Assessing and treating spinal cord injury

Abstract

This document provides methods and materials for assessing and / or treating mammals (e.g., humans) having, or suspected of having, a spinal cord injury. For example, the presence of (a) spinal cord-derived cell free DNA (cfDNA) and / or (b) an elevated level of one or more of a fatty acid binding protein 3 (FABP3) polypeptide, a RE1-silencing transcription factor (REST) polypeptide, an interleukin 6 (IL-6) polypeptide, and a neurofilament, heavy polypeptide (NF-H) polypeptide in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) can be used to identify that mammal as having a spinal cord injury. In some cases, a mammal (e.g., a human) identified as having a spinal cord injury as described herein can be administered one or more spinal cord injury treatments.

Description

[0001] ASSESSING AND TREATING SPINAL CORD INJURY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Patent Application Serial No. 63 / 678,650, filed on August 2, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0004] TECHNICAL FIELD

[0005] This document relates to methods and materials for assessing and / or treating mammals (e.g., humans) having, or suspected of having, a spinal cord injury. For example, the presence of (a) spinal cord-derived cell free DNA (cfDNA) and / or (b) an elevated level of one or more of a fatty acid binding protein 3 (FABP3) polypeptide, a RE1 -silencing transcription factor (REST) polypeptide, an interleukin 6 (IL-6) polypeptide, and a neurofilament, heavy polypeptide (NF-H) polypeptide in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) can be used to identify that mammal as having a spinal cord injury. In some cases, this document provides methods and materials for treating a mammal identified as having a spinal cord injury. For example, a mammal (e.g., a human) identified as having a fluid sample (e.g., a blood sample) that includes the presence of (a) spinal cord-derived cfDNA and / or (b) an elevated level of one or more of a FABP3 polypeptide, a REST polypeptide, an TL-6 polypeptide, and a NF-H polypeptide can be administered one or more spinal cord injury treatments.

[0006] BACKGROUND

[0007] Acute spinal cord injury (SCI) results in lasting neurological deficits, decreased life expectancy, and incurs an immense psychosocial toll on patients and caregivers (Ropper et al., N Engl J Med. 376(14): 1358-1369 (2017); Collaborators GBDSCI, Lancet Neurol. 22(11): 1026-1047 (2023); and Ahuja et al., Nat Rev Dis Primers, 3:17018 (2017)). SCI also results in significant socioeconomic burden, in the United States alone, SCI is estimated to cost more than $9.7 billion annually (Collaborators GBDSCI, Lancet Neurol. 22(11): 1026- 1047 (2023)). Prompt diagnosis with physical examination and advanced neuroimaging followed by urgent surgical decompression of the spinal cord is considered standard of care for optimizing long-term neurologic function by limiting secondary injury (Badhiwala et al., Lancet Neurol. 20(2): 117-126 (2021); Bozzo et al., J Neurotrauma. 28(8): 1401-11 (2011); and Ramakonar et al., Spinal Cord. 59(8):933- 934 (2021)). Currently, there is no clinically useful biomarker to expedite SCI diagnosis, quantify injury severity, predict therapeutic response, facilitate clinical trials, or enable effective prognostication (Azad et al., Biomarkers. 28(8):703-713 (2023)).

[0008] SUMMARY

[0009] This document provides methods and materials for assessing and / or treating mammals (e.g., humans) having, or suspected of having, a spinal cord injury. For example, the methods and materials provided herein can be used to determine if a mammal (e.g., a human) has a spinal cord injury. In some cases, a molecular profile of a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) can be used to determine if that mammal has a spinal cord injury. For example, a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury can be assessed to determine if that mammal has a spinal cord injury based, at least in part, on the molecular profile of the fluid sample. In some cases, the presence of (a) spinal cord-derived cfDNA and / or (b) an elevated level of one or more of a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, and a NF-H polypeptide in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) can be used to identify that mammal as having a spinal cord injury. In some cases, this document provides methods and materials for treating a mammal identified as having a spinal cord injury. For example, a mammal (e.g., a human) identified as having a spinal cord injury based, at least in part, on having a fluid sample (e.g., a blood sample) that includes the presence of (a) spinal cord-derived cfDNA and / or (b) an elevated level of one or more of a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, and a NF-H polypeptide can be administered one or more spinal cord injury treatments.

[0010] As demonstrated herein, the molecular profile of a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury can distinguish whether that mammal has a spinal cord injury. For example, a mammal having a spinal cord injury can have a fluid sample (e.g., a blood sample) having a molecular profile that includes (a) the presence of spinal cord-derived cfDNA and / or (b) the presence of an elevated level of one or more of a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, and a NF-H polypeptide (e.g., as compared to a mammal that does not have any spinal cord injury). Also as demonstrated herein, the molecular profile of a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having a spinal cord injury can distinguish the severity of the spinal cord injury. For example, the level of spinal cord- derived cfDNA in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having a spinal cord injury can be used to determine the severity of the spinal cord injury. These findings provide circulating molecular profiles for spinal cord injuries, which have the potential to enhance the diagnosis, risk assessment and management for patients with spinal cord injury. In addition, circulating molecular profiles described herein can be detected using peripheral blood draws, thereby providing a minimally invasive and widely accessible means of diagnosis, optionally, in a point-of-care setting.

[0011] In general, one aspect of this document features methods for assessing a mammal suspected of having a spinal cord injury. The methods can include, or consist essentially of, (a) determining (1) that a blood sample from a mammal suspected of having a spinal cord injury contains the presence of spinal cord-derived cfDNA, and (2) that the blood sample contains the presence of at least one of: an elevated level of a fatty acid binding protein 3 (FABP3) polypeptide or mRNA encoding the FABP3 polypeptide, an elevated level of a RE1 -silencing transcription factor (REST) polypeptide or mRNA encoding the REST polypeptide, an elevated level of an interleukin 6 (IL-6) polypeptide or mRNA encoding the IL-6 polypeptide, and an elevated level of a neurofilament, heavy polypeptide (NF-H) or mRNA encoding the NF-H polypeptide; (b) classifying the mammal as having a spinal cord injury if the presence of (1) is determined and the presence of (2) is determined. The mammal can be a human. The presence or absence of the elevated level of the FABP3 polypeptide or mRNA encoding the FABP3 polypeptide is determined. The presence or absence of the elevated level of the REST polypeptide or mRNA encoding the REST polypeptide is determined. The presence or absence of the elevated level of the IL-6 polypeptide or mRNA encoding the IL-6 polypeptide is determined. The presence or absence of the elevated level of the NF-H polypeptide or mRNA encoding the NF-H polypeptide is determined. The method can include determining the presence of each of the elevated level of the FABP3 polypeptide or mRNA encoding the FABP3 polypeptide, the elevated level of the REST polypeptide or mRNA encoding the REST polypeptide, the elevated level of the IL-6 polypeptide or mRNA encoding the IL-6 polypeptide, and the elevated level of the NF-H polypeptide or mRNA encoding the NF-H polypeptide. The blood sample can be a plasma sample.

[0012] In another aspect, this document features methods for determining the severity of a spinal cord injury within a mammal. The methods can include, or consist essentially of, determining the level of spinal cord-derived cfDNA in a blood sample from a mammal having a spinal cord injury, and (a) classifying the spinal cord injury as being an American Spinal Injury Association (ASIA) grade A spinal cord injury when the spinal cord-derived cfDNA is present at a level of from about 45 genome equivalents (hGE) per mL of the blood sample (hGE / mL) to about 1000 hGE / mL; (b) classifying the spinal cord injury as being an ASIA grade B spinal cord injury when the spinal cord-derived cfDNA is present at a level of from about 11 hGE / mL to about 44 hGE / mL; (c) classifying the spinal cord injury as being an ASIA grade C spinal cord injury when the spinal cord-derived cfDNA is present at a level of from about 5 hGE / mL to about 10 hGE / mL; or (d) classifying the spinal cord injury as being an ASIA grade D spinal cord injury when the spinal cord-derived cfDNA is present at a level of from about 0.1 hGE / mL to about 4.9 hGE / mL. The mammal can be a human. The blood sample can be a plasma sample.

