  • a precursor may comprise 70-100 nucleotides and comprise a nucleotide sequence selected from SEQ ID NOs: l through 17.
  • Corresponding cDNA forms of SEQ ID NOs: 1 through 17 may also be assayed.
  • miRNAs are endogenously transcribed from DNA, but not translated into protein.
  • the DNA sequence that codes for an miRNA generally includes the miRNA sequence and an approximate reverse complement.
  • the miRNA sequence and its reverse- complement base hybridize to form a double stranded RNA hairpin loop, this forming the primary miRNA structure (pri-miRNA).
  • a nuclear enzyme cleaves the base of the hairpin to form pre-miRNA.
  • the pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm where the Dicer enzyme cuts 20-25 nucleotides from the base of the hairpin to release the mature miRNA.
  • the miRNA can be released into the extracellular environment when the cell membrane is compromised during apoptotic or necrotic death, secreted when bound to lipoproteins or secreted in cell-derived plasma microvesicles such as exosomes.
  • miRNAs The standard nomenclature system is used herein to identify miRNA. For example, a list of miRNAs (miRs) from a homosapien (hsa) having particular sequences is provided in Table 1.
  • RNA or DNA based screening may be employed. Changes to the absolute levels of any of these markers is indicative of changes to the expression of the subject miRNA. Furthermore, ratios of expression changes are also useful in determining a diagnosis. Still further, the miRNA which is identified and measured may be a whole molecule or a fragment thereof. For example, one may identify only fragments of miRNA from a membranous microvesicle sample, depending on how it has been processed. Provided that the fragment comprises sufficient sequence to indicate its origin with a particular miRNA, fragmented miRNAs are useful in the context of the method of the subject assay.
  • nucleic acid molecule should be understood as a reference to both ribonucleic acid molecules and deoxyribonucleic acid molecules and fragments thereof.
  • the subject assay extends, therefore, to both directly screening for miRNA levels in a membranous microvesicle sample or screening for the complementary cDNA which has been reverse-transcribed from an miRNA population of interest. It is well within the skill of the person of skill in the art to design methodology directed to screening for either RNA or DNA.
  • fragment means a portion of the subject nucleic acid molecule. This is relevant with respect to screening for modulated miRNA levels in a fluid sample or membranous microvesicle samples which may have been chemically (e.g. enzymatically) or mechanically treated since the subject miRNA may have been degraded or otherwise fragmented. One may, therefore, actually be detecting fragments of the subject RNA molecule, which fragments are identified by virtue of the use of a suitably specific probe. Alternatively, one or more precursors of SEQ ID NOs: 1 through 17 may be detected.
  • Reference to a "membranous microvesicle” means any particle which is comprised of a cellular plasma membrane component. Such membranous microvesicles may adopt a structure which takes the form of a lumen surrounded by plasma membrane. Examples of membranous microvesicles include, but are not limited to, microparticles, exosomes, apoptotic blebs, apoptotic bodies, cellular blebs, extracellular vesicles and the like. In an embodiment, the membranous microvesicle is an exosome. Free circulating miRNAs may also be measured.
  • Reference to an "exosome” includes vesicles which are secreted by a wide variety of cell types.
  • the exosomes tested in the subject assay are generally enriched from a biological sample.
  • biological sample is meant any biological material derived from an individual.
  • the miRNA may be located in fluid medium or in a medium which comprises exosomes.
  • samples include, but are not limited to, a sample comprising or enriched from membranous microvesicles (e.g.
  • exosomes blood, serum, plasma, urine, lymph, cerebrospinal fluid, ascites, saliva, mucus, stool, biopsy specimens, breast milk, gastric juice, pleural fluid, semen, sweat, tears, hair, vaginal secretion, interstitial fluid and fluid which has been introduced into the body of an individual and subsequently removed such as, for example, the saline solution extracted from the lung following lung lavage or the solution retrieved from an enema wash.
  • the biological sample may be tested directly or undergo some form of pre-treatment prior to testing.
  • the sample may require the addition of a reagent, such as a buffer, to immobilise the miRNA in the sample.
