WO2026055162A2 - Procédés, kits et systèmes pour la détermination de l'état de sclérose en plaques et méthodes de traitement de la sclérose en plaques sur la base de ceux-ci - Google Patents

Procédés, kits et systèmes pour la détermination de l'état de sclérose en plaques et méthodes de traitement de la sclérose en plaques sur la base de ceux-ci

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Publication number
WO2026055162A2
WO2026055162A2 PCT/US2025/044541 US2025044541W WO2026055162A2 WO 2026055162 A2 WO2026055162 A2 WO 2026055162A2 US 2025044541 W US2025044541 W US 2025044541W WO 2026055162 A2 WO2026055162 A2 WO 2026055162A2
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subject
sample
genomic loci
modifications
fold
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WO2026055162A3 (fr
WO2026055162A8 (fr
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Matthew EATON
Jonathan BEAGAN
Rehan VERJEE
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Precede Biosciences Inc
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Precede Biosciences Inc
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Publication of WO2026055162A8 publication Critical patent/WO2026055162A8/fr
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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

Definitions

  • MS Multiple sclerosis
  • CNS central nervous system
  • migraine (22% of cases), fibromyalgia (15% of cases), nonspecific or nonlocalizing neurological symptoms with abnormal MRI presentation (12% of cases), conversion disorder/functional neurological disorder (11% of cases), and neuromyelitis optica (NMO) (6% of cases).
  • NMO neuromyelitis optica
  • MS myelin oligodendrocyte glycoprotein antibody disease
  • CNS vasculitis neurosarcoidosis
  • CNS manifestations of autoimmune diseases such as Sjögren’s syndrome, systemic lupus erythematosus (SLE), and antiphospholipid antibody syndrome
  • Sjögren’s syndrome systemic lupus erythematosus
  • SLE systemic lupus erythematosus
  • antiphospholipid antibody syndrome chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids
  • 12953047v1 Page 1 of 224 Attorney Docket: 2014191-0043 Beçhet’s syndrome.
  • MRI also does not provide insight into the biological mechanisms underlying MS in a subject, and therefore is limited in its ability to determine, e.g., which subjects are likely to respond well to treatment with a certain therapeutic, which subjects are more likely to exhibit worsening disease state, and which subjects are likely to be prone to relapse.
  • cfDNA cell-free DNA
  • the present disclosure also encompasses methods where chromatin accessibility and/or binding of one or more transcription factors are detected at the one or more genomic loci instead of (or in addition to) histone modifications and/or DNA methylation.
  • Liquid biopsies are now widely utilized in clinical oncology to detect cancer recurrence and inform therapeutic decisions.
  • most commercially available cfDNA assays only detect tumor genomic alterations and not all disease states have a characteristic genomic alteration that can be used for detection.
  • MS is associated with a number of environmental factors, and while certain alleles have been associated with an increased risk of MS, it is currently not possible (and may be impossible) to detect the presence of MS on the basis of genome sequencing alone, let alone characterizing disease states.
  • the present disclosure provides tools to analyze multiple epigenomic features from patient plasma, including DNA methylation, chromatin accessibility, and histone modifications.
  • the present disclosure demonstrates that epigenomic cfDNA profiling can be used to detect MS in patients as well as characterize disease severity. Diagnosing and monitoring of MS by cfDNA profiling would be immediately clinically actionable, as guidelines recommend that MS be treated with different drugs depending on disease severity.
  • the present disclosure includes, among other things, technologies for the determination of MS status and for the detection, monitoring, and/or treatment of MS based on MS status.
  • the present disclosure relates to the measurement of histone modifications in a sample obtained or derived from a subject to detect and/or treat MS based on 12953047v1 Page 2 of 224 Attorney Docket: 2014191-0043 MS status.
  • the present disclosure includes, among other things, histone modification measurements in cell-free DNA (cfDNA) that are characteristic of MS, and which in various embodiments are useful, e.g., for detecting, monitoring, selecting treatment for, and/or treating MS.
  • cfDNA cell-free DNA
  • the present disclosure includes, among other things, histone modification measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating MS.
  • histone modification measurements in cfDNA can be used to detect or determine resistance of MS in a subject to a therapy. In some embodiments, histone modification measurements in cfDNA can be used to monitor progression of MS (e.g., progression in a subject currently being administered a therapy). In some embodiments, histone modification measurements in cfDNA can be used to inform therapeutic selection for a subject with MS (e.g., determine an initial therapy, predict patients that are likely to respond to a given therapy, and/or determine when therapy should be changed for a subject).
  • histone modification measurements in cfDNA can be used as a complement to other methods (e.g., imaging methods and/or symptom-based methods) for monitoring MS (e.g., performed concurrently with other methods and/or performed subsequent to other methods, e.g., to monitor disease progression).
  • the present disclosure includes exemplary genomic loci that are differentially modified in MS vs. healthy subjects.
  • genomic loci differentially modified in cfDNA are or include one or more enhancers.
  • genomic loci differentially modified in cfDNA are or include one or more promoters.
  • a genomic locus is differentially modified if it is characterized by increased or decreased histone modification as compared to a reference (e.g., a sample from a healthy subject).
  • Increased or decreased histone modification can be or include, e.g., increased or decreased histone methylation (hypermethylation or hypomethylation, respectively) of one or more particular methylation marks, or a combination thereof; increased or decreased pan-methylation; increased or decreased histone acetylation (hyperacetylation or hypoacetylation, respectively) of one or more particular acetylation marks, or a combination thereof; and/or increased or decreased pan-acetylation (e.g., pan-H3 acetylation).
  • histone methylation can be or include histone methylation marks selected from H3K4me1, H3K4me2, H3K4me3, or a combination thereof.
  • histone 12953047v1 Page 3 of 224 Attorney Docket: 2014191-0043 methylation can be or include H3K4me3.
  • histone acetylation can be or include histone acetylation marks selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, or a combination thereof.
  • histone acetylation can be or include H3K27ac.
  • the present disclosure relates to the measurement of DNA methylation in a sample obtained or derived from a subject to detect and/or treat MS.
  • the present disclosure includes, among other things, DNA methylation measurements in cell-free DNA (cfDNA) that are characteristic of MS, and which in various embodiments are useful, e.g., for detecting, monitoring, selecting treatment for, and/or treating MS.
  • cfDNA cell-free DNA
  • the present disclosure includes, among other things, DNA methylation measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating MS.
  • the present disclosure includes, among other things, DNA methylation measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating MS.
  • DNA methylation measurements in cfDNA can be used to detect or determine resistance of MS to a therapy.
  • the present disclosure includes exemplary genomic loci that are differentially DNA methylated in MS.
  • a genomic locus is differentially modified if it is characterized by increased or decreased DNA methylation as compared to a reference (e.g., a sample from a healthy subject).
  • genomic loci differentially modified in cfDNA are or include one or more enhancers.
  • genomic loci differentially modified in cfDNA are or include one or more promoters.
  • the present disclosure further relates, in various embodiments, to the measurement of chromatin accessibility in cell-free DNA (cfDNA) to determine MS status.
  • cfDNA cell-free DNA
  • the present disclosure includes, among other things, chromatin accessibility measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating MS.
  • chromatin accessibility measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating MS.
  • chromatin accessibility measurements in cfDNA can 12953047v1 Page 4 of 224 Attorney Docket: 2014191-0043 be used to detect or determine resistance of MS to a therapy.
  • the present disclosure includes genomic loci that are differentially accessible in MS.
  • genomic loci differentially accessible in cfDNA are or include one or more enhancers.
  • genomic loci differentially accessible in cfDNA are or include one or more promoters.
  • histone methylation e.g., H3K4me3 corresponds and/or is correlated with chromatin accessibility.
  • histone acetylation corresponds and/or is correlated with chromatin accessibility.
  • DNA methylation corresponds and/or is correlated with chromatin accessibility.
  • a genomic locus is differentially accessible if it is characterized by increased or decreased chromatin accessibility as compared to a reference (e.g., a sample from a healthy subject). Increased or decreased histone modification can be or include, e.g., increased or decreased accessibility as determined by various chromatin accessibility assays known in the art.
  • the present disclosure further relates, in various embodiments, to the measurement of transcription factor binding in cell-free DNA (cfDNA) to determine MS status.
  • cfDNA cell-free DNA
  • the present disclosure includes, among other things, transcription factor binding measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and/or treating MS.
  • transcription factor binding measurements in cfDNA can be used to detect or determine resistance of MS in a subject to a therapy.
  • the present disclosure includes genomic loci that are differentially bound by transcription factors in MS.
  • genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more enhancers.
  • genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more promoters.
  • histone methylation e.g., H3K4me3
  • histone acetylation e.g., H3K27ac
  • DNA methylation corresponds and/or is correlated with transcription factor binding.
  • a genomic locus is differentially bound by transcription factors if it is characterized by increased or decreased transcription factor binding as compared to a reference (e.g., a sample from a healthy subject).
  • Increased or decreased transcription factor binding can be or include, e.g., increased or decreased transcription factor binding as determined by various transcription factor binding assays known in the art.
  • the present disclosure provides a method of determining MS status in a subject, the method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) binding of one or more transcription factors, and/or (iv) DNA methylation.
  • cfDNA cell-free DNA
  • the one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, and pan-acetylation.
  • the histone modification assay detects H3K4me3 modifications.
  • the histone modification assay detects H3K27ac modifications.
  • the histone modification assay is selected from ChIP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.
  • chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and a DNase hypersensitivity assay.
  • ATAC-seq Assay of Transpose Accessible Chromatin sequencing
  • NOMe-seq Nucleosome Occupancy and Methylome sequencing
  • FAIRE-seq Formmaldehyde-Assisted Isolation of Regulatory Elements sequencing
  • MNase-seq Merococcal Nuclease digestion with sequencing
  • DNase hypersensitivity assay a transcription factor binding assay.
  • the transcription factor binding assay is selected from ChIP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN 12953047v1 Page 6 of 224 Attorney Docket: 2014191-0043 (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.
  • DNA methylation is quantified using Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq).
  • a method comprises quantifying two or more of the following, each at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) transcription factor binding, and/or (iv) DNA methylation.
  • the method comprises quantifying two or more histone modifications, e.g., quantifying H3K4me3 and H3K27ac modifications.
  • a method comprises quantifying one or more histone modifications and DNA methylation, e.g., quantifying H3K4me3 and/or H3K27ac modifications and DNA methylation.
  • a method comprises quantifying H3K4me3 modifications, H3K27ac modifications and DNA methylation.
  • a biological sample is a liquid biopsy sample, e.g., a plasma sample, serum sample, or urine sample.
  • quantification of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at the one or more genomic loci as compared to a reference indicates that the subject has MS.
  • the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and/or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a cohort of healthy subjects.
  • a method comprises quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci in Tables 1-6.
  • a method comprises quantifying H3K4me3 modifications for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 1.
  • a method comprises quantifying H3K27ac modifications for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2.
  • a method comprises 12953047v1 Page 7 of 224 Attorney Docket: 2014191-0043 quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 3.
  • a method comprises quantifying H3K4me3 modifications for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 4. In some embodiments, a method comprises quantifying H3K27ac modifications for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 5. In some embodiments, a method comprises quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 6. [0025] In some embodiments, the activity of one or more transcription factors is determined by measuring transcription factor binding.
  • the activity of the one or more transcription factors is assessed using a method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) binding of one or more transcription factors, and/or (iv) DNA methylation.
  • cfDNA cell-free DNA
  • the one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, and pan-acetylation.
  • the histone modification assay detects H3K4me3 modifications.
  • the histone modification assay detects H3K27ac modifications.
  • a sample is obtained from a subject having MS wherein imaging of MS is not possible and/or feasible and/or when it is not possible to detect/diagnose MS using imaging or symptoms alone.
  • the present disclosure provides a method of detecting multiple sclerosis (MS) in a subject, the method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) binding of one or more transcription factors, and/or 12953047v1 Page 8 of 224 Attorney Docket: 2014191-0043 (iv) DNA methylation.
  • cfDNA cell-free DNA
  • one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, and pan-acetylation.
  • a histone modification assay detects H3K4me3 modifications.
  • a histone modification assay detects H3K27ac modifications.
  • a histone modification assay is selected from ChIP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.
  • chromatin accessibility can be quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and a DNase hypersensitivity assay.
  • ATAC-seq Assay of Transpose Accessible Chromatin sequencing
  • NOMe-seq Nucleosome Occupancy and Methylome sequencing
  • FAIRE-seq Formmaldehyde-Assisted Isolation of Regulatory Elements sequencing
  • MNase-seq Merococcal Nuclease digestion with sequencing
  • DNase hypersensitivity assay e.
  • a transcription factor binding assay is selected from ChIP- seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.
  • DNA methylation can be quantified using Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq).
  • methods described herein comprise quantifying two or more of the following, each at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) transcription factor binding, and/or (iv) DNA methylation. [0037] In some embodiments, methods described herein comprise quantifying two or more histone modifications. [0038] In some embodiments, methods described herein comprise quantifying H3K4me3 and H3K27ac modifications.
  • methods described herein comprise quantifying one or more histone modifications and DNA methylation.
  • methods described herein comprise quantifying H3K4me3 and/or H3K27ac modifications and DNA methylation.
  • methods described herein comprise quantifying H3K4me3 modifications, H3K27ac modifications and DNA methylation.
  • a liquid biopsy sample is a plasma sample, serum sample, or urine sample.
  • methods described herein comprise quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci in Tables 1-6.
  • an increase or decrease of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci indicates that the subject has MS.
  • methods described herein comprise quantifying: 12953047v1 Page 10 of 224 Attorney Docket: 2014191-0043
  • H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, or 700 genomic loci in Table 1
  • H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 genomic loci in Table 2
  • DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500, genomic loci in Table 3
  • H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 genomic loci in Table 4
  • H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,
  • one or more genomic loci comprise one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and/or an excitatory neuron cell identity genes, or one or more regulatory regions thereof, and an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at the one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and/or an excitatory neuron cell identity genes, or one or more regulatory regions thereof as compared to a reference indicates that the subject has MS.
  • one or more genomic loci comprise one or more enhancer regions associated with one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and/or an excitatory neuron cell, and an increase in enhancer signal at the one or more enhancer regions associated with the one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and/or an excitatory neuron cell identity gene indicates that the subject has MS.
  • the one or more cell identity genes of an oligodendrocyte progenitor cell comprise NSUN5, DAAM2, CNTN2, or any combination thereof.
  • one or more enhancer regions associated with NSUN5 include chr7:72713576- 72716059.
  • one or more enhancer regions of DAAM2 include chr6:39788740-39793623.
  • one or more genomic loci comprise one or more enhancer regions of NSUN5, DAAM2, CNTN2, or any combination thereof; wherein an increase in enhancer signal at the one or more enhancer regions of NSUN5, DAAM2, CNTN2, or any combination thereof indicates that a subject has MS.
  • one or more genomic loci comprise one or more gene markers of synaptic plasticity or one or more regulatory regions thereof, and an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at the one or more gene markers of synaptic plasticity or one or more regulatory regions thereof as compared to a reference indicates that a subject has MS.
  • one or more genomic loci comprise one or more promoter regions associated with one or more marker genes of synaptic plasticity, wherein an increase in promoter signal at one or more promoter regions associated with one or more marker genes of synaptic plasticity indicates that a subject has MS.
  • one or more marker genes of synaptic plasticity include SQSTM1 and/or LILRB2.
  • one or more genomic loci comprise one or more promoter regions of SQSTM1 and/or LILRB2, and an increase in promoter signal as compared to a reference indicates that the subject has MS.
  • one or more promoter regions of LILRB2 include chr19:54,782,258-54,785,464.
  • one or more promoter regions for SQSTM1 include chr5:179232387-179234388.
  • one or more genomic loci comprise one or more MS-risk alleles or one or more regulatory regions thereof, and an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at the MS-risk alleles or one or more regulatory regions thereof as compared to a reference indicates that a subject has MS.
  • one or more genomic loci comprise one or more promoter regions associated with one or more MS-risk alleles, wherein an increase in promoter signal at 12953047v1 Page 12 of 224 Attorney Docket: 2014191-0043 the one or more promoter regions associated with the one or more MS-risk alleles indicates that the subject has MS.