[0013] In another aspect, this document features methods for treating a mammal having a spinal cord injury where the methods can include, or consist essentially of, (a) determining that a blood sample from the mammal includes (1) the presence of spinal cord-derived cfDNA, and (2) the presence of at least one of an elevated level of a FABP3 polypeptide or mRNA encoding the FABP3 polypeptide, an elevated level of a REST polypeptide or mRNA encoding the REST polypeptide, an elevated level of an IL-6 polypeptide or mRNA encoding the IL-6 polypeptide, and an elevated level of a NF-H or mRNA encoding the NF-H polypeptide; and (b) administering a spinal cord injury treatment to the mammal. The determining step can be performed in less than 1 hour. In another aspect, this document features methods for treating a spinal cord injury where the methods can include, or consist essentially of, subjecting a mammal identified as having a blood sample including (1) the presence of spinal cord-derived cfDNA, and (2) the presence of at least one of an elevated level of a FABP3 polypeptide or mRNA encoding the FABP3 polypeptide, an elevated level of a REST polypeptide or mRNA encoding the REST polypeptide, an elevated level of an IL-6 polypeptide or mRNA encoding the IL-6 polypeptide, and an elevated level of a NF-H or mRNA encoding the NF-H polypeptide to a spinal cord injury treatment. The mammal can be a human. The blood sample can be a plasma sample. The spinal cord injury can be traumatic SCI, traumatic central cord syndrome, acute epidural spinal cord compression from cancer, acute epidural spinal cord compression from infection, or subacute to chronic spinal cord compression from degenerative cervical spondylosis.

[0014] In another aspect, this document features methods for detecting spinal cord-derived cfDNA in a blood sample. The methods can include, or consist essentially of, (a) determining that the blood sample including cfDNA contains the presence of (1) a hypom ethylated cg27088725 locus, (2) a hypomethylated cg07209034 locus, (3) a hypom ethylated cg24336338 locus, (4) a hypomethylated cg03776878 locus, or (5) a hypomethylated cg23617848 locus; and (b) classifying the cfDNA as spinal-cord derived cfDNA if the presence of any one of (1) - (5) is determined. The blood sample can be a human blood sample. The sample can be a plasma sample.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1. Overview of an exemplary study for detection of acute spinal cord injury via a multi-analyte assay of peripheral blood.

[0018] Figure 2. Cohort overview with patient, operative, and injury characteristics. Figures 3A-3E. Methylation-based cell-free DNA markers of spinal cord injury. Figure 3A) Structure at a genomic locus identified as spinal cord-specific biomarker. Lollipops represent CpG sites. Red highlights hypomethylated CpG sites identified in the Illumina Methyl ationEPIC 850K array. Arrows mark positions of PCR primers. Figure 3B) Spike-in experiment demonstrating the ddPCR assay sensitivity for detection of spinal cord- derived biomarkers. Human spinal cord gDNA was mixed with human leukocyte gDNA in the indicated proportions (0 to 5%), and the concentration (copies / uL) of fully unmethylated spinal cord-derived markers was determined. Figure 3C) Receiver operating curve demonstrating the ability of our hypomethylation-based ddPCR assay to discriminate between acute SCI patients and healthy controls. Figure 3D) Plasma concentration in genome equivalents (hGE / mL) of spinal cord-derived cfDNA in acute SCI patients. Figure 3E) Percent of spinal cord-derived cfDNA out of total measured cfDNA in acute SCI patients.

[0019] Figures 4A-4G. Proteomic profiling of plasma from patients with acute spinal cord injury. Figure 4A) Principal component analysis of protein biomarkers measured in acute spinal cord injury patients versus healthy controls. Figures 4B-4C) Difference in GFAP and NF-L concentrations between acute SCI patients detected using ddPCR and healthy controls. Figures 4D-4G) Difference in FABP3, REST, IL-6, and NF-H concentrations between acute SCI patients detected using ddPCR and healthy controls.

[0020] Figures 5A-5F. Development of the Spinal Cord Injury Index (SCII). Figure 5A) Correlation between concentrations of spinal cord-derived cfDNA and FABP3, REST, IL-6, and NF-H. Figure 5B) Receiver Operating Characteristic (ROC) curve for ability of SCII to discriminate between patients with acute SCI and healthy controls. Figure 5C) Distribution of SCII across injury severity. Yellow indicates patient who achieved 6-month ASIA conversion. Figure 5D) Receiver Operating Characteristic (ROC) curve for ability of preoperative SCII to predict 6-month ASIA conversion. Figure 5E) Vignette of ASIA B tSCI patient who achieved ASIA conversion by 3 months post SCI. Figure 5F) Vignette of ASIA B CCS patient who did not achieve ASIA conversion by 6 months post SCI.

[0021] Figures 6A-6D. Representative 2D ddPCR plots for (Figure 6A) spinal cord genomic DNA extracted from healthy patient rapid autopsy spinal cord tissue (Figure 6B) cell-free DNA extracted from healthy patient plasma (Figure 6C) cell-free DNA extracted from acute SCI patient plasma and (Figure 6D) genomic DNA extracted from acute SCI patient peripheral blood mononuclear cells.

[0022] Figure 7. Spike-in experiment using 3 CpG site assay demonstrates poor technical calibration for detection of spinal cord neuron-specific biomarkers. Human spinal cord gDNA was mixed with human leukocyte gDNA in the indicated proportions (0 to 5%), and the concentration (copies / uL) of fully unmethylated spinal cord neuron markers was determined.

[0023] Figures 8A-8B. Spike-in experiment using 2 CpG site assay (Figure 8A) and inverted 2 CpG site assay (Figure 8B). Human spinal cord gDNA was mixed with human leukocyte gDNA in the indicated proportions and the concentration (copies / uL) of unmethylated spinal cord loci (Figure 8A) or methylated spinal cord loci (Figure 8B) was determined.

[0024] Figures 9A-9C. Performance of integrated protein and spinal cord-specific cfDNA ("diagnosis") score tuned for acute SCI versus healthy control discrimination. Included proteins: NF-H, SAA1, S100A12, IL-10, NF-L. Figure 9A) Receiver Operating Characteristic (ROC) curve for ability of diagnosis score to discriminate between patients with acute SCI and healthy controls. Figure 9B) Distribution of diagnosis score across injury severity. Figure 9C) ROC curve for ability of preoperative diagnosis score to predict 6-month ASIA conversion.

[0025] Figure 10. Changes in NPQ levels for GFAP and NF-L, as well as SCII levels over the immediate perioperative course of a patient with cervicothoracic ependymoma. DETAILED DESCRIPTION

[0026] This document provides methods and materials for assessing and / or treating mammals (e.g., humans) having, or suspected of having, a spinal cord injury. In some cases, this document provides methods and materials that can be used to determine whether or not a mammal (e.g., a human) has a spinal cord injury. For example, a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury can be assessed for the molecular profile of the fluid sample. As described herein, a distinct molecular profile can be present in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having a spinal cord injury (e.g., as compared to a molecular profile that can be present in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) that does not have a spinal cord injury). This document also provides methods and materials for treating a mammal identified as having a spinal cord injury as described herein. For example, a mammal having a fluid sample (e.g., a blood sample) that includes (a) the presence of spinal cord-derived cfDNA and / or (b) the presence of an elevated level of one or more of a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, and a NF-H polypeptide can be administered one or more spinal cord injury treatments.

[0027] In some cases, the methods provided herein can be used to identify a mammal (e.g., a human) as having a spinal cord injury efficiently. For example, the methods provided herein can be used to identify a mammal (e.g., a human) as having a spinal cord injury in less than 16 hours (e.g., less than 12 hours, less than 10 hours, less than 8 hours, less than 5 hours, less than 3 hours, or less than 1 hour). For example, the methods provided herein can be used to identify a mammal (e.g., a human) as having a spinal cord injury in from about 8 hours to about 16 hours (e.g., from about 8 hours to about 12 hours, from about 8 hours to about 10 hours, from about 8 hours to about 9 hours, from about 9 hours to about 16 hours, from about 10 hours to about 16 hours, from about 12 hours to about 16 hours, from about 9 hours to about 15 hours, from about 10 hours to about 15 hours, or from about 11 hours to about 14 hours).

[0028] A mammal (e.g., a human) can be assessed to determine whether or not the mammal has a spinal cord injury by detecting the molecular profile of a fluid sample (e.g., a blood sample) obtained from the mammal. In some cases, a fluid sample (e.g., a blood sample) obtained from a mammal obtained from a mammal having, or suspected of having, a spinal cord injury can be assessed to determine if the mammal has a spinal cord injury based, at least in part, on the molecular profde of the fluid sample. For example, a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury can be assessed for the presence or absence of spinal cord-derived cfDNA to determine whether or not that mammal has a spinal cord injury. For example, a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury can be assessed for the presence or absence of an elevated level of one or more of a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, and a NF-H polypeptide to determine whether or not that mammal has a spinal cord injury.

[0029] Any type of mammal can be assessed and / or treated as described herein. Examples of mammals that can be assessed and / or treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats. In some cases, a human (e.g., a subject or a patient) can be assessed and / or treated as described herein.