  • sample which is the subject of testing may be freshly isolated or it may have been isolated at an earlier point in time and subsequently stored or otherwise treated prior to testing.
  • the sample may have been collected at an earlier point in time and frozen or otherwise preserved in order to facilitate its transportation to the site of testing.
  • the sample may be treated to neutralize any possible pathogenic infection, thereby reducing the risk of transmission of the infection to the technician.
  • the choice of sample may be dependent on the nature of the subject including age and overall health status.
  • the biological sample comprises or is enriched for membranous microvesicles or free miRNAs.
  • the sample is whole blood, plasma serum, cerebral fluid, spinal fluid, interstitial fluid or a fraction thereof.
  • the term "individual” should be understood to include a human of any age or at an age regarded as being at risk. Such age selection includes subjects from 40 to 49, 50 to 59, 60 to 69, 70 to 79, 80 to 89 and 90 to 100 years old. Whilst the subject assay is directed to humans, the assay may be trialled in an animal model such as a non-human primate or a rodent (e.g. mouse) model.
  • an animal model such as a non-human primate or a rodent (e.g. mouse) model.
  • the miRNA markers are defined by SEQ ID NOs: l through 17 although the preferred panel is SQ ID NOs: l through 16. Such markers include large precursor forms which comprise a sequence selected from SEQ ID NOs: l through 17 or their corresponding cDNA forms.
  • up-regulated levels of one or more of SEQ ID NOs: l through 13 and/or down-regulated levels of one or more of SEQ ID NOs: 14 through 16 relative to a healthy control is regarded as a signature of AD or a risk of developing AD.
  • the miRNA display a fold expression difference of more than 1.2 and a significance of p ⁇ 0.05. As indicated above, if a disease or unhealthy control is employed, the up- or down-regulation may be reversed. Also contemplated herein is a measurement of ratios of ACt values, fold changes and levels of expression between any 2 of SEQ ID NOs: 1 through 17 including any 2 of SEQ ID NOs: 1 through 16.
  • a method of screening for the onset, predisposition to the onset or monitoring the progress of AD in an individual comprising measuring the level of expression of SEQ ID NO: 16 (hsa-miRNA- 1306-5p) in a biological sample from individual wherein a higher level of expression of the miRNA relative to a healthy control level is indicative of the onset or predisposition of the onset of AD.
  • a "biological sample” in this context includes a sample comprising or enriched from membranous microvesicles (e.g. exosomes) as well as whole blood, serum, plasma and the like.
  • Reference to the "onset" of AD should be understood as a reference to the commencement of changes to the brain which are characteristic of AD, such as ⁇ plaque deposition or formation of neurofibrillary tangles. In this regard, these changes may be well advanced in that cognitive impairment has become evident. Alternatively, the physical changes to the brain may be at a very early stage such that symptoms of cognitive impairment are not yet evident. Assessment of an individual's predisposition to the development of AD is contemplated herein. Changed levels of the subject miRNA markers are proposed to be indicative of that individual's predisposition to developing AD.
  • Detection of converse changes in the levels of the miRNA marker may be desired under circumstances other diagnosis, for example, to monitor the effectiveness of therapeutic or prophylactic treatment directed to modulating AD onset or progression. For example, where elevated levels of the markers indicates that an individual has developed AD screening for a decrease in the levels of this markers subsequently to the onset of a therapeutic regime may be utilized to indicate reversal or other form of improvement of the subject individual's condition.
  • Contemplated herein is a method of monitoring treatment of a subject with or having a risk of developing AD or as a monitor of the effectiveness of therapeutic or prophylactic treatment regimes directed to inhibiting or otherwise slowing AD development.
  • mapping the modulation of miRNA marker expression levels in a biological sample is a valuable indicator of the status of an individual or the effectiveness of a therapeutic or prophylactic treatment regime which is currently in use.
  • a method contemplated herein extends to monitoring for changes in miRNA marker expression levels in an individual relative to their levels prior to treatment or during treatment, or relative to one or more earlier marker expression levels determined from a biological sample of the individual.
