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WO2025226885A1 - Biomarqueur de l'inflammation intestinale dans les selles pour aider à la détection et au traitement d'une maladie intestinale inflammatoire - Google Patents

Biomarqueur de l'inflammation intestinale dans les selles pour aider à la détection et au traitement d'une maladie intestinale inflammatoire

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Publication number
WO2025226885A1
WO2025226885A1 PCT/US2025/026089 US2025026089W WO2025226885A1 WO 2025226885 A1 WO2025226885 A1 WO 2025226885A1 US 2025026089 W US2025026089 W US 2025026089W WO 2025226885 A1 WO2025226885 A1 WO 2025226885A1
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WIPO (PCT)
Prior art keywords
mtdna
subject
amount
sample
intestinal inflammation
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English (en)
Inventor
Joe Brown
Kevin Zhu
Thomas Wallach
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University of North Carolina at Chapel Hill
Research Foundation of the State University of New York
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University of North Carolina at Chapel Hill
Research Foundation of the State University of New York
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Publication of WO2025226885A1 publication Critical patent/WO2025226885A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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 invention relates to a biomarker associated with intestinal inflammation, and more particularly, to methods for diagnosing or identifying a risk of developing intestinal inflammation using mitochondrial DNA and methods of treating disorders associated with intestinal inflammation.
  • IBD Inflammatory Bowel Disease
  • UC Ulcerative Colitis
  • CD Crohn’s Disease
  • FC fecal calprotectin
  • EED Environmental Enteropathic Dysfunction
  • NEC Necrotizing Enterocolitis
  • the present invention is based on the discovery that the levels of mitochondrial DNA (mtDNA) in stool samples can serve as a biomarker for the presence and/or risk of intestinal inflammation as it relates to various gastrointestinal disorders in a subject. Mitochondrial DNA levels can also serve to predict the risk of gastrointestinal disorders as well as staging of disease. In particular, the inventors have shown that the level of mtDNA is indicative of the presence, stage, risk, and/or responsiveness of a disease characterized by intestinal inflammation.
  • mtDNA mitochondrial DNA
  • one aspect of the present invention relates to a method for detecting intestinal inflammation in a subject, comprising: i) obtaining a stool sample from the subject; and ii) quantitating the amount of mitochondrial DNA (mtDNA) in the sample; wherein the presence of an increased amount of mtDNA in the sample relative to the amount in a control population indicates the presence of intestinal inflammation in the subject.
  • mtDNA mitochondrial DNA
  • the present invention relates to determining the severity of intestinal inflammation in a subject having or suspected of having intestinal inflammation, comprising: i) obtaining a stool sample from the subject; and ii) quantitating the amount of mtDNA in the sample; wherein the amount of mtDNA in the sample relative to the amount in a control population indicates the severity of intestinal inflammation in the subject.
  • the present invention relates to a method of determining the risk of intestinal inflammation in a subject, comprising: i) obtaining a stool sample from the subject; and ii) quantitating the amount of mtDNA in the sample; wherein the presence of an increased amount of mtDNA in the sample relative to the amount in a control population indicates the subject is at risk for intestinal inflammation.
  • the present invention relates to a method of treating intestinal inflammation in a subject in need thereof, comprising: i) obtaining a stool sample from the subject; ii) quantitating the amount of mtDNA in the sample; wherein the amount of mtDNA in the sample relative to the amount in a control population indicates the severity of intestinal inflammation in the subject; and iii) administering a therapy to the subject based on the severity of the inflammation.
  • the present invention relates to a method of monitoring the effectiveness of a treatment for intestinal inflammation in a subject in need thereof, comprising: i) obtaining a stool sample from the subject at two or more timepoints before, during, and/or after the treatment; and ii) quantitating the amount of mtDNA in each of the samples; wherein an increase or no change in the amount of mtDNA in the sample over time indicates that the treatment is ineffective and a decrease in the amount of mtDNA in the sample over time indicates that the treatment is effective.
  • the present invention relates to a method of guiding treatment for intestinal inflammation in a subject in need thereof, said method comprising: i) obtaining a stool sample from the subject at two or more timepoints before, during, and/or after the treatment; ii) quantitating the amount of mtDNA in each of the samples; wherein an increase or no change in the amount of mtDNA in the sample over time indicates that the treatment is ineffective and a decrease in the amount of mtDNA in the sample over time indicates that the treatment is effective; and iii) modifying the treatment administered to the subject based on the effectiveness of previous treatment.