  • one or more genomic loci comprise TNFRSF14 or one or more regulatory regions thereof, and wherein an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at TNFRSF14 or one or more regulatory regions thereof as compared to a reference indicates that a subject has MS.
  • one or more genomic loci comprise a TNFRSF14 promoter region, wherein an increase in promoter signal at the TNFRSF14 promoter region as compared to a reference indicates a subject has MS.
  • a reference is a predetermined threshold, a measurement from a liquid biopsy sample, and/or a normalized value.
  • a reference is a measurement from a liquid biopsy sample obtained from a subject or a cohort of subjects that have not been diagnosed with MS.
  • MS is progressive MS or relapsing remitting MS.
  • the present disclosure describes a method for determining MS disease severity in a subject, comprising: performing a method described herein for determining MS status in a subject, and comparing a value obtained from performing said method to a reference.
  • one or more genomic loci comprise one or more microglial cell identity genes or one or more regulatory regions thereof, and an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more microglial cell identity genes or one or more regulatory regions thereof as compared to a reference indicates that a subject has severe MS.
  • one or more genomic loci comprise one or more enhancer regions of one or more microglial cell identity genes, and an increase in enhancer signal (e.g., H3K27ac) in one or more enhancer regions of one or more microglial cell identity genes indicates a subject has severe MS.
  • enhancer signal e.g., H3K27ac
  • one or more microglial cell identity genes include MAP4K4.
  • one or more enhancer regions of MAP4K4 include chr2:102513113-102515404.
  • a reference is a predetermined threshold, a measurement from a liquid biopsy sample, and/or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a subject or a cohort of subjects that have diagnosed with moderate or severe MS (e.g., subjects having moderate or high TSPO PET signal, respectively).
  • the present disclosure describes a method of determining MS disease progression in a subject, the method comprising determining, at a first time point and a second time point, MS severity in the subject, wherein MS severity is determined at each point in time using a method described herein.
  • a method described herein can be used to predict disease flare and/or progression in a subject.
  • one or more genomic loci comprise one or more microglial cell identity genes or one or more regulatory regions thereof and/or one or more neutrophil cell identity genes or one or more regulatory regions thereof, and an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more microglial cell identity genes or one or more regulatory regions thereof and/or one or more neutrophil cell identity genes or one or more regulatory regions thereof as compared to a reference indicates that the subject has an increased likelihood of experiencing disease flare and/or progression as compared to a reference, wherein the reference is optionally a plasma sample obtained for a subject or cohort of subjects having MS and not exhibiting an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more microglial cell identity genes or one or more regulatory regions thereof and/or one or more neutrophil cell identity genes or one or more regulatory regions thereof.
  • one or more genomic loci comprise one or more enhancer regions of one or more microglial cell identity genes.
  • 12953047v1 Page 14 of 224 Attorney Docket: 2014191-0043
  • a subject has previously been determined to have MS (e.g., using MRI and/or PET imaging).
  • the present disclosure describes method of treating a subject, comprising determining the MS status of the subject using a method described herein, and: (a) if the subject is determined to have MS, administering an MS therapeutic; and (b) if the subject is not determined to have MS, not administering an MS therapeutic.
  • the present disclosure describes a method of treating a subject having MS, comprising determining MS severity in the subject using a method described herein, wherein: (a) if the subject is determined to have severe MS, administering a high or moderate efficacy MS therapeutic; and (b) if the subject is determined to have moderate disease severity, administering a low efficacy MS therapeutic.
  • the present disclosure describes a method of treating MS in a subject, comprising determining MS disease progression in the subject using a method described herein, wherein the subject is being administered a first MS therapeutic at the first time point and the second time point, and wherein: (a) if MS disease severity is determined to increase between the first and the second time points, administering a second MS therapeutic, wherein the second therapeutic has a higher efficacy than the first MS therapeutic; and (b) if MS disease severity is determined to stay the same and/or decrease between the first and the second time points, continuing treatment with the first MS therapeutic.
  • a first MS therapeutic is a low efficacy MS therapeutic and a second MS therapeutic is a moderate or high efficacy MS therapeutic.
  • a first MS therapeutic is a moderate efficacy MS therapeutic the second MS therapeutic is a high efficacy MS therapeutic.
  • a low efficacy MS therapeutic is an interferon (e.g., beta- o r beta- ), glatiramer acetate, or teriflunomide).
  • a moderate efficacy 12953047v1 Page 15 of 224 Attorney Docket: 2014191-0043 MS therapeutic is cladribine, an S1p inhibitor (e.g., fingolimod, siponimod, ozanimod, or ponesimod), or a fumarate (e.g., dimethyl, diroximel, or monomethyl fumarate).
  • a high efficacy MS therapeutic is Ocrelizumab, Ofatumumab, Natalizumab, or Alemtuzumab.
  • the present disclosure describes a kit comprising reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Tables 1-6.
  • a kit comprises reagents for quantifying: (a) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, or 700 genomic loci in Table 1; (b) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 genomic loci in Table 2; (c) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500, genomic loci in Table 3; (d) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 genomic loci in Table 4; (e) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, or 600 genomic loci in Table 5; (f) DNA methylation for at least 5, 10, 20,
  • a kit comprises one or more antibodies for use in ChIP- seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac- modified histones. 12953047v1 Page 16 of 224 Attorney Docket: 2014191-0043 [0076] In some embodiments, a kit comprises one or more methyl-binding domains for use in MBD-seq. In some embodiments, a kit comprises one or more antibodies that bind methylated DNA for use in MeDIP. [0077] In some embodiments, a kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample.
  • cfDNA cell-free DNA
  • a kit comprises reagents for library preparation for sequencing. [0079] In some embodiments, a kit comprises reagents for sequencing. [0080] In some embodiments, a kit comprises instructions for determining if a subject has MS. [0081] Among other things, the present disclosure describes a non-transitory computer readable storage medium encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method described herein. [0082] Among other things, the present disclosure describes a system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform a method described herein.
  • a system comprising a sequencer configured to generate a sequencing data set from a sample; and a non-transitory computer readable storage medium described herein and/or a computer system described herein.
  • a sequencer is configured to generate a Whole Genome Sequencing (WGS) data set from the sample.
  • WGS Whole Genome Sequencing
  • a system comprises a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample.
  • a sample preparation device comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.
  • cfDNA cell-free DNA
  • one or more genomic loci are selected from Tables 1-6.
  • a device comprises reagents for quantifying: (a) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, or 700 genomic loci in Table 1; (b) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 genomic loci in Table 2; (c) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500, genomic loci in Table 3; (d) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 genomic loci in Table 4; (e) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, or 600 genomic loci in Table 1; (b) H3K27ac modifications for at least
  • a system comprises one or more antibodies for use in ChIP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.
  • a system comprises reagents that comprise one or more methyl-binding domains for use in MBD-seq.
  • a system described herein comprises a device comprising reagents for isolation of cell-free DNA (cfDNA) from a biological sample, optionally a liquid biopsy sample.
  • a system described herein comprises a device comprising reagents for library preparation for sequencing.
  • a system described herein comprises a sequencer comprising reagents for sequencing.
  • BRIEF DESCRIPTION OF THE DRAWING [0092]
  • Fig.1 provides an outline of a comprehensive epigenomic platform described herein, that can offer dynamic resolution into target and pathway biology from 1 mL of plasma.
  • Cell free DNA exists in circulation as chromatin fragments that maintain epigenetic modifications on histones and DNA.
  • Binding agents that detect markers of active enhancers e.g., H3K27ac
  • active promoters e.g., H3K4me3
  • DNA methylation can be used to enrich for associated DNA fragments from a small volume of sample (e.g., 1 mL of plasma) and sequenced to define genome-wide epigenomic maps that capture the underlying transcriptional state of cells in a subject. Shown, for illustrative purposes, is an example of H3K27ac, H3K4me3, and DNAme signal obtained at the TROP2 locus and the loci of enhancers associated with TROP2. [0093] Fig.2: Identification of MS-associated Signals Across Genomic and Epigenomic Analytes.
  • FIG. 1 Plasma samples from Multiple Sclerosis (MS) patients display elevated abundance of cell free mitochondrial DNA, quantified by the percent of de-duplicated shallow whole genome reads mapping to the mitochondrial genome.
  • B Plasma samples from Multiple Sclerosis (MS) patients display elevated abundance of cell free mitochondrial DNA, quantified by the percent of de-duplicated shallow whole genome reads mapping to the mitochondrial genome.
  • B -(D) Promoter, enhancer, and DNA regions genome-wide were assessed for elevated/depleted signal in MS patients compared to healthy subjects, revealing hundreds of regions with MS-associated alterations in signal strength.
  • Fig.3 MS-Associated Promoter Signal Increases at Synaptic Pathways and Risk Genes.
  • TNFRSF14 displayed the highest promoter signal of any gene in the genome in 7/18 tested MS samples.
  • D Samples from rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients were tested to compare promoter and enhancer signals across autoimmune diseases. Enriched TNFRSF14 promoter signal was detected only in the MS patient population.
  • E Genes were rank ordered by their MS vs.
  • Fig.4 MS-Associated Enhancers Are Enriched For Neural Cell Type Genes.
  • A Differentially modified enhancer regions were assigned to their nearest gene (within 50 kb), after which genes were rank ordered by their most differential (MS vs healthy) enhancer signal and gene set enrichment analysis (GSEA) was performed utilizing the MSigDB cell type signature gene sets (C8). Terms with normalized enrichment scores greater than 1.3 and FDR- adjusted p-values below 0.01 are highlighted in purple and select neural cell types are labeled (with simplified term names).
  • B-C Enhancer signal at oligodendrocyte progenitor cell identity genes (NSUN5, DAAM2, and CNTN2) and housekeeping genes (ACTB, GAPDH) are visualized in (B) and quantified in (C).
  • the present disclosure is based, at least in part, on the demonstration that MS in a subject can be detected and characterized by detecting and quantifying the presence of histone modifications and/or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample obtained or derived from the subject.
  • cfDNA cell-free DNA
  • the present disclosure also encompasses methods where chromatin accessibility and/or binding of one or more transcription factors are detected at the one or more genomic loci instead of (or in addition to) histone modifications and/or DNA methylation.
  • MS status [0098] Multiple sclerosis (MS) is an immune-driven disease characterized by demyelination and axonal damage in the central nervous system (CNS). It is twice as common in females and most often diagnosed between 20–40 years of age. The MS prevalence in the United States has been steadily increasing over the past several decades, with approximately 58 cases per 100,000 persons in 1975 increasing to 309.2 cases per 100,000 persons (450.1 per 100,000 for females) during 2010–2019. This roughly translates to 1 in 300 people in the United States living with MS, with the incidence being the highest for Black people (10.2 per 100,000 person- years versus 6.2 in white people).
  • having simultaneous enhancing and nonenhancing lesions in the first attack fulfills the dissemination-in-time criteria without requiring a second clinical attack.
  • Oligoclonal bands a marker of inflammatory reaction chronicity, can also fulfill the dissemination-in-time criteria.
  • the revised criteria aim to increase sensitivity in detecting MS to expedite treatment and prevent disability.
  • MS lesions must be seen in at least two of the following four locations: cortical/juxtacortical, periventricular, infratentorial, and spinal cord.
  • Typical clinical syndromes for MS include optic neuritis, internuclear ophthalmoplegia, facial sensory loss or trigeminal neuralgia, ataxia, and partial transverse myelitis.
  • Electrophoresis is a method that can be used to separate ionic molecules under the influence of an electric field.
  • Chromatin accessibility can refer to the degree to which nuclear macromolecules are able to physically contact DNA and is determined in part by the occupancy and modification status of nucleosomes. Modified histones can regulate chromatin accessibility through a variety of mechanisms, such as altering transcription factor (TF) binding through steric hindrance and modulating nucleosome affinity for active chromatin remodelers.
  • TF transcription factor
  • nucleosomes across the genome are non-uniform: while histones can be densely arranged within facultative and constitutive heterochromatin, histones can be depleted at regulatory loci, 12953047v1 Page 34 of 224 Attorney Docket: 2014191-0043 including within enhancers, insulators and transcribed gene bodies. Active regulatory elements of the genome are generally accessible.
  • Differential accessibility of a genomic locus can refer to, or be determined by or detected as, a comparative difference or change in modification status of one or more genomic loci between a first sample, condition, disease, or state and a second or reference sample, condition, disease, or state.
  • a reference is typically produced by measurement using a methodology identical, similar, or comparable to that by which a compared non-reference measurement was taken.
  • a reference can be a value or set of values that are predetermined or derived from a sample or set of samples.
  • a reference can be a sample or set of samples.
  • a reference value can be a predetermined threshold value, a value that varies in accordance with circumstances (e.g., according to patient subpopulation, age, weight, or other variables), or a ratio.
  • Reference ratios can be ratios relating to the modification and/or accessibility of multiple loci within individual samples and/or references, or across or between samples and/or references.
  • a reference can have or represent a normal, non-diseased state.
  • a reference can have or represent a diseased state, e.g., MS.
  • a reference can represent MS by being obtained from a subject diagnosed as having MS (e.g., based on imaging, symptoms, and/or CSF analysis).
  • a reference is a non-contemporaneous sample from the same source, e.g., a prior sample from the same source, e.g., from the same subject.
  • a reference for the modification status of one or more genomic loci can be the modification status of the one or more genomic loci (e.g., one or more differentially modified genomic loci) in a sample (e.g., a sample from a subject), or a plurality of samples, known to represent a particular state (e.g., MS).
  • a reference for the accessibility status of one or more genomic loci can be the accessibility status of the one or more genomic loci (e.g., one or more differentially accessible genomic loci) in a sample (e.g., a sample from a subject), or a plurality of samples, known to represent a particular state (e.g., MS).
  • differential modification or differential accessibility can refer to a differential (e.g., between a sample and a reference) with an absolute log2(fold-change) that is greater than or equal to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or more, or any range in between, inclusive, e.g., as measured according to an assay provided herein.
  • Enhancers are genomic loci that can be differentially modified or differentially accessible in and/or between conditions, diseases, and other states.
  • Enhancers are cis-acting DNA regulatory regions that are thought to bind trans-acting proteins that contribute to expression patterns of associated genes. Chromatin ImmunoPrecipitation sequencing (ChIP-seq) of histone modifications (e.g., acetylation) have identified millions of enhancers in mammalian genomes. The number of active enhancers in any given cell type is estimated to be in the tens of thousands. Certain transcription factors (TFs), sometimes referred to as “master” transcription factors, associate with active enhancers with important impacts on gene expression and cell function. Certain such transcription factors preferentially associate with enhancers that regulate genes required for establishing cell identity and function, including enhancer domains known as “super-enhancers”.
  • master TFs can participate in inter-connected auto-regulatory circuitries or “cliques” that are self-reinforcing, show marked cell selectivity, and function to maintain cell state and/or cell survival.
  • Techniques for Detecting and Quantifying Histone Modifications and Transcription Factor Binding [0155] Various techniques of molecular biology are well known in the art and/or disclosed in the present application for detecting and quantifying histone modifications and/or transcription factor binding. In some embodiments, the methods, kits and systems of present disclosure involve the detection and quantification of histone modifications and/or transcription factor binding in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA.
  • Chromatin ImmunoPrecipitation is one technique of molecular biology useful in detecting and quantifying histone modifications and transcription 12953047v1 Page 36 of 224 Attorney Docket: 2014191-0043 factor binding in samples.
  • CUT&RUN or CUT&Tag are other more recent techniques that can also be used to detect and quantify histone modifications and transcription factor binding sites.
  • ChIP can involve various steps including one or more of fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. ChIP has become a very widely used tissue-based technique for determining the in vivo location of binding sites of various transcription factors and histones.
  • ChIP helps to detect DNA-protein interactions that take place in living cells. More importantly, ChIP can be coupled to many commonly used molecular biology techniques such as PCR and real-time PCR, PCR with single-stranded conformational polymorphism, Southern blot analysis, Western blot analysis, cloning, and microarray. The resulting versatility has increased the potential of this technique. [0157] ChIP of tissue samples usually involves cross-linking of the chromatin-bound proteins by formaldehyde, followed by sonication or nuclease treatment to obtain small DNA fragments. Immunoprecipitation can be then carried out using specific antibodies to the DNA- binding protein of interest.
