WO2024077282A2 - Biomarqueurs pour le diagnostic de la sclérose latérale amyotrophique - Google Patents

Biomarqueurs pour le diagnostic de la sclérose latérale amyotrophique Download PDF

Info

Publication number
WO2024077282A2
WO2024077282A2 PCT/US2023/076301 US2023076301W WO2024077282A2 WO 2024077282 A2 WO2024077282 A2 WO 2024077282A2 US 2023076301 W US2023076301 W US 2023076301W WO 2024077282 A2 WO2024077282 A2 WO 2024077282A2
Authority
WO
WIPO (PCT)
Prior art keywords
lateral sclerosis
amyotrophic lateral
als
sample
protein
Prior art date
Application number
PCT/US2023/076301
Other languages
English (en)
Inventor
Patrick Lilley
Gwendelyn LILLEY
Original Assignee
Neu Bio, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Neu Bio, Inc. filed Critical Neu Bio, Inc.
Publication of WO2024077282A2 publication Critical patent/WO2024077282A2/fr

Links

Classifications

    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/16—Primer sets for multiplex assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the invention relates to the diagnosis of disease using biomarkers, and more specifically, to a system and method of diagnosing amyotrophic lateral sclerosis (ALS) based on altered expression of one or more genes.
  • ALS amyotrophic lateral sclerosis
  • ALS Amyotrophic lateral sclerosis
  • MND motor neuron disease
  • Lou Gehrig's disease is a neurodegenerative disease that results in the progressive loss of motor neurons that control voluntary muscles. ALS causes progressive degeneration of motor nerve cells in the brain and spinal cord, resulting in atrophy of the innervated muscles. The onset of this disease typically manifests between ages 40 and 70, but cases have been reported in younger patients. As ALS progresses, symptoms become more wide-spread and some muscles become paralyzed. In late-stage ALS, most voluntary muscles are paralyzed.
  • ALS Early symptoms of ALS include stiff muscles, muscle twitches and gradual increasing weakness and muscle wasting. Limb-onset ALS begins with weakness in the arms or legs, while bulbar-onset ALS begins with difficulty speaking or swallowing. Half of the people with ALS develop at least mild difficulties with thinking and behavior, and about 15% develop frontotemporal dementia. Most people experience pain. The affected muscles are responsible for chewing food, speaking, and walking. Motor neuron loss continues until the ability to eat, speak, move, and finally the ability to breathe is lost. ALS eventually causes paralysis and early death, usually from respiratory failure. [0005] Despite decades of research and billions of dollars, much about ALS remains unknown.
  • ALS currently has no known cure and its prognosis is bleak. Fifty percent of affected patients live at least three or more years after diagnosis; 20 percent live five years or more; and only up to 10 percent will survive more than ten years. The goal of treatment is to improve symptoms. A medication called riluzole may extend life by about two to three months. Non-invasive ventilation may result in both improved quality and length of life. Mechanical ventilation can prolong survival but does not stop disease progression. The disease can affect people of any age, but usually starts around the age of 60 and in inherited cases around the age of 50.
  • ALS can be identified in one of four stages based on its progression. In stage one, early symptoms of ALS become evident. The physical appearance of muscles can change. Muscles will become softer, appear to be weaker and can become tight and spastic. Cramping and twitching are also common signs that ALS is in its early stages. Other symptoms include a single region being affected. A patient can experience fatigue often, will have a poor balance, have difficulty in talking, have weak grip along with other similar symptoms and signs.
  • NIV non-invasive ventilation
  • Palliative care should begin shortly after someone is diagnosed with ALS. Discussion of end-of-life issues gives people with ALS time to reflect on their preferences for end-of-life care and can help avoid unwanted interventions or procedures. Nursing care can improve symptom management at the end of life and increases the likelihood of a peaceful death. In the final days of life, opioids can be used to treat pain and dyspnea, while benzodiazepines can be used to treat anxiety.
  • ALS Alzheimer's disease
  • Biomarkers are a non-invasive and cost-effective means to aid in clinical management of patients, particularly in areas of disease detection, prognosis, monitoring and therapeutic stratification.
  • a serological biomarker to be useful for early detection, its presence in serum must be relatively low in healthy individuals and those with benign disease.
  • the biomarker should preferably be tissue specific, such that a change in serum level can be directly attributed to disease (e.g., ALS) of that tissue.
  • PSA serum PSA is commonly used for prostate cancer screening in men over 50, but its usage remains controversial due to serum elevation in benign disease as well as prostate cancer. Nevertheless, PSA represents one of the most useful serological markers currently available. PSA is strongly expressed in only the prostate tissue of healthy men, with low levels in serum established by normal diffusion through various anatomical barriers. These anatomical barriers are disrupted upon development of prostate cancer, allowing increased amounts of PSA to enter circulation.
  • CEA carcinoembryonic antigen
  • CA19.9 carbohydrate antigen 19.9
  • CEA carcinoembryonic antigen
  • CA19.9 carbohydrate antigen 19.9
  • CEA carcinoembryonic antigen
  • CA19.9 carbohydrate antigen 19.9
  • CEA CYFRA 21 -1 (cytokeratin 19 fragment)
  • NSE neuron-specific enolase
  • TSE tissue polypeptide antigen
  • pro-GRP progastrin-releasing peptide
  • SCC antigen for lung cancer
  • CA 125 for ovarian cancer
  • PSA prostate-specific antigen
  • Embodiments of the invention include methods of detecting and prognosing ALS using biomarkers.
  • the methods and assays disclosed herein are directed to the examination of the amount of one or more biomarkers in a biological sample, wherein the determination of that amount is predictive or indicative of the presence of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • Methods for detecting biomarkers to be assessed include protocols that examine the presence and/or expression of a desired nucleic acid.
  • Tissue or cell samples from mammals can be conveniently assayed for, e.g., genetic- marker mRNAs or DNAs using Northern, dot-blot, or polymerase chain reaction (PCR) analysis, array hybridization, RNase protection assay, or using DNA SNP chip microarrays, which are commercially available, including DNA microarray snapshots.
  • PCR polymerase chain reaction
  • array hybridization e.g., array hybridization, RNase protection assay, or using DNA SNP chip microarrays, which are commercially available, including DNA microarray snapshots.
  • RT-PCR real-time PCR
  • Methods also include assays to detect/quantify specific proteins such as immune-affinity assays (e.g., ELISA, Western blot, flow cytometry, etc.).
  • embodiments include methods of detecting ALS based on specific mRNAs that have altered expression levels.
  • the Applicant has identified 52 genes that have altered levels of expression in patients with ALS. Accordingly, mRNAs and/or protein levels can be assayed and used as biomarkers to distinguish healthy individuals from individuals affected with ALS.
  • the use of biomarkers is non-invasive and potentially more sensitive than conventional methods.
  • the biomarkers can also detect early-onset or asymptomatic ALS.
  • the biomarkers can also be used to monitor disease progress and distinguish among stages of the disease.
  • Embodiments also include methods of prognosis, patient monitoring and distinguishing between ALS and ailments that present signs/symptoms similar to ALS (e.g., ALS mimic syndromes).
  • the methods described herein can predict that a patient is likely to get ALS. In embodiments, the methods described herein can detect asymptomatic ALS. In embodiments, the methods described herein can be used to monitor progression of ALS. In embodiments, the methods described herein can be used to distinguish between stages of ALS (i.e. , stage 1 - stage 4).
  • the method includes one or more mRNA biomarkers. In one embodiment, the method includes one or more protein biomarkers. In one embodiment, the method includes a combination of one or more mRNA biomarkers and one or more protein biomarkers.
  • Embodiments include the use of one or more of the following genes (or transcribed mRNAs or translated proteins) as biomarkers: CAPZA2, LDLR, C7orf42, CHD9, NCALD, IL2RB, MGC24103, VPS13C, MEGF9, ZCCHC17, FAR1 , ABCA1 , HIPK2, IKBIP, PLA2G2D, MAP2K1 , TFIP11 , SCRN1 , HNRPUL2, C19orf59, MCM3, FSTL4, PSMC4, UBE2D2, DDHD2, ATP2B4, DPAGT1 , FER1 L3, CTSW, STUB1 , CLN5, WDR23, RPL10P6, BBS2, ZNF652, IP6K1 , PAPD4, TLR2, C1 orf144, NUDT18, DNAJA3, ATP5J, ARHGAP30, MIR29B2CHG, POLR3C, NME1 , NLRP1