  • the present invention relates to a method of determining if a subject being treated for intestinal inflammation is in histological remission, comprising: i) obtaining a stool sample from the subject; and ii) quantitating the amount of mtDNA in the sample; wherein the presence of an amount of mtDNA in the sample that is equal to or lower than the amount in a control population indicates that the subject is in histological remission.
  • kits for carrying out the disclosed methods comprise reagents for detecting mtDNA in a stool sample.
  • the kit comprises one or more primers and/or probes for amplifying mtDNA.
  • FIGS. 1A-1D show concentration of fecal calprotectin (FIGS. 1A and 1C) and fecal mtDNA (FIGS. IB and ID) with increasing severity of IBD from histological remission to severe disease from samples from 13 IBD patients (FIGS. 1A and IB).
  • Scatter plot shows non-parametric correlations between scoring from Robart’s Histopathology Index and fecal calprotectin (FIG. 1C) and fecal mtDNA (FIG. ID) from the same 13 samples.
  • FIG. 2 shows a summary table of individual patients in Example 1.
  • FIG. 3 shows the negative and positive bands by comparing them with the no template controls and positive controls run on each plate.
  • the midpoint value is between the peaks of the positive and negative bands and sets the threshold at the midpoint value.
  • FIG. 4 shows the negative extraction controls in the far left two wells, no template controls (UV-treated molecular grade water) in the middle two wells, and positive controls (human stools) in the far right two wells.
  • FIG. 5 shows fecal calprotectin and mtDNA concentrations with accompanying metadata of IBD patients.
  • FIG. 7 shows a principal component analysis on mtDNA and other biomarkers. Results show weak pairwise correlations with each of the EED biomarkers. The mtDNA eigenvector was roughly orthogonal to the other biomarker eigenvectors.
  • the term "about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified amount.
  • Treat” or “treating” or “treatment” refers to any type of action that imparts a modulating effect, which, for example, can be a beneficial effect, to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay or reduction in the progression of the condition, and/or change in clinical parameters, disease or illness, etc., as would be well known in the art.
  • Altered level or altered levels refer to an increased level (e.g., a 5, 10, 50, 100-fold increase, or more) or a decreased level (e.g., a one or two-fold decrease, or more) in the quantity of one or more biomarker detectable in or via a biological sample from a subject, as compared to a level or levels of one or more biomarker in a control (e.g., corresponding subject not afflicted with intestinal inflammation or a an average level in a population).
  • a presence or absence of a detectable amount mtDNA may also be considered an altered level.
  • Bio sample refers to any material taken from the body of a subject that may carry the target compound or compounds of the tests described herein, including both tissue samples and biological fluids such as blood samples, stool samples, saliva samples, urine samples, etc.
  • Stool sample refers to whole feces or stool and/or any fraction thereof that may contain detectable levels of the biomarker mtDNA therein (if the biomarker mtDNA is present in the whole feces/stool sample from which said fraction is obtained), and in particular embodiments, refers to a fecal sample.
  • Diagnosing means providing an indication that a subject may be afflicted with or at risk of developing a disease, particularly a gastrointestinal disorder causing inflammation in the intestine, and includes other terms such as screening for a disease, providing a risk assessment for disease, etc. It will be appreciated that no such technique is perfect and that such diagnosis, prognosis, or the like may be confirmed by other procedures such as physical examination, imaging, histological examination of tissue samples, etc.
  • prognosing includes providing an assessment or indication of disease in response to treatment (such as but not limited to antiinflammatory drugs, immunosuppressant drugs, biologies, antibiotics, dietary changes, surgery, and combinations thereof) after initial diagnosis, as an indication of the efficacy of the diagnostic and/or treatment, risk of the disease returning, severity of disease following treatment, or the like.
  • treatment such as but not limited to antiinflammatory drugs, immunosuppressant drugs, biologies, antibiotics, dietary changes, surgery, and combinations thereof
  • Marker or “biomarker” as used herein refers to mtDNA that can be detected, directly or indirectly in a biological sample from a subject, an increase or decrease of the amount of which, compared to amounts found in similar subjects without disease or in the general population (e.g., controls), is indicative of the presence or risk of intestinal inflammation.
