Network analysis of the cerebrospinal fluid proteome of sporadic and familial forms of amyotrophic lateral sclerosis and methods of treatment of the same

Abstract

In one aspect, the disclosure relates to methods for diagnosing a neurodegenerative disease associated with loss of motor neurons such as, for example, amyotrophic lateral sclerosis (ALS). In a further aspect, the disclosed method can also be used to distinguish between ALS subtypes In an aspect, the method involves obtaining a sample of cerebrospinal fluid or another biological sample from a patient, contacting the sample with one or more enzymes capable of C-terminal and / or N-terminal cleavage of lysine and / or arginine, and analyzing the cleaved proteins using mass spectrometry, including comparison of the cleaved proteins to one or more databases and quantifying the cleaved proteins in order to diagnose the neurodegenerative disease and identify the subtype of the neurodegenerative disease. Also disclosed are methods for treating ALS using antisense oligonucleotides (ASOs).

Description

NETWORK ANALYSIS OF THE CEREBROSPINAL FLUID PROTEOME OF SPORADIC AND FAMILIAL FORMS OF AMYOTROPHIC LATERAL SCLEROSIS AND METHODS OF TREATMENT OF THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 63 / 558,328, filed February 27, 2024, and U.S. Provisional Application Ser. No. 63 / 670,238, filed July 12, 2024, each of which is incorporated herein by reference in its entirety.CROSS REFERENCE TO SEQUENCE LISTING

[0002] The genetic components described herein are referred to by sequence identifier numbers (SEQ ID NO). The sequence listing in written computer readable format (CRF) as an xml file named “80503_2040_Sequence_Listing.xml” created on February 25, 2025, and having a size of 82,239 bytes, is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grant numbers NS084974, NS137434, and AG066511 awarded by The National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] Amyotrophic Lateral Sclerosis (ALS) is a heterogeneous motor neuron disease that typically results in death within 3-5 years following diagnosis. Clinical manifestations include a spectrum of upper and lower motor neuron involvement and wide variability in disease progression. Roughly 10% of ALS cases are driven by an inherited mutation, of which the most common are the C9orf72 hexanucleotide repeat expansion and point mutations in the gene for superoxide dismutase 1 (SOD1). Though the pathogenic mechanisms underlying the various genetic forms of ALS are likely to be distinct from sporadic disease, the clinical presentations are remarkably similar, making the discovery of biomarkers that distinguish the various forms of ALS of paramount importance. Also, in people who harbor disease-causing mutations but remain asymptomatic, comparison of biomarkers from the pre- and post-symptomatic phase will provide insight into potential markers of disease transition, and also mark an early time point when disease modifying therapies could be started. A focus on cerebrospinal fluid (CSF) biomarkers allows forinterrogation of CNS protein changes that may differentiate disease pathways among various pathogenic forms of ALS as well as provide tools allowing for early diagnosis and monitoring of disease activity.

[0005] Mass spectrometry-based proteomics coupled with systems biology approaches using coexpression network analysis is a valuable tool for discovery of disease biomarkers and pathways, including in ALS and Alzheimer’s Disease. Unbiased proteomics of human brain and CSF coupled with network analysis has emerged as a valuable tool for organizing proteome wide expression data into groups or “modules” of highly correlated proteins that reflect various biological functions linked to neurodegeneration. While ALS brain proteomic networks have been examined, ALS CSF proteomic networks from large cohorts that include both sporadic and familial ALS across different mutation carriers have been under-investigated.

[0006] C9orf72-linked amyotrophic lateral sclerosis (c9ALS) is caused by an intronic G4C2 repeat expansion in the C9orf72 gene. Several disease mechanisms by which the G4C2 expansion promotes neurodegeneration have been proposed, which include the transcription and translation of sense encoded G4C2 repeats into toxic repeat containing RNA transcripts and dipeptide repeat proteins (DPRs), respectively. However, new strategies that target the G4C2 sense strand are needed.

[0007] Despite advances in ALS research, there is still a scarcity of minimally-invasive methods for diagnosing ALS in living subjects, including those who are pre-symptomatic but have a genetic predisposition to ALS, as well as determining ALS type in diagnosed individuals. Following diagnosis and classification of ALS type, new strategies for treatment based on ALS type are needed. These needs and other needs are satisfied by the present disclosure.SUMMARY

[0008] In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to methods for diagnosing a neurodegenerative disease associated with loss of motor neurons such as, for example, amyotrophic lateral sclerosis (ALS). In a further aspect, the disclosed method can also be used to distinguish between ALS subtypes In an aspect, the method involves obtaining a sample of cerebrospinal fluid or another biological sample from a patient, contacting the sample with one or more enzymes capable of C-terminal and / or N-terminal cleavage of lysine and / or arginine, and analyzing the cleaved proteins using mass spectrometry, including comparison of the cleaved proteins to one or more databases and quantifying the cleaved proteins in order to diagnose theneurodegenerative disease and identify the subtype of the neurodegenerative disease. Also disclosed are methods for treating ALS using antisense oligonucleotides (ASOs).

[0009] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0011] FIGs. 1A-1 E show experimental workflow with differential expression of ALS versus control CSF proteomes. FIG. 1A: Schematic of experimental workflow to examine proteomic differences between subjects with ALS and controls in cerebrospinal fluid (CSF). FIGs. 1B-1D: Volcano plots showing differential abundance profiles comparing control CSF (n=44) to that from sALS (n=35), C9orf72 ALS (n=10) and asymptomatic C9orf72 carriers (n=6). Log2 fold change (x-axis) and one-way ANOVA with Benjamini-Hochberg corrected by disease -Iog10 p-values (y- axis). Note the commonly increased CSF proteins in patients with ALS such as NEFL, NEFM, CHIT1 , and GPNMB. FIG. 1E: Scatter plot showing differential expression of C9orf72 ALS versus asymptomatic C9orf72 carriers. Log2 fold change of C9orf72- Associated ALS (x-axis) and Log2 fold change of C9orf72 Asymptomatic carriers (y-axis) were compared for each protein (n=2105). Proteins are colored based on distance from the origin.

[0012] FIGs. 2A-2D show protein and peptide-specific SOD1 CSF levels for SOD1 ALS. FIG. 2A: Volcano plot representing differential protein abundance comparing SOD 1 -associated ALS cases (n=6) versus all other ALS cases (n=45). Log2 fold change (x-axis) and one-way ANOVA with Benjamini-Hochberg corrected by disease -Iog10 p-values (y-axis) are shown for each protein(n=2,105). FIG. 2B: Total SOD1 normalized protein abundance levels across all subgroups. Oneway ANOVA was used to determine if a difference was present between the ALS groups. Individual SOD1 mutations are depicted by color. FIG. 2C: SOD1 (SEQ ID NO. 1) specific-peptide level quantification across controls and disease subgroups. Peptides (SEQ ID NOs. 2-7) were visualized for overlap of the canonical SOD1 protein sequence (P00441). Only fully-tryptic peptides that were detected in all 6 SOD1 ALS patients were included. FIG. 2D: Boxplots for abundance of each peptide identified were evaluated with one-way ANOVA. Each datapoint in the SOD1 ALS group is annotated with the mutation associated with each patient. Peptides that overlap with SOD1 regions that have mutations (single amino acid substitutions) in this cohort are emphasized with the specific mutation noted.