  • the DNA can be then released from the proteins and analyzed using various methods. ChIP has also been used to study RNA-protein interactions. X-ChIP methods utilize fixed chromatin fragmented by sonication, while the N-ChIP methods utilize native chromatin, which can be unfixed and nuclease digested. [0158]
  • the first step of the technique can be the cross-linking of DNA and proteins. Formaldehyde is one of the most used cross-linking agents.
  • formaldehyde can be the ease of reversibility of the cross-links and its ability to form bonds that span approximately 2 angstroms. This means that formaldehyde can bind molecules in close association with each other.
  • formaldehyde can be added to the medium in the cell culture flask or plate. It enters the cells through the cell membrane and cross-links the proteins to the chromatin. Formaldehyde fixation of tumor tissues has also been done.
  • Other cross-linking agents include chemicals such as methylene blue and acridine orange, cisplatin, dimethylarsinic acid, potassium chromate, and ultraviolet (UV) light and lasers.
  • Harvested chromatin can be sonicated in one or more sonication cycles. DNA can be typically broken into to 100–500 bp fragments to pinpoint the location of the DNA sequence of interest.
  • Chromatin can be immunoprecipitated using one or more antibodies that bind a target epitope.
  • an antibody used in ChIP can selectively bind a particular transcription factor or one or more particular histone modifications, such as one or more particular histone acetylation modifications or histone methylation modifications.
  • an antibody used to bind a target epitope can be a “pan” antibody (e.g., a pan- acetylation antibody, a pan-methylation antibody, an antibody that binds a group of histone modifications associated with increased transcription activation, and/or an antibody that binds a group of histone modifications associated with increased transcription repression).
  • the antibody against the protein of interest is allowed to bind to the protein-DNA complex, and the complex can be then precipitated.
  • Immunosorbants commonly used to separate the antigen-antibody complex from the lysate include salmon sperm DNA-protein A-Sepharose®, protein G, magnetic beads, and other engineered immunoprecipitation systems known to those of skill in the art.
  • Immunoprecipitated DNA can be eluted. Once the DNA of interest is isolated, many detection and quantification methods can be used to study the isolated gene fragments. Commonly utilized methods include PCR, real-time PCR, slot blot hybridization, microarray techniques, and deep or next-generation sequencing. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. ChIP-seq can be used to map DNA-binding proteins, e.g., transcription factor binding sites and histone modifications in a genome-wide manner.
  • ChIP chromatin immunoprecipitation
  • Cell-free Chromatin ImmunoPrecipitation sequencing involves applying ChIP-seq to samples that include cell-free DNA, e.g., liquid biopsy samples including cfDNA such as plasma samples including cfDNA (e.g., see Sadeh et al., Nat Biotechnol (2021) 39: 586–598 and Jang et al., Life Sci Alliance (2023) 6(12):e202302003 the entire contents of each of which are incorporated herein by reference).
  • cfChIP-seq uses antibodies or antibody fragments that bind specific histone modifications (e.g., H3K4me3 and/or H3K27ac) and/or transcription factors that are coupled (covalently or non-covalently) to beads, e.g., magnetic beads such as Dynabeads® magnetic beads and incubated with a volume, e.g., about 1 mL of thawed plasma obtained from a subject.
  • specific histone modifications e.g., H3K4me3 and/or H3K27ac
  • transcription factors that are coupled (covalently or non-covalently) to beads, e.g., magnetic beads such as Dynabeads® magnetic beads and incubated with a volume, e.g., about 1 mL of thawed plasma obtained from a subject.
  • exemplary antibodies 12953047v1 Page 38 of 224 Attorney Docket: 2014191-0043 that bind H3K4me3 include PA5-27029 (available from Thermo Fisher Scientific in Waltham, MA) and C15410003 (available from Diagenode in Denville, NJ) and exemplary antibodies that bind H3K27ac include ab21623 or ab4729 (both available from Abcam in Cambridge, UK) and C15210016 (available from Diagenode in Denville, NJ).
  • the antibodies or antibody fragments can be covalently coupled to beads, e.g., epoxy beads.
  • the antibodies or antibody fragments can be non-covalently coupled to beads, e.g., Protein A or Protein G beads such as Dynabeads® Protein A or Dynabeads® Protein G beads.
  • beads e.g., Protein A or Protein G beads such as Dynabeads® Protein A or Dynabeads® Protein G beads.
  • a cfDNA library is then typically prepared from the captured cfDNA. Library preparation can be done on-bead or after releasing the captured cfDNA by digestion of bound histones, e.g., using proteinase K.
  • the cfDNA library is then sequenced to generate reads of captured cfDNA sequences, e.g., by next-generation sequencing (NGS) as is known in the art.
  • NGS next-generation sequencing
  • a cfChIP-seq bioinformatic pipeline can include, e.g., alignment of sequence reads to a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference.
  • CUT&Tag involves antibody-based binding of a target protein, e.g., transcription factor or histone modification of interest, where antibody incubation is directly followed by the shearing of the chromatin and library preparation (see Kaya-Okur et al., Nat Comm (2019) 10:1930).
  • Samples can then be incubated with assembled transposomes, which consist of Protein A fused to the Tn5 transposase enzyme that is conjugated to NGS adapters. After incubation, unbound transposome can be washed away using stringent conditions.
  • Tn5 is a Mg 2+ -dependent enzyme so Mg 2+ can be added to activate the reaction, which results in the chromatin being cut close to the protein binding site and 12953047v1 Page 39 of 224 Attorney Docket: 2014191-0043 simultaneous addition of the NGS adapter DNA sequences. Chromatin cleavage and library preparation can be achieved in one single step.
  • CUT&RUN is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing (see Skene and Henikoff, Elife (2017) 6:1-35, Skene et al., Nat Protoc (2016) 13:1006-1019). As only targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has low background levels.
  • a sample is incubated with an antibody or antibody fragment that binds the target protein, e.g., transcription factor or histone modification of interest.
  • the sample is then incubated with Protein-A-MNase after which CaCl 2 can be added to initiate the calcium dependent nuclease activity of MNase to cleave the DNA around the target protein.
  • the protein- A-MNase reaction can be quenched by adding chelating agents (EDTA and EGTA). Cleaved DNA fragments are then liberated, extracted, and used to construct a sequencing library.
  • chelating agents EDTA and EGTA
  • the methods, kits and systems of the present disclosure involve the detection and quantification of chromatin accessibility in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA.
  • ATAC-seq Assay of Transpose Accessible Chromatin sequencing
  • NOMe-seq Nucleosome Occupancy and Methylome sequencing
  • FAIRE-seq Formmaldehyde-Assisted Isolation of Regulatory Elements sequencing
  • MNase-seq Merococcal Nuclease digestion with sequencing
  • DNase hypersensitivity assays are exemplary techniques of molecular biology useful in detecting and quantifying chromatin accessibility in samples.
  • DNase hypersensitivity assays can use the non-specific DNA endonuclease Deoxyribonuclease I (DNase I), which selectively digests accessible DNA regions.
  • DNase I hypersensitivity sites (DHS) identified by DNase-seq include open chromatin regulatory regions.
  • a typical DNase hypersensitivity assay can include a first step in which nuclei are isolated from cells using lysis buffer, and nuclei are digested using DNase I. DNA fragment sizes are measured 12953047v1 Page 40 of 224 Attorney Docket: 2014191-0043 to identify optimal digestion using gel electrophoresis. Biotinylated linkers can be ligated to the ends of digested DNA after polishing to make blunt ends, and the DNA can then be isolated.
  • DNA with biotinylated linker can be digested by restriction endonuclease MmeI and captured by streptavidin coated Dynabeads® to generate short tags to which a second sequencing adaptor can be ligated.
  • a second linker can be ligated and amplified to generate a library for sequencing.
  • a DNase-seq bioinformatic pipeline can include, e.g., alignment of sequence reads to a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference.
  • MNase-seq determines chromatin accessibility with micrococcal nuclease (MNase) that preferentially digests nucleosome-free, protein-unbound DNA.
  • MNase micrococcal nuclease
  • a typical MNase- seq assay can include a first step in which nuclei are isolated from either native or crosslinked chromatin and digested using MNase with titration.
  • In vivo formaldehyde crosslinking step that is designed to capture the interaction between proteins and DNA. This crosslinking allows bound proteins to shield their associated DNA from digestion by MNase.
  • samples are digested with MNase, which can be specifically activated by addition of Ca2+ to the buffer. Digestion can be halted by chelating the reaction, at which point the samples are RNase treated, crosslinks are reversed, and proteins are digested away from the chromatin. DNA can then be isolated via a phenol-chloroform extraction.
  • FAIRE-seq is a method in which nucleosome-depleted regions of DNA (NDRs) are isolated from chromatin.
  • a typical FAIRE-seq assay can include a first step in which cells are fixed using formaldehyde so that histones are crosslinked to interacting DNA.
  • Crosslinked chromatin can then be sheared by sonication that generates protein-free DNA and protein- crosslinked DNA fragments.
  • Protein-free DNA can be isolated using a phenol–chloroform extraction: DNA crosslinked with protein stays in organic phase, while protein-free DNA stays in aqueous phase. Highly crosslinked DNA remains in the organic phase and the non-crosslinked 12953047v1 Page 41 of 224 Attorney Docket: 2014191-0043 DNA is pulled to the aqueous phase.
  • Non-crosslinked DNA from the aqueous phase can then be amplified and sequenced. Reads enriched in the sequencing pool tend to have lower nucleosome and transcription factor binding and are therefore inferred to come from accessible regions.
  • NOMe-seq is a method to identify nucleosome-depleted regions of DNA (NDRs) with M.CviPI methyltransferase that methylates cytosine in GpC dinucleotides not protected by nucleosomes or other proteins.
  • NDRs nucleosome-depleted regions of DNA
  • M.CviPI methyltransferase that methylates cytosine in GpC dinucleotides not protected by nucleosomes or other proteins.
  • GpC m in the human genome does not occur naturally in most cell types.
  • GpC m levels at open chromatin regions can be compared to background signals and used to detect and quantify NDRs.
  • a typical NOMe-seq protocol can include a step in which samples are treated with M.CviPI and S-adenosylhomocysteine (SAM) to methylate accessible GpC sites.
  • SAM S-adenosylhomo
  • M.CviPI treated DNA can be sheared using a sonicator, so that DNA fragments can be sequenced.
  • DNA is treated with bisulfite, which converts unmethylated cytosine to uracil using sodium bisulfite, while methylated cytosine is unaffected.
  • a library is generated using adapters and sequenced. Accessible chromatin is expected to have high levels of GpC m but low levels of C m pG. Therefore, NOMe-seq identifies NDRs using the two separate methylation analyses that serve as independent (but opposite) measures, providing matched chromatin designations for each regulatory element.
  • ATAC-seq uses hyperactive Tn5 transposase that preferentially cuts accessible chromatin regions and simultaneously inserts adapters to the fragmented region (Buenrostro et al., Nat Methods (2013) 10(12):1213-1218 the entirety of which is incorporated herein by reference).
  • a typical ATAC-seq assay can include a first step in which samples are incubated with Tn5 transposase. DNA can then be isolated and purified. DNA fragmented and tagged by Tn5 transposase can be purified and then amplified to generate a library and sequenced for analysis.
  • the methods, kits and systems of the present disclosure involve the detection and quantification of chromatin accessibility in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA.
  • DNA methylation typically refers to the methylation of the 5’ position of cytosine (mC) by DNA methyltransferases (DNMT). It is a major epigenetic modification in humans and many other species.
  • MeDIP-seq was first reported by Weber et al., Nat Genet (2005) 37:853–862. In a typical MeDIP-seq protocol, antibody or antibody-fragment that binds 5-methylcytidine (5mC) is used to enrich methylated DNA fragments, then these fragments are sequenced and analyzed.
  • 5mC 5-methylcytidine
  • MBD-seq Methyl-CpG-Binding Domain sequencing
  • the present disclosure provides methods for obtaining a classifier, e.g., a validated classifier that can be used to determine MS status.
  • a subject is determined to have a validated epigenetic profile indicative of MS based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject, wherein the presence of the validated epigenetic profile has been determined using a validated classifier.
  • the validated classifier may be obtained by: (a) determining a genomic profile of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation in biological samples obtained from a first cohort of subjects who have previously been determined to have an MS; (b) determining a genomic profile of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation in biological samples obtained from a second cohort of healthy subjects or subjects who have previously been determined not to have MS; (c) comparing the genomic profile determined in step (a) and the genomic profile determined in step (b), to identify genomic loci that have statistically different histone modification, chromatin accessibility, binding of transcription factor, and/or DNA methylation levels (“differential loci”); (d) training a classifier on histone modification, chromatin accessibility, binding of transcription factor, and/or DNA methylation levels in the differential loci to distinguish between (i
  • Genomic Loci The present disclosure includes the identification of exemplary genomic loci that are differentially modified and/or differentially accessible in MS. See Tables 1-6 which show the chromosomal coordinates of each genomic locus, a measure of signal detected at each locus (BaseMean), fold-change observed in MS vs. healthy controls (log2FoldChange), statistical measures of significance (pvalue and padj) and the gene each loci is associated with (Symbol). The genomic loci are grouped by gene.
  • Tables 1-3 list loci for which an increase in epigenetic modifications was observed in MS subjects as compared to healthy subjects.
  • Tables 4-6 list loci for which a decrease in epigenetic modifications was observed in MS subjects as compared to healthy subjects.
  • the present disclosure is not limited to methods that use the exact same chromosomal coordinates that are recited in Tables 1-6.
  • the present disclosure encompasses methods that use any of the genomic loci in Table 1-6 and also subregions thereof, i.e., references herein to methods that involve detecting and/or quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci of Table 1-6 encompasses methods that detect these marks anywhere within these genomic loci including within any subregions.
  • Table 1 references chr1:10056254-10058255 as a genomic locus for detecting and/or quantifying H3K4me3 modification
  • this encompasses methods that detect and/or quantify H3K4me3 modification at any position or sub-region of chr1:10056254-10058255, e.g., methods that detect 12953047v1 Page 45 of 224 Attorney Docket: 2014191-0043 and/or quantify H3K4me3 modification within chr1:10056354-10058155, etc.
  • a subregion may span at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 or at least 3000 contiguous base pairs that are located between the lower and upper coordinates of a genomic locus recited in Tables 1-6. In some embodiments, a subregion may span less than 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 or at least 3000 contiguous base pairs that are located between the lower and upper coordinates of a genomic locus recited in Tables 1-6. In some embodiments, a subregion may have the same central coordinate as a genomic locus recited in Tables 1-6.
  • a subregion may have a different central coordinate as a genomic locus recited in Tables 1-6. It is also to be understood that the lower/upper coordinates of the genomic loci in Tables 1-6 are approximate and that the present disclosure encompasses methods where any one or more of the genomic loci are expanded by increasing the size of the genomic locus by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or up to 50% in one or both directions.
  • a classifier is generated using a set of differentially modified and/or differentially accessible genomic loci that are correlated with MS. Sequence reads that fall into each selected genomic locus are analyzed and counted, e.g., as described herein including the Examples.
  • counts from genomic loci that are correlated with MS are aggregated.
  • Other ways of using the genomic loci and related sequencing data to generate and apply a classifier to determine MS status are described herein and known in the art, e.g., without limitation, methods that use a learning statistical classifier system or a combination of learning statistical classifier systems.
  • exemplary genomic loci from one or more of Tables 1-6 are used in a monomodal classifier, e.g., a classifier that uses a single histone modification (e.g., H3K4me3 or H3K27ac) or DNA methylation at one or more genomic loci for purposes of determining MS status.
  • exemplary genomic loci from any one of Table 1- 6, or any combination thereof are used in combination in a multimodal classifier, e.g., a classifier that uses more than one histone modification (e.g., H3K4me3 and H3K27ac) or one or more histone modifications (e.g., H3K4me3 and/or H3K27ac) and DNA methylation at one or more genomic loci for purposes of determining MS status.
  • a multimodal classifier e.g., a classifier that uses more than one histone modification (e.g., H3K4me3 and H3K27ac) or one or more histone modifications (e.g., H3K4me3 and/or H3K27ac) and DNA methylation at one or more genomic loci for purposes of determining MS status.