  • Embodiments also include the use of the mRNAs and proteins identified herein as therapeutic targets for treating ALS.
  • Embodiments include a method of detecting ALS or determining a prognosis of a subject with ALS, that includes steps of a) measuring the expression level of at least one mRNA in a test sample of the subject; b) comparing the expression level of the mRNA in the test sample to a level in a base sample; and c) detecting or determining the prognosis of ALS based on altered expression the mRNA in the test sample.
  • the method can be used with blood, serum other bodily fluids including saliva. In other embodiments, the method also compares levels of at least one protein between the base sample and the test sample.
  • the method can also include a step of treating a patient for ALS.
  • Embodiments include a method of detecting ALS or determining a prognosis of a subject with ALS, that includes steps of a) measuring the expression level of at least one protein in a test sample of the subject; b) comparing the expression level of the protein in the test sample to a level in a base sample; and c) detecting or determining the prognosis of ALS based on altered expression the protein in the test sample.
  • the method can be used with blood, serum other bodily fluids including saliva. In other embodiments, the method also compares levels of at least mRNA between the base sample and the test sample.
  • the method can also include a step of treating a patient for ALS.
  • Embodiments also include a method of detecting ALS or determining a prognosis of a test subject with ALS, that includes steps of: a) measuring expression levels of two or more mRNAs in samples from subjects with ALS; b) measuring expression levels of the same mRNAs in samples from healthy subjects; c) comparing the expression levels of the mRNAs in the samples from the subjects with ALS to the levels in the samples from the healthy subjects; d) identifying mRNAs that have altered levels of expression in the samples from the subjects with ALS; e) creating a biomarker fingerprint from the mRNAs with altered levels of expression; and f) diagnosing or determining the prognosis of ALS in the test subject by comparing of levels of mRNAs from the test subject to those in the biomarker fingerprint.
  • the method compares levels of two or more proteins between healthy subjects and subjects with ALS to create the biomarker fingerprint.
  • the methods can also include a step of treating a patient for ALS.
  • Embodiments also include a diagnostic kit for diagnosing ALS or determining a prognosis of a subject with ALS.
  • the kit can include a plurality of nucleic acid molecules, each nucleic acid molecule encoding a mRNA sequence.
  • the nucleic acid molecules identify variations in expression levels of one or more mRNAs in a plasma or saliva sample from a test subject.
  • the expression levels of one or more mRNAs can represent a nucleic acid expression fingerprint that is indicative for the presence of ALS.
  • FIG. 1 is a detailed list of biomarkers for detection and/or prognosis of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • FIG. 2 is a list of the top twenty diagnostic biomarkers for detection and/or prognosis of amyotrophic lateral sclerosis (ALS v. Healthy Controls).
  • FIG. 3 is a list of the top twenty diagnostic biomarkers for detection and/or prognosis of amyotrophic lateral sclerosis (ALS v. ALS-Mimicking Diseases).
  • FIG. 4 is a list of the top twenty diagnostic biomarkers for detection and/or prognosis of amyotrophic lateral sclerosis (ALS v. Combined Healthy and Mimics).
  • FIG. 5 is a list of the top twenty causal biomarkers for detection and/or prognosis of amyotrophic lateral sclerosis (ALS v. Healthy Controls).
  • references in this specification to "one embodiment/aspect” or “an embodiment/aspect” means that a particular feature, structure, or characteristic described in connection with the embodiment/aspect is included in at least one embodiment/aspect of the disclosure.
  • the use of the phrase “in one embodiment/aspect” or “in another embodiment/aspect” in various places in the specification are not necessarily all referring to the same embodiment/aspect, nor are separate or alternative embodiments/aspects mutually exclusive of other embodiments/aspects.
  • various features are described which may be exhibited by some embodiments/aspects and not by others.
  • various requirements are described which may be requirements for some embodiments/aspects but not other embodiments/aspects.
  • Embodiment and aspect can in certain instances be used interchangeably.
  • algorithm refers to a specific set of instructions or a definite list of well-defined instructions for carrying out a procedure, typically proceeding through a well-defined series of successive states, and eventually terminating in an end-state.
  • biomarker refers generally to a DNA, RNA, protein, carbohydrate, or glycolipid-based molecular marker, the expression or presence of which in a subject's sample can be detected by standard methods (or methods disclosed herein) and is predictive or prognostic of an ailment such as ALS. Biomarkers may be present in a test sample but absent in a control sample, absent in a test sample but present in a control sample, or the amount of biomarker can differ between a test sample and a control sample.
  • biomarkers assessed can be present in such a sample, but not in a control sample, or certain biomarkers are seropositive in the sample, but seronegative in a control sample. Also, optionally, expression of such a biomarker may be determined to be higher than that observed for a control sample.
  • markers and “biomarker” are used herein interchangeably.
  • altered expression or "differential level" of a biomarker may include either an increased or decreased level.
  • an altered expression level can refer to a biomarker level that is decreased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 51 %, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% (i.e., indicative of the absence of the biomarker molecule).
  • an altered expression level refers to a level that is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 51 %, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200% or more.
  • ALS mimic syndromes refers to unrelated disorders that have a similar presentation and clinical features to ALS or its variants. Because the prognosis of ALS and closely related subtypes of motor neuron disease are generally poor, neurologists may carry out investigations to evaluate and exclude other diagnostic possibilities. Disorders of the neuromuscular junction, such as myasthenia gravis (MG) and Lambert-Eaton myasthenic syndrome, can also mimic ALS, although this rarely presents diagnostic difficulty over time. Benign fasciculation syndrome and cramp fasciculation syndrome can also, occasionally, mimic some of the early symptoms of ALS. Nonetheless, the absence of other neurological features that develop inexorably with ALS means that, over time, the distinction will not present any difficulty to the experienced neurologist; where doubt remains, EMG may be helpful.
  • MG myasthenia gravis
  • Lambert-Eaton myasthenic syndrome can also mimic ALS, although this rarely presents diagnostic difficulty over time. Benign fasciculation syndrome and cramp fasciculation syndrome can also, occasionally, mimic some of the early symptoms of
  • Riluzole or “Rilutek®” refers to a drug for treating ALS. In recent studies, it was found to modestly prolong survival (e.g., by about two to three months). It may have a greater survival benefit for those with bulbar-onset ALS. Riluzole may work by decreasing release of the excitatory neurotransmitter glutamate from pre- synaptic neurons. The most common side effects are nausea and a lack of energy (asthenia). It is generally recommended that people with ALS begin treatment with riluzole as soon as possible following their diagnosis.
  • Edavone or “Radicava®” refers to a drug for treating ALS. Edaravone has been shown to modestly slow the decline in function in a small group of people with early-stage ALS. It may work by protecting motor neurons from oxidative stress. The most common side effects are bruising and gait disturbance. Treatment with edaravone can be expensive and requires daily hour-long IV infusions for ten days in a two-week period.
  • additional biomedical information refers to one or more evaluations of an individual, other than using any of the biomarkers described herein, that are associated with ALS risk.