[0013] FIGs. 3A-3D show CSF Network modules associate with brain cell-types ALS disease subtypes. FIG. 3A: Cluster dendrogram indicates similarity of WGCNA network modules based on correlation of eigenproteins (first principal component). FIG. 3B: Relationship between ALS disease subgroup (sporadic, SOD1 , Asymptomatic C9orf72 and symptomatic C9orf72) with individual protein modules was evaluated by cross referenced trait values with module proteins using a Biweight midcorrelation (BiCor) analysis. Significance as determined by BiCor are denoted by overlain asterisks; *p < 0.05, **p < 0.01 , ***p < 0.001 . Note the relatedness of modules 7 and 10 as well as the overlap in significance between these modules by disease subtype compared to control group. FIG. 3C: Cell-type enrichment was characterized by comparing module proteins with a list of proteins known to be enriched in astrocytes, microglia, neurons, oligodendrocytes, and endothelia; respectively (see methods). Significance levels determined by one-tailed Fisher’s exact test are denoted by overlain asterisks; *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 3D: Top gene ontology (GO) terms were selected from significant GO annotations.

[0014] FIGs. 4A-4B show CSF Network Modules vary across ALS subgroups. FIG. 4A: Eigenproteins for each CSF proteome modules (n=12) were compared across control and ALS disease type by one-way ANOVA. Hub proteins and GO terms for each module are highlighted FIG. 4B: Differentially abundant proteins from each subgroup compared to controls for C9 ALS, Sporadic ALS and C9 Asymptomatic were mapped by module. Symptomatic vs asymptomatic C9orf72 differences were also included. The height of the bars represents the fraction of module member proteins that were differentially abundant. The bars show a heatmap for average Iog2 difference in abundance.

[0015] FIGs. 5A-5F show Emory ALS CSF network changes are preserved in an independentdataset. FIG. 5A: Schematic of experimental workflow to quantitatively evaluate similarities and differences across proteomes generated from two different data sources (Emory ALS Set1 and Oh et al. ALS Set 2), indicating sample size, number of proteins quantified, and number of modules calculated. FIG. 5B: Volcano plot of Oh et al. (2023) Set2 proteome indicating differential expression of ALS (n=20), versus control (n=20). FIG. 5C: Venn diagram representing the number of proteins that were quantified in the Emory Set1 and Set 2 ALS datasets. FIG. 5D: Module preservation of Emory Set1 and Set2 dataset. Number of proteins in each module (x-axis) is compared across Zsummary, and overall measurement of preservation, (y-axis). The line at Zsummary=10 (q=1 * 10'23) indicates Emory ALS modules are highly preserved in the replication proteome. The line at Zsummary=2 (q=0.05) indicates Emory ALS modules are preserved independent dataset. FIG. 5E: Scatter plot representing differential abundance of Emory ALS module eigenprotein sALS - Control (y-axis) versus abundance of Oh et al. ALS synthetic eigenprotein ALS - Control (x-axis). FIG. 5F: Several paired Emory ALS module eigenprotein and Set2 ALS synthetic module eigenprotein comparisons are presented. Synthetic eigenproteins were constructed for Emory ALS dataset and measured by disease type in Emory ALS and Oh et al. ALS datasets. A minimum of four proteins from the top 20% of module membership by kME (correlation to module eigenprotein) were used to assess synthetic eigenprotein value (y-axis) and compared across disease type (y-axis). Those that are significant (p<0.05) by one way ANOVA p-values are represented in red. Modules are identified by module number and top GO terms in FIGs. 4A-4D.

[0016] FIGs. 6A-6F show assessing disease specificity of ALS CSF network and protein changes. FIG. 6A: Schematic of experimental workflow processing quantitative proteomics datasets with tandem-mass-tag (TMT) chemistry. FIG. 6B: Scatter plot indicating differential expression of sporadic ALS (n=35) versus the Alzheimer’s disease dataset (AD; n=149). Log2 fold change of sALS (x-axis) and Log2 fold change of AD (y-axis) were compared for each protein (n=1 ,871). Proteins are colored corresponding to module. FIG. 6C: Venn diagram shows overlap of top ten module hub proteins, the proteins that most highly correlated with each module, (n=120) that significantly varied across disease type and control by one-way ANOVA. FIG. 6D: Heatmap indicating which datasets had proteins associated with disease both present and significantly different from controls (red), present and not significantly different from controls (black), or not present in the dataset (white). FIG. 6E: A selection of well characterized markers shared across neurodegenerative disease. For each marker the normalized, Iog2-transformed abundance (y- axis) and disease type (x-axis) are shown for each of the datasets in A, alongside p-values fromone way ANOVA. FIG. 6F: ALS-specific changes, shared across ALS disease type and control but not AD.

[0017] FIG. 7 shows synthetic eigenproteins were constructed for Emory ALS dataset and measured by disease type in Emory ALS dataset (Set 1) and the replications ALS dataset (Set2). ALS datasets. A minimum of four proteins from the top 20% of module membership by kME (correlation to module eigenprotein) were used to assess synthetic eigenprotein value (y-axis) and compared across disease type (y-axis). Where significant (p<0.05) by one way ANOVA p- values are represented in red. Modules are identified by module number and top GO terms.

[0018] f show poly(GP) DPRs are reduced in the CSF of c9ALS patients treated with sense strand targeting ASO, BIIB078. (FIG. 8A) Timeline and summary of BIIB078 dosing, CSF poly(GP) measurements, and ALSFRS-R scores. CSF was collected prior to BIIB078 dosing at each timepoint. Left axis shows relative CSF poly(GP) abundance normalized to a baseline poly(GP) abundance collected immediately prior to first dose (100% depicted by dotted line). Right axis shows ALSFRS-R scores recorded contemporaneously. (FIG. 8B) Slope plot of raw poly(GP) abundance (A.U. = arbitrary units) in CSF as measured by immunoassay at baseline and last dose for each case. (FIG. 8C) Slope plot of ALSFRS-R measured at most proximal visit preceding first dose and last visit after terminal BIIB078 dose. (FIG. 8D) Slope plot of neurofilament light chain (NfL) abundance measured in CSF as measured by immunoassay at visit preceding first BIIB078 dose and last dose for each case.

[0019] FIGs. 9A-9G show Intrathecally delivered BIIB078 ASOs are broadly distributed throughout the CNS parenchyma of BIIB078-treated c9ALS cases. (FIG. 9A) Anti-ASO immunohistochemical staining and miRNAscope in situ hybridization (ISH) assessment of BIIB078 distribution in the spinal cord of control, drug naive c9ALS (“Naive”), and BIIB078-5 treated cases. BIIB078 is present in neurons of the ventral horn. (Days) represent duration between delivery of last BIIB078 dose and autopsy. Scale bar = 30 pm. (FIGs. 9B-9G) Macro and high magnification images of BIIB078 distribution as detected by miRNAscope ISH in subarachnoid regions (motor cortex, frontal cortex, cerebellar cortex) and periventricular regions (hippocampus, basal ganglia, medulla). Scale bar = 30 pm.