[0030] A mammal (e.g., a human) being assessed and / or treated as described herein can have, or can be suspected of having, any type of spinal cord injury. A spinal cord injury can be a complete spinal cord injury or an incomplete spinal cord injury. A spinal cord injury can include damage to the spinal cord itself and / or to one or more bones that surround the spinal cord. In some cases, a spinal cord injury can be an acute spinal cord injury. Examples of spinal cord injuries include, without limitation, traumatic SCI, traumatic central cord syndrome, and acute epidural spinal cord compression (e.g., acute epidural spinal cord compression from cancer, acute epidural spinal cord compression from infection, and subacute to chronic spinal cord compression from degenerative cervical spondylosis).

[0031] A mammal (e.g., a human) being assessed and / or treated as described herein can have, or can be suspected of having, a spinal cord injury in any area of the spinal cord. Examples of spinal cord areas within a mammal that can have a spinal cord injury that can be assessed and / or treated as described herein can include, without limitation, cervicomedullary spinal cord, cervical spinal cord, thoracic spinal cord, and thoracolumbar spinal cord. Any appropriate fluid sample from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury can be assessed as described herein (e.g., assessed for (a) the presence of spinal cord-derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide) in a fluid sample (e.g., a blood sample) obtained from a mammal such as a human). In some cases, a fluid sample can be a biological sample. For example, a fluid sample can include peripheral blood mononuclear cells (PBMCs). For example, a fluid sample can include one or more biological molecules (e g., nucleic acids such as DNA (e g., cfDNA) and RNA, polypeptides, carbohydrates, lipids, hormones, and / or metabolites). Examples of fluid samples that can be assessed as described herein include, without limitation, blood samples (e.g., whole blood samples, serum samples, and plasma samples), urine, saliva, cerebral spinal fluid (CSF), lymphatic fluid, and surgical drain fluid. A fluid sample can be a fresh sample or a frozen sample. In some cases, a fluid sample can be processed to isolate or extract one or more biological molecules. For example, a blood sample can be obtained from a mammal having, or suspected of having, a spinal cord injury and can be assessed to determine the molecular profile of the blood sample.

[0032] In some cases, a fluid sample that can be assessed as described herein (e.g., assessed for (a) the presence of spinal cord-derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide) in a fluid sample (e.g., a blood sample) obtained from a mammal such as a human) is not a CSF sample.

[0033] A fluid sample (e.g., a blood sample) that can be assessed as described herein (e.g., assessed for (a) the presence of spinal cord-derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide) in a fluid sample (e.g., a blood sample) obtained from a mammal such as a human) can be obtained from a mammal (e.g., a human) at any appropriate time. In some cases, a fluid sample can be obtained from a mammal after the mammal has sustained a spinal cord injury. For example, a fluid sample can be obtained from a mammal within 7 days of the mammal sustaining a spinal cord injury. In some cases, a sample can be obtained from a mammal after the mammal has sustained a spinal cord injury, but before the mammal is subjected to a surgery (e g., surgical decompression of the spinal cord). In some cases, a sample can be obtained from a mammal after the mammal has been subjected to a surgery (e.g., surgical decompression of the spinal cord).

[0034] In some cases, two or more (e.g., two, three, four, five, six, or more) samples can be obtained from a single mammal (e.g., a single human). For example, a pre-operative sample can be obtained from a mammal after the mammal has sustained a spinal cord injury and before the mammal is subjected to a surgical decompression of the spinal cord, two or more (e.g., two, three, four, five, or more) peri-operative samples can be obtained from the mammal during the surgical decompression of the spinal cord, and, optionally, a postoperative sample can be obtained from the mammal following surgical decompression of the spinal cord. In some cases, serially obtained peri-operative samples can be used to monitor the progress of a neurosurgery (e.g., surgical decompression of a spinal cord or resection of a tumor intrinsic to the spinal cord). For example, serially obtained peri-operative samples can be used to detect changes in the molecular profile of the fluid sample in real time, thereby allowing real time monitoring of the progress of a neurosurgery (e.g., surgical decompression of a spinal cord or resection of a tumor intrinsic to the spinal cord).

[0035] A fluid sample (e.g., a blood sample) that can be assessed as described herein (e.g., assessed for (a) the presence of spinal cord-derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide) in a fluid sample (e.g., a blood sample) obtained from a mammal such as a human) can include any appropriate amount of biological molecules. In some cases, a fluid sample that can be assessed as described herein can include an amount of cfDNA of less than about 15 nanograms (ng) per mb of fluid sample (ng / mL). For example, a fluid sample that can be assessed as described herein can include an amount of cfDNA of from about 1 ng / mL to about 15 ng / mL.

[0036] A molecular profile described herein can include any appropriate number of biomarkers. In some cases, a molecular profile described herein can include a single biomarker. In some cases, a molecular profile described herein can include a panel of biomarkers. A panel of biomarkers can include any number of biomarkers. For example, a panel of biomarkers can include any two or more (e.g., two, three, four, five, six, seven, or more) biomarkers. In some cases, a biomarker can include methylation of one or more chromosomal loci on a cfDNA molecule. In some cases, a biomarker can include the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide). In some cases, a molecular profile can include (a) the presence of spinal cord-derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide).

[0037] In some cases, a molecular profile used to determine whether or not a mammal (e.g., a human) has a spinal cord injury as described herein (e.g., based, at least in part, on (a) the presence of spinal cord-derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide in a fluid sample (e.g., a blood sample) obtained from the mammal) can include the presence of spinal cord-derived cfDNA. For example, a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury can be assessed for the presence or absence of spinal cord-derived cfDNA to determine whether or not the mammal has a spinal cord injury. In some cases, a mammal suspected of having a spinal cord injury can be identified as having a spinal cord injury based, at least in part, on a molecular profile that includes the presence of spinal cord-derived cfDNA in a fluid sample (e.g., a blood sample) obtained from the mammal.

[0038] Any appropriate method can be used to detect the presence, absence, or amount of spinal cord-derived cfDNA. In some cases, the methylation status of one or more chromosomal loci (e.g., chromosomal loci containing CpG sites) on a cfDNA can be used to determine whether that cfDNA molecule is a spinal cord-derived cfDNA molecule. For example, a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury can be assessed for the methylation status of one or more chromosomal loci (e.g., chromosomal loci containing CpG sites) on a cfDNA to determine whether or not the sample includes spinal cord-derived cfDNA. In some cases, a mammal suspected of having a spinal cord injury can be identified as having a spinal cord- derived cfDNA based, at least in part, on the presence of one or more hypomethylated chromosomal loci (e.g., chromosomal loci containing CpG sites) on a cfDNA in a fluid sample (e.g., a blood sample) obtained from the mammal. For example, hypomethylation of one or more of cg27088725, cg07209034, cg24336338, cg03776878, and cg23617848 (e.g., as numbered in assembly hgl9 of the UCSC Genome Browser) can be used to identify that cfDNA molecule as a spinal cord-derived cfDNA molecule. The term “hypomethylated” as used herein with respect to a level of methylation of a chromosomal locus on a cfDNA a sample refers to any number of methylated cytosine bases at that locus that is lower than the number of methylated cytosine bases at that locus typically observed in a control sample. Control samples are samples obtained from mammals that do not have any spinal cord injury (e.g., a healthy mammal such as a healthy human). It will be appreciated that the methylation status from comparable samples is used when determining whether or not a particular methylation status is hypomethylated.

[0039] In cases where a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury is determined to lack spinal cord- derived cfDNA as described herein (e.g., based, at least in part, on the methylation status of one or more chromosomal loci (e.g., chromosomal loci containing CpG sites) on a cfDNA), the fluid sample can contain a non-detectable level of spinal cord-derived cfDNA. For example, a fluid sample obtained from a mammal having, or suspected of having, a spinal cord injury that is determined to lack spinal cord-derived cfDNA as described herein may contain a level of spinal cord-derived cfDNA that is exceedingly low and may represent normal cellular turnover.