  • Additional biomedical information includes any of the following: physical descriptors of an individual, the height and/or weight of an individual, the gender of an individual, the ethnicity of an individual, smoking history, occupational history, exposure to known toxins/carcinogens (e.g., exposure to pesticides), family history of ALS. Additional biomedical information can be obtained from an individual using routine techniques known in the art, such as from the individual themselves by use of a routine patient questionnaire or health history questionnaire, etc., or from a medical practitioner, etc.
  • additional biomedical information can be obtained from routine imaging techniques, including CT imaging (e.g., low-dose CT imaging) and X-ray. Testing of biomarker levels in combination with an evaluation of any additional biomedical information may, for example, improve sensitivity, specificity, and/or AUC for detecting ALS as compared to biomarker testing alone or evaluating any particular item of additional biomedical information alone.
  • CT imaging e.g., low-dose CT imaging
  • X-ray e.g., X-ray.
  • AUC area under the curve
  • ROC receiver operating characteristic
  • the feature data across the entire population e.g., the cases and controls
  • the true positive and false positive rates for the data are calculated.
  • the true positive rate is determined by counting the number of cases above the value for that feature and then dividing by the total number of cases.
  • the false positive rate is determined by counting the number of controls above the value for that feature and then dividing by the total number of controls.
  • ROC curves can be generated for a single feature as well as for other single outputs, for example, a combination of two or more features can be mathematically combined (e.g., added, subtracted, multiplied, etc.) to provide a single sum value, and this single sum value can be plotted in a ROC curve. Additionally, any combination of multiple features, in which the combination derives a single output value, can be plotted in a ROC curve. These combinations of features may comprise a test.
  • the ROC curve is the plot of the true positive rate (sensitivity) of a test against the false positive rate (1 -specificity) of the test.
  • detecting or “determining” with respect to a biomarker value includes the use of both the instrument required to observe and record a signal corresponding to a biomarker value and the materials required to generate that signal.
  • the biomarker value is detected using any suitable method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection methods, nuclear magnetic resonance, quantum dots, and the like.
  • prognosis refers to the forecast or likely outcome of a disease. As used herein, it refers to the probable outcome of ALS, including whether the disease will respond to treatment or mitigation efforts and/or the likelihood that the disease will progress.
  • fingerprint refers to a plurality or pattern of biomarkers that have elevated or reduced levels in a subject with disease.
  • a fingerprint can be generated by comparing subjects with the disease to healthy subjects and used for screening/diagnosis of the disease.
  • microRNA refers to small endogenous RNA molecules that can be used as serum diagnostic biomarkers for diseases including ALS.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • Methods for obtaining (e.g., producing, isolating, purifying, synthesizing, and recombinantly manufacturing) polypeptides are well known to one of ordinary skill in the art.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • the term “medicament” refers to an active drug to treat ALS, or the signs or symptoms or side effects of ALS.
  • plasma refers to the liquid portion of the blood that carries cells and proteins throughout the body. Plasma can be separated from the blood by spinning a tube of fresh blood containing an anticoagulant in a centrifuge until the blood cells fall to the bottom of the tube.
  • PCR or “polymerase chain reaction” refers to a common method used to make many copies of a specific DNA segment. Variations of the technique can be used to determine the presence and amount of one or more mRNAs in a sample.
  • RT-qPCR quantitative reverse-transcription PCR
  • sample refers to a biological sample obtained from an individual, body fluid, body tissue, cell line, tissue culture, or other source.
  • Body fluids are, for example, lymph, sera, whole fresh blood, peripheral blood mononuclear cells, frozen whole blood, plasma (including fresh or frozen), urine, saliva, semen, synovial fluid and spinal fluid. Samples also include synovial tissue, skin, hair follicle, and bone marrow. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.
  • subject refers to any single animal, more preferably a mammal (including such non-human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates) for which treatment is desired. Most preferably, the patient herein is a human.
  • An mRNA that is “upregulated” generally refers to an increase in the level of expression of the mRNA in response to a given treatment or condition.
  • An mRNA that is “downregulated” generally refers to a decrease in the level of expression of the mRNA in response to a given treatment or condition. In some situations, the mRNA level can remain unchanged upon a given treatment or condition.
  • An mRNA from a patient sample can be “upregulated,” i.e.
  • the level of mRNA can be increased, for example, by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 90%, about 100%, about 200%, about 300%, about 500%, about 1 ,000%, about 5,000% or more of the comparative control mRNA level or a reference level.
  • an mRNA can be “downregulated,” i.e., the level of mRNA level can be decreased, for example, by about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 2%, about 1 % or less of the comparative control mRNA level or a reference level.
  • the level of a polypeptide, protein, or peptide from a patient sample can be increased as compared to a control or a reference level. This increase can be about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 90%, about 100%, about 200%, about 300%, about 500%, about 1 ,000%, about 5,000% or more of the comparative control protein level or a reference level.
  • the level of a protein biomarker can be decreased.
  • This decrease can be, for example, at a level of about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 2%, about 1 % or less of the comparative control protein level or a reference level.
  • nucleic acid probe or “oligonucleotide probe” refers to a nucleic acid capable of binding to a target nucleic acid of complementary sequence, such as the mRNA biomarkers provided herein, through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a probe may include natural (e.g., A, G, C, or T) or modified bases (7- deazaguanosine, inosine, etc.).
  • the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
  • the probes are preferably directly labeled with isotopes, for example, chromophores, lumiphores, chromogens, or indirectly labeled with biotin to which a streptavidin complex may later bind.
  • isotopes for example, chromophores, lumiphores, chromogens, or indirectly labeled with biotin to which a streptavidin complex may later bind.
  • treating refers to one or more of (1 ) inhibiting the disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); and (2) ameliorating the disease condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease.
  • Treatments for ALS can include physical/occupational therapy, speech therapy, respiratory therapy, psychotherapy, diet, supplements, CBT therapy and medications.
  • Medications include tofersen (QalsodyTM), Riluzole and edaravone.
  • standard ID refers to a standardized identification for genes provided by the Human Gene Nomenclature Committee (HGNC).
  • accession or “accession ID” refers to a unique identifier for a sequence record in a database such as GenBank.
  • An accession number applies to the complete record and is usually a combination of a letters and numbers, such as a single letter followed by five digits (e.g., 1112345) or two letters followed by six digits (e.g., AF123456).
  • synthetic protein-binding moiety refers to molecules capable of mimicking the binding and/or functional sites of proteins. They can be constructed by, for example, using nonantibody molecular scaffolds, recombinant protein synthesis or by means of chemical peptide synthesis.
  • the Applicant has recognized mRNA profiling in subjects with ALS.
  • Specific mRNAs or “biomarkers” are aberrantly expressed in tissues affected by ALS as compared to healthy tissue.
  • biomarker expression can provide insights into cellular processes involved in the progression of ALS.
  • mRNA expression levels can also be used for prognosis.
  • the technology provides diagnostic methods for predicting and/or prognosticating the effectiveness of treatment.
  • the present invention is based on the finding that ALS can be reliably identified based on particular biomarker expression profiles with high sensitivity and specificity.