[0020] FIGs. 10A-10D show BIIB078 is differentially abundant in different CNS regions. Concentration of BIIB078 ASO in the spinal cord (FIG. 10A) and motor cortex (FIG. 10B) of control (“Con”), drug-naive c9ALS (“Naive”), and BIIB078 treated c9ALS cases (“ASO”). Spinal Cord: n=6 control, n=8 drug-naive c9ALS, n=6 BIIB078 treated c9ALS cases. Motor Cortex: n=6 control,n=10 drug-naive c9ALS, n=6 BIIB078 treated c9ALS cases. (FIG. 10C) Relative BIIB078 ASO concentration across various brain regions normalized to spinal cord. n=6 cases per region. (FIG. 10D) Heatmap showing correlation of measured BIIB078 ASO concentration across brain regions measured in (FIG. 10C). L.I.=Last Injection. Values represent Kendall T Rank correlation coefficients.

[0021] FIGs. 11A-11B show C9orf72 expression in the spinal cord of BIIB078 treated c9ALS cases. (FIG. 11 A) qPCR for total, variant 1 , and variant 3 C9orf72 expression. n=8 control, n=14 drug-naive c9ALS, n=6 BIIB078 treated c9ALS cases. *P < 0.05, **P < 0.01, ***P < 0.001 , ****P < 0.0001, Kruskal-Wallis one-way analysis of variance followed by Conover-Iman multiple comparison test. (FIG. 11 B) Correlation of total and transcript specific C9orf72 expression and BIIB078 ASO concentration. A strong correlation between C9orf72 transcript V1 and V3 expression with BIIB078 ASO concentration was observed.

[0022] FIGs. 12A-12E show G4C2 associated neuropathological hallmarks are present in postmortem tissue from BIIB078-treated c9ALS patients. (FIGs. 12A-12B) Immunopositive poly(GA) and poly(GP) inclusions were detected in the spinal cord, motor cortex, and frontal cortex of drug- naive (“Naive”) and BIIB078 treated (“ASO”) cases. Scale bar = 30 pm. (FIG. 12C) poly(GP) and (FIG. 12D) poly(GA) abundance, as measured by quantitative MSD immunoassays, is indistinguishable between drug-naive and BIIB078 treated c9ALS cases. Only comparison between drug-naive and BIIB078 treated c9ALS cases were considered; Mann-Whitney test (FIG. 12E) BIIB078 ASO and poly(GA) inclusions co-occur in the same cell as detected by double staining immunohistochemistry in BIIB078 treated cases. (FIGs. 12C-12D) Spinal Cord: n=11 control, n=18 drug-naive c9ALS, n=6 BIIB078 treated c9ALS cases. Motor Cortex: n=17 control, n=21 drug-naive c9ALS, n=6 BIIB078 treated c9ALS cases. Frontal Cortex: n=12 control, n=18 drug-naive c9ALS, n=6 BIIB078 treated c9ALS cases.

[0023] FIGs. 13A-13B show abundant pTDP-43 pathology in BIIB078-treated c9ALS patients. (FIG. 13A) Quantitative assessment of pTDP-43 in the spinal cord, motor cortex, and frontal cortex of control (“Con”), drug naive c9ALS (“Naive”), and BIIB078-treated cases (“ASO”). Spinal Cord: n=11 control, n=18 drug-naive c9ALS, n=6 BIIB078 treated c9ALS cases. Motor Cortex: n=17 control, n=21 drug-naive c9ALS, n=6 BIIB078 treated c9ALS cases. Frontal Cortex: n=12 control, n=18 drug-naive c9ALS, n=6 BIIB078 treated c9ALS cases. (FIG. 13B) Relative qPCR expression for tSTMN2 in the spinal cord, motor cortex, and frontal cortex of control, drug naive c9ALS, and Bl I B078-treated cases. Spinal Cord: n=8 control, n=14 drug-naive c9ALS, n=6BIIB078 treated c9ALS cases. Motor Cortex: n=11 control, n=16 drug-naTve c9ALS, n=6 BIIB078 treated c9ALS cases. Frontal Cortex: n=8 control, n=15 drug-naTve c9ALS, n=6 BIIB078 treated c9ALS cases. *P < 0.05, **P < 0.01 , ***P < 0.001 , ****p < 0.0001 , Kruskal-Wallis one-way analysis of variance followed by Conover-Iman multiple comparison test.

[0024] FIGs. 14A-14E show box plots for peptide sequences for exemplary protein NEFL indicating abundance in different ALS types relative to controls. In each of these instance, a significant interaction between peptide abundance (y-ais) and Disease group (x-axis) was observed, as measured by Analysis of Variance (ANOVA). Similar plots can be constructed for other relevant full-length proteins as discussed herein.

[0025] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0026] Disclosed herein is a method for evaluating the CSF proteome in order to diagnose and classify shared and distinct alterations associated with CNS cell-types and pathways across sporadic and genetic ALS subgroups.

[0027] In one aspect, disclosed herein is a method for performing unbiased CSF proteomics from sporadic ALS (sALS), C9orf72 ALS, C9orf72 asymptomatic carriers, SOD1 ALS, and healthy controls. In a further aspect, separation of subjects into these cohorts enabled measurement of differentially abundant proteins (DAPs) among i) each ALS subgroup and control; ii) symptomatic and asymptomatic C9orf72; and iii) genetic and sporadic ALS. In a further aspect, changes in the CSF proteome from the Emory ALS cohort were validated with a published, independent ALS CSF proteomic dataset. In a still further aspect, to address disease specificity, potential biomarkers in these ALS cohorts were compared to Alzheimer’s disease (AD). In one aspect, network modules found to change significantly with ALS type were associated with Module 5 (M5)- Extracellular matrix / Heparin binding, M7-Cytoskeleton / Microglial, and M10- Ubiquitination / Gluconeogenesis. In still another aspect, differentially abundant proteins, relative to controls, were identified in each group. In an aspect, of particular interest were proteins that differentiated C9orf72 ALS from asymptomatic C9orf72 carriers, which may be used to identify atransition from asymptomatic to symptomatic disease. In any of these aspects, these findings suggest that while sporadic and genetic forms of ALS display largely overlapping CSF proteomes, differences point to unique pathogenic pathways. In one aspect, sporadic ALS may be associated with an unknown mutation.