[0040] Any appropriate method can be used to detect the methylation status of one or more chromosomal loci (e.g., chromosomal loci containing CpG sites) on a cfDNA. In some cases, methylation-based droplet digital polymerase chain reaction (ddPCR) can be used to detect the methylation status of one or more chromosomal loci (e.g., chromosomal loci containing CpG sites) on a cfDNA in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury. In some cases, targeted DNA methylation analysis via nanopore sequencing can be used to detect the methylation status of one or more chromosomal loci (e.g., chromosomal loci containing CpG sites) on a cfDNA in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury. In some cases, the methylation status of one or more chromosomal loci (e.g., chromosomal loci containing CpG sites) on a cfDNA can be detected as described in Example 1. In some cases, a molecular profile used to determine whether or not a mammal (e.g., a human) has a spinal cord injury as described herein (e.g., based, at least in part, on (a) the presence of spinal cord-derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide in a fluid sample (e.g., a blood sample) obtained from the mammal) can include an elevated level of one or more polypeptides (or mRNA encoding such polypeptides). For example, a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury can be assessed for the presence or absence of an elevated level of one or more polypeptides (or mRNA encoding such polypeptides) to determine whether or not the mammal has a spinal cord injury. In some cases, a mammal suspected of having a spinal cord injury can be identified as having a spinal cord injury based, at least in part, on a molecular profile that includes the presence of an elevated level of one or more polypeptides (or mRNA encoding such polypeptides) in a fluid sample (e.g., a blood sample) obtained from the mammal. The term “elevated level” as used herein with respect to a level of a polypeptide (or mRNA encoding that polypeptide) in a sample refers to any level that is higher than a reference level of the polypeptide (or mRNA). The term “reference level” as used herein with respect to a level of a polypeptide (or mRNA) refers to the level of the polypeptide (or mRNA) typically observed in a control sample. Control samples are samples obtained from mammals that do not have any spinal cord injury (e g., a healthy mammal such as a healthy human). It will be appreciated that levels of polypeptides (or mRNAs) from comparable samples are used when determining whether or not a particular level is an altered level of a polypeptide (or mRNA).

[0041] In some cases, a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury can be assessed for the presence, absence, or level of any appropriate polypeptides (or mRNAs encoding the polypeptides). Examples of polypeptides that can be present at an elevated level in a fluid sample (e.g., a blood sample) obtained from a mammal having a spinal cord injury include, without limitation, a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, a NF-H polypeptide, a neurofilament light (NF-L) polypeptide, an interleukin- 10 (IL- 10) polypeptide, a serum amyloid Al (SAA1) polypeptide, and a S100 calcium-binding protein A12 (S100A12) polypeptide. Examples of mRNAs that can be present at an elevated level in a fluid sample (e.g., a blood sample) obtained from a mammal having a spinal cord injury include, without limitation, mRNAs encoding a FABP3 polypeptide, mRNAs encoding a REST polypeptide, mRNAs encoding an IL-6 polypeptide, mRNAs encoding a NF-H polypeptide, mRNAs encoding a NF-L polypeptide, mRNAs encoding an IL-10 polypeptide, mRNAs encoding a SAA1 polypeptide, and mRNAs encoding a S100A12 polypeptide.

[0042] In some cases, a molecular profile of a fluid sample (e.g., a blood sample) obtained from a mammal having a spinal cord injury can include an elevated level of a FABP3 polypeptide (or an increased level of an mRNA encoding a FABP3 polypeptide). Examples of FABP3 polypeptides and nucleic acid sequences encoding a FABP3 polypeptide include, without limitation, those set forth in the UniProt Knowledgebase (UniProtKB; see, e.g., The UniProt Consortium, Nucleic Acids Research , 51(D1):D523-D531 (2023)) at, for example, accession no. P05413. For example, an elevated level of a FABP3 polypeptide (or an mRNA encoding a FABP3 polypeptide) can be any level that is higher than a reference level of the FABP3 polypeptide (or a reference level of an mRNA encoding a FABP3 polypeptide). For example, an elevated level of a FABP3 polypeptide can be at least 5% (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more) higher than a reference level of a FABP3 polypeptide. For example, an elevated level of a FABP3 polypeptide can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold greater than a reference level of a FABP3 polypeptide. For example, an elevated level of an mRNA encoding a FABP3 polypeptide can be a level that is at least 5% (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more) higher than a reference level of mRNA encoding a FABP3 polypeptide. For example, an elevated level of an mRNA encoding a FABP3 polypeptide can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold greater than a reference level of mRNA encoding a FABP3 polypeptide.

[0043] In some cases, a molecular profile of a fluid sample (e.g., a blood sample) obtained from a mammal having a spinal cord injury can include an elevated level of a REST polypeptide (or an increased level of an mRNA encoding a REST polypeptide). Examples of REST polypeptides and nucleic acid sequences encoding a REST polypeptide include, without limitation, those set forth in the UniProtKB at, for example, accession no. Q 13127. For example, an elevated level of a REST polypeptide (or an mRNA encoding a REST polypeptide) can be any level that is higher than a reference level of the REST polypeptide (or a reference level of an mRNA encoding a REST polypeptide). For example, an elevated level of a REST polypeptide can be at least 5% (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more) higher than a reference level of a REST polypeptide. For example, an elevated level of a REST polypeptide can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold greater than a reference level of a REST polypeptide. For example, an elevated level of an mRNA encoding a REST polypeptide can be a level that is at least 5% (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more) higher than a reference level of mRNA encoding a REST polypeptide. For example, an elevated level of an mRNA encoding a REST polypeptide can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold greater than a reference level of mRNA encoding a REST polypeptide.

[0044] In some cases, a molecular profile of a fluid sample (e.g., a blood sample) obtained from a mammal having a spinal cord injury can include an elevated level of an IL-6 polypeptide (or an increased level of an mRNA encoding an IL-6 polypeptide). Examples of IL-6 polypeptides and nucleic acid sequences encoding an IL-6 polypeptide include, without limitation, those set forth in the UniProtKB at, for example, accession no. P05231. For example, an elevated level of an IL-6 polypeptide (or an mRNA encoding an IL-6 polypeptide) can be any level that is higher than a reference level of the IL-6 polypeptide (or a reference level of an mRNA encoding an IL-6 polypeptide). For example, an elevated level of an IL-6 polypeptide can be at least 5% (e g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more) higher than a reference level of an IL-6 polypeptide. For example, an elevated level of an IL- 6 polypeptide can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold greater than a reference level of an IL-6 polypeptide. For example, an elevated level of an mRNA encoding an IL-6 polypeptide can be a level that is at least 5% (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more) higher than a reference level of mRNA encoding an IL-6 polypeptide. For example, an elevated level of an mRNA encoding an IL-6 polypeptide can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold greater than a reference level of mRNA encoding an IL-6 polypeptide.

[0045] In some cases, a molecular profde of a fluid sample (e.g., a blood sample) obtained from a mammal having a spinal cord injury can include an elevated level of a NF-H polypeptide (or an increased level of an mRNA encoding a NF-H polypeptide). Examples of NF-H polypeptides and nucleic acid sequences encoding a NF-H polypeptide include, without limitation, those set forth in the UniProtKB at, for example, accession no. P12036. For example, an elevated level of a NF-H polypeptide (or an mRNA encoding a NF-H polypeptide) can be any level that is higher than a reference level of the NF-H polypeptide (or a reference level of an mRNA encoding a NF-H polypeptide). For example, an elevated level of a NF-H polypeptide can be at least 5% (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more) higher than a reference level of a NF-H polypeptide. For example, an elevated level of a NF-H polypeptide can be a level that is at least 2 (e g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold greater than a reference level of a NF-H polypeptide. For example, an elevated level of an mRNA encoding a NF-H polypeptide can be a level that is at least 5% (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more) higher than a reference level of mRNA encoding a NF-H polypeptide. For example, an elevated level of an mRNA encoding a NF-H polypeptide can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold greater than a reference level of mRNA encoding a NF-H polypeptide.

[0046] Any appropriate method can be used to determine the presence, absence, or level of a polypeptide or mRNA encoding that polypeptide. In some cases, the presence, absence, or level of a polypeptide can be assessed by detecting and / or quantifying the polypeptide. Examples of methods that can be used to detect and / or quantify polypeptides include, without limitation, immunohistochemistry (IHC) techniques, mass spectrometry techniques (e.g., proteomics-based mass spectrometry assays or targeted quantification-based mass spectrometry assays), western blotting techniques, proximity ligation assays, and enzyme- linked immunosorbent assays. In some cases, the presence, absence, or level of an mRNA encoding a polypeptide can be assessed by detecting and / or quantifying the mRNA encoding a polypeptide. Examples of methods that can be used to detect and / or quantify mRNA include, without limitation, RT-PCR techniques (e.g., quantitative RT-PCR techniques), and RNASeq. In some cases, the presence, absence, or level of a polypeptide and / or mRNA encoding that polypeptide can be identified as described in Example 1.

[0047] In some cases, the molecular profile of a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) suspected of having a spinal cord injury can be used to identify the mammal as having a spinal cord injury. For example, a molecular profile that (a) includes the presence of spinal cord-derived cfDNA and / or (b) includes the presence of an elevated level of one or more of a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, and a NF-H polypeptide in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) suspected of having a spinal cord injury can be used to identify the mammal as having the spinal cord injury.

[0048] In some cases, the molecular profile of a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) suspected of having a spinal cord injury can be used to identify the mammal as not having a spinal cord injury. For example, a molecular profile that (a) lacks the presence of spinal cord-derived cfDNA and (b) lacks the presence of an elevated level of each of a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, and a NF-H polypeptide in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) suspected of having a spinal cord injury can be used to identify the mammal as not having the spinal cord injury.