  • the expression of biomarkers typically includes both up- and down- regulated levels of mRNAs/proteins.
  • An analysis of mRNA/protein expression biomarkers allows for creation of a “fingerprint” by analyzing mRNA/protein expression patterns in diseased and healthy subjects. Thereafter, individual mRNA/protein expression levels can be used for the detection of ALS at early stages of the disease (e.g., when asymptomatic). It is also possible to monitor progression of ALS (e.g., between early, moderate and late stages) and distinguish ALS from diseases that present similar signs/symptoms such as ALS mimic syndromes.
  • ALS Early detection of ALS is important to effective treatment. Medications and treatments can be more effective if they begin in early stages of ALS, before progression of signs/symptoms. Further, the biomarkers can be used as therapeutic targets and to identify pathways for study of the disease.
  • embodiments include diagnostic markers or a molecular fingerprint, for quick and reliable identification and/or treatment of cells exhibiting or having a predisposition to develop ALS.
  • Embodiments further include methods of diagnosing ALS based on specific mRNAs that have altered expression levels. While individual mRNAs can be monitored, the invention includes 52 mRNAs of particular value as biomarkers to screen or distinguish healthy individuals from individuals affected with disease.
  • the mRNAs of particular interest include: CAPZA2, LDLR, C7orf42, CHD9, NCALD, IL2RB, MGC24103, VPS13C, MEGF9, ZCCHC17, FAR1 , ABCA1 , HIPK2, IKBIP, PLA2G2D, MAP2K1 , TFIP11 , SCRN1 , HNRPUL2, C19orf59, MCM3, FSTL4, PSMC4, UBE2D2, DDHD2, ATP2B4, DPAGT1 , FER1 L3, CTSW, STUB1 , CLN5, WDR23, RPL10P6, BBS2, ZNF652, IP6K1 , PAPD4, TLR2, C1orf144, NUDT18, DNAJA3, ATP5J, ARHGAP30, MIR29B2CHG, POLR3C, NME1 , NLRP1 , LSM5, SOD1 , C14ORF159, EB
  • Embodiments further include methods of diagnosing ALS based on specific proteins that have altered expression levels. While individual proteins can be monitored, proteins translated from the mRNAs above (and listed in Table 1 ) are of particular value as biomarkers.
  • the methods and materials can be used for assessing subjects (e.g., human patients) for ALS.
  • embodiments include materials and methods for using identifiable markers to assist clinicians in assessing ALS disease activity, assessing the likelihood of response and outcomes of therapy, and predicting long-term disease outcomes.
  • subjects with ALS can be diagnosed based on the presence of certain diagnostic indicators in samples from the subject.
  • the technology allows for the diagnosis of ALS based on one or more combinations of markers.
  • kits for predicting, diagnosing or monitoring responsiveness of an ALS treatment or therapy wherein the kit is calibrated to measure marker levels in a sample from the patient.
  • the amount of biomarkers can be determined by using, for example, a reagent that specifically binds with the biomarker protein or a fragment thereof, (e.g., an antibody, a fragment of an antibody, an antibody derivative or synthetic protein-binding moiety).
  • a reagent that specifically binds with the biomarker protein or a fragment thereof e.g., an antibody, a fragment of an antibody, an antibody derivative or synthetic protein-binding moiety.
  • the level of expression can be determined using a method common in the art such as proteomics, flow cytometry, immunocytochemistry, immunohistochemistry, enzyme-linked immunosorbent assay, multi-channel enzyme linked immunosorbent assay, and variations thereof.
  • the expression level of a biomarker in the biological sample can also be determined by detecting the level of expression of a transcribed biomarker polynucleotide or fragment thereof encoded by a biomarker gene, which may be cDNA, mRNA or heterogeneous nuclear RNA (hnRNA).
  • the step of detecting can include amplifying the transcribed biomarker polynucleotide and can use the method of quantitative reverse transcriptase polymerase chain reaction (rtPCR).
  • the expression level of a biomarker can be assessed by detecting the presence of the transcribed biomarker polynucleotide or a fragment thereof in a sample with a probe which anneals with the transcribed biomarker polynucleotide or fragment thereof under stringent hybridization conditions.
  • compositions and kits for practicing the methods.
  • reagents e.g., primers, probes
  • sets e.g., sets of primers pairs for amplifying a plurality of markers.
  • Additional reagents for conducting a detection assay may also be provided (e.g., enzymes, buffers, positive and negative controls for conducting QuARTS, PCR, sequencing, bisulfite, or other assays).
  • the kits containing one or more reagent necessary, sufficient, or useful for conducting a method are provided.
  • reactions mixtures containing the reagents.
  • master mix reagent sets containing a plurality of reagents that may be added to each other and/or to a test sample to complete a reaction mixture.
  • the technology described herein is associated with a programmable machine designed to perform a sequence of arithmetic or logical operations as provided by the methods described herein.
  • some embodiments of the technology are associated with (e.g., implemented in) computer software and/or computer hardware.
  • the technology relates to a computer comprising a form of memory, an element for performing arithmetic and logical operations, and a processing element (e.g., a microprocessor) for executing a series of instructions (e.g., a method as provided herein) to read, manipulate, and store data. Therefore, certain embodiments employ processes involving data stored in or transferred through one or more computer systems or other processing systems.
  • Embodiments disclosed herein also relate to apparatus for performing these operations.
  • This apparatus may be specially constructed for the required purposes, or it can be a general-purpose computer (or a group of computers) selectively activated or reconfigured by a computer program and/or data structure stored in the computer.
  • a group of processors performs some or all of the recited analytical operations collaboratively (e.g., via a network or cloud computing) and/or in parallel.
  • a microprocessor is part of a system for determining the presence of one or more mRNA or proteins associated with ALS; generating standard curves; determining a specificity and/or sensitivity of an assay or marker; calculating an ROC curve; sequence analysis; all as described herein or is known in the art.
  • a microprocessor is part of a system for determining the amount, such as concentration, of one or more mRNAs associated with a ALS; generating standard curves; determining a specificity and/or sensitivity of an assay or marker; calculating an ROC curve; sequence analysis; all as described herein or is known in the art.
  • the amount of one or more mRNAs can be determined by abundance, measured per mole or millimole.
  • the amount of mRNAs can be determined by fluorescence, other measurement using an optical signal or other measurement known to one of skill to measure levels of mRNAs.
  • a microprocessor or computer uses an algorithm to measure the amount of an mRNA or multiple mRNAs.
  • the algorithm can include a mathematical interaction between a marker measurement or a mathematical transform of a marker measurement.
  • the mathematical interaction and/or mathematical transform can be presented in a linear, nonlinear, discontinuous or discrete manner.
  • a software or hardware component receives the results of multiple assays and determines a single value result to report to a user that indicates an ALS risk based on the results of the multiple assays.
  • Related embodiments calculate a risk factor based on a mathematical combination (e.g., a weighted combination, a linear combination) of the results from multiple assays as disclosed herein.
  • Some embodiments include a storage medium and memory components.
  • Memory components e.g., volatile and/or nonvolatile memory find use in storing instructions (e.g., an embodiment of a process as provided herein) and/or data (e.g., a work piece such as methylation measurements, sequences, and statistical descriptions associated therewith).
  • Some embodiments relate to systems also comprising one or more of a CPU, a graphics card, and a user interface (e.g., comprising an output device such as display and an input device such as a keyboard).
  • Programmable machines associated with the technology comprise conventional extant technologies and technologies in development or yet to be developed (e.g., a quantum computer, a chemical computer, a DNA computer, an optical computer, a spintronics based computer, etc.).
  • the technology includes a wired (e.g., metallic cable, fiber optic) or wireless transmission medium for transmitting data.
  • a wired e.g., metallic cable, fiber optic