[0028] In an aspect, recently, a randomized, placebo controlled, quadruple-blind, dose-escalating clinical trial (NCT03626012; NCT04288856) using an antisense oligonucleotide (ASO) targeting G4C2 repeat-containing transcripts (BIIB078) was discontinued due to failure to meet secondary clinical efficacy endpoints despite evidence of target engagement, namely a reduction in poly(GP) DPR abundance in the cerebrospinal fluid of treated patients. In a further aspect, these observations raised questions about the importance of G4C2 sense strand-associated toxic mechanisms, and whether neuropathological hallmarks associated with G4C2 repeat expansions are decreased in disease-relevant central nervous system (CNS) parenchyma of c9ALS patients treated with BIIB078. However, in one aspect, post-mortem G4C2 repeat-associated neuropathological hallmarks were examined in brain and spinal cord from six c9ALS patients treated with BIIB078. Further in this aspect, while there was widespread distribution of the ASO drug, C9orf72 mRNA transcript variants and G4C2-containing intronic transcripts were not significantly different between drug naive and BIIB078-treated c9ALS cases. Herein it is demonstrated that poly(GA) and poly(GP) DPRs are robustly detected in several regions of the CNS, including the primary motor cortex and spinal cord in BIIB078-treated c9ALS cases and that their relative abundance is indistinguishable from drug-naive c9ALS cases. In yet another aspect, these findings suggest that CSF biomarkers do not completely reflect target engagement and ASO efficacy in the CNS parenchyma; consequently, ceasing strategies that target the G4C2 sense strand was premature.Method for Diagnosing a Neurodegenerative Disease

[0029] In one aspect, disclosed herein is a method of diagnosing a neurodegenerative disease associated with loss of motor neurons, the method including at least the steps of (i) providing a sample from a patient, (ii) measuring one or more proteins in the sample, thereby providing measured relative quantities of each of the one or more proteins, (iii) comparing the measured relative quantities of the one or more proteins to reference relative quantities of the one or more proteins associated with a specific neurodegenerative disease associated with loss of motor neurons, and (iv) diagnosing the patient with a specific neurodegenerative disease associated with loss of motor neurons when the measured relative quantities of the one or more proteins aresubstantially similar to reference relative quantities of the one or more proteins associated with a specific neurodegenerative disease associated with loss of motor neurons. In a further aspect, the sample can be from cerebrospinal fluid (CSF).

[0030] In one aspect, the neurodegenerative disease associated with loss of motor neurons can be Amyotrophic Lateral Sclerosis (ALS), and the measured relative quantities of the one or more proteins correlate with disease onset, progression, or both disease onset and progression in an ALS subgroup. In a further aspect, the ALS subgroup can be associated with an inherited mutation.

[0031] In one aspect, the inherited mutation can be a c9orf72 hexanucleotide repeat and the patient can be a symptomatic carrier or asymptomatic carrier of the c9orf72 hexanucleotide repeat. In another aspect, the inherited mutation can be a superoxide dismutase 1 gene (SOD1) mutation. In one aspect, the ALS subgroup can be sporadic ALS, which is, in some aspects, associated with one or more unknown mutations.Exemplary Proteins

[0032] In an aspect, the one or more proteins can be neurofilament medium protein (NEFM) and neurofilament light chain protein (NEFL). In another aspect, the one or more proteins can be chitinase 1 protein (CHIT1) and chitinase-3 like-protein-1 (CHI3L1). In still another aspect, the one or more proteins can be superoxide dismutase protein (SOD1). Further in this aspect, a low abundance of the SOD1 protein can indicate the presence of an A5T mutation in the SOD1 protein. Proteins can have from 1 to 100 characteristic peptides. Exemplary peptides for SOD1 are shown in FIG. 2C. Exemplary peptides for NEFL (see also FIGs. 14A-14E), NEFM, CHIT1 ,CHI3L1, CHI3L2, and UCHL1 are shown in Table 1 below:

[0033] In one aspect, the one or more proteins can be ALS-specific proteins such as, for example, insulin-like growth factor 2 (IGF2), retinoic acid receptor responder 2 (RARRES2), galactosespecific lectin 3 (LGALS3), and lysozyme (LYZ).

[0034] In another aspect, the one or more proteins can include Glycoprotein nonmetastatic melanoma protein B (GPNMB). In still another aspect, the one or more proteins can be or include aldo-keto reductase family 1 , member B1 (AKR1 B1), Transketolase (TKT), cofilin 1 (CFL1), cofilin 2 (CFL2), peptidylprolyl isomerase A (PPIA), and fatty-acid-binding protein 3 (FABP3).

[0035] In still another aspect, the one or more proteins can include lactate dehydrogenase A (LDHA). In yet another aspect, the one or more proteins can be elastin microfibril interfacer 1 (EMILIN1) and DnaJ heat shock protein family (Hsp40) member C3 (DNAJC3).

[0036] In one aspect, the one or more proteins can be or include Histone H1.5 protein (HIST1 H1 B), Histone H4 (HIST1 H4A), Histone H2A (HIST1 H2AB), Histone H1.4 (HIST1H1 E), Histone H2A.Z (H2AFZ), and Histone H2B type 2-F HIST2H2BF.Method for Measuring Proteins

[0037] In one aspect, measuring the one or more proteins in the sample providing measured relative quantities of each protein includes one or more of: (a) contacting the sample with an enzyme capable of C-terminal cleavage of lysine, arginine, or both; and (b) contacting the sample with an enzyme capable of N-terminal cleavage of lysine, arginine, or both; wherein performing (a), (b), or both (a) and (b) provides cleaved proteins; analyzing the cleaved proteins by mass spectrometry, wherein mass spectrometry generates fragmentation spectra from peptides; and analyzing cleavage of the cleaved proteins.

[0038] In one aspect, analyzing cleavage of the cleaved proteins includes at least mass selection of one or more target peptide ions, fragmentation of the one or more target peptide ions, and mass-to-charge (m / z) analysis of the one or more target peptide ions. In another aspect, the method further includes searching fragmentation spectra of the cleaved proteins against genomic or metagenomic databases to deduce amino acid sequences of the cleaved proteins, thereby providing positive identifications and measured relative quantities for the one or more proteins in the sample.Biodistribution of a Drug Targeting C9orf72

[0039] Amyotrophic lateral sclerosis (ALS) is a progressive, ultimately fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons in the primary motor cortex and spinal cord, respectively. Known genetic causes of ALS are present in approximately 12% of patients, the most common of which is an expanded G4C2 repeat in the first intron of the gene C9orf72. Hexanucleotide expansions in C9orf72 are also the most common genetic cause of frontotemporal dementia (FTD). The specific mechanism(s) by which the intronic G4C2 repeat expansion promotes neurodegeneration remain unknown; however, several lines of evidence demonstrate that toxic gain of function mechanisms associated with the bidirectional transcription and translation of expanded G4C2 repeats underlie neurodegeneration in C9ALS / FTD. Sense (G4C2) or antisense (C4G2) repeat containing RNAs can sequester RNA binding proteins (RBP) resulting in dysregulated RBP function. Translation across repeat-containing transcripts produce aberrant dipeptide repeat (DPR) proteins shown to be toxic through myriad mechanisms. Accordingly, therapeutic strategies have focused on silencing the expression of repeat-containing transcripts. Motivated by preclinical and clinical success in other diseases, RNA-based therapies including siRNA and antisense oligonucleotides (ASOs) have emerged as leading therapeutic approaches to reduce expression of repeat-containing transcripts and thereby mitigate toxicity in C9ALS / FTD.