[0049] In some cases, the molecular profile of a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having a spinal cord injury can be used to determine the severity of the spinal cord injury. For example, a molecular profile that (a) includes the presence of spinal cord-derived cfDNA and / or (b) includes the presence of an elevated level of one or more of a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, and a NF- H polypeptide in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having a spinal cord injury can be used to determine the severity of the spinal cord injury. In some cases, a level of spinal cord-derived cfDNAin a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having a spinal cord injury can be used to determine the severity of the spinal cord injury. For example, when a mammal having a spinal cord injury is identified as having a fluid sample (e.g., a blood sample) including from about 45 hGE / mL spinal cord-derived cfDNA to about 1000 hGE / mL spinal cord-derived cfDNA, that mammal can be determined to have an American Spinal Injury Association (ASIA) grade A spinal cord injury. In another example, when a mammal having a spinal cord injury is identified as having a fluid sample (e.g., a blood sample) including from about 11 hGE / mL to about 44 hGE / mL spinal cord-derived cfDNA, that mammal can be determined to have an ASIA grade B spinal cord injury. In yet another example, when a mammal having a spinal cord injury is identified as having a fluid sample (e.g., a blood sample) including from about 5 hGE / mL to about 10 hGE / mL spinal cord-derived cfDNA, that mammal can be determined to have an ASIA grade C spinal cord injury. In still another example, when a mammal having a spinal cord injury is identified as having a fluid sample (e.g., a blood sample) including from about 0.1 hGE / mL to about 4.9 hGE / mL spinal cord-derived cfDNA, that mammal can be determined to have an ASIA grade D spinal cord injury.

[0050] In some cases, the molecular profile of a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having, or suspected of having, a spinal cord injury can be used to determine a spinal cord injury index (SCII). For example, a molecular profile that (a) includes the presence of spinal cord-derived cfDNA and (b) includes the presence of an elevated level of one or more of a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, and a NF-H polypeptide in a fluid sample (e.g., a blood sample) obtained from a mammal (e.g., a human) having a spinal cord injury can be used to determine a SCII. Any appropriate method can be used to determine a SCII. In some cases, a SCII can be determined as described in Example 1.

[0051] In some cases, a SCII determined as described herein (e.g., based, at least in part, on

[0052] (a) the presence of spinal cord-derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide in a fluid sample (e.g., a blood sample) obtained from a mammal) can be used to determine the severity of a spinal cord injury. In some cases, a SCII can be used to determine the severity of the spinal cord injury. For example, when a mammal having a spinal cord injury is identified as having a SCII of from about 15 to about 25 (e.g., from about 15 to about 22, from about 15 to about 20, from about 15 to about 18, from about 17 to about 25, from about 20 to about 25, from about 22 to about 25, or from about 18 to about 22), that mammal can be determined to have an ASIA grade A spinal cord injury. In another example, when a mammal having a spinal cord injury is identified as having a SCII of from about 11 to about 19 (e.g., from about 11 to about 17, from about 11 to about 15, from about 13 to about 19, from about 15 to about 19, or from about 13 to about 15), that mammal can be determined to have an ASIA grade B spinal cord injury. In yet another example, when a mammal having a spinal cord injury is identified as having a SCII of from about 10 to about 15 (e.g., from about 10 to about 13, from about 12 to about 15, or from about 12 to about 14), that mammal can be determined to have an ASIA grade C spinal cord injury. In still another example, when a mammal having a spinal cord injury is identified as having a SCII of from about 10 to about 15 (e.g., from about 10 to about 12, from about 11 to about 15, or from about 11 to about 12), that mammal can be determined to have an ASIA grade D spinal cord injury.

[0053] In some cases, when a mammal (e.g., a human) is identified as having a spinal cord injury as described herein (e.g., based, at least in part, on (a) the presence of spinal cord- derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide in a fluid sample (e.g., a blood sample) obtained from the mammal), the mammal can be selected to receive one or more spinal cord treatments. For example, a mammal (e.g., a human) identified as having a fluid sample (e.g., a blood sample) having a molecular profile that (a) includes the presence of spinal cord-derived cfDNA and / or (b) includes the presence of an elevated level of one or more of a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, and a NF-H polypeptide can be selected to received one or more spinal cord treatments.

[0054] In some cases, when a mammal (e.g., a human) is identified as having a spinal cord injury as described herein (e.g., based, at least in part, on (a) the presence of spinal cord- derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide in a fluid sample (e.g., a blood sample) obtained from the mammal), the mammal can be administered (or instructed to self-administer) one or more spinal cord treatments. For example, a mammal (e.g., a human) identified as having a fluid sample (e g., a blood sample) having a molecular profile that (a) includes the presence of spinal cord- derived cfDNA and / or (b) includes the presence of an elevated level of one or more of a FABP3 polypeptide, a REST polypeptide, an IL-6 polypeptide, and a NF-H polypeptide can be administered one or more spinal cord treatments.

[0055] When a mammal (e.g., a human) identified as having a spinal cord injury as described herein (e.g., based, at least in part, on (a) the presence of spinal cord-derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide in a fluid sample (e.g., a blood sample) obtained from the mammal) is administered one or more spinal cord treatments, the one or more spinal cord treatments can include any appropriate spinal cord treatment(s). In some cases, a spinal cord treatment can be designed to maintain the ability of a mammal to breathe. In some cases, a spinal cord treatment can be designed to prevent shock. In some cases, a spinal cord treatment can be designed to prevent further spinal cord damage (e.g., immobilizing one or more portions of the spine such as the neck). In some cases, a spinal cord treatment can be designed to avoid possible complications (e.g., stool or urine retention, respiratory or cardiovascular conditions, and the formation of deep vein blood clots). In some cases, a spinal cord treatment can be designed to reduce or eliminate one or more symptoms of a spinal cord injury. Examples of symptoms of a symptoms of a spinal cord injury include, without limitation, loss of movement, loss of sensation (e.g., loss of the ability to feel heat, cold, and / or touch), a change in sensation (e.g., a change in the ability to feel heat, cold, and / or touch), loss of bowel control, loss of bladder control, exaggerated reflex activities or spasms, pain in the spinal cord, a stinging sensation in the spinal cord, trouble breathing (e.g., trouble breathing after injury), coughing, clearing secretions from the lungs, extreme back pain, weakness in any part of the body, incoordination in any part of the body, loss of control in any part of the body, numbness (e.g., in the hands, fingers, feet, and / or toes), tingling (e.g., in the hands, fingers, feet, and / or toes), loss of feeling (e.g., in the hands, fingers, feet, and / or toes), trouble with balance, and trouble with walking. Examples of spinal cord injury treatments include, without limitation, administering to the mammal one or more corticosteroids (e.g., methylprednisolone), administering to the mammal one or more neuroprotective agents (e.g., riluzole and hypertonic saline), immobilizing the mammal, putting the mammal in traction (e.g., to stabilize or align the spine), performing surgery (e.g., to remove fragments of bones, foreign objects, herniated disks, and / or fractured vertebrae that may compress the spine, and / or to stabilize the spine and prevent future pain or complications) on the mammal, performing hemodynamic augmentation on the mammal, and modulating intraspinal pressure in the mammal.

[0056] In some cases, when a mammal (e.g., a human) is identified as having a spinal cord injury as described herein (e.g., based, at least in part, on (a) the presence of spinal cord- derived cfDNA and / or (b) the presence of an elevated level of a polypeptide (or an mRNA encoding a polypeptide in a fluid sample (e.g., a blood sample) obtained from the mammal) the determination can be confirmed using one or more additional diagnostic techniques. Examples of techniques that can be used to identify the presence of a spinal cord injury include, without limitation, neuroimaging techniques (e.g., computerized tomography (CT) scanning (e.g., CT coIonography), x-ray techniques, and magnetic resonance imaging (MRI) techniques), and physical examinations.

[0057] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0058] EXAMPLES

[0059] Example 1: A multi-analyte blood test for acute spinal cord injury

[0060] This Example describes the integration of spinal cord-derived cell-free DNA (cfDNA) and proteomics into a multi-analyte blood test that can inform diagnosis and prognosis of a spinal cord injury (SCI; e.g., an acute SCI). For example, this Example describes the development of a SCI index which can be used to accurately diagnose SCI (AUC: 0.91, 95% CI 0.82-0.99, P<0.0001), to determine SCI severity fP<0.0001), and to predict six-month neurologic improvement (AUC: 0.77, 95% CI 0.61-0.93, P=0.006). RESULTS

[0061] Cohort overview

[0062] 50 patients with acute SCI were assessed (Figure 2, Table 1). SCI type included traumatic SCI (tSCI, N= 14), traumatic central cord syndrome (CCS, N= 19), and acute epidural spinal cord compression (ESCC, N= 17). Presenting neurologic status (American Spinal Injury Association (ASIA) Impairment Scale (AIS)) was A in 12%, B in 24%, C in 26%>, and D in 38%). Most patients were male (68%), sustained a cervical level injury (60%), and underwent surgery via a posterior approach (9%).