  • a medical protocol to treat a subject the protocol comprising assessing or stratifying the subject with respect to AD by the assay herein described and then providing therapeutic, psychological and/or behavioral intervention to ameliorate any symptoms of AD.
  • the membranous microvesicles may be derived from any suitable biological sample and may be either isolated from that sample or enriched therein. Methods for performing isolation or enrichment are known and it is within the skill of the person in the art to select and apply a method appropriate to the particular circumstances.
  • the microvesicles may be enriched for by subjecting the biological sample of which they are part to mechanical or chemical disrupture such that nucleic acid material is release.
  • the nucleic acid material would include miRNA.
  • RNA amplification or probing steps require use of primers.
  • Reference to a "primer” or an “oligonucleotide primer” should be understood as a reference to any molecule comprising a sequence of nucleotides, or functional derivatives or analogues thereof, the function of which includes hybridization to a region of a nucleic acid molecule of interest.
  • the primer may comprise non-nucleic acid components.
  • the primer may also comprise a non-nucleic acid tag such as a fluorescent or enzymatic tag or some other non-nucleic acid component which facilitates the use of the molecule as a probe or which otherwise facilitates its detection or immobilization.
  • the primer may also comprise additional nucleic acid components, such as the oligonucleotide tag.
  • the primer may be a protein nucleic acid which comprises a peptide backbone exhibiting nucleic acid side chains. The design and synthesis of primers suitable for use in the subject assay would be well known to those of skill in the art.
  • Various techniques can be used to analyse an amplification product in order to determine relative miRNA expression levels. Their operational characteristics, such as ease of use or sensitivity, vary so that different techniques may be useful for different purposes. They include but are not limited to sequencing, pyrosequencing, enzyme digestion, microarray analysis, denaturing gradient gel electrophoresis, agarose gel based separation, melt curve analysis on real-time PCR cyclers, quantitative real-time PCR, denaturing high performance liquid chromatography, mass spectrometry, primer extension, oligonucleotide-ligation, mutation specific polymerase chain reaction, denaturing gradient, electrophoresis (DGGE), temperature gradient denaturing electrophoresis, constant denaturing electrophoresis, single strand conformational electrophoresis and denaturing high performance liquid chromatography (DUPLC).
  • DGGE electrophoresis
  • DPLC denaturing high performance liquid chromatography
  • an algorithm-based screening assay to screen biological samples from subjects for levels of selected miRNAs including a panel of miRNA.
  • input data in the form of expression levels are collected based on miRNAs defined by SEQ ID NOs: l through 16 or precursor forms thereof or their corresponding cDNA forms and subjected to an algorithm to assess the statistical significance of any elevation or reduction in levels which information is then output data.
  • Computer software and hardware for assessing input data are encompassed by the present invention.
  • the samples include a sample comprising or enriched for membranous microvesicles, blood, serum, plasma, urine, lymph, cerebrospinal fluid, ascites, saliva, mucus, stool, biopsy specimens, breast milk, gastric juice, pleural fluid, semen, sweat, tears, hair, vaginal secretion or interstitial fluid.
  • Another aspect of the present invention contemplates a method of stratifying a subject with respect to AD a female, the method comprising subjecting the subject to a diagnostic assay to determine the levels of one or more of miRNAs defined by SEQ ID NOs: l through 16 or precursor forms thereof or their corresponding cDNA forms to generate an index of probability of the subject having a stage of AD or is at risk of developing AD.
  • the present invention further provides the use the levels of one or more miRNAs selected from SEQ ID NOs: l through 16 or precursor forms thereof or their corresponding cDNA forms in the generation of an index of probability for use in a diagnostic assay to predict the presence of AD or a risk of developing same.
  • the assay of the present invention permits integration into existing or newly developed pathology architecture or platform systems.
  • the present invention contemplates a method of allowing a user to determine the status of a subject with respect to AD, the method including:
  • the method generally further includes:
  • the base station can include first and second processing systems, in which case the method can include:
  • the method may also include:
  • references to an "algorithm” or “algorithmic functions” as outlined above includes the performance of a multivariate analysis function.