  • Mitochondrial DNA "human Mitochondrial DNA” or "fecal mitochondrial DNA” as used herein refers to a circular molecule of about 16,600 base pairs long and comprising 37 genes, which encode 13 proteins, 22 tRNAs, and 2 rRNAs. The vast majority of about 3,000 proteins involved in mitochondrial functions, however, are encoded by the nuclear genome.
  • Cytochrome b refers to the region of mtDNA encoding cytochrome b and in some embodiments can be the region targeted for measurement of mtDNA.
  • Subjects as described herein include human subjects and “patients” as well as other mammals in veterinarian or research settings.
  • the subjects may be male or female and may be of any race or ethnicity, including but not limited to Caucasian, African American, African, Asian, Hispanic, Indian, etc.
  • the subjects may be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric.
  • Subjects may also include animal subjects, particularly mammalian subjects such as dog, cat, horse, mouse, rat, etc., e.g., screened for veterinary medicine or pharmaceutical drug development purposes.
  • Subjects include but are not limited to those who may have, possess, have been exposed to, or have been previously diagnosed as afflicted with one or more risk factors for intestinal inflammation.
  • Risk factors include age, gender, race, smoking, diet, obesity, diabetes, work exposure, and family history. These risk factors may be considered in combination with the disclosed methods of detecting intestinal inflammation for a diagnosis, prognosis, or screening. The disclosed methods of detecting intestinal inflammation for a diagnosis, prognosis or screening may also be used in combination with other diagnostic methods.
  • a "value" may be an amount of mtDNA in a sample, a score based on the detected amounts of biomarkers mtDNA in a sample, a percentile based on a population of patients, a quantitative amount or semi-quantitative amount of mtDNA in a sample, or other suitable quantities based on the detected biomarkers.
  • the present invention is based on the discovery that the levels of mtDNA can serve as a biomarker for the presence and/or risk of intestinal inflammation as it relates to various gastrointestinal disorders in a subject.
  • mtDNA levels may serve to predict the risk of gastrointestinal disorders as well as staging of disease and monitoring of treatment.
  • levels of mtDNA may be indicative of the presence, stage, risk, and/or responsiveness of a disease characterized by intestinal inflammation.
  • elevated levels of extracellular mtDNA may serve as a biomarker of inflammation, e.g., as mtDNA is released into the extracellular environment upon cellular stress, damage, or early immune activation, and often precedes detectable histological or clinical signs of inflammation.
  • mtDNA levels may increase before overt inflammation, thereby providing predictive value for identifying individuals at risk of developing inflammatory conditions.
  • elevated mtDNA levels when elevated mtDNA levels are detected in conjunction with other biomarkers of immune activation or epithelial barrier dysfunction, elevated mtDNA levels may reflect the presence of active inflammation. It will be further appreciated that elevated levels can refer to predictive and/or diagnostic contexts in which mtDNA levels are analyzed in relation to temporal changes, baseline measurements, and accompanying clinical or molecular features indicative of inflammation severity or onset.
  • One advantage of the present invention is the ability of the disclosed methods to detect intestinal inflammation without invasive and/or expensive techniques. Another advantage is the ability of the disclosed methods to detect intestinal inflammation at different severity stages (e.g., at a potentially more treatable stage) then current techniques.
  • some embodiments of the present invention relate to a method for detecting intestinal inflammation in a subject, comprising: i) obtaining a stool sample from the subject; and ii) quantitating the amount of mitochondrial DNA (mtDNA) in the sample; wherein the presence of an increased amount of mtDNA in the sample relative to the amount in a control population indicates the presence of intestinal inflammation in the subject.
  • a control subject or a control population may comprise healthy individuals without clinical or histological evidence of intestinal inflammation, e.g., whose stool mtDNA levels represent a baseline reference level.
  • a control subject or a control population may be, e.g., age-, gender-, race-, and/or geography-matched to a subject, and confirmed to be devoid or substantially free of enteric infection or gastrointestinal symptoms at a time of sample collection.
  • a control subject or a control population may comprise individuals with normal levels of established fecal inflammatory markers, as would be understood by the skilled artisan in the relevant art, such as calprotectin or lactoferrin, and no recent history of antibiotic or anti-inflammatory medication use.