[0063] Table 1.

[0064] Identification of spinal cord-specific methylation loci

[0065] Illumina MethylationEPIC 850K array-based methylation profiling was performed on fresh, frozen human spinal cord tissue obtained from an institutional rapid autopsy program (N=3 unique patients) and on formalin fixed, paraffin embedded human spinal cord tissue

[0066] (N=3 unique patients) without spinal cord pathology. The results in silico from these six samples were compared to methylation profiles from 25 other tissue and cell types, including cultured human cortical neurons, to identify CpG sites differentially methylated in spinal cord tissue.

[0067] Two candidate regions were selected in silica (Assay 1, 2 CpG sites; Assay 2, 3 CpG sites) and bespoke droplet digital polymerase chain reaction (ddPCR) was designed to amplify these regions if all included CpG sites were hypomethylated (Figure 3A). When applied to bisulfite converted genomic DNA (gDNA) from human spinal cord tissue and peripheral blood mononuclear cells (PBMCs) from non-SCI control patients, it was observed that Assay 1 had robust detection of spinal cord tissue gDNA (z.e., when target CpG sites were hypomethylated) and no detection of spinal-cord specific cfDNA in non-SCI control cfDNA. (Figures 6 A-6B). A standard curve was established by spiking known concentrations of spinal cord gDNA into control PBMC gDNA, further demonstrating that Assay 1 had markedly better calibration at low spinal cord gDNA concentrations and demonstrated linearity (Assay 1: Spearman p =0.80, P<0.0001, Figure 3B; Assay 2: Spearman p=0.64, <0.01 Figure 7). It was further confirmed that Assay 1 did not amplify when applied to acute SCI patient PBMC gDNA (Figure 6D). As a result, all further ddPCR analyses were performed utilizing Assay 1. To conduct further technical validation, a “reverse assay” was designed where the CpG sites of interest were inverted ( / .(?., assay designed to detect hypermethylation at target sites, rather than hypomethylation) and likely to represent non-spinal cord derived DNA. As predicted, detection using the reverse assay was inversely correlated with amount of spiked-in spinal cord gDNA (Figures 8A-8B).

[0068] Detection of spinal cord-derived cfDNA in peripheral blood

[0069] The spinal cord-derived ddPCR assay was applied to bisulfite converted cfDNA from 50 patients with acute SCI and 11 non-SCI control patients. Median time from reported neurologic deficit or injury to blood draw was 1 day (IQR, 0-2 days) for SCI patients and all draws were obtained prior to surgery. The absolute plasma concentration of spinal cord- derived cfDNA was measured in haploid genome equivalents per milliliter of plasma (hGE / mL). Spinal cord-specific cfDNA was detected in 39 of 50 patients with acute SCI and in 0 of 11 non-SCI control patients, yielding a sensitivity of 78% and a specificity of 100% for diagnosis of acute SCI (Figure 3C). The concentration of spinal cord-derived cfDNA did not significantly differ across SCI etiologies (Figure 3D) but did vary with SCI severity (Figure 3E). Significantly higher levels of cfDNA were measured in ASIA A injuries compared to ASIA B (P=0.04), ASIA C ( =0.009), and ASIA D injuries (P<0.001). Median spinal cord-derived concentrations for ASIA A patients were 118.7 hGE / mL (IQR, 27.5 - 9907 hGE / mL), 15.1 hGE / mL (IQR, 4.87 - 47.6 hGE / mL) for ASIA B patients, 5.67 hGE / mL (IQR, 2.62 - 41.6 hGE / mL) for ASIA C patients, and 4.60 hGE / mL (IQR, 0 - 9.43 hGE / mL) for ASIA D patients).

[0070] Proteomic profiling of plasma from SCI patients

[0071] Next, proteomic profiling of 119 CNS-related proteins was performed using a proximity ligation assay, NUcleic acid Linked Immuno-Sandwich Assay (NULISA, Alamar Biosciences), optimized for circulating biomarker development. Proteomic profiling was performed in a subset of 34 acute SCI patients and 12 non-SCI controls. The proteomic profiling was performed from the same physical sample from which cfDNA was extracted for all SCI patients.

[0072] Protein levels were normalized to an internal control, rescaled, log2 transformed, and represented as NULISA protein quantification (NPQ) units. Principal component analysis (PCA) demonstrated distinct separation of proteomic signatures between injured versus healthy subjects (Figure 4A). Previously, glial fibrillary acidic protein (GFAP) and neurofilament light (NF-L) were measured in serum and CSF of patients with tSCI and were found to be associated with injury severity and six-month neurologic outcomes (Stukas et al., Neurology. 100(12):el221-el233 (2023)). Similarly, it was found that GFAP and NF-L levels were significantly higher in SCI patients, relative to non-SCI controls. However, it was observed that GFAP and NF-L levels did not significantly differ between SCI patients in whom spinal cord-derived cfDNA was detected and SCI patients in whom spinal cord- derived cfDNA was not detected (Figure 4B-C). Both GFAP (Spearman p = 0.42, P = 0.004) and NF-L (Spearman p =0.70, <0.0001 ) levels significantly correlated with concentrations of spinal cord-derived cfDNA.

[0073] Parsimonious sets of proteins associated with the main clinical outcome of interest: six-month ASIA conversion were identified. To achieve this, proteins were ranked based on difference in median NPQ values between SCI patients who converted and those who did not. Forward selection was then performed in a logistic regression model to identify a set of proteins associated with six-month ASIA conversion. This approach identified four proteins: Fatty Acid Binding Protein 3 (FABP3), REl-Silencing Transcription factor (REST), Interleukin 6 (IL-6), and Neurofilament, Heavy polypeptide (NF-H). Levels of all four proteins were significantly higher in SCI patients in whom spinal cord-derived cfDNA was detected, compared to non-SCI controls. Levels of REST, IL-6, and NF-H were higher in SCI patients without detection of spinal cord-derived cfDNA, relative to non-SCI controls (Figure 4D-G).

[0074] Development of the Spinal Cord Injury Index

[0075] Next, the association between concentration of spinal cord-derived cfDNA with these four proteins was assessed (Figure 5A). Consistent positive correlations were found: FABP3 (Spearman p=0.47, <0.001), REST (Spearman p=0.62, O.OOl), IL-6 (Spearman p=0.41, P=0.004), and NF-H (Spearman p=0.30, =0.04).

[0076] To develop a composite score reflecting neuroglial injury after SCI, we linearly combined NPQ values of the four selected proteins using coefficients from the logistic regression used for candidate selection and the log 10 scaled spinal cord-derived cfDNA concentration. This integrated value is summarized as the Spinal Cord Injury Index (SCII). Though non-SCI controls were not used to inform SCII development, it was observed that the SCII reliably distinguished acute SCI patients from non-SCI controls (AUC=0.91, <0.0001) (Figure 5B). The SCII demonstrated a step-wise decrease with decreasing injury severity (Kruskal -Willis, P<0.0001) with relatively tight clustering within AIS groups (Figure 5C). Finally, it was sought to confirm that the SCII predicted six-month ASIA conversion, observing that the SCII discriminated between SCI patients who converted (N=18) and those who did not (N=16) with an AUC of 0.77 (P=0.005, Figure 5D). Two vignettes are presented to illustrate the importance of considering both the SCII and spinal cord-derived cfDNA dynamics. Figure 5E depicts a 78-year-old male patient who sustained an ASIA B cervical tSCI. The patient’s preoperative SCII suggested that the patient may achieve ASIA conversion and remained roughly stable following surgery. However, concentration of spinal cord-derived cfDNA became undetectable following decompressive surgery, suggesting improvement of ongoing injury to the spinal cord. The patient achieved ASIA conversion from B to D by 3 months post-injury. In contrast, Figure 5F depicts a 56- year-old male patient who sustained an ASIA B cervical tSCI. The patient’s pre- and postoperative SCII suggested ASIA conversion would be unlikely, this remained stable following surgery. Concentration of spinal cord-derived cfDNA increased after uncomplicated decompressive surgery, suggesting ongoing injury to the spinal cord, and the patient remained ASIA B six months following the operation.