[0040] BIIB078 (Tadnersen) is a mixed-backbone, 4-8-6 “gapmer” ASO composed of a central unmodified DNA gap and flanking 2'-O-methoxyethyl (MOE) modified RNA wings (GSRS UNI I: OSO6W71 NMN). Designed to reduce G4C2 repeat-containing transcripts through RNase-H mediated cleavage, BIIB078 is complementary to an 18bp sequence in the first intron of C9orf72. Several preclinical studies utilizing both in vitro and in vivo models provided strong evidence that C9orf72 targeting ASOs can silence expression of G4C2 repeat-containing transcripts and mitigate associated toxic mechanisms. A seminal preclinical study demonstrated that a single bolus of BIIB078 delivered by intracerebroventricular (ICV) injection in 9-month-old transgenic c9BAC mice silenced expression of G4C2 repeat transcripts and resulted in a sustained reduction of DPR burden in vivo. These studies supported the initiation of a randomized, placebo controlled, quadruple-blind, dose-escalating clinical trial to assess the tolerability and efficacy of intrathecally administered BIIB078 in c9ALS (NCT03626012; NCT04288856). Topline results from this trial released in March 2022 indicated that BIIB078 did not provide clinical benefit, despite indirect evidence of target engagement as demonstrated by the finding that poly(GP) and poly(GA) DPRs are reduced in the cerebrospinal fluid of patients with c9ALS treated with BIIB078. However, several critical questions remained unaddressed: 1) did intrathecally administered BIIB078 distribute broadly through the CNS? 2) did BIIB078 target engagement occur in the CNS parenchyma and reduce G4C2 repeat containing transcripts? And 3) did BIIB078 treatment modify neuropathological hallmarks associated with c9ALS in disease relevant brain regions?

[0041] To address these questions, an in-depth molecular and neuropathological assessment of the biodistribution and efficacy of BIIB078 in cerebrospinal fluid (CSF) and post-mortem tissue samples from c9ALS patients treated with BIIB078 was performed.Methods for Treating ALS

[0042] In one aspect, disclosed herein is method for treating ALS in a subject, such as a human, the method including at least the step of administering a pharmaceutical composition containing at least one antisense oligonucleotide (ASO) to the subject, wherein the ASO selectively binds to a target nucleotide sequence having one or more G4C2 repeats. In a further aspect, the ALS can be c9ALS and the ASO can be BIIB078, although other ASOs targeting other forms of ALS are also contemplated and should be considered disclosed. In one aspect, the pharmaceutical composition is administered intrathecally.

[0043] In an aspect, performing the method reduces poly(GP) in cerebrospinal fluid (CSF) of the subject by at least about 50% or by at least about 75%. In some aspects, the method is performedonce or is performed 2 or more times, and wherein each dose of the pharmaceutical composition is separated by a time interval of from about 2 weeks to about 4 weeks.

[0044] In another aspect, the pharmaceutical composition comprises from about 10 mg to about 90 mg of the ASO, or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mg of the ASO per dose..

[0045] In one aspect, the ASO localizes to a CNS region affected by ALS such as, for example, the spinal cord, the motor cortex, the subarachnoid brain region, or the periventricular brain region. In another aspect, the ASO persists in the CNS region for from at least 10 days to at least 500 days, or for at least 10 days, 20, days 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, at least 400 days, or at least 500 days.. In some aspects, elevated levels of the ASO may or may not be detected in the CSF of the patient. Herein, it has been unexpectedly discovered that even when the ASO does not persist in the CSF of the patient, or remains at low levels, that the ASO can still be found post-mortem in the cells and tissues of the CNS and can thus have a therapeutic effect despite low or no levels in the CSF.

[0046] In any of these aspects, performing the method reduces at least one C9orf72 transcript in the CSF of the subject. Further in this aspect, the at least one C9orf72 transcript includes one or more G4C2 repeats and can be C9orf72 variant 1 , C9orf72 variant 3, or both.

[0047] In one aspect, the method further includes administering at least one additional ALS treatment to the subject, such as, for example, a second pharmaceutical composition, stem cell therapy, gene therapy, an immunomodulatory therapy, or any combination thereof. Further in this aspect, the second pharmaceutical composition can be or include riluzole, edaravone, sodium phenylbutyrate / taurursodiol, tofersen, or any combination thereof.

[0048] In one aspect, performing the method reduces at least one symptom of ALS including, but not limited to, muscle weakness, fatigue, pain, loss of fine motor function, slurred speech, difficulty swallowing, muscle cramps, muscle atrophy, shortness of breath, coughing, speech impairment, cognitive changes, emotional changes, excessive salivation, difficulty controlling bowel movements, tripping, falling, difficulty walking, difficulty standing, or any combination thereof.

[0049] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosedand that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0050] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0051] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0052] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0053] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0054] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0055] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0056] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.Definitions

[0057] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0058] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cleaved protein,” “a mutation,” or “an enzyme,” include, but are not limited to, mixtures, combinations, or series of two or more such cleaved proteins, mutations, or enzymes, and the like.

[0059] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can beexpressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0060] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. 'about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0061] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1 % to 5%” should be interpreted to include not only the explicitly recited values of about 0.1 % to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0062] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal valueindicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0063] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0064] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).

[0065] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.ASPECTS

[0066] The present disclosure can be described in accordance with the following numbered aspects, which should not be confused with the claims.

[0067] Aspect 1. A method of diagnosing a neurodegenerative disease associated with loss of motor neurons comprising providing a sample from a patient, measuring one or more proteins in the sample, thereby providing measured relative quantities of each of the one or more proteins, comparing the measured relative quantities of the one or more proteins to reference relative quantities of the one or more proteins associated with a specific neurodegenerative disease associated with loss of motor neurons, and diagnosing the patient with a specific neurodegenerative disease associated with loss of motor neurons when the measured relative quantities of the one or more proteins are substantially similar to reference relative quantities of the one or more proteins associated with a specific neurodegenerative disease associated with loss of motor neurons.

[0068] Aspect 2. The method of aspect 1, wherein the sample is from cerebrospinal fluid (CSF).

[0069] Aspect 3. The method of aspect 1 wherein the neurodegenerative disease associated with loss of motor neurons comprises Amyotrophic Lateral Sclerosis (ALS) and where the measured relative quantities of the one or more proteins correlate with disease onset, progression, or both disease onset and progression in an ALS subgroup.

[0070] Aspect 4. The method of aspect 3, wherein the ALS subgroup is associated with an inherited mutation.

[0071] Aspect s. The method of aspect 4, wherein the inherited mutation is a c9orf72 hexanucleotide repeat.

[0072] Aspect 6. The method of aspect 5, wherein the patient is a symptomatic carrier or asymptomatic carrier of the c9orf72 hexanucleotide repeat.

[0073] Aspect 7. The method of aspect 4, wherein the inherited mutation is a superoxide dismutase 1 gene (SOD1) mutation.

[0074] Aspect 8. The method of aspect 3, wherein the ALS subgroup is sporadic ALS.

[0075] Aspect 9. The method of aspect 1 , wherein the one or more proteins comprise neurofilament medium protein (NEFM) and neurofilament light chain protein (NEFL).

[0076] Aspect 10. The method of aspect 1 , wherein the one or more proteins comprise chitinase 1 protein (CHIT1) and chitinase-3 like-protein-1 (CHI3L1).

[0077] Aspect 11. The method of aspect 1 , wherein the one or more proteins comprise superoxide dismutase protein (SOD1).

[0078] Aspect 12. The method of aspect 11 , wherein a low abundance of the SOD1 protein indicates an A5T mutation is present in the SOD1 protein.