  • a control subject or a control population may be established by obtaining a stool sample from the subject or each member of the control population, and quantitating the amount of mitochondrial DNA (mtDNA) in the sample.
  • a stool sample can be obtained from each member of a population of healthy individuals without clinical or histological evidence of intestinal inflammation, and the amount of mtDNA in each sample quantitated to determine the level of mtDNA therein, e.g., averaging mtDNA levels among all members of the control population to determine a reference level.
  • a reference level of mtDNA may be established by obtaining a stool sample from a subject (e.g., each member of a control population), and quantitating the amount of mitochondrial DNA (mtDNA) in the sample.
  • intestinal inflammation may be related to various gastrointestinal disorders, including without limitation, Inflammatory Bowel Disease (IBD), which comprises Ulcerative Colitis (UC) and Crohn’s disease (CD), Necrotizing Enterocolitis (NEC), and Environmental Enteropathic Dysfunction (EED).
  • IBD Inflammatory Bowel Disease
  • UC Ulcerative Colitis
  • CD Crohn’s disease
  • NEC Necrotizing Enterocolitis
  • EED Environmental Enteropathic Dysfunction
  • a subject may be a subject having IBD, that has had IBD, or may be at risk for IBD.
  • a subject at risk for IBD may be one that has a family history of the disease, may be genetically predisposed to the disease, or may have symptoms, habits, or other disease(s) known to lead to said disease.
  • a subject may have NEC, may have had NEC, or may be at risk for NEC.
  • a subject may have EED, may have had EED, or may become at risk for EED.
  • a subject at risk for EED may be a subject that has a family history of EED, is genetically predisposed to EED, has recurrent exposure to enteropathogens, or has symptoms, habits, or other disease known to lead to EED.
  • the present invention relates to a method of determining the severity of intestinal inflammation in a subject having or suspected of having intestinal inflammation, comprising: i) obtaining a stool sample from the subject; and ii) quantitating the amount of mtDNA in the sample; wherein the amount of mtDNA in the sample relative to the amount in a control population indicates the severity of intestinal inflammation in the subject.
  • a method of determining the severity of intestinal inflammation in a subject may be used to identify the severity of intestinal inflammation related to a gastrointestinal disorder in a subject at risk for intestinal inflammation, that has exhibited symptoms of intestinal inflammation, or is known to have intestinal inflammation.
  • mtDNA levels in subjects with active inflammation can be about 0.5 to 2 orders of magnitude, or any value therebetween, greater than mtDNA levels in a control population.
  • mtDNA levels may be at least 2 orders of magnitude greater than a control population, including 2, 3, 4, 5 or greater orders of magnitude greater than a control population.
  • mtDNA levels may be at least 100-fold (i.e., 10,000%) greater than a control population, including increases of approximately 100-fold (10,000%), 1,000-fold (100,000%), 10,000-fold (1,000,000%), 100,000-fold (10,000,000%) or greater relative to a control population.
  • the method can provide an indication of severity of IBD that correlates with any of the known scoring systems.
  • the Pediatric Ulcerative Colitis Activity Index (PUCAI) for ulcerative colitis evaluates the severity of ulcerative colitis on a scale of less than 10: the disease is not present; 10-34: mild; 35-64: moderate; 65 or more: severe.
  • PUCAI Pediatric Ulcerative Colitis Activity Index
  • PCDAI Pediatric Crohn Disease Activity Index
  • PCDAI score of >30 has acceptable sensitivity and specificity to indicate disease of moderate/ severe activity.
  • a PCDAI decrease of 12.5 points or greater following therapeutic intervention accurately reflects a clinically significant response.
  • Another common scoring system for intestinal inflammation is Robarts Histopathology Index (RHI), which assesses four characteristics of mucosal activity, inflammatory infiltrate, lamina intestinal neutrophils, neutrophils in epithelium, and erosion or ulceration, all of which are rated on a scale of 0 to 3. Each characteristic is weighted (e.g., using respective multiplicative weights of 1, 2, 3 and 5) to produce a total score ranging from 0 (no disease activity) to 33 (most severe disease activity).
  • RHI Robarts Histopathology Index
  • fecal mtDNA may offer additional information and potential treatment options for lower and intermediate values of severity, where fecal calprotectin is insufficient for interpreting severity.