[0077] MATERIALS AND METHODS

[0078] Patient selection

[0079] Patients at least 18 years of age who were diagnosed with acute SCI were recruited. Diagnosis of acute SCI was based on patient report of new neurologic deficit and evidence of SCI on magnetic resonance imaging (MRI). Included SCI etiologies were traumatic SCI (tSCI), traumatic central cord syndrome (CCS), and acute epidural spinal cord compression (ESCC). Patients who presented beyond seven days from new neurologic deficit were excluded. Patients who received blood product transfusions prior to preoperative blood sample procurement were also excluded.

[0080] Sample collection and processing

[0081] Blood samples were obtained from arterial or venous access lines within seven days of the initial injury and prior to surgical intervention. Between 10 and 30 mL of peripheral blood were collected in cfDNA blood collection tubes (Streck). Tubes were centrifuged for 10 minutes at 814g to separate the plasma. Plasma was transferred to a standard 15 mL tube and centrifuged a second time at 5,662g for 30 minutes. Supernatant containing cfDNA was stored at -80°C.

[0082] Identification of spinal cord-specific CpG sites

[0083] Genome-wide methylation sequencing using Illumina Methyl ationEPIC 850K arrays was performed on fresh, frozen (N=3) and formalin fixed paraffin embedded (FFPE) human spinal cord tissue (N=3) from control patients. DNA methylation analysis was performed using the minfi package in R v4.1.0 (The R Foundation for Statistical Computing, Auckland, New Zealand). An atlas of DNA methylation data for 25 different healthy tissue types was compiled from publicly available data (see Table 2). A beta value (0) was generated for each CpG site, where 0 = 0 indicated a completely unmethylated signal and 0 = 1 indicated a completely methylated signal. CpG sites that were differentially methylated between spinal cord tissue and other tissues were determined using the dmpFinder function in minfi. To measure differential methylation effect size, the mean and median beta value difference between spinal cord tissue and all other tissues were computed. CpG sites that were significantly differentially methylated and that had large methylation difference effect sizes were selected as candidate CpG sites. Furthermore, CpG sites within 100-200 base pairs of one another were prioritized for downstream analysis.

[0084] Table 2. References for DNA methylation tissue atlas. Primer / probe design for ddPCR

[0085] Custom ddPCR primer / probe pairs were designed to target two chromosomal loci containing CpGs which were differentially hypomethylated in spinal cord tissue. Assay 1 targeted chromosome 16 and 2 CpG sites (cg27088725, cg07209034), with one probe covering each locus. Assay 2 targeted chromosome 12 and 3 CpG sites (cg24336338, cg03776878, cg23617848), with one probe covering two loci and the other probe covering one locus. With this approach, double positive fluorescent indicated identification of all target loci hypomethylated on a single cfDNA molecule.

[0086] Cell-free DNA extraction and bisulfite conversion

[0087] Plasma from banked blood samples were removed from -80°C storage and thawed on ice. The BioChain cfPure Cell Free DNA extraction kit was used to extract cfDNA from plasma. Plasma was centrifuged at 5200g for 30 minutes, at 4°C. Supernatant containing cfDNA was transferred for extraction. Supernatant containing cfDNA was enzymatically lysed to remove bound proteins and prepared with buffers to inhibit DNases. Solution was combined with magnetic beads to bind cfDNA. Beads were washed with washing buffers and ethanol. Nuclease-free water was used to elute cfDNA into a working solution. Concentration of cfDNA (ng / uL) was measured using Qubit. Purity of cfDNA was assessed using the Agilent 2100 Bioanalyzer system and High Sensitivity DNA Assay protocol. Bisulfite conversion of extracted cfDNA was performed using the EpiTect Plus DNA Bisulfite kit.

[0088] Digital droplet PCR

[0089] Reaction volumes of 20 pl, consisting of 19 pl mastermix (11 pl Supermix for Probes (no deoxyuridine triphosphate), 7 pl of nuclease-free water, 1 pl of each primer / probe mix for both the Fam and Hex probes), and 1 pl cfDNA sample of patient plasma, were prepared and used for droplet generation. Droplets were transferred to a 96-well plate for DNA amplification and thermal cycling. Droplet Digital PCR was performed using the QX200 ddPCR system according to manufacturer’s instructions (Bio-Rad Laboratories). QuantaSoft vl.7.4.0917 (Bio-Rad Laboratories) software was used for data analysis.

[0090] Prior to plasma sample testing, thermal gradient experiments were performed on genomic DNA extracted from spinal cord tissue to determine optimal amplification conditions thermal cycling. Based on clearest separation of negative and positive droplet clusters, thermal cycling conditions were set at 95 °C for 10 min (1 cycle), 94 °C for 30 s and 55 °C for 60 s (40 cycles), and infinite hold at 12 °C. For quality control, wells with total droplet counts of less than 10,000 were considered invalid and excluded from analysis. Samples containing genomic DNA from human spinal cord tissue were used to verify assay performance. Thresholds in fluorescence values were determined using negative control wells containing genomic DNA from human leukocytes and positive control wells containing genomic DNA from human spinal cord tissue.

[0091] Thresholds were applied to ddPCR reads of patient plasma samples to determine the number of double positive droplets. Unique plasma samples were analyzed in triplicate. Estimated target DNA concentrations (copies / pl) was calculated using the formula C = - n(Nneg / Ntot) / V droplet, such that C = sample concentration (copies / pl), Nneg = number of negative droplets, Ntot = total number of droplets, Vdroplet = volume of droplet (1 nl). Plasma concentration in hGE / mL were calculated using the formula PC=C -RV -(EV / TV) / PV, such that PC = plasma concentration (copies / ml), C = sample concentration (copies / pl), RV = PCR reaction volume (20 pl), EV = volume in which cfDNA was eluted (15 pl); TV = volume of cfDNA added to the PCR reaction (1 pl), PV = volume of plasma used for cfDNA extraction (5 ml). Conversion factor 1 ng = 303 hGE of cfDNA was used to calculate the plasma concentration of spinal cord-derived cfDNA in ng / mL. Percent of spinal cord- derived cfDNA was calculated by dividing the plasma concentration of spinal cord-derived cfDNA (ng / mL) over the total plasma concentration of cfDNA measured via Qubit prior to bisulfite conversion.

[0092] Proteomic profiling

[0093] The NULISA 119-plex neurologic panel was designed to quantify proteins involved in neurological diseases and achieved attomolar level detection. Plasma volumes of 50uL were subjected to analysis via the NULISA proteomic platform. Briefly, this platform employed a unique dual-DNA-barcode strategy, wherein capture antibodies (Abs) were tagged with double-stranded DNA featuring a poly(A) tail and disease-specific barcode, and detection Abs were tagged with a complementary biotinylated barcode. The presence of the target protein prompts the formation of an immunocomplex. This complex was then isolated using paramagnetic oligo(dT) beads, leveraging dT-poly(A) hybridization. A wash process in a salt-sensitive environment subsequently released these complexes into a low-salt buffer. Streptavidin-coated beads were then introduced, effecting a secondary capture phase for the complexes and eliminating non-specifically bound Abs through further washing, culminating in the isolation of near-pure immunocomplexes. In the final step, the binding of a specialized DNA ligation sequence via T4 DNA ligase created a novel DNA reporter molecule that embodied the unique target-specific barcodes. These reporters were quantitatively analyzed by next-generation sequencing. Data normalization — achieved using internal controls — addressed technical variability, and a rescaling and log2 transformation process yielded the final NULISA protein quantification (NPQ) units.

[0094] Derivation of the Spinal Cord Injury Index

[0095] Proteins were ranked based on difference in median NPQ values between SCI patients who achieved six-month ASIA conversion and those who did not. Forward selection in a logistic regression model was performed to identify a set of proteins associated with six- month ASIA conversion. The coefficients of identified proteins were linearly combined with log 10 scaled spinal cord-derived cfDNA concentration to define a composite Spinal Cord Injury Index (SCII).

[0096] The same methodology was used, but comparing SCI patients with non-SCI controls, to define a composite score specifically aimed for SCI diagnosis. This identified five proteins (NF-H, NF-L, Interleukin- 10 (IL-10), Serum amyloid Al (SAA1), and S100 calcium-binding protein A12 (S100A12)) that were linearly combined with loglO scaled spinal cord-derived cfDNA concentration. As depicted in Figure 9, this approach expectedly achieves robust performance for discriminating between SCI patients and non-SCI controls (AUC, 0.95), but demonstrated suboptimal association with injury severity and prediction of six-month ASIA conversion (AUC, 0.57).

[0097] Example 2: Molecular Monitoring of Intramedullary Spinal Cord Tumor Surgery

[0098] This Example describes demonstrates that blood-based assays provided herein can be used to quantify intra-operative neuroglial damage in real time.