[0079] Aspect 13. The method of aspect 1 , wherein the one or more proteins comprise ALS- specific proteins, wherein the ALS-specific proteins comprise insulin-like growth factor 2 (IGF2), retinoic acid receptor responder 2 (RARRES2), galactose-specific lectin 3 (LGALS3), and lysozyme (LYZ).

[0080] Aspect 14. The method of aspect 1 , wherein the one or more proteins comprise Glycoprotein nonmetastatic melanoma protein B (GPNMB).

[0081] Aspect 15. The method of aspect 1 , wherein the one or more proteins comprise aldoketo reductase family 1 , member B1 (AKR1 B1), Transketolase (TKT), cofilin 1 (CFL1), cofilin 2 (CFL2), peptidylprolyl isomerase A (PPIA), and fatty-acid-binding protein 3 (FABP3).

[0082] Aspect 16. The method of aspect 1 , wherein the one or more proteins comprise lactate dehydrogenase A (LDHA).

[0083] Aspect 17. The method of aspect 1 , wherein the one or more proteins comprise elastin microfibril interfacer 1 (EMILIN1) and DnaJ heat shock protein family (Hsp40) member C3 (DNAJC3).

[0084] Aspect 18. The method of aspect 1 , wherein the one or more proteins comprise Histone H1.5 protein (HIST1H1B), Histone H4 (HIST1H4A), Histone H2A (HIST1 H2AB), Histone H1.4 (HIST1 H1E), Histone H2A.Z (H2AFZ), and Histone H2B type 2-F HIST2H2BF.

[0085] Aspect 19. The methods of any of one aspects 1-18, wherein measuring the one or more proteins in the sample providing measured relative quantities of each protein comprises one or more of:(a) contacting the sample with an enzyme capable of C-terminal cleavage of lysine, arginine, or both;(b) contacting the sample with an enzyme capable of N-terminal cleavage of lysine, arginine, or both; wherein performing (a), (b), or both (a) and (b) provides cleaved proteins; analyzing the cleaved proteins by mass spectrometry, wherein mass spectrometry generates fragmentation spectra from peptides; and analyzing cleavage of the cleaved proteins.

[0086] Aspect 20. The method of aspect 19, wherein analyzing cleavage of the cleaved proteins comprises mass selection of one or more target peptide ions, fragmentation of the one or more target peptide ions, and mass-to-charge (m / z) analysis of the one or more target peptide ions.

[0087] Aspect 21. The method of aspect 19, further comprising searching fragmentation spectra of the cleaved proteins against genomic or metagenomic databases to deduce amino acid sequences of the cleaved proteins, thereby providing positive identifications and measured relative quantities for the one or more proteins in the sample.

[0088] Aspect 22 A method for treating ALS in a subject, the method comprising administering a pharmaceutical composition comprising at least one antisense oligonucleotide (ASO) to the subject, wherein the ASO selectively binds to a target nucleotide sequence comprising one or more G4C2 repeats.

[0089] Aspect 23. The method of aspect 22, wherein the subject is a human.

[0090] Aspect 24. The method of aspect 22 or 23, wherein the ALS comprises c9ALS and the ASO comprises BIIB078.

[0091] Aspect 25. The method of any one of aspects 22-24, wherein the pharmaceutical composition is administered intrathecally.

[0092] Aspect 26. The method of any one of aspects 22-25, wherein performing the method reduces poly(GP) in cerebrospinal fluid (CSF) of the subject by at least about 50%.

[0093] Aspect 27. The method of aspect 26, wherein performing the method reduces poly(GP) in cerebrospinal fluid (CSF) of the subject by at least about 75%.

[0094] Aspect 28. The method of any one of aspects 22-27, wherein the method is performed once.

[0095] Aspect 29. The method of any one of aspects 22-27, wherein the method is performed 2 or more times, and wherein each dose of the pharmaceutical composition is separated by a time interval.

[0096] Aspect 30. The method of aspect 29, wherein the time interval comprises from about 2 weeks to about 4 weeks.

[0097] Aspect 31. The method of any one of aspects 22-30, wherein the pharmaceutical composition comprises from about 10 mg to about 90 mg of the ASO.

[0098] Aspect 32. The method of any one of aspects 22-31 , wherein the ASO localizes to a CNS region affected by ALS.

[0099] Aspect 33. The method of aspect 32, wherein the CNS region comprises the spinal cord, the motor cortex, the subarachnoid brain region, or the periventricular brain region.

[0100] Aspect 34. The method of aspect 32 or 33, wherein the ASO persists in the CNS region for from at least 10 days to at least 500 days.

[0101] Aspect 35. The method of any one of aspects 22-34, wherein performing the method reduces at least one C9orf72 transcript in the CSF of the subject.

[0102] Aspect 36. The method of aspect 35, wherein the at least one C9orf72 transcript comprises one or more G4C2 repeats.

[0103] Aspect 37. The method of aspect 35 or 36, wherein the at least one C9orf72 transcript comprises variant 1 , variant 3, or both.

[0104] Aspect 38. The method of any one of aspects 22-37, further comprising administering at least one additional ALS treatment to the subject.

[0105] Aspect 39. The method of aspect 38, wherein the at least one additional ALS treatment comprises administration of a second pharmaceutical composition, stem cell therapy, gene therapy, an immunomodulatory therapy, or any combination thereof.

[0106] Aspect 40. The method of aspect 39, wherein the second pharmaceutical composition comprises riluzole, edaravone, sodium phenylbutyrate / taurursodiol, tofersen, or any combination thereof.

[0107] Aspect 41. The method of any one of aspects 22-40, wherein performing the method reduces at least one symptom of ALS.

[0108] Aspect 42. The method of aspect 41 , wherein the at least one symptom comprises muscle weakness, fatigue, pain, loss of fine motor function, slurred speech, difficulty swallowing, muscle cramps, muscle atrophy, shortness of breath, coughing, speech impairment, cognitive changes, emotional changes, excessive salivation, difficulty controlling bowel movements, tripping, falling, difficulty walking, difficulty standing, or any combination thereof.EXAMPLES

[0109] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.Example 1 : Materials and Methods for Diagnostic ApplicationsCSF Samples

[0110] All CSF samples were collected as part of ongoing research at the Emory ALS Center in Atlanta, Georgia. Research participants provided informed consent under protocols approved by the Institutional Review Board at Emory University. The Emory ALS CSF cohort contained samples from C9orf72 ALS (n=10), C9orf72 asymptomatic carriers (n=6), SOD1 ALS (n=6), andsALS (n=35), as well as age matched healthy controls (n=44). Characteristics of the Oh et al. ALS and Emory AD (disease specificity) CSF cohorts were previously published. Characteristics of the Emory ALS cohort are summarized in Table 2.Protein Digestion and Tandem Mass Tag (TMT) Labeling of CSF

[0111] In order to sample the CSF in an unbiased manner and given that have been previously shown that immunodepletion resulted in only a marginal improvement in proteomic coverage, the CSF samples were not immunodepleted prior to digestion. First, 50 pL of CSF was transferred to 1 mL deep well plates for digestion with lysyl endopeptidase (LysC) and trypsin. The samples were then reduced and alkylated with 1 pL of 0.5 M tris-2(-carboxyethyl)-phosphine (ThermoFisher) and 5 pL of 0.4 M chloroacetamide in a 90 °C water bath for 10 min followed with 5 min bath sonication. After the sample was cooled down on ice, 56 pL of 8 M urea buffer (8 M urea, 10 mM Tris, 100 mM NaFhPC , pH 8.5) with 12.5 mAU of LysC (Wako), was added to each sample, resulting in a final urea concentration of 4 M.