  • the present invention relates to a method of determining a risk of intestinal inflammation in a subject, comprising: i) obtaining a stool sample from the subject; and ii) quantitating the amount of mtDNA in the sample; wherein the presence of an increased amount of mtDNA in the sample relative to the amount in a control population indicates the subject is at risk for intestinal inflammation.
  • the method may be used to analyze the current condition of a subject (e.g., level of intestinal inflammation) and provide a determination of the potential for the subject to develop intestinal inflammation. Such a determination can be used to develop a treatment plan for the subject, such as when to start treatment and which treatment option to select or change.
  • a subject e.g., level of intestinal inflammation
  • Such a determination can be used to develop a treatment plan for the subject, such as when to start treatment and which treatment option to select or change.
  • the present invention relates to a method of treating intestinal inflammation in a subject in need thereof, comprising: i) obtaining a stool sample from the subject; ii) quantitating the amount of mtDNA in the sample; wherein the amount of mtDNA in the sample relative to the amount in a control population indicates the severity of intestinal inflammation in the subject; and iii) administering a therapy to the subject based on the severity of the inflammation.
  • a method as presented herein may be used to develop a treatment plan for the subject based on severity of inflammation.
  • anti-inflammatory drugs such as, without limitation, aminosalicylates, mesalamine (DELZICOL®, ROWASA®), balsalazide (COLAZAL®) and olsalazine (DIPENTUM®); corticosteroids; immunosuppressants such as azathioprine (AZASAN®, IMURAN®), mercaptopurine (PURINETHOL®, PURIXAN®) and methotrexate (TREXALL®); small molecules such as tofacitinib (XELJANZ), upadacitinib (RINVOQ®) and ozanimod (ZEPOSIA®); biologies, such as anti-TNF-alpha (e.g., REMICADE®, HUMIRA®, SIMPONI®), anti-interleukin (e.g
  • the present invention relates to a method of monitoring effectiveness of a treatment for intestinal inflammation in a subject in need thereof, comprising: i) obtaining a stool sample from a subject at two or more timepoints before, during, and/or after the treatment; and ii) quantitating the amount of mtDNA in each of the samples; wherein an increase or no change in the amount of mtDNA in the sample over time indicates that the treatment is ineffective and a decrease in the amount of mtDNA in the sample over time indicates that the treatment is effective.
  • a time point may be about 24hrs, 48hrs, 72hrs, 96hrs, 120hrs, 144hrs, 168hrs, 192hrs, 216hrs, 240hrs, 264hrs, 288hrs, 312hrs, 336hrs, or any value therebetween, before, during, and/or after the treatment.
  • a time point may be about from 24hrs to 72hrs, from 48hrs to 96hrs, from 72hrs to 120hrs, from 96hrs to 144hrs, from 120hrs to 168hrs, from 144hrs to 192hrs, from 168hrs to 216hrs, from 192hrs to 240hrs, from 216hrs to 264hrs, from 240hrs to 288hrs, from 264hrs to 312hrs, from 288hrs to 336hrs, or any range of values therebetween.
  • two or more time points can refer to obtaining a stool sample from a subject before and after treatment, such as 24hrs before treatment and 72hrs after treatment.
  • the method can also be used to monitor effectiveness of a treatment for IBD after said treatment has been started.
  • the present invention relates to a method of guiding treatment for intestinal inflammation in a subject in need thereof, said method comprising: i) obtaining a stool sample from the subject at two or more timepoints before, during, and/or after the treatment; ii) quantitating the amount of mtDNA in each of the samples; wherein an increase or no change in the amount of mtDNA in the sample over time indicates that the treatment is ineffective and a decrease in the amount of mtDNA in the sample over time indicates that the treatment is effective; and iii) modifying the treatment administered to the subject based on the effectiveness of previous treatment.
  • the method comprises obtaining one or more samples before initiation of treatment to establish a baseline mtDNA level, followed by obtaining one or more samples during the course of treatment, wherein an increase in mtDNA levels relative to baseline indicates an ineffective treatment response and prompts adjustment or escalation of therapy.
  • the method comprises obtaining one or more samples following cessation of treatment, wherein persistent elevation or rebound in mtDNA levels post-treatment indicates residual or recurring inflammation, thereby guiding decisions to resume or extend therapy.