[0099] Case Report

[0100] A 36-year-old woman with no prior medical history presented with progressive righthand clumsiness and thenar wasting. Electrodiagnostic testing excluded carpal tunnel syndrome, prompting cervical MRI, which demonstrated a well-circumscribed intramedullary mass extending from mid-C7 to upper T3 (cranio-caudal length ~ 5.5 cm). The spinal cord was expanded with rostral T2 hyperintensity to C2 and caudal extension to T6. She underwent C7-T3 laminoplasty for tumor biopsy and resection. Intraoperative neuromonitoring potentials remained stable. Microsurgical technique enabled gross total resection, and pathology confirmed WHO grade II ependymoma. Immediate postoperative MRI showed expected surgical changes with reduced edema.

[0101] Postoperatively, the patient had transient left leg motor weakness which recovered to baseline rapidly. Sensory disturbances in both legs persisted despite intact proprioception. Six months later the patient remained full strength, reporting only residual gait imbalance related to sensory loss. One-year MRI demonstrated no residual tumor and complete syrinx resolution.

[0102] Methods

[0103] Peripheral blood (5 mL) was obtained at five peri-operative time points — skin incision, dural opening, initial myelotomy, completion of tumor resection, and postoperative day 1 — using cfDNA-stabilizing tubes (Streck). Plasma was isolated by double-spin centrifugation and stored at -80 °C. Spinal -cord-derived cfDNA was extracted, bisulfite- converted, and quantified with spinal-specific droplet-digital PCR as reported elsewhere (Azad, J Clin Invest 135 (2025)). Protein profiling was performed on 50 pL aliquots with the NULISA 119-plex neurologic panel (Alamar Biosciences). The Spinal Cord Injury Index (SCII) was calculated for each time point using the original weighting coefficients that integrate cfDNA and the four-protein signature (FABP-3, REST, IL-6, NF-H). SC11 is a composite measure that is dimensionless. GFAP and NF-L were measured on the same targeted proteomics panel and are presented in NULISA Protein Quantification (NPQ) units.

[0104] Molecular Assessments

[0105] Figure 10 depicts biomarker kinetics. SCII remained relatively stable, from 12.2 at skin incision to 14.3 after dural opening, and began rising after myelotomy was initiated. SCII values peaked at 16.5 on completion of tumor resection and fell to 14. 1 on postoperative day 1. The median SCII value observed in acute SCI patients with American Spinal Injury Association Impairment Scale (AIS) C injury severity was 14.2 while the median SCII value in acute SCI patients with AIS B injury severity was 15.4.

[0106] In contrast, intra-operative levels of GFAP, steadily rose from initial myelotomy and continued to sharply rise after completion of surgery and into postoperative day 1. Similarly, NF-L levels only began rising after completion of tumor resection and continued to rise on postoperative day 1, despite the patient returning to their baseline neurologic function. The temporal dissociation suggested that SCII is sensitive to immediate neuroglial injury, whereas classical astroglial and axonal proteins may represent lagging indicators.

[0107] Together, these results show that SCII allows for high temporal fidelity, molecular monitoring that parallels surgical manipulation, and precedes classical protein biomarker elevations. Accordingly, these results demonstrate that the methods provided herein can be used as a real-time liquid biopsy for spinal cord monitoring during neurosurgical procedures such that serial monitoring of the SCII can be used to dynamically track progress during spinal cord tumor surgery.

[0108] OTHER EMBODIMENTS

[0109] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for assessing a mammal suspected of having a spinal cord injury, wherein said method comprises:(a) determining (1) that a blood sample from said mammal contains the presence of spinal cord-derived cfDNA, and (2) that said blood sample contains the presence of at least one of: an elevated level of a fatty acid binding protein 3 (FABP3) polypeptide or mRNA encoding said FABP3 polypeptide, an elevated level of a REl-silencing transcription factor (REST) polypeptide or mRNA encoding said REST polypeptide, an elevated level of an interleukin 6 (IL-6) polypeptide or mRNA encoding said IL-6 polypeptide, and an elevated level of a neurofilament, heavy polypeptide (NF-H) or mRNA encoding said NF-H polypeptide;(b) classifying said mammal as having a spinal cord injury if said presence of (1) is determined and said presence of (2) is determined.

2. The method of claim 1, wherein said mammal is a human.

3. The method of any one of claims 1-2, wherein said presence or absence of said elevated level of said FABP3 polypeptide or mRNA encoding said FABP3 polypeptide is determined.

4. The method of any one of claims 1-2, wherein said presence or absence of said elevated level of said REST polypeptide or mRNA encoding said REST polypeptide is determined.

5. The method of any one of claims 1-2, wherein said presence or absence of said elevated level of said IL-6 polypeptide or mRNA encoding said IL-6 polypeptide is determined.

6. The method of any one of claims 1-2, wherein said presence or absence of said elevated level of said NF-H polypeptide or mRNA encoding said NF-H polypeptide is determined.

7. The method of any one of claims 1-2, wherein said method comprises determining the presence of each of said elevated level of said FABP3 polypeptide or mRNA encoding said FABP3 polypeptide, said elevated level of said REST polypeptide or mRNA encoding said REST polypeptide, said elevated level of said IL-6 polypeptide or mRNA encoding said IL-6 polypeptide, and said elevated level of said NF-H polypeptide or mRNA encoding said NF-H polypeptide.

8. The method of any one of claims 1-7, wherein said blood sample is a plasma sample.

9. A method for determining the severity of a spinal cord injury within a mammal, wherein said method comprises determining the level of spinal cord-derived cfDNAin a blood sample from said mammal; and(a) classifying said spinal cord injury as being an American Spinal Injury Association (ASIA) grade A spinal cord injury when said spinal cord-derived cfDNA is present at a level of from about 45 genome equivalents (hGE) per mL of said blood sample (hGE / mL) to about 1000 hGE / mL;(b) classifying said spinal cord injury as being an ASIA grade B spinal cord injury when said spinal cord-derived cfDNA is present at a level of from about 11 hGE / mL to about 44 hGE / mL;(c) classifying said spinal cord injury as being an ASIA grade C spinal cord injury when said spinal cord-derived cfDNA is present at a level of from about 5 hGE / mL to about 10 hGE / mL; or(d) classifying said spinal cord injury as being an ASIA grade D spinal cord injury when said spinal cord-derived cfDNA is present at a level of from about 0.1 hGE / mL to about 4.9 hGE / mL.

10. The method of claim 9, wherein said mammal is a human.

11. The method of any one of claims 9-10, wherein said blood sample is a plasma sample.

12. A method for treating a mammal having a spinal cord injury, wherein said method comprises:(a) determining that a blood sample from said mammal comprises (1) the presence of spinal cord-derived cfDNA, and (2) the presence of at least one of: an elevated level of a FABP3 polypeptide or mRNA encoding said FABP3 polypeptide, an elevated level of a REST polypeptide or mRNA encoding said REST polypeptide, an elevated level of an IL-6 polypeptide or mRNA encoding said IL-6 polypeptide, and an elevated level of a NF-H or mRNA encoding said NF-H polypeptide;(b) administering a spinal cord injury treatment to said mammal.

13. The method of claim 12, wherein said determining step is performed in less than 1 hour.

14. A method for treating a spinal cord injury, wherein said method comprises subjecting a mammal identified as having a blood sample comprising (1) the presence of spinal cord- derived cfDNA, and (2) the presence of at least one of: an elevated level of a FABP3 polypeptide or mRNA encoding said FABP3 polypeptide, an elevated level of a REST polypeptide or mRNA encoding said REST polypeptide, an elevated level of an IL-6 polypeptide or mRNA encoding said LL-6 polypeptide, and an elevated level of a NF-H or mRNA encoding said NF-H polypeptide to a spinal cord injury treatment.

15. The method of any one of claims 12-14, wherein said mammal is a human.

16. The method of any one of claims 12-15, wherein said blood sample is a plasma sample.

17. The method of any one of claims 12-16, wherein said spinal cord injury is selected from the group consisting of traumatic SCI, traumatic central cord syndrome, acute epidural spinal cord compression from cancer, acute epidural spinal cord compression from infection, and subacute to chronic spinal cord compression from degenerative cervical spondylosis.

18. A method for detecting spinal cord-derived cell-free DNA (cfDNA) in a blood sample, said method comprising:(a) determining that said blood sample comprising cfDNA contains the presence of:(1) a hypomethylated cg27088725 locus, (2) a hypomethylated cg07209034 locus, (3) a hypomethylated cg24336338 locus, (4) a hypomethylated cg03776878 locus, or (5) a hypomethylated cg23617848 locus; and(b) classifying said cfDNA as spinal-cord derived cfDNA if said presence of any one of said (1) - said (5) is determined.

19. The method of claim 18, wherein said blood sample is a human blood sample.

20. The method of any one of claims 18-19, wherein said sample is a plasma sample.

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