  • a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and/or binding of a transcription factor at one or more loci provided in one or more of Tables 1-6.
  • a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and/or binding of a transcription factor at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 600, 800, 1,000, 1,500, 2,000, 3,000, 4,000, or more loci listed in one or more of Tables 1-6 (e.g., 1-200, 5- 200, 10-200, 15-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 1-150, 5-150, 10-150, 15-150, 20-150, 30-150, 40-150, 50-150, 60-150, 70-150, 80-150, 90-150, 100-150, 1-100, 5-100, 10-100, 15-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-150, 100-150, 1-
  • a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and/or binding of a transcription factor at each of the loci provided in one or more of Tables 1-6. In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and/or binding of a transcription factor for at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in one or more of Tables 1-6.
  • a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and/or binding of a transcription factor for at least a percent of loci identified in any one of Tables 1-6 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100%.
  • Differential H3K4me3 modification [0185] Exemplary genomic loci demonstrating differential H3K4 methylation (in particular H3K4 trimethylation, H3K4me3) in MS vs.
  • Tables 1 and 4 which shows the chromosomal coordinates of each genomic locus.
  • Table 1 lists exemplary loci in which, in some embodiments, H3K4me3 modifications are increased in MS subjects vs. healthy subjects
  • Table 4 lists loci in which, in some embodiments, H3K4me3 modifications are decreased in MS subjects vs. healthy subjects.
  • the genomic loci are sorted by 12953047v1 Page 47 of 224 Attorney Docket: 2014191-0043 fold increase in Tables 1 and 4. “Symbol” corresponds to the gene each differentially modified loci was associated with.
  • “lfcSE” and “stat” correspond to the standard error and test statistic values calculated by DEseq in a differential test.
  • Entrez is the entrez gene ID.
  • a person of skill in the art will recognize that the methods disclosed herein do not require that every genomic locus listed in Tables 1 and 4 be assessed for H3K4me3 modification. Instead, a subset of loci may be assessed for H3K4me3 modification.
  • Subsets of the genomic loci of Tables 1 and 4 can be selected (e.g., for use in determining MS status) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and/or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)).
  • Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier.
  • subsets of loci of Tables 1 and 4 are together, individually, and/or in randomly selected subsets, at least as informative (e.g., as statistically significant and/or reliable) for uses disclosed herein, e.g., for determining MS status.
  • the present disclosure particularly includes, among other things, subsets of the genomic loci of Tables 1 and 4, which have an absolute log2(fold-change) of 3.0 or higher, 2.9 or higher, 2.8 or higher, 2.7 or higher, 2.6 or higher, 2.5 or higher, 2.4 or higher, 2.3 or higher, 2.2 or higher, 2.1 or higher, 2.0 or higher, 1.9 or higher, 1.8 or higher, 1.7 or higher, 1.6 or higher, 1.5 or higher, 1.4 or higher, 1.3 or higher, 1.2 or higher, 1.1 or higher, 1.0 or higher, 0.9 or higher, 0.8 or higher, 0.7 or higher, 0.6 or higher, or 0.5 or higher.
  • the present disclosure also includes subsets of the genomic loci of Tables 1 and 4, which have an absolute log2(fold-change) of 2.8 to less than 3.0, 2.6 to less than 2.8, 2.4 to less than 2.6, 2.2 to less than 2.4, 2.0 to less than 2.2, 1.8 to less than 2.0, 1.6 to less than 1.8, 1.4 to less than 1.6, 1.2 to less than 1.4, 1.0 to less than 1.2, 0.8 to less than 1.0, 0.6 to less than 0.8, 0.4 to less than 0.6, or 0.4 to less than 0.6.
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, or 700 loci identified in Tables 1 and 4 (or any subset thereof) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject.
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least a number of loci identified in a Tables 1 and 12953047v1 Page 48 of 224 Attorney Docket: 2014191-0043 4 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, or 700 is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject.
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Tables 1 and 4 (e.g., about 1 to about 700, about 5 to about 700, about 10 to about 700, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least a percent of loci identified in Tables 1 and 4 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Tables 1 and 4).
  • a reference e.g., a sample
  • a subject 12953047v1 Page 49 of 224 Attorney Docket: 2014191-0043 from which the sample is obtained or derived is determined to have a particular MS status if at least one of the top 10 loci identified in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least one of the top 50 loci identified in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 10 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference.
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one of the top 10 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified in Tables 1 and 4 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 1 and 4 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, 12953047v1 Page 50 of 224 Attorney Docket: 2014191-0043 or 3000 loci identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one of the top 50 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) identified in Tables 1 and 4 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 1 and 4 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 1 and 4 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least one or more promoter regions associated with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 of the genes listed in Tables 1 and 4 (or any subset thereof) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a 12953047v1 Page 51 of 224 Attorney Docket: 2014191-0043 subject from which the sample is obtained or derived is determined to have a particular MS status if at least one or more promoter regions associated with one of more of the genes listed in Tables 1 and 4 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if one or more promoter regions associated with at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 genes identified in Tables 1 and 4 (e.g., about 1 to about 1000, about 5 to about 1000, about 10 to about 1000, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if one or more promoter regions of at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of the genes identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least promoter regions of a percent of genes identified in Tables 1 and 4 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more promoter regions associated with at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the genes having the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 12953047v1 Page 52 of 224
  • Attorney Docket: 2014191-0043 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top”
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least one or more promoter regions of one or more of the genes identified as having one of the top 10 loci in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if one or more promoter regions of one or more of the genes having one of the top 25 loci identified in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at one or more promoter regions of one or more genes identified as having one of the top 50 loci identified in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more promoter regions of at least five of the genes identified as having the top 10 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if one or more promoter regions of at least five genes identified as having one of the top 25 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more promoter regions of at least five of the genes identified as having one of top 50 loci in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference.
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more promoter regions of one or more genes (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 12953047v1 Page 53 of 224 Attorney Docket: 2014191-0043 or 10) identified as having one of the top 10 loci identified in Tables 1 and 4 and one or more promoter regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 genes identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., at least 1, at least 2, at
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one or more promoter regions of one of more genes identified as having one of the top 25 loci in Tables 1 and 4 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and one or more promoter regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 genes identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more promoter regions of one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) genes identified as having one of the top 50 loci identified in Tables 1 and 4 and one or promoter regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 genes identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more promoter regions of five or more genes identified as having one of the top 25 loci in Tables 1 and 4 and one or more promoter regions associated with at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 genes identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a 12953047v1 Page 54 of 224 Attorney Docket: 2014191-0043 sample or subject from which the sample is derived is determined to have a particular MS status if one or more promoter regions of at least five the genes identified in Tables 1 and 4 as having one of the top 50 loci and one or more promoter regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 genes identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more promoter regions of one or more marker genes of synaptic plasticity (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10; e.g., one or more marker genes of synaptic plasticity listed in Table 1) are differentially H3K4me3 modified (e.g., has increased H3K4me3 modifications) as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more promoter regions of SQSTM1 (e.g., chr5:179232387- 179234388), LILRB2 (e.g., chr19:54783399-54785400; chr19:54783483-54785484; chr19:54783738-54785739; or chr19:54784033-54786034), or a combination thereof is differentially H3K4me3 modified (e.g., has increased H3K4me3 modifications) as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more promoter regions of one or more MS- risk alleles (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10; e.g., one or more MS-risk alleles listed in Table 1) are differentially H3K4me3 modified (e.g., have increased H3K4me3 modifications) as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more promoter regions of TNFRSF14 (e.g., chr1:2486803-2488804) is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • differentially H3K4me3 modified refers to a methylation status characterized by an increase or decrease in a value measuring methylation (e.g., of read 12953047v1 Page 55 of 224 Attorney Docket: 2014191-0043 counts and/or normalized read counts for a given genomic locus), and/or a mean, median and/or mode thereof, and/or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%,
  • an increase or decrease in a value measuring methylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1-fold to 10- fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0- fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold, 1.2-fold to 4.0-fold, 1.4-fold to 4.0-fold, 1.6
  • a promoter region refers to a region a certain number of nucleotides upstream of a gene (e.g., 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 nucleotides upstream of a gene). In some embodiments, a promoter region refers to a region identified in Tables 1 and 4.
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, or 700 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject).
  • a reference e.g., a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject).
  • a reference e.g., a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if (a) H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, or 700 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and (b) H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 loci identified
  • a sample or subject from which the sample is obtained or derived is determined to have a particular status if: H3K4me3 modifications for at least 1 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 1 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject). H3K4me3 modifications for at least 5 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 5 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject).
  • H3K4me3 modifications for at least 10 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 10 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject).
  • H3K4me3 modifications for at least 25 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject)
  • H3K4me3 modifications for 12953047v1 Page 57 of 224 Attorney Docket: 2014191-0043 at least 25 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject).
  • H3K4me3 modifications for at least 50 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 50 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject).
  • H3K4me3 modifications for at least 75 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 75 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject).
  • H3K4me3 modifications for at least 100 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 100 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject).
  • a reference e.g., a healthy subject
  • H3K4me3 modifications for at least 100 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject).
  • Differential H3K27ac modification [0200] Exemplary genomic loci demonstrating differential H3K27ac modification in MS vs. subjects not having MS are provided in Tables 2 and 5 which shows the chromosomal coordinates of each genomic locus. Table 2 lists exemplary loci in which, in some embodiments, H3K27ac modifications are increased in MS subjects vs.
  • Table 5 lists exemplary loci in which, in some embodiments, H3K27ac modifications are decreased in MS subjects vs. healthy subjects.
  • the genomic loci are sorted by fold increase in Tables 2 and 5. “Symbol” corresponds to the gene each differentially modified loci was associated with. “lfcSE” and “stat” correspond to the standard error and test statistic values calculated by DEseq in a differential test. “Entrez” is the entrez gene ID. [0201]
  • a person of skill in the art will recognize that the methods disclosed herein do not require that every genomic locus listed in Tables 2 and 5 be assessed for H3K27ac modification. Instead, a subset of loci may be assessed for H3K27ac modification.
  • Subsets of the genomic loci of Tables 2 and 5 can be selected (e.g., for use in determining MS status) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and/or a particular threshold of differential between 12953047v1 Page 58 of 224 Attorney Docket: 2014191-0043 relevant states (e.g., a measured log2(fold-change)).
  • Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier.
  • subsets of loci of Tables 2 and 5, and loci included in such subsets are together, individually, and/or in randomly selected subsets, at least as informative (e.g., as statistically significant and/or reliable) for uses disclosed herein, e.g., for determining MS status.
  • the present disclosure also includes subsets of the genomic loci of Tables 2 and 5, which have an absolute log2(fold-change) of 4.0 to less than 4.2, 3.8 to less than 4.0, 3.6 to less than 3.8, 3.4 to less than 3.6, 3.2 to less than 3.4, 3.0 to less than 3.2, 2.8 to less than 3.0, 2.6 to less than 2.8, 2.4 to less than 2.6, 2.2 to less than 2.4, 2.0 to less than 2.2, 1.8 to less than 2.0, 1.6 to less than 1.8, 1.4 to less than 1.6, 1.2 to less than 1.4, 1.0 to less than 1.2, 0.8 to less than 1.0, 0.6 to less than 0.8, or 0.4 to less than 0.6.
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 loci identified in Tables 2 and 5 (or any subset thereof) are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject.
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least a number of loci identified in a Tables 2 and 5 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to 12953047v1 Page 59 of 224 Attorney Docket: 2014191-0043 have a particular MS status if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Tables 2 and 5 (e.g., about 1 to about 1000, about 5 to about 1000, about 10 to about 1000, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least a percent of loci identified in Tables 2 and 5 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Tables 2 and 5).
  • a reference e.g., a sample from a healthy subject
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least one of the top 10 loci identified in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy 12953047v1 Page 60 of 224 Attorney Docket: 2014191-0043 subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least one of the top 50 loci identified in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 10 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one of the top 10 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified in Tables 2 and 5 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 2 and 5 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000 loci identified in Tables 2 and 5 in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status 12953047v1 Page 61 of 224 Attorney Docket: 2014191-0043 if at least one of the top 50 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) identified in Tables 2 and 5 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000loci identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 2 and 5 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000 loci identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 2 and 5 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000loci identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if one or more enhancer regions associated with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 of the genes listed in Tables 2 and 5 (or any subset thereof) are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if one or more enhancer regions associated with one of more of the genes listed in Tables 2 and 5 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to 12953047v1 Page 62 of 224 Attorney Docket: 2014191-0043 have a particular MS status if one or more enhancer regions associated with at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 genes identified in Tables 2 and 5 (e.g., about 1 to about 1000, about 5 to about 1000, about 10 to about 1000, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if one or more enhancer regions of at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of the genes identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least enhancer regions of a percent of genes identified in Tables 2 and 5 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more enhancer regions associated with at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the genes having the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Tables 2 and 5).
  • a reference e.g., a sample from a healthy subject
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least one or more enhancer regions of one or more of the genes identified as having one of the top 10 loci in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if one or more enhancer regions 12953047v1 Page 63 of 224 Attorney Docket: 2014191-0043 of one or more of the genes having one of the top 25 loci identified in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at one or more enhancer regions of one or more genes identified as having one of the top 50 loci identified in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more enhancer regions of at least five of the genes identified as having the top 10 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if one or more enhancer regions of at least five genes identified as having one of the top 25 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more enhancer regions of at least five of the genes identified as having one of top 50 loci in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference.
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more enhancer regions of one or more genes (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified as having one of the top 10 loci identified in Tables 2 and 5 and one or more enhancer regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 genes identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one or more enhancer regions of one of more genes identified as having one of the top 25 loci in Tables 2 and 5 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and one or more enhancer regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 12953047v1 Page 64 of 224 Attorney Docket: 2014191-0043 500, 600, 700, 800, 900, 1000, or 1100 genes identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more enhancer regions of one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) genes identified as having one of the top 50 loci identified in Tables 2 and 5 and one or enhancer regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 genes identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more enhancer regions of five or more genes identified as having one of the top 25 loci in Tables 2 and 5 and one or more enhancer regions associated with at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 genes identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more enhancer regions of at least five the genes identified in Tables 2 and 5 as having one of the top 50 loci and one or more enhancer regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 genes identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more enhancer regions of one or more marker genes of synaptic plasticity (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10; e.g., one or more marker genes of synaptic plasticity listed in Table 2) are differentially H3K27ac modified (e.g., to have increased H3K27ac modifications) as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more enhancer regions of SQSTM1 (e.g., chr5:179232387- 179234388), LILRB2 (e.g., chr19:54783399-54785400; chr19:54783483-54785484; chr19:54783738-54785739; or chr19:54784033-54786034), or a combination thereof is differentially H3K27ac modified (e.g., to have increased H3K27ac modifications) as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more enhancer regions of one or more MS- risk alleles (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10; e.g., one or more MS-risk alleles listed in Table 2) are differentially H3K27ac modified (e.g., to have increased H3K27 modifications) as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more enhancer regions of TNFRSF14 (e.g., chr1:2486803-2488804) is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more enhancer regions of one or more cell identity genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) of an oligodendrocyte progenitor cell, an astrocyte, and/or an excitatory neuron cell are differentially H3K27ac modified (e.g., to have increased H3K27ac modifications) as compared to a reference (e.g., a sample from a healthy subject).
  • cell identity genes e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more
  • H3K27ac modified e.g., to have increased H3K27ac modifications
  • the one or more one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and/or an excitatory neuron cell are selected from genes provided in Table 2.
  • the one or more one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and/or an excitatory neuron cell comprise NSUN5, DAAM2, CNTN2, or any combination thereof.
  • the one or more enhancer regions of NSUN5 include chr7:72713576-72716059.
  • the one or more enhancer regions of DAAM2 include chr6:39788740-39793623.
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more enhancer regions of one or more cell identity genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) of a microglial cell is differentially 12953047v1 Page 66 of 224 Attorney Docket: 2014191-0043 H3K27ac modified (e.g., to have increased H3K27ac modifications) as compared to a reference (e.g., a sample from a healthy subject).
  • the one or more one or more cell identity genes of a microglial cell comprise MAP4K4.