  • some embodiments relate to data transmission over a network (e.g., a local area network (LAN), a wide area network (WAN), an ad-hoc network, the Internet, etc.).
  • a network e.g., a local area network (LAN), a wide area network (WAN), an ad-hoc network, the Internet, etc.
  • programmable machines are present on such a network as peers and in some embodiments the programmable machines have a client/server relationship.
  • data are stored on a computer-readable storage medium such as a hard disk, flash memory, memory stick, optical media, a floppy disk, etc.
  • the technology provided herein is associated with a plurality of programmable devices that operate in concert to perform a method as described herein.
  • a plurality of computers e.g., connected by a network
  • may work in parallel to collect and process data e.g., in an implementation of cluster computing or grid computing or some other distributed computer architecture that relies on complete computers (with onboard CPUs, storage, power supplies, network interfaces, etc.) connected to a network (private, public, or the Internet) by a conventional network interface, such as Ethernet, fiber optic, or by a wireless network technology.
  • a network private, public, or the Internet
  • some embodiments provide a computer that includes a computer-readable medium.
  • the embodiment includes a random access memory (RAM) coupled to a processor.
  • the processor executes computer-executable program instructions stored in memory.
  • processors may include a microprocessor, an ASIC, a state machine, or other processor, and can be any of a number of computer processors, such as processors from Intel Corporation of Santa Clara, Calif, and Motorola Corporation of Schaumburg, III.
  • processors include, or may be in communication with, media, for example computer-readable media, which stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.
  • Embodiments of computer-readable media can include an electronic, optical, magnetic, or other storage or transmission device capable of providing a processor with computer-readable instructions.
  • suitable media include, but are not limited to, a floppy disk, CD-ROM, DVD, magnetic disk, memory chip, ROM, RAM, an ASIC, a configured processor, all optical media, all magnetic tape or other magnetic media, or any other medium from which a computer processor can read instructions.
  • various other forms of computer-readable media may transmit or carry instructions to a computer, including a router, private or public network, or other transmission device or channel, both wired and wireless.
  • the instructions may comprise code from any suitable computer-programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, and JavaScript.
  • Computers are connected in some embodiments to a network.
  • Computers may also include a number of external or internal devices and peripherals such as a mouse, a CD-ROM, DVD, a keyboard, a display, or other input or output devices.
  • Examples of computers are personal computers, digital assistants, personal digital assistants, cellular phones, mobile phones, smart phones, pagers, digital tablets, laptop computers, internet appliances, and other processor-based devices.
  • the computers related to aspects of the technology provided herein may be any type of processor-based platform that operates on any operating system, such as Microsoft Windows, Linux, UNIX, Mac OS X, etc., capable of supporting one or more programs comprising the technology provided herein.
  • Some embodiments comprise a personal computer executing other application programs (e.g., applications).
  • the applications can be contained in memory and can include, for example, a word processing application, a spreadsheet application, an email application, an instant messenger application, a presentation application, an Internet browser application, a calendar/organizer application, and any other application capable of being executed by a client device.
  • embodiments could be accomplished as computer signals embodied in a carrier wave, as well as signals (e.g., electrical and optical) propagated through a transmission medium.
  • signals e.g., electrical and optical
  • the various types of information discussed above could be formatted in a structure, such as a data structure, and transmitted as an electrical signal through a transmission medium or stored on a computer readable medium.
  • the disclosure provides a system for predicting progression of ALS.
  • the disease can be asymptomatic, early-stage, mid-stage or latestage ALS.
  • ALS can be identified in an individual using a system that includes: an apparatus configured to determine expression levels of nucleic acids, proteins, peptides or other molecule from a biological sample taken from the individual; and hardware logic designed or configured to perform operations including: (a) receiving expression levels of a collection of signature genes/proteins/peptides from a biological sample taken from said individual, wherein the collection of signature genes/proteins/peptides includes at least one mRNA from those listed in Table 1.
  • Information relevant to the patient's diagnosis include, but are not limited to, age, ethnicity, tumor localization, pertinent past medical history related to co-morbidity, other oncological history, family history for ALS, physical exam findings, radiological findings, biopsy date, biopsy result, local vs. distant disease recurrence and survival outcome. These clinical variables may be included in the predictive model in various embodiments.
  • a biomarker or biomarker panel can be used in a method for diagnosing ALS in an individual.
  • a biomarker or biomarker panel is selected, a method for diagnosing an individual that may be suffering from ALS and can include one or more of the following steps: 1 ) collect or otherwise obtain a biological sample; 2) perform an analytical method to detect and measure the biomarker or biomarkers in the panel in the biological sample; 3) perform any data normalization or standardization required for the method used to collect biomarker values; 4) calculate a biomarker score; 5) combine the biomarker scores to obtain a total diagnostic score; and 6) report the individual's diagnostic score.
  • the diagnostic score may be a single number determined from the sum of all the marker calculations that is compared to a preset threshold value that is an indication of the presence or absence of disease.
  • the diagnostic score may be a series of bars that each represent a biomarker value and the pattern of the responses may be compared to a pre-set pattern for determination of the presence or absence of disease.
  • the nucleic acid can be isolated from a saliva, plasma or blood sample.
  • the DNA or RNA can be extracellular or extracted from a cell in the plasma or blood sample.
  • the DNA or RNA can also be extracted from a cellular biopsy, including from a tumor, including, a solid tumor in the breast.
  • a protein or peptide or other biological molecule such can be isolated from a saliva, plasma or a blood sample.
  • the protein or peptide or other biological molecule can be extracellular or extracted from a cell in the saliva, plasma or a blood sample.
  • the protein or peptide or other biological molecule can also be extracted from a cellular biopsy, including from a tumor, including, a solid tumor in the breast.
  • the ALS biomarker analysis system can provide functions and operations to complete data analysis, such as data gathering, processing, analysis, reporting and/or diagnosis.
  • the computer system can execute the computer program that may receive, store, search, analyze, and report information relating to the biomarkers.
  • the computer program may comprise multiple modules performing various functions or operations, such as a processing module for processing raw data and generating supplemental data and an analysis module for analyzing raw data and supplemental data to generate ALS status and/or diagnosis. Diagnosing the status of ALS can include generating or collecting any other information, including additional biomedical information, regarding the condition of the individual relative to the disease, identifying whether further tests may be desirable, or otherwise evaluating the health status of the individual.
  • the ALS biomarker analysis system can provide functions and operations to complete data analysis, such as data gathering, processing, analysis, reporting and/or diagnosis.
  • the computer system can execute the computer program that may receive, store, search, analyze, and report information relating to the ALS biomarkers.
  • the computer program can include multiple modules performing various functions or operations, such as a processing module for processing raw data and generating supplemental data and an analysis module for analyzing raw data and supplemental data to generate an ALS status and/or diagnosis.