  • samples are collected both during and after treatment, allowing for assessment of both immediate and sustained treatment efficacy.
  • a progressive decrease in mtDNA concentration across two or more time points may be indicative of a favorable therapeutic response, whereas stable or increasing levels across two or more time points may suggest inadequate treatment of inflammation and advise a change in the therapeutic approach
  • the method can also be used to monitor the effectiveness of a treatment after it has been started and modify the treatment based on the severity of the inflammation.
  • the present invention relates to a method of determining if a subject being treated for intestinal inflammation is in histological remission, comprising: i) obtaining a stool sample from the subject; and ii) quantitating the amount of mtDNA in the sample; wherein the presence of an amount of mtDNA in the sample that is equal to or lower than the amount in a control population indicates that the subject is in histological remission. It will be appreciated by one of skill in the relevant art that histological remission is an important therapeutic objective in treatment of intestinal inflammation.
  • histological remission can reflect amelioration or disappearance of microscopic tissue damage and can be associated with improved long-term clinical outcomes, including without limitation, reduced relapse rates and complications.
  • Current methods for assessing histological remission typically rely on invasive procedures, including without limitation, endoscopy and/or biopsy, which may be burdensome, costly, and/or unsuitable for frequent monitoring.
  • one goal of the present invention is to address these challenges for assessing histological remission by enabling non- invasive detection of histological remission through quantitation of mtDNA in stool samples, e.g., to offer a simpler, safer, and more accessible approach to assess mucosal healing and guide clinical decision-making.
  • methods disclosed herein may be used to monitor effectiveness of a treatment after it has started and/or ended, and to determine if a subject is or remains in histological remission.
  • methods disclosed herein may be used to screen for necrotizing enterocolitis (NEC) in premature infants.
  • Premature infants fall within four categories: late preterm: infant bom at 34 and 36 weeks; moderately preterm: infant bom between 32 and 34 weeks; very preterm: infant bom at less than 32 weeks; extremely preterm: infant born at or before 25 weeks.
  • Such a screening can be used to develop a treatment plan for the premature infant, such as when to start treatment and which treatment option to select or change.
  • NEC is a severe, lifethreatening gastrointestinal disease that primarily affects premature infants, particularly those born before 32 weeks of gestation
  • NEC is characterized by inflammation and necrosis of the intestinal tissue and is one of the leading causes of morbidity and mortality in neonatal intensive care units.
  • Early detection of NEC is critical, as the disease can progress rapidly and lead to intestinal perforation, systemic infection, and death.
  • current diagnostic methods rely heavily on non-specific clinical signs and radiographic findings, which often appear after significant tissue damage has occurred.
  • another aim of the present invention is to provide methods for screening for NEC using non-invasive biomarkers, such as mtDNA levels in stool, so as to detect and/or prevent NEC earlier and more accurately. For example, earlier detection can facilitate timely clinical intervention and improve outcomes, such as decreased mortality.
  • treatment options for NEC may vary depending on disease severity and may include, without limitation, bowel rest with cessation of enteral feeding, administration of broad-spectrum antibiotics, and supportive care such as fluid and electrolyte management.
  • Early identification of NEC through the methods of the invention may allow for more personalized treatment planning, including without limitation, improved determination of time(s) to initiate treatment, and to select or to adjust therapeutic strategies based on a condition of a subject.
  • a biological sample may be any tissue or fluid that contains mtDNA.
  • a biological sample may be a stool sample.
  • a level of mtDNA may be measured by any method known in the art.
  • an amplification assay may be used.
  • polymerase chain reaction PCR
  • quantitative PCR may be used to quantify a target nucleic acid (e.g., mtDNA or fragments thereof) with a high degree of precision.
  • quantitative PCR is commonly performed in real-time (e.g., quantitative real-time PRC) and may use fluorescent dyes that include, but are not limited to, SYBR® GREENTM, EVAGREEN® and/or fluorophore-containing nucleic acid probes (e.g., tetrachloro-6- carboxyfluorescein, TET), such as TAQMAN®, to measure the amount of amplified product in real time.
  • a mtDNA target may be any nucleic acid or fragment thereof originating in the mitochondrial genome of a subject.