[0112] Samples were then mixed well, gently spun down, and incubated overnight at 25 °C for digestion with LysC. The following day, samples were diluted to 1 M urea with a mixture of 360 pL of 50 mM ammonium bicarbonate and 5 pg of Trypsin (ThermoFisher). The samples were subsequently incubated overnight at 25 °C for digestion with trypsin. The next day, the digestedpeptides were acidified to a final concentration of 1 % formic acid and 0.1% trifluoroacetic acid. This was immediately followed by desalting on 30 mg HLB columns (Waters) and then eluted with 1 mL of 50% acetonitrile (ACN) as previously described. To normalize protein quantification across batches 150 pL of elution was taken from all CSF samples and then combined to generate a pooled sample as previously described. This pooled sample was split with 850 pL each as global internal standards (GIS). All individual samples and the GIS standards were then dried using a speed vacuum. Six TMT batches were balanced for diagnosis, age, and sex using ARTS (automated randomization of multiple traits for study design). Using an 18-plex Tandem Mass Tag (TMT-pro) kit (ThermoFisher, Lot# UK297033 and WI336758), 17 channels were allocated for CSF samples with the remaining channel (126) containing a GIS pool.High pH Peptide Fractionation

[0113] Dried samples were re-suspended in high pH loading buffer (0.07% vol / vol NH4OH, 0.045% vol / vol FA, 2% vol / vol ACN) and loaded onto a Waters BEH column (2.1 mm x 150 mm with 1.7 pm particles). A Vanquish UPLC system (ThermoFisher Scientific) was used to carry out the fractionation. Solvent A consisted of 0.0175% (vol / vol) NH4OH, 0.01125% (vol / vol) FA, and 2% (vol / vol) ACN; solvent B consisted of 0.0175% (vol / vol) NH4OH, 0.01125% (vol / vol) FA, and 90% (vol / vol) ACN. The sample elution was performed over a 25 min gradient with a flow rate of 0.6 mL / min with a gradient from 0 to 50% solvent B. A total of 96 individual equal volume fractions were collected across the gradient. Fractions were concatenated to 48 fractions and dried to completeness using vacuum centrifugation.Mass Spectrometry Analysis and Data Acquisition

[0114] All samples (~ 1 pg for each fraction) were loaded and eluted by an Ultimate 3000 RSLCnano (Thermo Scientific) with an in-house packed 20 cm, 150 pm i.d. capillary column with 1.7 pm CSH (Waters) over a 22 min gradient. Mass spectrometry was performed with a high-field asymmetric waveform ion mobility spectrometry (FAIMS) Pro front-end equipped Orbitrap Eclipse (Thermo) in positive ion mode using data-dependent acquisition with 1.5 s top speed cycles for each FAIMS compensative voltage. Each cycle consisted of one full MS scan followed by as many MS / MS events that could fit within the given 1 s cycle time limit. MS scans were collected at a resolution of 120,000 (410-1600 m / z range, 4 x 105AGC, 50 ms maximum ion injection time, FAIMS compensative voltage of -45 and -65). Only precursors with charge states between 2 + and 6+ were selected for MS / MS. All higher energy collision-induced dissociation (HCD) MS / MS spectra were acquired at a resolution of 30,000 (0.7 m / z isolation width, 35% collision energy,1 x io5AGC target, 54 ms maximum ion time, turboTMT on). Dynamic exclusion was set to exclude previously sequenced peaks for 20 s within a 10-ppm isolation window.Database Search and Protein Quantification

[0115] Database searches and protein quantification was performed on 576 RAW files (96 RAW files / fractions per batch) using FragPipe (version 19.1). The FragPipe pipeline relies on MSFragger (version 3.7) for peptide identification, MSBooster, and Percolator for FDR filtering and downstream processing. MS / MS spectra were searched against all canonical Human proteins downloaded from Uniprot (20,402; accessed 02 / 11 / 2019), as well as common contaminants (51 total), and all reverse sequences (20,453). The workflow used in FragPipe followed default TMT-18 plex (i.e. , TMTpro) parameters. Briefly, precursor mass tolerance was - 20 to 20 ppm, fragment mass tolerance of 20 ppm, mass calibration and parameter optimization were selected, and isotope error was set to -1 / 0 / 1 / 2 / 3. Enzyme specificity was set to strict-trypsin with up to two missing trypsin cleavages allowed. Cleavage was set to semi Peptide length was allowed to range from 7 to 35 and peptide mass from either 200 to 5,000 Da. Variable modifications that were allowed in the search included: oxidation on methionine, N-terminal acetylation on protein and peptide, TMT labeling reagent modifications on serine, threonine, and histidine with a maximum of 3 variable modifications per peptide. The false discovery rate (FDR) threshold was set to 1%. A total of 49,762 peptides which mapped to 2,568 proteins were detected. After filtering out proteins that were absent in 50% or more of specimens 23,743 peptides and 2,105 proteins were retained. One dataset of Emory ALS peptides and one dataset of Emory ALS proteins as well as two other previously published datasets were used. The ALS dataset from Oh et al. (1831 proteins) and the Emory Alzheimer’s disease (disease specificity) dataset (2146 proteins) were retrieved and processed in FragPipe as above, for proteins only, with default parameters for 11 and 16 plex TMT, respectively. Oh et al. ALS and Emory Alzheimer’s peptides were not processed.Bioinformatics Processing and Statistical Analysis

[0116] A Tunable Approach for Median Polish of Ratio (TAMPOR) and removal of peptides or proteins absent in 50% of cases or greater was employed, as previously published. To ensure the reliability of this data, the analysis was initiated by identifying and removing potential outliers, although none were present. Parallelized ordinary, nonparametric, bootstrapping regression was then performed to remove variation due to age, sex, and batch effect using an established pipeline. Fast parallel one-way ANOVA with Benjamini-Hochberg correction for multiplecomparisons was conducted within each disease group using an in-house script to identify peptides and proteins that were differentially expressed. Differential abundance is presented as volcano plots, which were generated with the ggplot2 package.Protein Network Analysis

[0117] Weighted Gene Co-expression network analysis (WGCNA) was used to construct modules of co-expressing proteins as previously published. Briefly, the blockwiseModules function from the WGCNA package in R was utilized with the following parameters for Emory ALS CSF samples: soft threshold power beta = 4, deepSplit = 4, minimum module size = 15, merge cut height = 0.07, and a signed network with partitioning around medoids (mapping a distance matrix to k clusters, where K is data-adaptively selected). Module correlation to disease type was evaluated with Biweight midcorrelation (BiCor) analysis. A Fisher’s exact test (FET) was performed for each module member against the merged human cell type marker list to determine cell type enrichment using an in-house script. Similarly, to determine module gene ontology (GO) a FET was performed for each module member against the Bader Lab’s GMT formatted ontology lists from February 8, 2023. One way ANOVA was conducted across disease type for each module eigenprotein.