  • Diagnosing ALS status can include generating or collecting any other information, including additional biomedical information, regarding the condition of the individual relative to the disease, identifying whether further tests may be desirable, or otherwise evaluating the health status of the individual.
  • a "computer program product” refers to an organized set of instructions in the form of natural or programming language statements that are contained on a physical media of any nature (e.g., written, electronic, magnetic, optical or otherwise) and that may be used with a computer or other automated data processing system. Such programming language statements, when executed by a computer or data processing system, cause the computer or data processing system to act in accordance with the particular content of the statements.
  • Computer program products include without limitation: programs in source and object code and/or test or data libraries embedded in a computer readable medium.
  • the computer program product that enables a computer system or data processing equipment device to act in pre-selected ways may be provided in a number of forms, including, but not limited to, original source code, assembly code, object code, machine language, encrypted or compressed versions of the foregoing and any and all equivalents.
  • a computer program product for indicating a likelihood of ALS.
  • the computer program product includes a computer readable medium embodying program code executable by a processor of a computing device or system, the program code comprising: code that retrieves data attributed to a biological sample from an individual, wherein the data comprises biomarker values that each correspond to one of at least N biomarkers in the biological sample selected from the group of biomarkers provided in Table 1 and code that executes a classification method that indicates a ALS status of the individual as a function of the biomarker values.
  • a computer program product for indicating a likelihood of ALS.
  • the computer program product includes a computer readable medium embodying program code executable by a processor of a computing device or system, the program code comprising: code that retrieves data attributed to a biological sample from an individual, wherein the data comprises a biomarker value corresponding to a biomarker in the biological sample selected from the group of biomarkers provided in Table 1 ; and code that executes a classification method that indicates a ALS status of the individual as a function of the biomarker value.
  • the kit i.e. , diagnostic kit
  • the kit can include reagents for determining, from a plasma sample of a subject, the amount of mRNAs or mutations in a gene based on assaying the nucleic acids, proteins, peptides or other biological molecule isolated from ALS, a circulating cell or the remnants of a circulating cell present in plasma, including a protein, peptide or other biological molecule.
  • the nucleic acid can be a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA) and/or an artificial nucleic acid, including an artificial nucleic acid analogue.
  • RNAs include non-coding RNA (ncRNA), transfer RNA (tRNA), messenger RNA (mRNA), small interfering RNA (siRNA), PIWI RNA (piRNA), small nuclear RNA (snoRNA), small nuclear (snRNA), extracellular RNA (exRNA), and ribosomal RNA (rRNA).
  • ncRNA non-coding RNA
  • tRNA transfer RNA
  • mRNA messenger RNA
  • siRNA small interfering RNA
  • piRNA small nuclear RNA
  • snoRNA small nuclear RNA
  • snRNA small nuclear RNA
  • snRNA small nuclear RNA
  • snRNA extracellular RNA
  • rRNA ribosomal RNA
  • the disclosed methods and assays provide for convenient, efficient, and potentially cost-effective means to obtain data and information useful in assessing appropriate or effective therapies for treating patients.
  • the kit can use conventional methods for detecting the biomarkers, whether a protein, peptide, other biological molecule or an RNA or a DNA to be assessed include protocols that examine the presence and/or expression of a desired nucleic acid, for example a SNP, in a sample.
  • Tissue or cell samples from mammals can be conveniently assayed for, e.g., RNA-Seq, genetic-marker RNA, including in an embodiment an mRNA or DNAs using Northern, dot-blot, or polymerase chain reaction (PCR) analysis, array hybridization, RNase protection assay, or using DNA SNP chip microarrays, which are commercially available, including DNA micro array snapshots.
  • RNA-Seq genetic-marker RNA
  • mRNA or DNAs including in an embodiment an mRNA or DNAs using Northern, dot-blot, or polymerase chain reaction (PCR) analysis, array hybridization, RNase protection assay, or using DNA SNP chip microarrays, which are commercially available, including DNA micro array snapshots.
  • RT-PCR real-time PCR
  • Probes used for PCR can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme.
  • a detectable marker such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme.
  • Such probes and primers can be used to detect the presence of a mutation in a DNA, an RNA and in one embodiment, an mRNA in a sample and as a means for detecting a cell expressing the mRNA.
  • a great many different primers and probes can be prepared based on known sequences and used effectively to amplify, clone, and/or determine the presence and/or levels of mRNAs.
  • FISH fluorescence in situ hybridization
  • Other methods include protocols that examine or detect a mutation in a DNA or an RNA. These other methods include protocols that examine or detect mRNAs in a tissue or cell sample by microarray technologies.
  • test and control RNAs including in an embodiment, mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes that have potential to be expressed in certain disease states can be arrayed on a solid support.
  • Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.
  • Differential gene expression analysis of disease tissue can provide valuable information.
  • Microarray technology utilizes nucleic acid hybridization techniques and computing technology to evaluate the mRNA expression profile of thousands of genes within a single experiment.
  • the biomarkers are particularly useful in ALS diagnosis as their expression patterns is different when comparing healthy subjects with subjects that have ALS.
  • the expression of biomarkers typically includes both up- and down-regulated levels of mRNAs.
  • the biomarkers set forth herein can determine if a patient has ALS or does not have ALS.
  • progression of ALS can be determined (e.g., asymptomatic, early-stage, mid-stage or late-stage ALS).
  • Table 1 includes a list of mRNA biomarkers for detecting Amyotrophic lateral sclerosis.
  • FIG. 2 is a list of the top twenty diagnostic biomarkers for detection and/or prognosis of amyotrophic lateral sclerosis (ALS v. Healthy Controls).
  • the short-list includes the following markers: ABCA1 , ATP2B4, ZNF652, CAPZA2, LDLR, VPS13C, MGC24103, PSMC4, IL2RB, MEGF9, NUDT18, TFIP11 , UBE2D2, PLA2G2D, HNRPUL2, ZCCHC17, FAR1 , MAP2K1 , SCRN1 and CTSW.
  • FIG. 3 is a list of the top twenty diagnostic biomarkers for detection and/or prognosis of amyotrophic lateral sclerosis (ALS v. ALS-mimicking diseases).
  • the short-list includes the following markers: MIR29B2CHG, SCRN1 , ATP5J, POLR3C, NLRP1 , CLN5, EBNA1 BP2, MRPS33, LSM5, FSTL4, CHD9, FER1 L3, DNAJA3, CTSW, PAPD4, TFIP11 , NME1 , FAR1 , NUDT18 and TLR2.
  • FIG. 4 is a list of the top twenty diagnostic biomarkers for detection and/or prognosis of amyotrophic lateral sclerosis (ALS v. combined healthy and ALS-mimicking diseases).
  • the short-list includes the following markers: SCRN1 ABCA1 , CAPZA2, IL2RB, ATP2B4, LDLR, ZNF652, MEGF9, ARHGAP30, VPS13C, HNRPUL2, ZCCHC17, NUDT1 8, PSMC4, MGC24103, MAP2K1 , TFIP11 , FAR1 , PLA2G2D and ATP5J.
  • FIG. 5 is a list of these markers and includes: ABCA1 , C19orf59, CTSW, IL2RB, FER1 L3, CHD9, IP6K1 , CAPZA2, PSMC4, MEGF9, VPS13C, NUDT18, NCALD, HIPK2, LDLR, FAR1 , TFIP11 , IKBIP, SCRN1 and FSTL4.
  • Levels of biomarkers can be determined from techniques known in the art. Other methods for determining the level of the biomarker besides RT-PCR or another PCR-based method include proteomics techniques, as well as individualized genetic profiles. Individualized genetic profiles can be used to treat ALS based on patient response at a molecular level.
  • the specialized microarrays herein, e.g., oligonucleotide microarrays or cDNA microarrays
  • the one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers can be stored in a liquid or in a dry form, including, following lyophilization.
  • biomarkers are stored dry, the one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26,
  • biomarkers can be resuspended using water or a solution one of skill in the art would know would know would result in the stable resuspension of the one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers.