  • a range of different architectures and platforms may be implemented in addition to those described above. It will be appreciated that any form of architecture suitable for implementing the present invention may be used.
  • one beneficial technique is the use of distributed architectures.
  • a number of end stations may be provided at respective geographical locations. This can increase the efficiency of the system by reducing data bandwidth costs and requirements, as well as ensuring that if one base station becomes congested or a fault occurs, other end stations could take over. This also allows load sharing or the like, to ensure access to the system is available at all times.
  • This particular method is also amenable for high throughput screening of ACt levels based on qPCR wherein a ACt value is selected for cutoff in terms of a subject having or is at risk of developing AD or who is healthy.
  • the end stations can be hand-held devices, such as PDAs, mobile phones, or the like, which are capable of transferring the subject data to the base station via a communications network such as the Internet, and receiving the reports.
  • a communications network such as the Internet
  • the term “data” means the levels or concentrations of the biomarkers.
  • the "communications network” includes the internet. When a server is used, it is generally a client server or more particularly a simple object application protocol (SOAP).
  • SOAP simple object application protocol
  • HC healthy controls
  • MCI mild cognitive impairment
  • AD Alzheimer's disease
  • NINCDA-ADRDA National Institute of Neurological and Communicative Diseases and Stroke-Alzheimer's Disease and Related Disorders Association
  • Serum-gel tubes are processed within two hours of collection and serum is snap frozen in liquid nitrogen.
  • serum from 49 patients (HC, 23; MCI, 3; AD, 23) are collected for deep sequencing and serum from 60 patients (HC, 36; MCI, 8; AD, 16) are collected for validation. All individuals included are assessed for full blood pathology testing (Melbourne Health and PathWest Laboratory Medicine, Melbourne, Australia), apolipoprotein ⁇ 4 ( ⁇ 4) genotyping and assessment of cognitive functions (mini-mental state examination, MMSE) at the time of collection (Ellis et al. (2009) supra). Of these, 83 (discovery set, 23 and validation set, 60) patients hadAmyloid-PET neuroimaging data available for assessment of cerebral ⁇ accumulation.
  • ⁇ burden is expressed as (Cheng et al. (2013) supra) C-PiB standardized uptake value ratio (SUVR) as previously described (Villenmagne et al. (2013) supra). Patient data from both baseline and 54-month time points are available for analyses.
  • Serum exosomal RNA is isolated by using the Plasma/serum exosomal RNA isolation kit (Norgen Biotek, Canada) from 1 ml serum per participant whereby the manufactures' protocol is followed. The total exosomal RNA yield, composition and quality is analyzed by the Agilent 2100 Bioanalyser using the Small RNA kit (Agilent). Exosomal RNA is converted into cDNA libraries using the Ion Total RNA-Seq Kit V2 (Life Technologies, Australia) and prepared for sequencing as described previously (Cheng et al. (2013) Kidney International). Pooled libraries with unique barcodes are loaded on 318 sequencing chips and run on the Ion Torrent Personal Genome Machine (Life Technologies, Australia) [Cheng et al.
  • the sequences are then assessed for quality and primer-adapter sequences are trimmed by the Torrent Suite software (version 3 -4- 1), followed by alignment to the human reference genome (HG19).
  • the trimmed and aligned data is transferred to Partek Genomics Suite (Partek, Singapore) and mapped to known miRNA using miRBase V-20.Bioinformatics analysis and differential expression is performed using Partek Genomics Suite.
  • the panel of candidate miRNA found highly associated with AD are used for validation on a new set of serum samples collected from AIBL. Serum samples for the validation study are spiked with synthetic C. elegan miR-39 (Qiagen, Australia) during exosomal RNA extraction to monitor extraction efficiency and for normalization purposes.
  • exosomal miRNA is converted to cDNA (TaqMan MicroRNA Reverse Transcription Kit, Applied Biosystems, Australia) according to the manufacturers' protocol with a primer pool containing 23 miRNA assays (TaqMan microRNA assays, 5x, Applied Biosystems).