  • differentially H3K27ac modified refers to an acetylation status characterized by an increase or decrease in a value measuring acetylation (e.g., of read counts and/or normalized read counts for a given genomic locus), and/or a mean, median and/or mode thereof, and/or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, or greater, or any
  • an increase or decrease in a value measuring acetylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1-fold to 10- fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0- fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold.1.2-fold to 4.0-fold.1.4-fold to 4.0-fold, 1.6
  • one or more enhancer regions of a recited gene are provided in Tables 2 and 5.
  • one or more enhancer regions of a recited gene corresponds to: (i) one or more loci with increased or decreased H3K27ac modifications as 12953047v1 Page 67 of 224 Attorney Docket: 2014191-0043 compared to a reference (e.g., a sample from a healthy subject) within a certain number of nucleotides (e.g., 50,000 nucleotides) of the recited gene; and/or (ii) one or more loci with increased or decreased H3K27ac modifications as compared to a reference (e.g., a sample from a healthy subject) that are closest to the recited gene in the genome.
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, or 500 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject).
  • a reference e.g., a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject).
  • a reference e.g., a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if (a) H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, or 500 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and (b) H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject).
  • a reference e.g
  • a sample or subject from which the sample is obtained or derived is determined to have a particular status if: H3K27ac modifications for at least 1 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 1 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject).
  • H3K27ac modifications for at least 5 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 5 12953047v1 Page 68 of 224 Attorney Docket: 2014191-0043 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject).
  • H3K27ac modifications for at least 10 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 10 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject).
  • H3K27ac modifications for at least 25 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 25 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject).
  • H3K27ac modifications for at least 50 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 50 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject).
  • H3K27ac modifications for at least 75 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 75 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject).
  • H3K27ac modifications for at least 100 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 100 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject).
  • Exemplary genomic loci demonstrating differential DNA methylation in MS are provided in Tables 3 and 6 which show the chromosomal coordinates of each genomic locus.
  • Table 3 lists exemplary loci in which, in some embodiments, increased DNA methylation is increased in MS subjects vs. healthy subjects
  • Table 6 lists exemplary loci in which, in some embodiments, DNA methylation is decreased in MS subjects vs. healthy subjects.
  • the genomic loci are sorted by fold increase in Tables 3 and 6. “Symbol” corresponds to the gene each 12953047v1 Page 69 of 224 Attorney Docket: 2014191-0043 differentially modified loci was associated with.
  • “lfcSE” and “stat” correspond to the standard error and test statistic values calculated by DEseq in a differential test. “Entrez” is the entrez gene ID.
  • “Entrez” is the entrez gene ID.
  • Subsets of the genomic loci of Tables 3 and 6 can be selected (e.g., for use in determining MS status) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and/or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)).
  • Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier.
  • an algorithm e.g., during the process of obtaining a classifier.
  • loci of Tables 3 and 6, and loci included in such subsets are together, individually, and/or in randomly selected subsets, at least as informative (e.g., as statistically significant and/or reliable) for uses disclosed herein, e.g., for determining MS status. See also the Examples of the present disclosure for experiments showing that informative classifiers can be generated using many different combinations of the loci.
  • the present disclosure particularly includes, among other things, subsets of the genomic loci of Tables 3 and 6, which have an absolute log2(fold-change) of 10.0 or higher, 6.5 or higher, 6.0 or higher, 5.5 or higher, 5.0 or higher, 4.5 or higher, 4.0 or higher, 3.5 or higher, 3.0 or higher, 2.5 or higher, 2.0 or higher, 1.9 or higher, 1.8 or higher, 1.7 or higher, 1.6 or higher, 1.5 or higher, 1.4 or higher, 1.3 or higher, 1.2 or higher, 1.1 or higher, 1.0 or higher, 0.9 or higher, 0.8 or higher, 0.7 or higher, 0.6 or higher, or 0.5 or higher.
  • the present disclosure also includes subsets of the genomic loci of Tables 3 and 6, which have an absolute log2(fold-change) of 10.0 or higher, 6.5 or higher, 6.0 or higher, 5.5 to less than 6.0, 5.0 to less than 5.5, 4.5 to less than 5.0, 4.0 to less than 4.5, 3.8 to less than 4.0, 3.6 to less than 3.8, 3.4 to less than 3.6, 3.2 to less than 3.4, 3.0 to less than 3.2, 2.8 to less than 3.0, 2.6 to less than 2.8, 2.4 to less than 2.6, 2.2 to less than 2.4, 2.0 to less than 2.2, 1.8 to less than 2.0, 1.6 to less than 1.8, 1.4 to less than 1.6, 1.2 to less than 1.4, 1.0 to less than 1.2, 0.8 to less than 1.0, or 0.6 to less than 0.8.
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) are differentially 12953047v1 Page 70 of 224 Attorney Docket: 2014191-0043 DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least a number of loci identified in a Tables 3 and 6 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy).
  • a reference e.g., a sample from a healthy
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Tables 3 and 6 (e.g., about 1 to about 650, about 5 to about 650, about 10 to about 650, about 25 to about 200, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci identified in Tables 3
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if at least a percent of loci identified in Tables 3 and 6 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one (e.g., at least 2, at least 3, at least 4, at 12953047v1 Page 71 of 224 Attorney Docket: 2014191-0043 least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Tables 3 and 6).
  • a reference e.g., a sample from a healthy subject
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least one of the top 10 loci identified in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least one of the top 50 loci identified in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 10 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one of the top 10 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified in Tables 3 and 6 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a 12953047v1 Page 72 of 224 Attorney Docket: 2014191-0043 sample from a healthy subject).
  • a reference e.g., a 12953047v1 Page 72 of 224 Attorney Docket: 2014191-0043 sample from a healthy subject.
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 3 and 6 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least one of the top 50 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) identified in Tables 3 and 6 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 3 and 6 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 3 and 6 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if one or more regions in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 or more) are differentially DNA methylated as 12953047v1 Page 73 of 224 Attorney Docket: 2014191-0043 compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if one or more regions in one or more of the genes listed in Tables 3 and 6 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if one or more regions associated with at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 genes identified in Tables 3 and 6 (e.g., about 1 to about 500, about 5 to about 500, about 10 to about 500, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if one or more regions of at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of the genes identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if one or more regions of at a percent of genes identified in Tables 3 and 6 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more regions of at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the genes having the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 12953047v1 Page 74 of 224
  • Attorney Docket: 2014191-0043 300, 350, 400, 450, 500, 550, 600, or 650 identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Tables 3 and 6).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if one or more regions of one or more of the genes identified as having one of the top 10 loci in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions of one or more of the genes having one of the top 25 loci identified in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if one or more regions of one or more genes identified as having one of the top 50 loci identified in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions of at least five of the genes identified as having the top 10 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a subject from which the sample is obtained or derived is determined to have a particular MS status if one or more regions of at least five genes identified as having one of the top 25 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions of at least five of the genes identified as having one of top 50 loci in Tables 3 and 6 are differentially DNA methylated as compared to a reference.
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more regions of one or more genes (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified as having one of the top 10 loci identified in Tables 3 and 6 and one or more enhancer regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 genes identified in Tables 3 and 6 (or any subset thereof) in total are 12953047v1 Page 75 of 224 Attorney Docket: 2014191-0043 differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject.
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more regions of at least one gene identified as having one of the top 25 loci in Tables 3 and 6 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and one or more regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 genes identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more regions of at least one (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) genes identified as having one of the top 50 loci identified in Tables 3 and 6 and one or more regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 genes identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more regions of five or more genes identified as having one of the top 25 loci in Tables 3 and 6 and one or more regions associated with at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 genes identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • a sample or subject from which the sample is derived is determined to have a particular MS status if one or more regions of at least five the genes identified in Tables 3 and 6 as having one of the top 50 loci and one or more regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 genes identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).
  • a reference e.g., a sample from a healthy subject
  • differentially DNA methylated refers to a methylation status characterized by an increase or decrease in a value measuring methylation (e.g., of read counts and/or normalized read counts for a given genomic locus), and/or a mean, median and/or mode thereof, and/or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to
  • an increase or decrease in a value measuring methylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase of 0.1-fold to 10-fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0-fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold.1.2-fold to 4.0-fold.1.4-fold to 4.0-fold, 1.6-fold to 0.1-fold to
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if DNA methylation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, or 500 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject).
  • a reference e.g., a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if DNA methylation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 12953047v1 Page 77 of 224 Attorney Docket: 2014191-0043 140, or 150 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject).
  • a reference e.g., a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular MS status if (a) DNA methylation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, or 500 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and (b) DNA methylation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject).
  • a reference e.g., a healthy subject
  • a sample or subject from which the sample is obtained or derived is determined to have a particular status if: DNA methylation for at least 1 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 1 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject). DNA methylation for at least 5 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 5 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject).
  • DNA methylation for at least 10 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 10 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject).
  • DNA methylation for at least 25 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 25 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject).
  • DNA methylation for at least 50 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 50 loci 12953047v1 Page 78 of 224 Attorney Docket: 2014191-0043 identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject).
  • DNA methylation for at least 75 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 75 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject).
  • Genomic loci provided in Tables 1-6 can also demonstrate differential chromatin accessibility or transcription factor binding in MS.
  • histone methylation e.g., H3K4me3 corresponds and/or is correlated with chromatin accessibility.
  • histone acetylation corresponds and/or is correlated with chromatin accessibility.
  • DNA methylation corresponds and/or is correlated with chromatin accessibility.
  • chromatin accessibility corresponds and/or is correlated with H3K4me3 modifications.
  • MS status may be determined by detecting and quantifying chromatin accessibility at one or more genomic loci in Tables 1 and 3 in accordance with the section above discussing exemplary genomic loci with differential H3K4me3 modifications.
  • chromatin accessibility corresponds and/or is correlated with H3K27ac modifications.
  • MS status may be determined by detecting and quantifying chromatin accessibility at one or more genomic loci in Tables 2 and 5 in accordance with the section above discussing exemplary genomic loci with differential H3K27ac modifications.
  • 12953047v1 Page 79 of 224 Attorney Docket: 2014191-0043
  • chromatin accessibility corresponds and/or is correlated with DNA methylation.
  • MS status may be determined by detecting and quantifying chromatin accessibility at one or more genomic loci in Tables 3 and 6 in accordance with the section above discussing exemplary genomic loci with differential DNA methylation.
  • histone methylation e.g., H3K4me3
  • histone acetylation e.g., H3K27ac
  • DNA methylation corresponds and/or is correlated with transcription factor binding.
  • binding of RNA pol II corresponds and/or is correlated with H3K4me3 modifications.
  • MS status may be determined by detecting and quantifying binding of RNA pol II at one or more genomic loci in Tables 1 and 4 in accordance with the section above discussing exemplary genomic loci with differential H3K4me3 modifications.
  • binding of p300, mediator complex, cohesin complex or RNA pol II corresponds and/or is correlated with H3K27ac modifications.
  • MS status may be determined by detecting and quantifying binding of p300, mediator complex, cohesin complex or RNA pol II at one or more genomic loci in Tables 2 and 5 in accordance with the section above discussing exemplary genomic loci with differential H3K27ac modifications.
  • Methods, kits and systems of the present disclosure include analysis of differentially modified and/or differentially accessible genomic loci to determine MS status. Methods, kits and systems of the present disclosure can be used in any of a variety of applications.
  • methods, kits and systems of the present disclosure can be used in detecting and/or treating MS. Methods, kits and systems of the present disclosure can also be used to detect or determine resistance of MS to a therapy or transformation of MS. 12953047v1 Page 80 of 224 Attorney Docket: 2014191-0043 [0236] In various embodiments, methods, kits and systems of the present disclosure can be applied to an asymptomatic human subject.
  • a subject can be referred to as “asymptomatic” if the subject does not report, and/or demonstrate by non-invasively observable indicia (e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or autoimmune screening), sufficient characteristics of MS to support a medically reasonable suspicion that the subject is likely suffering from MS. Detection of early- stage MS can be achieved using methods, kits and systems of the present disclosure, with attendant medical benefits including potential for early treatment and attendant improvement in therapeutic outcomes. [0237] In various embodiments, methods, kits and systems of the present disclosure can be applied to a symptomatic human subject.
  • a subject can be referred to as “symptomatic” if the subject report, and/or demonstrates by non-invasively observable indicia (e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or MS screening), sufficient characteristics of MS to support a medically reasonable suspicion that the subject is likely suffering from MS.
  • a sample from a subject, where the subject is suspected of having MS can be assayed according to one or more embodiments of the present disclosure to determine if the subject in fact has MS.
  • methods, kits and systems of the present disclosure can be used to determine that a subject has MS that correlates with a prior determination of MS (e.g., based on imaging, symptoms, and/or CSF analysis).
  • methods, kits and systems of the present disclosure can be used to validate or confirm a prior determination that a subject has MS.
  • regular, preventive, and/or prophylactic screening to determine MS status improves diagnosis of MS, including and/or particularly early-stage MS.
  • the present disclosure provides, among other things, methods, kits and systems particularly useful for the diagnosis and treatment of early-stage MS.
  • MS detection in accordance with the present disclosure is carried out annually, and/or in which a subject is asymptomatic at time of detecting, methods, kits and systems of the present disclosure are especially likely to detect early-stage MS.
  • 12953047v1 Page 81 of 224 Attorney Docket: 2014191-0043
  • detecting in accordance with methods, kits and systems of the present disclosure reduces MS symptoms.
  • MS status determination in accordance with the present disclosure is performed once for a given subject or multiple times for a given subject.
  • MS status determination in accordance with the present disclosure is performed on a regular basis, e.g., every six months, annually, every two years, every three years, every four years, every five years, or every ten years.
  • methods, kits and systems disclosed herein provide a determination of MS status. In other instances, methods, kits and systems disclosed herein will be indicative of MS status but not definitive for MS status. In various instances in which methods, kits and systems of the present disclosure are used to determine MS status, the same can be followed by a further confirmatory assay, which further assay can confirm, support, undermine, or reject a determination resulting from a prior determination, e.g., a determination in accordance with the present disclosure.
  • a confirmatory assay can be a test that is currently recognized by medical practitioners, e.g., based on symptoms, imaging, CSF analysis, or other testing.
  • MS status determination according to one or more methods, kits and/or systems disclosed herein is followed by treatment of MS.
  • treatment of MS includes administration of a therapeutic regimen including one or more therapies provided herein, including without limitation one or more of a low, moderate, or high efficacy MS therapy.
  • treatment of MS includes administration of a therapeutic regimen including one or more treatments provided herein as available, appropriate, and/or preferred for a particular MS status.
  • methods, kits and systems can be used to determine whether a particular subject is likely to be and/or is characterized as responsive to a MS therapeutic agent. In some such embodiments, methods, kits and systems can be followed by treatment of the subject with a MS therapeutic agent. [0245] In various embodiments, methods, kits and systems can be used to determine whether a particular subject is likely to be and/or is characterized as resistant to, non-responsive to, or not recommended treatment with an MS therapeutic agent. In some such embodiments, 12953047v1 Page 82 of 224 Attorney Docket: 2014191-0043 methods, kits and systems can be followed by treatment with a higher efficacy MS therapeutic agent.
  • Responsiveness can refer to the ability or likelihood of a therapy to cause a reduction in the number and/or size of MS lesions, improved symptoms, slowing of disease progression, shortening of flare duration, lessened disability, reduced disease activity, and/or an increased likelihood of achieving no evidence of disease activity status. Responsiveness can refer to improvement in prognosis. Responsiveness can refer to achievement of a treatment benefit, including e.g., improvement in one or more symptoms of MS.
  • Responsiveness can be measured quantitatively (e.g., as in the case of MS lesion size and/or numbers; as in the case of measurement of histone modification, chromatin accessibility, transcription factor binding, or DNA methylation at one or more genomic loci; or as in the calculation of clinical benefit (CBR)), or qualitatively (e.g., by measures such as “pathological complete response” (pCR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD), or other qualitative criteria).
  • CBR clinical benefit
  • Resistance can refer to the inability or unlikelihood of a therapy to achieve a desired therapeutic effect (e.g., a reduction MS lesions, lessened disability, reduced symptoms, or decreased flare frequency), or other treatment benefit such as, e.g., improvement in one or more symptoms of MS) in a subject.