  • One or more of the biomarkers can be used in a method of diagnosing amyotrophic lateral sclerosis (ALS) or determining a prognosis of a test subject with ALS.
  • ALS amyotrophic lateral sclerosis
  • one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers can be used in a method of diagnosing ALS or determining a prognosis of a test subject with ALS.
  • At least one biomarker or a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers can be used in a method of diagnosing ALS or determining a prognosis of a test subject with ALS.
  • biomarkers can be used in a method of diagnosing ALS or determining a prognosis of a test subject with ALS. In this manner, about one biomarker or a combination of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 ,
  • biomarkers can be used in a method of diagnosing ALS or determining a prognosis of a test subject with ALS.
  • the expression levels of one or more mRNAs are measured in blood (or saliva) samples from subjects with ALS.
  • the expression levels of one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers is used to generate a footprint or signature for subsequent diagnosis of patients.
  • the expression levels of at least one biomarker or a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers is used to generate a footprint or signature for subsequent diagnosis of patients.
  • the expression levels of no more than one biomarker or a combination of no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers is used to generate a footprint or signature for subsequent diagnosis of patients.
  • the expression levels of about one biomarker or a combination of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers is used to generate a footprint or signature for subsequent diagnosis of patients.
  • expression levels of the same nucleic acids including DNA and/or RNA and further are measured in a sample (e.g., blood or saliva) from healthy subjects. This is used as a control. Thereafter, samples from healthy patients can be compared to identify mRNAs that have altered levels of expression in the plasma samples from the subjects with ALS.
  • a biomarker fingerprint or signature can be created from the mRNAs with altered levels of expression. This can be used for diagnosing or determining the prognosis of ALS in the test subject by comparing of levels of mRNAs from the sample of the test subject. Conventional statistical analysis can be used to determine, for example, confidence levels. The same approach can be used to identify protein biomarkers, generate a biomarker footprint and diagnose or determine the prognosis of ALS in a test subject by comparing of levels of protein biomarkers.
  • Embodiments include a method of identifying an initial set of peripheral diagnostic biomarkers for detecting ALS and validating these biomarkers on additional datasets.
  • the method can be implemented by the steps of: a) Producing binary classifiers to distinguish between patients with a confirmed ALS diagnosis and patients with a confirmed negative ALS diagnosis; b) Identifying the first minimal set of biomarkers that maximizes diagnostic accuracy; c) Setting aside the first minimal set and repeat the analysis with the remaining biomarkers, to determine the next minimal set; and d) Repeating the above step until observing a significant decline in diagnostic accuracy.
  • markers e.g., four or less
  • ALS amyotrophic lateral sclerosis
  • the doctor draws a sample of blood and sends it to a lab to test for ALS.
  • the blood sample is prepared and the plasma is obtained.
  • the plasma is then tested to identify the presence of biomarkers associated with ALS.
  • the lab uses one or more of the following mRNA biomarkers in its test: CAPZA2, LDLR, C7orf42, CHD9, NCALD, IL2RB, MGC24103, VPS13C, MEGF9, ZCCHC17, FAR1 , ABCA1 , HIPK2, IKBIP, PLA2G2D, MAP2K1 , TFIP11 , SCRN1 , HNRPUL2, C19orf59, MCM3, FSTL4, PSMC4, UBE2D2, DDHD2, ATP2B4, DPAGT1 , FER1 L3, CTSW, STUB1 , CLN5, WDR23, RPL10P6, BBS2, ZNF652, IP6K1 , PAPD4, TLR2, C1orf144, NUDT18, DNAJA3, ATP5J, ARHGAP30, MIR29B2CHG, P0LR3C, NME1 , NLRP1 , LSM5, S0D1 , C14ORF159
  • the lab determines that the one or more biomarkers used to test for ALS are indicative of the presence of ALS.
  • the lab sends the test results to the doctor.
  • the patient Upon a subsequent visit to the doctor, the patient is informed of the result.
  • the patient can be administered therapeutics to help prevent ALS signs/symptoms.
  • the doctor draws a sample of blood and sends it to a lab to test for ALS.
  • the blood sample is prepared and the plasma is obtained.
  • the plasma is then tested to identify the presence of biomarkers associated with ALS.
  • the lab uses one or more of the following mRNA biomarkers listed above.
  • the lab determines that the one or more biomarkers used to test for ALS are indicative early stages of ALS (i.e. , stage 1 ). Upon a subsequent visit to the doctor, the patient is informed of the result. The patient can be administered therapeutics to help prevent the disease from progressing.
  • the patient is re-tested on a monthly basis to monitor progress of the disease. As the disease progresses, a group of healthcare professionals focuses on treating symptoms and providing supportive care.
  • Embodiments of the invention can be compiled into a diagnostic kit for diagnosing ALS.
  • the kit can identify one or more target cells that have the biomarkers for ALS in blood (whole blood, plasma or serum) from a test subject.
  • the kit can include a collection of nucleic acid molecules such that each nucleic acid molecule encodes a mRNA sequence.
  • the nucleic acid molecules can be used to identify variations in expression levels of one or more mRNAs in a plasma sample from a test subject.
  • the expression levels of the mRNAs can be used in a comparison/analysis of test samples with a fingerprint indicative of the presence of ALS.
  • the same approach can be used to identify protein biomarkers, generate a biomarker footprint and diagnose or determine the prognosis of ALS in the test subject by comparing of levels of protein biomarkers.
  • kits for diagnosing ALS can include one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers disclosed herein.
  • the skilled artisan will appreciate that the number of biomarkers may be varied without departing from the nature of the present disclosure, and thus other combinations of biomarkers are also encompassed by the present disclosure.
  • biomarkers or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers to use based on the symptoms of the patient suffering from ALS.
  • a kit includes the one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers disclosed herein.
  • the kit is for diagnosing ALS.
  • the kit can further optionally include instructions for use.
  • the kit can further optionally include (e.g., comprise, consist essentially of, consist of) tubes, applicators, vials or other storage container with the above-mentioned biomarker and/or vials containing one or more of the biomarkers.
  • each biomarker is in its own tube, applicator, vial or storage container or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,
  • biomarkers are in a tube, applicator, vial or storage container.
  • kits regardless of type, will generally include one or more containers into which the one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14,
  • kits 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 biomarkers are placed and, preferably, suitably aliquotted.
  • the components of the kits may be packaged either in aqueous media or in lyophilized form.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Evolutionary Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medical Informatics (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Theoretical Computer Science (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Des modes de réalisation de l'invention comprennent un système et un procédé d'utilisation de biomarqueurs dans la détection ou le diagnostic de la sclérose latérale amyotrophique (SLA). Un sujet peut faire l'objet d'un dépistage de la SLA sur la base de l'expression modifiée d'un ou de plusieurs biomarqueurs dans le sang, le plasma ou la salive du sujet. Les marqueurs peuvent distinguer la SLA d'autres affections avec des signes/symptômes similaires (c'est-à-dire, des analogues de SLA). Des modes de réalisation comprennent 52 biomarqueurs d'ARNm spécifiques pour dépister ou distinguer des individus sains d'individus atteints de la SLA. Les biomarqueurs peuvent également être utilisés pour déterminer le pronostic d'un sujet atteint de la maladie et identifier une SLA précoce et/ou asymptomatique.
PCT/US2023/076301 2022-10-07 2023-10-06 Biomarqueurs pour le diagnostic de la sclérose latérale amyotrophique WO2024077282A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263414453P 2022-10-07 2022-10-07
US63/414,453 2022-10-07