  • cDNA samples are pre-amplified (TaqMan PreAmp Master Mix Kit, Applied Biosystems) and qRT-PCR (TaqMan Fast Advanced Master Mix, Applied Biosystems) is performed using individual miRNA assays (TaqMan microRNA assays, 20x, Applied Biosystems) and run on the ViiA (Trade Mark) 7 Real- Time PCR System (Life Technologies) across a 384-well format. Reverse transcription and pre-amplification no template controls using primer pools and individual assays are also prepared to ensure there is no background amplification of miRNA assays.
  • Raw data are uploaded to DataAssist (Applied Biosystems) to calculate delta Ct (ACt) and normalization against controls further statistical analysis.
  • the number of reads of each miRNA are normalized to reads per million (RPM) across all samples. Low abundant miRNAs with less than 50 read counts across all samples are removed thus high abundant miRNAs are analyzed.
  • Initial statistical analysis of miRNA expression changes is performed using the Partek Genomics Suite. Selection of miRNAs is based upon ANOVA comparing healthy control and AD groups (clinical classification at the time of collection). Probability values are adjusted for multiple testing using the False Discovery Rate (FDR) method.
  • Significant changes in miRNA expression are expressed in fold change [LOG 2 ] and defined as p (AD Vs HC) ⁇ 0 05, p (AD, MCI and HC) ⁇ 0 05 and ⁇ 1 2 fold change.
  • 17 miRNA are found to be significantly deregulated (p ANOVA (AD Vs HC) ⁇ 0 05, ANOVA (AD, MCI and HC) ⁇ 0 05 and ⁇ 1 -2 fold change, Figure 1); 14 miRNA are found to be up- regulated (hsa-miR-361-5p, hsa-miR-30e-5p, hsa-miR-93-5p, hsa-miR-15a-5p, hsa-miR- 143-3p, hsa-miR-335-5p, hsa-miR-106b-5p, hsa-miR-101-3p, hsa-miR-424-5p, hsa-miR- 106a-5p, hsa-miR-18b-5p, hsa-miR-3065-5p, hsa-miR-20a-5p, and
  • Receiver Operating Characteristic (ROC) analyses statistics for each miRNA marker are shown in Table 5.
  • the ROC analyses is performed using the raw sequence count data to estimate the performance of each miRNA to predict AD.
  • Criterion cut points are chosen by taking the closest point on the curve to the top left corner of the ROC curve; approximating the most optimum performance for each marker.
  • exosomes compared to cell-free or whole blood has a number of disease specific advantages for diagnostic purposes.
  • isolating the enriched miRNA in exosomes from biological samples of AD patients removes the saturation of insignificant miRNA expressed in both AD and HC patients thus, providing a better performing predictive AD diagnosis.
  • miRNA originating from the brain have been demonstrated to cross the endothelial cellular layers of the blood brain barrier by transcytosis of exosomes across the endothelial layer in order to communicate between the brain and distant organs via biological fluids (Haqqani et al. (2013) Fluids barriers CNS 10:4).
  • exosomes serve as a RNase-protective vesicle which shield miRNA from RNase-rich environments such as the circulatory system (Huang et al. (2013) Bmc Genomics 14319). Furthermore, specific packaging of miRNA into exosomes (Gibbings et al. (2009) Nat Cell Biol 11.1143-1149) and increased secretion of microvesicles into peripheral blood of cancer patients compared to healthy patients (Mitchell et al. (2008) supra) suggests the importance of exosomes in the role of extracellular communication during disease. These factors allow the possibility of profiling disease specific miRNA that are found enriched from exosomes.
  • This study produced a large coverage of miRNA (1419 known miRNA) extracted from exosomal serum samples isolated for AD biomarker discovery by next-generation sequencing (NGS).
  • NGS next-generation sequencing
  • the advantage of NGS is its ability to detect absolute counts of all miRNA present in samples.
  • miRNAs need to lie within the detectable range of qRT-PCR. Using the workflow carried out in this study, it is estimated that approximately 50 RPM must be obtained across the majority of biological samples for successful biomarker validation by qRT-PCR.