  • a TAQMAN® assay (e.g., hCYTB484) may be used to target a nucleic acid comprising a human cytochrome b or fragment thereof using, e.g., a droplet digital (ddPCR) platform.
  • ddPCR droplet digital
  • stool samples are prepared for histological analysis by any methods known in the art and as described herein.
  • aliquots of mtDNA may be made and stored, e.g., in cryovials without any buffer.
  • DNA can be extracted from aliquots resulting in biological replicates for each, and at least one extraction blank can be performed with each batch of extractions.
  • methods of the invention as disclosed herein may comprise further steps based on an outcome of a determination.
  • methods when a determination indicates that a subject has an increased amount of mtDNA, methods may further comprise the step of indicating the presence of intestinal inflammation in a subject.
  • methods of the invention may further comprise the step of administering a therapy to a subject based on severity of inflammation.
  • methods of the invention may further comprise the step of modifying a treatment administered to a subject based on effectiveness of previous treatment.
  • methods of the present invention may further comprise the step of selecting an appropriate treatment or avoidance of treatment based on a risk or a responsiveness.
  • methods of the present invention may further comprise the step of administering to a subject an additional test to confirm the presence and/or severity of the intestinal inflammation. Additional tests may include, without limitation, biopsies, blood tests, and imaging techniques (e.g., X-rays, MRI, CT scans, endoscopic procedures, and the like).
  • methods of the present invention may further comprise the step of choosing not to administer a treatment or choosing a less severe treatment.
  • kits performing any of the disclosed methods.
  • the kit may comprise reagents for detecting mtDNA in a stool sample.
  • a kit may further comprise other components, such as therapeutic agents, carriers, buffers, containers, materials for collecting a stool sample, device for administration, and the like.
  • a kit may comprise one or more primers and/or probes for amplifying mtDNA.
  • a kit may be designed for therapeutic use, diagnostic use and/or research use and the additional components may be a kit suitable for an intended use.
  • the kit may further comprise labels and/or instructions, e.g., for diagnostic or treatment of a disorder.
  • Patients and sample preparation The study included 26 pediatric and young adult patients (ages 5 to 22 years) with planned endoscopic procedures and collection of stool sample for fecal calprotectin. See Table 1 and FIG. 2.
  • the stool samples were collected using at-home collection kits, stored at about 4°C until patient appointment, and stored at -80°C upon receipt. Stool sample ranged in consistency from firm to loose. After cataloguing received stool samples, three aliquots for mtDNA analysis were made and stored at -80°C in cryovials without any buffer. Calprotectin was measured by ELISA via Quest Diagnostics Clinical Laboratory as pg per gram of stool.
  • DNA extraction' DNA was extracted in biological triplicates using the QIAGEN® DNEASY® POWERSOIL® PROTM following manufacturer’s instructions and quantified mtDNA copies using hCYTB484, an assay targeting a human-specific region of the cytochrome b gene, on the QIAGEN® QIACUITY® digital PCR system. Copies of the mtDNA marker to mg of stool were normalized. The quantity of nucleic acids was assessed using an INVITROGEN® QUBIT 4 FLUOROMETERTM and the broad-range double-stranded DNA kit. Mean yields were 120 ng of dsDNA / pL of extract with a standard deviation of 97 ng of dsDNA/pL.
  • the hCYTB484 starts at the 484 basepair location within the cytochrome b gene of the human mtDNA genome.
  • the hCYTB484 amplicon is 121 basepair in length.
  • the primer sequence is hCYTB484F: CAATGAATCTGAGGAGGCTAC (SEQ ID NO:1); hCYTB604R: CGTGCAAGAATAGGAGGTG (SEQ ID NO:2); and a probe sequence of hCYTB520TM: ACCCTCACACGATTCTTTACCTTTCACT (SEQ ID NO:3).
  • the hCYTB484 assays has a ZEN QUENCHERTM (INTEGRATED DNA TECHNOLOGIES®, Coraville, IA, USA) located in approximately the middle of the probes. All primers and probes used in this study were manufactured by INTEGRATED DNA TECHNOLOGIES® (Coraville, IA, USA).
  • the dPCR used was QIAGEN® QIACUITY 4 SYSTEM® and the QIACUITY PROBE PCR KIT® (1 ml) (Cat. No. / ID: 250101) and QIACUITY NANOPLATE 8.5K 24-WELL® (10) (Cat. No. / ID: 250011).