Publications (1)

Publication Number Publication Date
WO2024077282A2 true WO2024077282A2 (fr) 2024-04-11

Family

ID=90609082

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/076301 WO2024077282A2 (fr) 2022-10-07 2023-10-06 Biomarqueurs pour le diagnostic de la sclérose latérale amyotrophique

Country Status (1)

Country Link
WO (1) WO2024077282A2 (fr)

Similar Documents

Publication Publication Date Title
Bergemalm et al. Systemic inflammation in preclinical ulcerative colitis
Planell et al. Usefulness of transcriptional blood biomarkers as a non-invasive surrogate marker of mucosal healing and endoscopic response in ulcerative colitis
EP3169814B1 (fr) Procédés pour évaluer le stade d'un cancer du poumon
EP3304093B1 (fr) Validation de mesure de biomarqueurs
CN106978480A (zh) 用于癌症的分子诊断试验
EP2942724A2 (fr) Procédé pour le diagnostic in vitro d'une maladie complexe
CN105603101B (zh) 检测8个miRNA表达量的系统在制备诊断或辅助诊断肝细胞癌产品中的应用
EP3408413B1 (fr) Prédiction d'une réponse thérapeutique dans des états inflammatoires
CN110383070A (zh) 癌生物标记
WO2013049152A2 (fr) Procédés pour évaluer le statut du cancer du poumon
US20230348980A1 (en) Systems and methods of detecting a risk of alzheimer's disease using a circulating-free mrna profiling assay
CN114875149A (zh) 检测生物标志物的试剂在制备预测胃癌预后产品中的应用
JP2023504334A (ja) 肺がんの診断のためのバイオマーカー
CN114990215A (zh) microRNA生物标志物在肺癌诊断或预测预后中的应用
CN102089443A (zh) 用于测定受试者中结肠直肠癌的概率的方法和装置
WO2024077282A2 (fr) Biomarqueurs pour le diagnostic de la sclérose latérale amyotrophique
US20230295727A1 (en) Biomarkers for the Diagnosis of Parkinson's Disease
US20240035090A1 (en) mRNA BIOMARKERS FOR DIAGNOSIS OF LIVER DISEASE
US20190127800A1 (en) Salivary Extracellular RNA Biomarkers for Gingivitis
EP3146455A2 (fr) Signatures moléculaires pour distinguer des rejets de greffe hépatique ou des lésions hépatiques
WO2020021028A1 (fr) Biomarqueurs de diagnostic et/ou de pronostic de fragilité
US20170121774A1 (en) Methods and compositions for assessing predicting responsiveness to a tnf inhibitor
JP7491847B2 (ja) 疼痛のための精密医療：診断バイオマーカー、薬理ゲノミクス、およびリパーパス薬
JP2011004743A (ja) 関節リウマチ患者におけるインフリキシマブ薬効の有効性を判別する方法
US20240071622A1 (en) Clinical classifiers and genomic classifiers and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23875881

Country of ref document: EP

Kind code of ref document: A2