  • Biomarkers studies attempting to validate a large panel consisting of low abundant miRNAs will result in unsuccessful validation of miRNAs and/or changes in direction of expression (Lei dinger et al. (2013) Genome Biol 7 :R78; Kumar et al. (2013) PLoS ONE 5:e69807).
  • 220 highly abundant miRNA are present in all samples across the study.
  • Seventeen significantly differentially expressed exosomal miRNA are found in AD patients and 16 miRNA are successfully validated displaying consistent expression changes with AD and HC across both NGS and qRT-PCR data sets.
  • the commonly used comparative delta Ct approach in qRT-PCR may not be applicable for diagnostic use as a healthy control group is required for every run.
  • normalized delta Ct is applied into a Random Forest model to predict clinical classification. Such a model is able to predict clinical classification (at 54 months) with high accuracy (87% sensitivity and 77% specificity).
  • a possible limitation in the assay is an ability to diagnose those with late AD where the rate of pathogenesis, in particular ⁇ deposition, slows in the later stages of AD when patients experience cerebral atrophy and severe cognitive impairment (Villenmagne et al. (2013) supra).
  • diagnosis is less critical and the assay may be used with other markers.
  • Patients of the AIBL study are diagnosed according to NINCDS- ADRDA criteria, which is heavily reliant on cognitive and neuropsychological testing for a clinical diagnosis of probable AD.
  • the diagnosis obtained by miRNA analysis is compared with the participant's classification by NINCDS-ADRDA criteria.
  • the additional strength of this study comes from the extensive database of metadata collected at baseline through to 54 months.
  • Amyloid-PET and ⁇ 4 genotyping information for those HC patients incorrectly classified with AD suggested progression towards AD. Taking into consideration that these HC patients could actually be prodromal AD, the predicted specificity would increase to 91 -4%.
  • the objective and purpose of the exosomal miRNA biomarker test is to predict future cognitive decline in asymptomatic or presymptomatic individuals and during the progression of patients with early dementia. This study represents a significant step towards developing a cost-effective, non-invasive and low risk diagnostic test to detect the onset and monitor various stages of AD in order for physicians to provide optimal care for patients.
  • serum samples are analyzed for exosomal miRNA associated with AD together with metadata which included cognitive and amyloid-PET imaging.
  • A16-miRNA expression profile is generated for the diagnosis of AD with sensitivity and specificity of 87% and 77%, respectively.
  • the exosomal miRNA AD signature is able to predict cognitively healthy patients presenting with high amyloid burden as probable AD.
  • MMSE mini-mental state examination.
  • APOE apolipoprotein E. ⁇ Compared with healthy controls
  • the average number of total reads obtained per sample was 780, 371. Reads were mapped to 1419 known mature miRNA using miRBase V-20. miRNA with lower than 50 reads across all samples were removed leaving 225 abundant miRNA for analysis. Raw reads were normalized to read per million (RPM). RPM values for each sample. ANOVA analysis was performed by comparing cohort groups with healthy controls. *P values are the result of Generalized Linear Modeling pre and post adjustment with age, sex and APOE ⁇ 4 allele. miRBase v.20 accession numbers. # Hsa-miR-3065 -5p was not used in the validation set as it was found to be undetectable using qRT-PCR
  • a UC Area under receiver operating curve.
  • CI Confidence interval.
  • PiB Carbon- 11 -labelled Pittsburgh compound B.

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Abstract

La présente invention concerne des biomarqueurs microARN, dont le niveau d'expression renseigne sur la maladie d'Alzheimer (AD), notamment en termes d'identification de début de la maladie d'Alzheimer et de différents stades de ladite maladie d'Alzheimer, ses diverses manifestations cliniques et des états associés à l'amyloïdose.
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WO2023216293A1 (fr) * 2022-05-11 2023-11-16 杭州青果医疗科技有限责任公司 Système et procédé de prédiction d'une démence ou d'un trouble cognitif léger

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