  • the hCYTB484 had a primer concentration of 200 nM and a probe concentration of 100 nM.
  • the pre-reaction volume was 13 pL per well and composed of 3.25 pL of the QIACUITY® PROB PCR ENZYMETM, 0.037 pL of hCYTB484 probe, 0.74 pL of forward and reverse primers, 6.23 pL of molecular-grade water, and 2 pL of template.
  • the thermocycling parameters included 2 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 30 seconds at 59°C.
  • the hCYTB484 on the QIACUITY® 4 is optimized by running combinations of primer and probe concentrations to achieve a positive band around 100 RFUs and a negative band around 40 RFUs. Testing for inhibition was determined by running samples undiluted and at 1 : 10 dilutions. No evidence of inhibition was observed.
  • Results' Of the 26 patients with paired endoscopy results and stool samples, an IBD status was determined for 13 patients, 7 UC and 6 CD. The results suggest that concentrations of fecal mtDNA in children and young adults with IBD increase with increasing severity of disease, with several orders of magnitude difference between histological remission versus moderate to severe cases. For example, about 10 4 copies mtDNA/mg of stool was detected for individuals experiencing active inflammation and about 10 3 copies/mg of stool for individuals that were healthy.
  • the mtDNA assay utilizes now widespread PCR technology, can be completed in-house at higher speed and lower cost than enzyme-linked immunoassay (ELISA), and due to the fairly high dynamic range of the assay, appear to be more accurate at low disease severity states.
  • ELISA enzyme-linked immunoassay
  • the study demonstrates at least equal overall fidelity to disease state as FC, however as FC has had challenges in identifying histologic remission, the clinical implications of higher accuracy at low disease states are substantial for mtDNA.
  • EED Environmental Enteropathic Dysfunction
  • Concentrations of fecal mtDNA were quantified using hCYTB484, an assay targeting the cytochrome b gene on the human mitochondrial genome, on the BioRad QX200 droplet digital polymerase chain reaction (PCR) and QIAGEN® QIACUITY® digital PCR platforms.
  • PCR BioRad QX200 droplet digital polymerase chain reaction
  • QIAGEN® QIACUITY® digital PCR platforms Following extraction for nucleic acids using QIAGEN® 96 Virus and POWERFECAL® QIACUBE® HT kits, 447 child stools collected from the United States, 120 from Bangladesh, 66 from Mozambique, and 1,586 from Cambodia were analyzed.
  • Using the stools from Mozambique correlations between concentrations of fecal mtDNA versus current widely used, non-invasive biomarkers of EED were also investigated.
  • the biomarkers Performing a principal component analysis on mtDNA and the biomarkers, the biomarkers captured similar information to each other, but the mtDNA eigenvector was roughly orthogonal to the biomarker eigenvectors suggesting that mtDNA may capture information different from the specific inflammatory responses associated with EED biomarkers. See FIG. 7.

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Abstract

La présente invention concerne un biomarqueur associé à une inflammation intestinale et, plus particulièrement, des méthodes de diagnostic ou d'identification d'un risque de développer une inflammation intestinale à l'aide d'ADN mitochondrial et des méthodes de traitement de troubles associés à une inflammation intestinale.
PCT/US2025/026089 2024-04-25 2025-04-24 Biomarqueur de l'inflammation intestinale dans les selles pour aider à la détection et au traitement d'une maladie intestinale inflammatoire Pending WO2025226885A1 (fr)

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Citations (2)

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US20140099648A1 (en) * 2011-04-29 2014-04-10 Universitaetsklinikum Freiburg Mitochondrial nucleic acid as a marker for autoimmune and autoinflammatory diseases
US20230121867A1 (en) * 2019-11-26 2023-04-20 Cedars-Sinai Medical Center Compositions and methods for treating diseases and conditions by depletion of mitochondrial or genomic dna from circulation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140099648A1 (en) * 2011-04-29 2014-04-10 Universitaetsklinikum Freiburg Mitochondrial nucleic acid as a marker for autoimmune and autoinflammatory diseases
US20230121867A1 (en) * 2019-11-26 2023-04-20 Cedars-Sinai Medical Center Compositions and methods for treating diseases and conditions by depletion of mitochondrial or genomic dna from circulation

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