  • Resistance includes natural resistance.
  • resistance includes the extent to which one or more desired therapeutic benefits results from administration of a therapy to a subject is less than that expected and/or achieved in a reference (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of benefit achieved in a reference).
  • methods, kits and systems can be used to detect the clinical efficacy of a course of therapy for MS.
  • methods and/or compositions of the present disclosure could be used to determine the presence, absence, or MS status of a subject over the course of treatment.
  • Methods and/or compositions of the present disclosure could be used in conjunction with, or confirmed by, other means of determining the presence, absence, or MS status of a subject including, for example measurements of lesion size or character by techniques such as MRI, measurements of immune activity using PET imaging, or by various qualitative, quantitative, or semi quantitative scoring systems including without limitation based on) in a qualitative fashion like “pathological complete response” (pCR), “clinical complete 12953047v1 Page 83 of 224 Attorney Docket: 2014191-0043 remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), or “clinical progressive disease” (cPD).
  • methods, kits and systems for MS status determination provided herein can inform treatment and/or payment (e.g., reimbursement for or reduction of cost of medical care, such as detecting or treatment) decisions and/or actions, e.g., by individuals, healthcare facilities, healthcare practitioners, health insurance providers, governmental bodies, or other parties interested in healthcare cost.
  • payment e.g., reimbursement for or reduction of cost of medical care, such as detecting or treatment
  • decisions and/or actions e.g., by individuals, healthcare facilities, healthcare practitioners, health insurance providers, governmental bodies, or other parties interested in healthcare cost.
  • methods, kits and systems for MS status determination provided herein can inform decision making relating to whether health insurance providers reimburse a healthcare cost payer or recipient (or not), e.g., for (1) MS status determination itself (e.g., reimbursement for detecting otherwise unavailable, available only for periodic/regular detecting, or available only for temporally- and/or incidentally- motivated detecting); and/or for (2) treatment, including initiating, maintaining, and/or altering therapy, e.g., based on the determined MS status.
  • methods, kits and systems for MS status determination provided herein are used as the basis for, to contribute to, or support a determination as to whether a reimbursement or cost reduction will be provided to a healthcare cost payer or recipient.
  • a party seeking reimbursement or cost reduction can provide results of MS status determination conducted in accordance with the present disclosure together with a request for such reimbursement or reduction of a healthcare cost.
  • a party making a determination as to whether or not to provide a reimbursement or reduction of a healthcare cost will reach a determination based in whole or in part upon receipt and/or review of results of MS status determination conducted in accordance with the present disclosure.
  • MS status determination using methods, kits and systems disclosed herein can be used in classifying subjects and/or samples (e.g., MS subjects and/or samples).
  • methods, kits and systems disclosed herein can be used to generate a set of subjects, and/or samples identified according to the present methods, kits and systems each classified as corresponding to a particular MS status, and optionally using two or more of such classified subjects, and/or samples to identify biomarkers that distinguish 12953047v1 Page 84 of 224 Attorney Docket: 2014191-0043 the classes (i.e., distinguish the subjects, and/or samples according to their class, e.g., according to their MS status).
  • samples obtained from a subject e.g., a liquid biopsy sample including cfDNA, e.g., a plasma sample including cfDNA
  • ChIP-seq for a histone modification e.g., H3K4me3 and/or H3K27ac
  • ChIP-seq sequence reads are aligned to human genome build hg19, e.g., using the Burrows-Wheeler Aligner (BWA).
  • BWA Burrows-Wheeler Aligner
  • MACS v2.1.1.20140616 can be used for ChIP-seq peak calling with a q-value (FDR) threshold of 0.01.
  • ChIP-seq data quality can optionally be evaluated by any of one or more of a variety of measures, including total peak number, FriP (fraction of reads in peak) score, number of high- confidence peaks (e.g., enriched > ten-fold over background), and percent of peak overlap with “blacklist” DHS peaks derived from the ENCODE project (Amemiya et al., Sci Rep (2019) 9(1):9354). If the ChIP-seq data quality is below a particular threshold the data may be discarded and the assay repeated.
  • ChIP-seq peaks that overlap with selected genomic loci that are differentially modified as provided herein for the relevant histone modification can then be used to determine MS status.
  • the number of reads overlapping the selected genomic loci for the relevant histone modification are summed, e.g., in some embodiments all the genomic loci that are differentially modified with an absolute log2(fold- .
  • the average number of reads in the local background of each ChIP-seq peak is subtracted to improve signal to noise.
  • the data can then be log2-transformed and quantile normalized to match the distribution of the data used to train the classifier.
  • the normalized data can then be used as input into a classifier that was trained using the same histone modification and selected genomic loci.
  • the classifier can then use the inputted data to determine ⁇ S status of the subject. It will be appreciated that this or similar approaches can be applied to assays of the present disclosure that quantify chromatin accessibility, transcription factor binding and/or DNA methylation. [0252] For the avoidance of any doubt, those of skill in the art will appreciate from the present disclosure that methods, kits and systems for MS status determination of the present 12953047v1 Page 85 of 224 Attorney Docket: 2014191-0043 disclosure are at least for in vitro use. Accordingly, all aspects and embodiments of the present disclosure can be performed and/or used at least in vitro.
  • methods of the present disclosure can be implemented on and/or in conjunction with a computer program and computer system.
  • methods of the present disclosure can be implemented on and/or in conjunction with a non-transitory computer readable storage medium encoded with the computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform the method.
  • a computer system can also store and manipulate data generated by methods of the present disclosure that comprise a plurality of genomic locus modification status and/or accessibility status changes/profiles, which data can be used by a computer system in implementing methods disclosed herein.
  • a computer system receives modification status and/or accessibility status data; (ii) stores the data; and (iii) compares the data in any number of ways described herein (e.g., analysis relative to appropriate references), e.g., to determine MS status.
  • a computer system (i) compares the genomic locus modification and/or accessibility status to a reference; and (ii) outputs an indication of whether the modification status and/or accessibility status of the genomic locus is significantly different from the reference and/or provides a determination regarding MS status.
  • Numerous types of computer systems can be used to implement methods of the present disclosure according to knowledge possessed by a skilled artisan in the bioinformatics and/or computer arts.
  • the software components can comprise both software components that are standard in the art and components that are special to the present disclosure (e.g., dCHIP software described in Lin et al., Bioinformatics (2004) 20:1233-1240, incorporated herein by reference in its entirety; radial basis machine learning algorithms (RBM) known in the art).
  • Methods of the present disclosure can also be programmed or modeled in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including specific algorithms to be used, thereby freeing a user of the need to procedurally program individual equations and algorithms.
  • a computer system comprises a database for storage of genomic locus modification status and/or accessibility status data. Such stored profiles can be accessed and used to perform comparisons of interest at a later point in time.
  • an algorithm can be a single learning statistical classifier system.
  • learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., a panel of genomic loci of interest) and making decisions based upon such data sets.
  • a single learning statistical classifier system such as a classification tree (e.g., random forest) is used.
  • a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem.
  • learning statistical classifier systems include, but are not limited to, those described in the Examples and also those using inductive learning (e.g., decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming.
  • inductive learning e.g., decision/classification trees such as random forests, classification and
  • methods of the present disclosure can include sending classification results to a medical practitioner, e.g., an oncologist.
  • a therapeutic agent or regimen is administered to a subject based on the MS status.
  • the therapeutic agent or regimen provided herein will be available, appropriate, and/or preferred for the determined MS status.
  • compositions for delivery of one or more therapeutic agents to a subject include pharmaceutical compositions for delivery of one or more therapeutic agents to a subject.
  • a pharmaceutical composition may be in any form known in the art, including formulations for administration according to any route known in the art. A suitable means of administration can be selected based on the age and condition of a subject.
  • Pharmaceutical composition forms of the present disclosure can include, e.g., liquid, semi-solid and solid dosage forms.
  • composition forms of the present disclosure can include, e.g., liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, and liposomes. Selection or use of any particular form may depend, in part, on the intended mode of administration and therapeutic application. Accordingly, the compositions can be formulated for administration by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection) or a non-parenteral mode. As used herein, parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection or infusion.
  • parenteral mode e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection
  • parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection or infusion.
  • compositions provided herein are present in unit dosage form, which unit dosage form can be suitable for self-administration.
  • a unit dosage form may be provided within a container, e.g., a pill, vial, cartridge, prefilled syringe, or disposable pen.
  • a pharmaceutical composition of the present disclosure can be in an injectable or infusible form.
  • the present disclosure includes sterile formulations for injection or 12953047v1 Page 88 of 224 Attorney Docket: 2014191-0043 infusion, which can be formulated in accordance with conventional pharmaceutical practices.
  • Sterile solutions can be prepared by incorporating a composition described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization.
  • Solutions can be formulated, e.g., using distilled water, physiological saline, or an isotonic solution containing glucose and other supplements such as D- sorbitol, D-mannose, D-mannitol, or sodium chloride as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, an alcohol such as ethanol and/or a polyalcohol such as propylene glycol or polyethylene glycol, and/or a nonionic surfactant such as polysorbate 80TM or HCO-50, and the like.
  • a suitable solubilizing agent for example, an alcohol such as ethanol and/or a polyalcohol such as propylene glycol or polyethylene glycol, and/or a nonionic surfactant such as polysorbate 80TM or H
  • sterile powders for the preparation of sterile injectable solutions methods for preparation include vacuum drying and freeze-drying that yield a powder of a composition described herein plus any additional desired ingredient (see below) from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin.
  • a pharmaceutical composition can be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage, e.g., at 2-8°C (e.g., 4°C).
  • a pharmaceutical composition of the present disclosure can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration.
  • dispersions are prepared by incorporating a composition described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a pharmaceutical composition can be formulated to include a pharmaceutically acceptable carrier or excipient.
  • compositions can be formulated with a carrier that will protect the therapeutic agent against rapid release, such as a controlled release formulation, 12953047v1 Page 89 of 224 Attorney Docket: 2014191-0043 including implants and microencapsulated delivery systems.
  • a carrier that will protect the therapeutic agent against rapid release, such as a controlled release formulation, 12953047v1 Page 89 of 224 Attorney Docket: 2014191-0043 including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known in the art.
  • Route of administration can be parenteral, for example, administration by injection.
  • Administration by injection can be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection.
  • Administration can be systemic or local.
  • a composition described herein can be therapeutically delivered to a subject by way of local administration.
  • local administration or “local delivery,” can refer to delivery that does not rely upon transport of the composition or therapeutic agent to its intended target tissue or site via the vascular system.
  • the composition may be delivered by injection or implantation of the composition or therapeutic agent or by injection or implantation of a device containing the composition or therapeutic agent.
  • the composition or therapeutic agent, or one or more components thereof may diffuse to an intended target tissue or site that is not the site of administration.
  • a pharmaceutical composition can be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid.
  • a pharmaceutical composition can be formulated by suitably combining the therapeutic molecule with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices.
  • pharmaceutically acceptable vehicles or media such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder.
  • examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent.
  • a buffer such as a phosphate buffer, or sodium acetate buffer
  • a soothing agent such as procaine hydrochloride
  • a stabilizer such as benzyl alcohol or phenol
  • an antioxidant an antioxidant.
  • the formulated injection can be packaged in a suitable ampule.
  • subcutaneous administration can be accomplished by means of a device, such as a syringe, a prefilled syringe, an auto-injector (e.g., disposable or reusable), a pen injector, a patch injector, a wearable injector, an ambulatory syringe infusion pump with subcutaneous infusion sets, or other device for combining with a therapeutic agent for subcutaneous injection.
  • a device such as a syringe, a prefilled syringe, an auto-injector (e.g., disposable or reusable), a pen injector, a patch injector, a wearable injector, an ambulatory syringe infusion pump with subcutaneous infusion sets, or other device for combining with a therapeutic agent for subcutaneous injection.
  • An injection system of the present disclosure may employ a delivery pen as described in U.S. Pat. No.5,308,341. Pen devices, most commonly used for self-delivery of insulin to patients with diabetes, are well known in the
  • Such devices can include at least one injection needle, are typically pre-filled with one or more therapeutic unit doses of a solution that includes the therapeutic agent and are useful for rapidly delivering solution to a subject with as little pain as possible.
  • One medication delivery pen includes a vial holder into which a vial of a therapeutic or other medication may be received.
  • the pen may be an entirely mechanical device or it may be combined with electronic circuitry to accurately set and/or indicate the dosage of medication that is injected into the user. See, e.g., U.S. Pat. No.6,192,891.
  • the needle of the pen device is disposable and the kits include one or more disposable replacement needles.
  • Pen devices suitable for delivery of any one of the presently featured compositions are also described in, e.g., U.S.
  • a microneedle-based pen device is described in, e.g., U.S. Pat. No.7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also the Precision Pen Injector (PPI) device, MOLLY TM , manufactured by Scandinavian Health Ltd.
  • PPI Precision Pen Injector
  • MOLLY TM manufactured by Scandinavian Health Ltd.
  • Nucleic acids encoding a therapeutic agent described herein can be incorporated into a gene construct to be used as a part of a gene therapy protocol to deliver nucleic acids that can be used to express and produce therapeutic agent within cells.
  • Expression constructs of such components may be administered in any therapeutically effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo.
  • Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1 (HSV-1), or 12953047v1 Page 91 of 224 Attorney Docket: 2014191-0043 recombinant bacterial or eukaryotic plasmids.
  • Viral vectors can transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized, polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO 4 precipitation.
  • a composition can be formulated for storage at a temperature below 0°C (e.g., -20°C or -80°C).
  • the composition can be formulated for storage for up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, or 2 years) at 2-8°C (e.g., 4°C).
  • compositions described herein are stable in storage for at least 1 year at 2-8°C (e.g., 4°C).
  • a pharmaceutical composition can include a therapeutically effective amount of a therapeutic agent described herein. Such effective amounts can be readily determined by one of ordinary skill in the art. A therapeutically effective amount can be an amount at which any toxic or detrimental effects of the composition are outweighed by therapeutically beneficial effects. In some embodiments, a dose can also be chosen to reduce or avoid production of antibodies or other host immune responses against a therapeutic agent. Those of skill in the art will appreciate that data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the amount of active ingredient included in a pharmaceutical composition is such that a suitable dose within the designated range can be administered to subjects.
  • the dose and method of administration can vary depending on weight, age, condition, and other characteristics of a patient, and can be suitably selected as needed by those skilled in the art.
  • Pharmaceutical compositions including certain therapeutic agents, e.g., therapeutic antibodies, can be administered as a fixed dose, or in a milligram per kilogram (mg/kg) dose.
  • an exemplary single dose of certain pharmaceutical compositions described herein can include certain therapeutic agents as described herein in an amount equal to, e.g., 0.001 to 1000 mg/kg, 1-1000 mg/kg, 1-100 mg/kg, 0.5-50 mg/kg, 0.1-100 mg/kg, 0.5-25 mg/kg, 1-20 mg/kg, and 1-10 mg/kg body weight.
  • Exemplary 12953047v1 Page 92 of 224 Attorney Docket: 2014191-0043 dosages of a composition described herein include, without limitation, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 4 mg/kg, 8 mg/kg, or 20 mg/kg. The present disclosure is not limited to such ranges or dosages.
  • the present disclosure further includes methods of preparing pharmaceutical compositions of the present disclosure and kits including pharmaceutical compositions of the present disclosure.
  • therapeutic agents of the present disclosure can be administered to a subject in a course of treatment that further includes administration of one or more additional therapeutic agents or therapies that are not therapeutic agents (e.g., surgery or radiation).
  • Combination therapies of the present disclosure can include simultaneous exposure of a subject to therapeutic agents of two or more therapeutic regimens.
  • a therapeutic agent as described herein can be administered together with (e.g., at the same time and/or in the same composition as) an additional agent or therapy.
  • a therapeutic agent of the present disclosure can be administered separately from an additional therapeutic agent or therapy (e.g., at a different time and/or in a different composition than the additional therapeutic agent or therapy). Dosing regimens of a therapeutic agent and one or more additional therapeutic agents with which it is administered in combination can be coordinated or independently determined. In various embodiments, an additional therapeutic agent or therapy administered in combination with a therapeutic agent as described herein can be administered at the same time as therapeutic agent, on the same day as therapeutic agent, or in the same week as therapeutic agent.
  • an additional therapeutic agent or therapy administered in combination with a therapeutic agent as described herein can be administered such that administration of the therapeutic agent and the additional therapeutic agent or therapy are separated by one or more hours before or after, one or more days before or after, one or more weeks before or after, or one or more months before or after administration of the therapeutic agent.
  • the administration frequency and/or dosage of one or more additional therapeutic agents can be the same as, similar to, or different from the administration frequency of a therapeutic agent.
  • the two or more regimens can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all “doses” of a first 12953047v1 Page 93 of 224 Attorney Docket: 2014191-0043 regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such therapeutic agents are administered in overlapping dosing regimens.
  • administration of a therapeutic agent can be to a subject having previously received, scheduled to receive, or in the course of a treatment regimen including an additional MS therapy. Administration of a therapeutic agent can, in some instances, improve delivery or efficacy of another therapeutic agent or therapy with which it is administered in combination.
  • therapeutic agent combination therapies can demonstrate synergy and/or greater-than-additive effects between a therapeutic agent and one or more additional therapeutic agents with which it is administered in combination.
  • a therapeutic agent can be administered in any effective amount as determined independently or as determined by the joint action of therapeutic agent and any of one or more additional therapeutic agents or therapies administered. Administration of the therapeutic agent may, in some embodiments, reduce the therapeutically effective dosage, required dosage, or administered dosage of the additional therapeutic agent or therapy relative to a reference regimen for administration of additional therapeutic agent or therapy or therapy absent the therapeutic agent.
  • a composition described herein can replace or augment other previously or currently administered therapy.
  • kits for detecting modification and/or accessibility of one or more genomic loci The present disclosure includes kits for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci. Kits of the present disclosure can include, e.g., reagents such as buffers and/or antibodies useful in the detection and quantification of histone modifications.
  • a kit of the present disclosure can include at least one antibody that selective binds a histone modification selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, or H3K4me3, or pan 12953047v1 Page 94 of 224 Attorney Docket: 2014191-0043 acetylation.
  • a kit of the present disclosure can include at least one antibody that selective binds H3K4me3 modifications.
  • a kit of the present disclosure can include at least one antibody that selective binds H3K27ac modifications.
  • kits of the present disclosure can include instructional materials disclosing or describing the use of the kit in a method of determining MS status and/or treatment disclosed herein.
  • a kit of the present disclosure can include one or more therapeutic agents useful in the treatment of MS, e.g., as disclosed herein, optionally in combination with instruction materials for treatment of MS.
  • a kit of the present disclosure comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from those provided in Tables 1-6.
  • the kit comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 1. In some embodiments, the kit comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 4. In some embodiments, the kit comprises reagents for quantifying H3K27ac for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2. In some embodiments, the kit comprises reagents for quantifying H3K27ac for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 5.
  • the kit comprises one or more antibodies for use in ChIP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.
  • the kit comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 3.
  • the kit comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 6.
  • the kit comprises one or more methyl-binding domains (e.g., for use in MBD-seq).
  • the kit comprises one or more antibodies that can bind methylated DNA (e.g., for use in MeDIP).
  • the kit comprises reagents for measuring chromatin accessibility via an ATAC-seq assay.
  • the kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample.
  • the kit comprises reagents for 12953047v1 Page 95 of 224 Attorney Docket: 2014191-0043 library preparation for sequencing.
  • the kit comprises reagents for sequencing.
  • the kit comprises instructions for determining if a subject has MS.
  • the present disclosure includes systems for detecting modification and/or accessibility of one or more genomic loci.
  • the present disclosure provides systems for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci.
  • Systems of the present disclosure can include a sequencer configured to generate a sequencing data set from a sample; and a non-transitory computer readable storage medium and/or a computer system.
  • the non-transitory computer readable storage medium is encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method of the present disclosure.
  • the computer system comprises a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform a method of the present disclosure.
  • the sequencer is configured to generate a Whole Genome Sequencing (WGS) data set from the sample.
  • the system also includes a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample.
  • the sample preparation device may include reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.
  • Systems of the present disclosure can include, e.g., reagents such as buffers and/or antibodies useful in the detection and quantification of histone modifications.
  • a system of the present disclosure can include at least one antibody that selective binds a histone modification selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, or H3K4me3, or pan acetylation.
  • a system of the present disclosure can include at least one antibody that selective binds H3K4me3 modifications.
  • a system of the present disclosure can include at least one antibody that selective binds H3K27ac modifications.
  • a system of the present disclosure can include instructional materials disclosing or describing the use of the system in a method of determining MS status and/or treatment disclosed herein.
  • a system of the present disclosure comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and/or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Tables 1-6.
  • the system comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 1.
  • the system comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2. In some embodiments, the system comprises reagents for quantifying H3K27ac for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2. In some embodiments, the system comprises reagents for quantifying H3K27ac for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 5. In some embodiments, the system comprises one or more antibodies for use in ChIP- seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac- modified histones.
  • the system comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 3. In some embodiments, the system comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 6. In some embodiments, the system comprises one or more methyl- binding domains (e.g., for use in MBD-seq). In some embodiments, the system comprises one or more antibodies that can bind methylated DNA (e.g., for use in MeDIP). [0291] In some embodiments, the system comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample.
  • cfDNA cell-free DNA
  • the sequencer comprises reagents for library preparation for sequencing. In some embodiments, the sequencer comprises 12953047v1 Page 97 of 224 Attorney Docket: 2014191-0043 reagents for sequencing. In some embodiments, the system comprises instructions for determining if a subject has MS. [0292] In some embodiments, the system comprises reagents for measuring chromatin accessibility via an ATAC-seq assay. Definitions [0293] “A” or “An”: The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” refers to one element or more than one element.
  • Accessibility Status or “Chromatin Accessibility Status”: As used herein, “accessibility status” or “chromatin accessibility status” of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of accessible chromatin. Accessibility status can be determined by various assays known in the art, including without limitation ChIP-seq as one example. Where two samples are separately analyzed by the same assay or comparable assays for detection of accessible DNA sequences, differences in chromatin accessibility status of genomic loci can be detected. Accessibility status can be compared to a standard or reference. A sample that has an accessibility status that differs in accessibility status from a standard or reference can be referred to as differentially modified.
  • Suitable assays for determining chromatin accessibility are known in the art.
  • Exemplary assays include ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), Mnase-seq (Micrococcal Nuclease digestion with sequencing), and/or a Dnase hypersensitivity assay.
  • the term “administration” typically refers to the administration of a disease appropriate (e.g., MS appropriate) treatment.
  • the disease appropriate treatment may comprise administering a composition to a subject, for example to achieve delivery of an agent that is, is included in, or is otherwise delivered by, the composition.
  • the disease appropriate treatment may comprise administering an appropriate surgical procedure or radiological procedure, optionally in combination with administration of a composition.
  • agent may refer to any chemical or physical entity, including without limitation any of one or more of an atom, e.g., a radioactive atom, molecule, compound, conjugate, polypeptide, polynucleotide, polysaccharide, lipid, cell, or combination or complex thereof.
  • Antibody refers to a polypeptide that includes one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen (e.g., a heavy chain variable domain, a light chain variable domain, and/or one or more CDRs).
  • antibody includes, without limitation, human antibodies, non-human antibodies, synthetic and/or engineered antibodies, fragments thereof, and agents including the same.
  • Antibodies can be naturally occurring immunoglobulins (e.g., generated by an organism reacting to an antigen). Synthetic, non-naturally occurring, or engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those of skill in the art.
  • each heavy chain includes a heavy chain variable domain (VH) and a heavy chain constant domain (CH).
  • VH heavy chain variable domain
  • CH heavy chain constant domain
  • the heavy chain constant domain includes three CH domains: CH1, CH2 and CH3.
  • a short region known as the “switch”, connects the heavy chain variable and constant regions.
  • the “hinge” connects CH2 and CH3 domains to the rest of the immunoglobulin.
  • Each light chain includes a light chain variable domain (VL) and a light chain constant domain (CL), separated from one another by another “switch.”
  • Each variable domain contains three hypervariable loops 12953047v1 Page 99 of 224 Attorney Docket: 2014191-0043 known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • CDR1, CDR2, and CDR3 three hypervariable loops 12953047v1 Page 99 of 224 Attorney Docket: 2014191-0043 known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4).
  • CDR1, CDR2, and CDR3 three hypervariable loops 12953047v1 Page 99 of 224
  • FR1, FR2, FR3, and FR4 four somewhat invariant “framework” regions
  • variable regions of a heavy and/or a light chain are typically understood to provide a binding moiety that can interact with an antigen. Constant domains can mediate binding of an antibody to various immune system cells (e.g., effector cells and/or cells that mediate cytotoxicity), receptors, and elements of the complement system. Heavy and light chains are linked to one another by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed.
  • an antibody is a polyclonal, monoclonal, monospecific, or multispecific antibody (e.g., a bispecific antibody).
  • an antibody includes at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at least one heavy chain-light chain dimer, or a tetramer that includes two heavy chain monomers and two light chain monomers.
  • antibody can include (unless otherwise stated or clear from context) any art-known constructs or formats utilizing antibody structural and/or functional features including without limitation intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), Small Modular ImmunoPharmaceuticals (SMIPs), single chain or Tandem diabodies (TandAb®), VHHs
  • SMIPs single
  • an antibody includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR) or variable domain.
  • an antibody can be a covalently modified (“conjugated”) antibody (e.g., an antibody that includes a polypeptide including one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked with one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule).
  • conjugated antibody e.g., an antibody that includes a polypeptide including one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked with one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule.
  • antibody sequence elements are humanized, primatized, chimeric, etc.,
  • An antibody including a heavy chain constant domain can be, without limitation, an antibody of any known class, including but not limited to, IgA, secretory IgA, IgG, IgE and IgM, based on heavy chain constant domain amino acid sequence (e.g. include but are not limited to human IgG1, IgG2, IgG3 and IgG4.
  • immunotype refers to the Ab class or subclass (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.
  • a “light chain” can be of a distinct type, e.g. amino acid sequence of the light chain constant domain.
  • an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human immunoglobulins.
  • Naturally produced immunoglobulins are glycosylated, typically on the CH2 domain.
  • affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification.
  • an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally.
  • antibodies produced and/or utilized in accordance with the present disclosure include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation.
  • an antibody can be specific for a particular histone modification (e.g., an antibody can bind one histone modification, e.g., H3K27ac with a higher affinity than other histone modifications, under conditions that are commonly used in ChIP-seq 12953047v1 Page 101 of 224 Attorney Docket: 2014191-0043 experiments).
  • an antibody is specific for an H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, or H3K4me3 modification.
  • an antibody is specific for an H3K27ac modification.
  • an antibody is specific for an H3K4me3 modification.
  • an antibody is a “pan” antibody.
  • pan antibody refers to an antibody that can bind a group of histone modifications having one or more features that are similar.
  • a pan antibody is a pan-methylation antibody (e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one methylated lysine, wherein the at least one methylated lysine can be at any one of a plurality of amino acid positions, e.g., in some embodiments, a pan-methylation antibody can bind an H3 protein comprising a methylated lysine at any position).
  • a pan-methylation antibody e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one methylated lysine, wherein the at least one methylated lysine can be at any one of a plurality of amino acid positions, e.g., in some embodiments, a pan-methylation antibody can bind an H3 protein comprising a methylated lysine at any position).
  • a pan antibody is a pan-acetylation antibody (e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one acetylated lysine, wherein the at least one acetylated lysine can be at any one of a plurality of amino acid positions, e.g., a pan-acetylation antibody can bind an H3 protein comprising an acetylated lysine at any position).
  • a pan antibody can bind one or more histone modifications that are associated with transcription activation.
  • a pan antibody can bind one or more histone modifications that are associated with transcription silencing.
  • an “antibody fragment” refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigen-binding portion or variable region thereof.
  • An antibody fragment can be produced by any means. For example, in some embodiments, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment can be recombinantly produced, i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment can be wholly or partially synthetically produced.
  • an antibody fragment (particularly an antigen-binding antibody fragment) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids.
  • 12953047v1 Page 102 of 224 Attorney Docket: 2014191-0043 [0306] Associated with: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other.
  • a particular entity e.g., an epigenetic profile comprising one or more histone modifications at a set of genomic loci, etc.
  • a particular disease, disorder, or condition if its presence, level and/or form correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g., across a relevant population).
  • two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
  • two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non- covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, or a combination thereof.
  • Between” or “From” As used herein, the term “between” refers to content that falls between indicated upper and lower, or first and second, boundaries, inclusive of the boundaries. Similarly, the term “from”, when used in the context of a range of values, indicates that the range includes content that falls between indicated upper and lower, or first and second, boundaries, inclusive of the boundaries.
  • biological sample typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell) of interest, as described herein.
  • a biological source is or includes an organism, such as a human subject.
  • a biological sample is or includes a biological tissue or fluid.
  • a biological sample can be or include cells, tissue, or bodily fluid.
  • Bodily fluids refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., blood, serum, plasma, Cowper’s fluid or pre- ejaculate fluid, chyle, chyme, stool, interstitial fluid, intracellular fluid, lymph, menses, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vitreous humor, vomit).
  • a biological sample can be or include blood, blood components, cell-free DNA (cfDNA), ascites, biopsy samples, surgical specimens, cell-containing body fluids, sputum, saliva, feces, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, gynecological 12953047v1 Page 103 of 224 Attorney Docket: 2014191-0043 fluids, secretions, excretions, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings or lavages such as a ductal lavages or bronchoalveolar lavages, aspirates, scrapings, or bone marrow.
  • cfDNA cell-free DNA
  • a biological sample is a liquid biopsy sample obtained from a bodily fluid.
  • a biological sample is or includes DNA obtained from a single subject or from a plurality of subjects.
  • a biological sample can be a “primary sample” obtained directly from a biological source or can be a “processed sample”, i.e., a sample that was derived from a primary sample, e.g., via dilution, purification, mixing with one or more reagents, or any other processing step(s) as described herein.
  • Blood component refers to any component of whole blood, including red blood cells, white blood cells, plasma, platelets, endothelial cells, mesothelial cells, epithelial cells, and cell-free DNA (cfDNA). Blood components also include the components of plasma, including proteins, metabolites, lipids, nucleic acids, and carbohydrates, and any other cells that can be present in blood, e.g., due to pregnancy, organ transplant, infection, injury, or disease.
  • Combination therapy refers to administration to a subject of two or more therapeutic agents or therapeutic regimens such that the two or more therapeutic agents or therapeutic regimens together treat a disease, condition, or disorder of the subject.
  • the two or more therapeutic agents or therapeutic regimens can be administered simultaneously, sequentially, or in overlapping dosing regimens.
  • combination therapy includes but does not require that the two therapeutic agents or therapeutic regimens be administered together in a single composition, nor at the same time.
  • the term “corresponding to” may be used to designate the position/identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition.
  • a monomeric residue in a polymer may be identified as “corresponding to” a residue in an appropriate reference polymer.
  • residues in a provided polypeptide or polynucleotide sequence are often designated (e.g., numbered or 12953047v1 Page 104 of 224 Attorney Docket: 2014191-0043 labeled) according to the scheme of a related reference sequence (even if, e.g., such designation does not reflect literal numbering of the provided sequence).
  • a reference sequence includes a particular amino acid motif at positions 100-110
  • a second related sequence includes the same motif at positions 110-120
  • the motif positions of the second related sequence can be said to “correspond to” positions 100-110 of the reference sequence.
  • corresponding positions can be readily identified, e.g., by alignment of sequences, and that such alignment is commonly accomplished by any of a variety of known tools, strategies, and/or algorithms, including without limitation software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, Hhpred/Hhsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE.
  • software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, Hhpred/Hhsearch, IDF, Infernal, KLAST, USEARCH, parasail,
  • Two sequences can be identified as corresponding if they are identical or if they share substantial identity, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more residues.
  • a nucleic acid sequence can correspond to a sequence that is identical or substantially identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the complement of the nucleic acid sequence, e.g., over a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more nucleic acid residues.
  • diagnosis includes the act, process, and/or outcome of determining whether, and/or the qualitative of quantitative probability that, a subject has or will develop the condition, disease, or related state.
  • diagnosing can include a determination relating to prognosis and/or likely response to one or more general or particular therapeutic agents or regimens.
  • Differentially accessible describes a genomic locus for which chromatin accessibility status differs between a first condition or sample and a second condition or sample (e.g., a standard or reference).
  • a differentially accessible genomic locus can include a greater or smaller measured accessibility 12953047v1 Page 105 of 224 Attorney Docket: 2014191-0043 under a selected condition of interest, such as MS, as compared to a reference state, such as a healthy subject.
  • Differentially modified describes a genomic locus for which histone modification status and/or DNA methylation status differs between a first condition or sample and a second condition or sample (e.g., a standard or reference).
  • a differentially modified genomic locus can include a greater or smaller number or frequency of histone modification and/or DNA methylations under a selected condition of interest, such as MS, as compared to a reference state, such as a healthy subject.
  • Identity refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules) and/or between polypeptide molecules.
  • % sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between protein and nucleic acid sequences as determined by the match between strings of such sequences.
  • Identity (often referred to as “similarity”) can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M. ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W. ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A.
  • calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences (or the complement of one or both sequences) for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non- identical sequences can be disregarded for comparison purposes).
  • the nucleotides or amino acids at corresponding positions are then compared.
  • a position in the first sequence is occupied 12953047v1 Page 106 of 224 Attorney Docket: 2014191-0043 by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally accounting for the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a computational algorithm, such as BLAST (basic local alignment search tool). Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin).
  • GCG Genetics Computer Group
  • BLASTP BLASTN
  • BLASTX Altschul et al., J Mol Biol (1990) 215:403-410
  • DNASTAR DNASTAR, Inc., Madison, Wisconsin
  • FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput Methods Genome Res [Proc Int Symp] (1994), Meeting Date 1992, 111-120. Eds. Suhai, Sandor. Plenum, New York, NY (the contents of each of which is separately incorporated herein by reference in its entirety).
  • Methylation status of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of DNA methylated sequences and/or the density (e.g., the measured density) of DNA methylation corresponding to the genomic locus.
  • Methylation status can be determined by various assays known in the art, including without limitation Bisulfite 12953047v1 Page 107 of 224 Attorney Docket: 2014191-0043 sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq). Where two samples are separately analyzed by the same assay or comparable assays for detection of DNA methylated sequences, differences in methylation status of genomic loci can be detected. Methylation status can be compared to a standard or reference.
  • Modification status or “histone modification status” of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of DNA sequences associated with histones bearing one or more histone modifications (e.g., one or more particular histone modifications) and/or the density (e.g., the measured density) of histone modifications (e.g., one or more particular histone modifications) corresponding to the genomic locus.
  • histone modifications e.g., one or more particular histone modifications
  • density e.g., the measured density
  • Modification status can be determined by various assays known in the art, including without limitation ChIP-seq as one example.
  • Other well-known assays include CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing and CUT&Tag (Cleavage Under Targets and Tagmentation).
  • CUT&RUN Cleavage Under Targets and Release Using Nuclease
  • CUT&Tag Cleavage Under Targets and Tagmentation
  • Modification status can be compared to a standard or reference.
  • a sample that has a modification status that differs in modification status or histone modification status from a standard or reference can be referred to as differentially modified.
  • a regulatory sequence is a nucleic acid sequence that controls expression of a coding sequence, e.g., a promoter sequence or an enhancer sequence. In some embodiments, a regulatory sequence can control or impact one or more aspects of gene expression (e.g., cell- type-specific expression, inducible expression, etc.).
  • Subject As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human).
  • a subject is suffering from a disease, disorder or 12953047v1 Page 108 of 224 Attorney Docket: 2014191-0043 condition (e.g., MS).
  • a subject is susceptible to a disease, disorder, or condition.
  • a subject displays one or more symptoms or characteristics of a disease, disorder or condition.
  • a subject is not suffering from a disease, disorder or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
  • a subject is a subject that has been tested for a disease, disorder, or condition, and/or to whom therapy has been administered.
  • a human subject can be interchangeably referred to as a “patient” or “individual”.
  • Therapeutic agent refers to any agent that elicits a desired pharmacological effect when administered to a subject.
  • an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population.
  • the appropriate population can be a population of model organisms or a human population.
  • an appropriate population can be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc.
  • a therapeutic agent is a substance that can be used for treatment of a disease, disorder, or condition (e.g., MS).
  • a therapeutic agent is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans.
  • a therapeutic agent is an agent for which a medical prescription is required for administration to humans.
  • Therapeutically effective amount refers to an amount that produces the desired effect for which it is administered.
  • the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition (e.g., MS) in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition.
  • a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
  • a therapeutically effective amount does not in fact require successful treatment be achieved in a particular 12953047v1 Page 109 of 224 Attorney Docket: 2014191-0043 individual.
  • a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.
  • reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.).
  • tissue e.g., a tissue affected by the disease, disorder or condition
  • fluids e.g., blood, saliva, serum, sweat, tears, urine, etc.
  • a therapeutically effective amount of a particular agent or therapy may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
  • treatment also “treat” or “treating” refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result.
  • such treatment can be of a subject who does not exhibit signs of the relevant disease, disorder, or condition and/or of a subject who exhibits only early signs of the disease, disorder, or condition (e.g., MS). Alternatively, or additionally, such treatment can be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, or condition.
  • a “prophylactic treatment” includes a treatment administered to a subject who does not display signs or symptoms of a condition to be treated or displays only early signs or symptoms of the condition to be treated such that treatment is administered for the purpose of diminishing, preventing, or decreasing the risk of developing the condition. Thus, a prophylactic treatment functions as a preventive treatment against a condition.
  • a “therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of a condition and is administered to the subject for the purpose of reducing the severity or progression of the condition.
  • Example 1 Materials and Methods
  • the present Example describes exemplary materials and methods that can be used to generate the sequencing data characterized in Example 2 to detect and characterize MS.
  • Materials Plasma samples [0326] Plasma samples were prepared from whole blood collected in EDTA blood collection tubes or Streck cell-free DNA BCT within 4-6 hours of collection and plasma was stored at - multiple sclerosis (MS) patients and healthy patients under a protocol approved by an IRB.
  • Chromatin immunoprecipitation (ChIP) Chromatin immunoprecipitation for histone marks (H3K4me3 and H3K27ac) in plasma samples was performed using methods similar to those previously described in Sadeh et al., Nat Biotechnol (2021) 39: 586-598 and Jang et al., Life Sci Alliance (2023) 6(12):e202302003. Briefly, about 1 mL frozen plasma was thawed and then prepared for ChIP.
  • On-target sites were identified from the 18-state chromHMM maps generated by Epimap (website: egg2.wustl.edu/roadmap/web_portal/chr_state_learning.html#exp_18state; accessed 10/4/21).
  • 200bp windows were selected with any of the following “active” chromatin states in > 50% of tissues in Epimap: 1_TssA, 2_TssFlnk, 3_TssFlnkU, 4_TssFlnkD, 8_EnhG2, and 14_TssBiv.
  • bioRxiv were used to assess for enrichment of Gene Ontology (GO) and MsigDB biological process / cell type signature annotations among genes near CREs.
  • Assessment of gene promoter activity based on H3K4me3 [0335] To estimate gene promoter activity, H3K4me3 was quantified within 1 kb of transcription start sites. Each peak was corrected for local background signal and the distribution of promoter counts were quantile normalized across samples. Differential promoter signals were quantified using Deseq2.
  • Assessment of gene enhancer activity based on H3K27ac [0336] H3K27ac cfChIP-seq peak calls present in 3 or greater MS/healthy samples were merged into a single Granges object and reduced to non-overlapping intervals.
  • Genomic loci that had differential analyte signal between MS and healthy subjects were determined using DESeq2 (Love et al., Genome Biol (2014) 15(12):550). These differential loci are shown in Table 1 (loci for which H3K4me3 modifications were found to increase), Table 4 (loci for which H3K4me3 modifications were found to decrease), Table 2 (loci for which H3K27ac modifications were found to increase), Table 5 (loci for which H3K27ac modifications were found to decrease), Table 3 (loci for which DNA methylation was found to increase) and Table 6 (loci for which DNA methylation was found to decrease) and grouped in accordance with the status they correlated with, i.e., Genomic locus (MS) or Genomic locus (healthy).
  • MS Genomic locus
  • Genomic locus healthy
  • autoimmune disorders e.g., MS, Rheumatoid Arthritis (RA), and systemic lupus erythematosus (SLE)
  • Promoter H3K4me3
  • enhancer signal H3K27ac
  • DNA methylation signal was also compared between MS subjects and healthy subjects, and differentially modified regions were identified.
  • Fig.2(B)-2(D) show volcano plots for all loci sequenced for each of H3K4me3, H3K27ac, and DNAme.
  • Fig.2(B)-2(D) a number of loci were identified that were differentially modified in MS subjects.
  • Differentially modified promoter loci were then analyzed to identify biological mechanisms underlying the MS disease state.
  • Figs.3(A)-(F) provide results of this analysis.
  • TNFRSF14 was among the genes found to be differentially modified.
  • 12953047v1 Page 116 of 224 Attorney Docket: 2014191-0043
  • MS subjects were found to have a statistically significant increase in promoter signal at the TNFRSF14 locus as compared to healthy subjects.
  • the data provided in Fig.3 indicates that upregulation of genes associated with synaptic plasticity (e.g., promoter signal associated with SQSTM1 and LILRB2) can be useful for detection and/or characterization of MS.
  • the particular genomic loci used for LILRB was chr19:54,782,258-54,785,464 and the particular genomic loci used from SQSTM1 was chr5:179232387-179234388.
  • 12953047v1 Page 117 of 224 Attorney Docket: 2014191-0043 [0344] Enhancer regions were assigned to their nearest gene within 50 kb. Genes were then rank ordered by MS vs.
  • Fig.4 MSigDB cell type signature gene sets (C8) to characterize cell types with which the detected loci were associated.
  • Fig.4(A) oligodendrocyte progenitor cells, fetal muscle Schwann cells, fetal cerebrum astrocytes, fetal cerebellum astrocytes, and embryonic ctx excitatory neurons (Ex 4) were all found to be enriched. These results were consistent with certain MS biological processes, including oligodendrocyte differentiation, neurogenesis, spinal cord development, neuron projection development, and glial cell differentiation. The enriched cell types also likely indicate preferential cell damage/death of the detected cell types.
  • Fig.4(A) again shows that technologies provided herein can detect biologically significant MS disease pathways and also provides several exemplary markers that can be used, e.g., to detect and/or characterize MS in a subject.
  • Figs.4(B) and 4(C) provide quantification of three genes that were found to have differential enhancer signal: NSUN5, DAAM2, and CNTN2, each of which are markers of oligodendrocyte development. As shown in Fig.4(C), statistically significant increases in enhancer signal was observed for each of these genes.
  • Fig.4(C) demonstrates that NSUN5, DAAM2, and CNTN2 activity (e.g., enhancer activity) can each be useful for characterizing MS disease state in a subject.
  • Fig.4(C) Also provided in Fig.4(C) is quantification of NSUN5, DAAM2, and CTN2 enhancer signal for RA and SLE; the data provided in Fig.4(C) shows that increased NSUN5, DAAM2, and CNTN2 enhancer signal was specific to MS.
  • enhancer signal was found to be under enriched for certain immune related signals in MS. See results provided in Fig.3(D), showing that enhancer signal decreased for genes associated with B lymphocyte Ovary CL18, Bone Marrow Na ⁇ ve T cell, Fetal Lung Mature B cells, and Bone Marrow Folicular B cells. Similar results were observed for promoter signals. Interestingly, under enrichment of promoter signal was not observed for all immune cells.
  • T cell associated genes e.g., TNFRSF14
  • upregulation of certain T cell associated genes was found to be increased in MS.
  • Fig.3(E) the under enrichment in enhancer signal observed in MS was not observed in other autoimmune disorders (in particular RA and SLE), again demonstrating that technologies provided herein detect MS-specific signal.
  • 12953047v1 Page 118 of 224 Attorney Docket: 2014191-0043 [0346]
  • Samples from MS subjects were also divided on the basis of TSPO-PET signal into “Moderate TPSO-PET Signal” and “Highest TSPO-PET Signal” groups, and the two groups were then analyzed to identify loci with differential epigenetic modifications.
  • Fig.5(A) shows the range of TSPO PET Signal (HRRT NAWM PK11195 DVR) observed in the MS subjects studied in the present experiment.
  • HRRT NAWM PK11195 DVR TSPO PET Signal
  • a bimodal distribution of signal was observed.
  • patients were divided into moderate or highest PET signal groups.
  • Fig.5(B) provides a volcano plot, identifying loci with differential enhancer signal in the moderate and highest TSPO-PET cohorts. Loci were analyzed to determine an associated cell state (e.g., using methods described above).
  • genomic loci associated with microglial and neutrophil cell identity genes were among the genomic loci found to exhibit the largest changes in enhancer signal in the two MS disease sates.
  • the data presented in Fig.5 demonstrates that MS subjects with different disease states (e.g., high and moderate PSMO-PET signal) exhibit epigenomic changes, which can be detected in the cfDNA using methods provided herein. Moreover, the data also provides exemplary loci that exhibit differential epigenomic signal and demonstrate that they can be used to determine MS disease severity in a subject (e.g., distinguish subjects with a moderate MS disease burden with those having a high MS disease burden).
  • a neutrophil-to-lymphocyte ratio can be used as a marker for MS disease activity, as it has been observed to be elevated in MS subjects and higher in MS subjects experiencing relapse compared to remission (see, e.g., Kivisäkk et al., “The neutrophil-to-lymphocyte ratio as a disease activity marker in multiple sclerosis and optic neuritis.” Journal of Neuroimmunology 309:73-79 (2017), the contents of which are incorporated by reference herein in their entirety).
  • a microglial and/or neutrophil enhancer signature using cfDNA 12953047v1 Page 119 of 224 Attorney Docket: 2014191-0043 can be used to indicate and/or predict MS disease severity and progression (disease flare and progression).
  • Tables identify exemplary genomic loci that are differentially modified and/or differentially accessible in MS vs. healthy subjects.
  • Table 1 is based on differential H3K4me3 modifications (in particular, H3K4me3 modifications that were found to increase in MS subjects as compared to healthy subjects).
  • Table 2 is based on differential H3K27ac modifications (in particular, H3K27ac modifications that were found to increase in MS subjects as compared to healthy subjects).
  • Table 3 is based on differential DNA methylation (in particular, DNA methylation that was found to increase in MS subjects as compared to healthy subjects).
  • Table 4 is based on differential H3K4me3 modifications (in particular, H3K4me3 modifications that were found to decrease in MS subjects as compared to healthy subjects).
  • Table 5 is based on differential H3K27ac modifications (in particular, H3K27ac modifications that were found to decrease in MS subjects as compared to healthy subjects).
  • Table 6 is based on differential DNA methylation (in particular, DNA methylation that was found to decrease in MS subjects as compared to healthy subjects).

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Abstract

La présente divulgation concerne, entre autres, des procédés, des kits et des systèmes permettant de déterminer l'état de la sclérose en plaques (SEP) chez un sujet. Dans divers modes de réalisation, la présente divulgation concerne l'utilisation d'une ou de plusieurs modifications d'histone, l'accessibilité de la chromatine, la liaison d'un ou plusieurs facteurs de transcription et/ou la méthylation de l'ADN qui sont caractéristiques de l'état de SEP. Dans certains modes de réalisation, des modifications différentielles et/ou une accessibilité différentielle sont détectées et quantifiées au niveau d'un ou plusieurs loci génomiques d'un échantillon biologique, par exemple, dans de l'ADN acellulaire (ADNcf) provenant d'un échantillon de biopsie liquide obtenu ou dérivé d'un sujet atteint de SEP. Dans divers modes de réalisation, un état déterminé est utile, par exemple, dans la sélection d'un traitement pour et/ou le traitement de la SEP.
PCT/US2025/044541 2024-09-03 2025-09-02 Procédés, kits et systèmes pour la détermination de l'état de sclérose en plaques et méthodes de traitement de la sclérose en plaques sur la base de ceux-ci Pending WO2026055162A2 (fr)

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