[0118] Module preservation of the Emory ALS dataset in that from Oh et al. was used to assess datasets collectively using the modulepreservation function of WGCNA with 500 permutations. Synthetic module eigenproteins were constructed using a consensus network template, of Emory ALS and Oh et al. ALS datasets, with a kMEintramodule. Among the top 20th percentile of kMEs, a synthetic module composed of at least 4 members, was curated and synthetic module eigenprotiens were calculate using the moduleEigengenes function of WGCNA as in.Example 2: Results for Diagnostic ApplicationsExperimental Workflow Identified Differentially Expressed Proteins Across Disease Subgroups

[0119] CSF proteomes from sALS (n=35), C9orf72 ALS (n=10), C9orf72 asymptomatic carriers (n=6), SOD1 ALS (n=6), and healthy controls (n=44) were compared with the goals of discovering differences between ALS and control, and also protein signatures that may differentiate the ALS groups (FIG. 1A). Following QC, TMT-MS proteomic analysis 2,105 proteins across all samples. Protein abundance was adjusted for batch, age, and sex. As expected, protein levels of neurofilaments (NEFM and NEFL) were increased in both sporadic and genetic ALS samples compared to controls, consistent with neurodegeneration. Furthermore, an increase in chitinases(CHIT1) and CHI3L1 linked to inflammation and previously shown to increase in ALS was observed.

[0120] Comparison of CSF samples between sporadic and C9orf72 ALS demonstrated increases in many of the same proteins as compared to controls (FIGs. 1B-1C). These increased proteins included proteins with axonal regeneration ontology as well as proteins with metabolic pathways associated with L-ascorbic acid, cellular ketone, and cellular aldehyde. Proteins related to chemical synaptic transmission and assembly of postsynaptic elements in C9orf72 ALS CSF were significantly decreased in abundance relative to sALS.

[0121] Analysis of CSF from asymptomatic C9orf72 carriers found 251 proteins that were differentially abundant from controls (FIG. 1 D). In comparison to those with C9orf72 ALS, asymptomatic mutation carriers showed reduced abundance of proteins related to amino sugar catabolism and keratin binding (FIG. 1E). Conversely, C9orf72 mutation carriers with ALS showed reduced abundance of proteins associated with keratinization, keratinocyte differentiation, and intermediate filament organization as compared to asymptomatic C9orf72 carriers. Many of the protein differences become more significant in symptomatic C9orf72 ALS suggesting that the C9orf72 repeat expansion induces protein change in pre-symptomatic stages of disease progression.

[0122] SOD1 protein was surprisingly decreased in abundance in CSF from SOD1 patients compared to all other forms of ALS as well as compared to controls (FIG. 2A). Total SOD1 was characterized to confirm the decreased abundance observed in FIG. 2A. Total SOD1 was decreased in abundance across all mutation groups, with A5T patients demonstrating the lowest levels of SOD1 (FIG. 2B). Fully-tryptic peptides from all 6 individuals covered 64.9% of the SOD1 protein (FIG. 2C). In all but one peptide, a significant difference in abundance was present and in all but one SOD1 was the lowest average abundance. Patients with mutation A5T consistently had the lowest peptide abundance (FIG. 2D). In addition, several histones were also decreased in abundance in SOD1 ALS patients, a difference also identified in C9orf72 mutation carriers. In summary, the protein markers identified are associated with each form of ALS, genetic versus sporadic causes of ALS, as well as proteins that may indicate a shift from symptomatic to asymptomatic disease states.Network Analysis of the ALS CSF Proteome Reveals Modules Related to Pathways, Brain Cell- Types, and Genetic Background

[0123] Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify modules or ‘communities’ of proteins that are highly correlated with across CSF samples. These modules in CSF reflect various biological functions linked to brain cell types and ontologies. Using the first principal component of all proteins in a module, or the module eigenprotein, the abundance of each module can be related to disease phenotypes.

[0124] Here, the network modules ranged from 476 (M1) to 29 (M12) member proteins with 409 proteins not nesting into a module. The 12 network modules generally divided into three branches of relatedness allowing inference of which modules were most similar (FIG. 3A). Correlation between specific disease cohorts was compared to network module co-expression to determine that five modules were associated with C9orf72, three with SOD1 , and two with sALS, with overlap in module identity. Module membership and proteins associated with specific brain cell-types were also compared for overlap in order to ascertain overarching trends. Three modules had significant overlap with neurons (M1 , M4, and M12) and microglia (M7, M8, and M9), two with oligodendrocytes (M1 and M4), and one with endothelia (M2; FIG. 3B). Modules showing a high degree of correlation did not necessarily have more overlap with proteins associated with cell type. Specifically, M1 and M4 were significantly enriched with neuronal and oligodendrocyte associated proteins but were more closely correlated to modules nine and 12; respectively (FIG. 3C). The three largest modules corresponded to M1- Neuronal, M2-Complement activation enriched with markers specific to endothelia, and M3-Adaptive immune response ontologies (FIG. 3D). Smaller modules were associated with M4-Neuron development, M5-Extracellular matrix / Heparin binding, M6-Lysosomal / Vesicle, M7-Cytoskeleton / Microglial, M8-lnflammatory Response, M9-Lysosomal, M10-Ubiquitination / Gluconeogenesis, M11-Postsynaptic membrane / Signaling, and M12-Nervous system development. Modules associated with MS- Extracellular matrix / Heparin binding (p=0.0084), M7-Cytoskeleton / Microglial (p=0.025), and M10- Ubiquitination / Gluconeogenesis (p=2.4 x w7) varied significantly among control and ALS groups (FIG. 4A). To reinforce these findings, most of the increased differentially abundant proteins (DAPs) in ALS cases irrespective of genetic cause mapped to M7, M5 and M10, whereas decreased DAPs in ALS were distributed to M4, M11 , and M12 (FIG. 4B). DAPs in C9orf72 patients, which were increased in abundance, also contributed to these modules, with significantly lower abundance proteins belonging to modules associated with M4-Neuron development and M12-Nervous system development. Proteins increased in abundance in asymptomatic C9orf72 cases were more commonly clustered into the module associated with M5-Extracellular matrix / Heparin binding and proteins that were significantly increased in C9orf72 ALS relative tosporadic cases, including neurofilaments, chitinases, and deubiquitinase belonged to the module associated with M10-Ubiquitination / Gluconeogenesis (FIG. 4B). Module overarching ontologies and correlation helped identify broad protein functions more closely associated with C9orf72 (MS- Extracellular matrix / Heparin binding) and SOD1 (M8-lnflammatory Response) as well as the degree to which these functions overlapped (i.e. all ALS subgroups correlating to M7- Cytoskeleton / Microglial and M10-Ubiquitination / Gluconeogenesis). Overall, network analysis effectively organizes the CSF proteome into protein modules that are strongly linked to hallmark ALS biomarkers (NEFL).Validation of Protein Abundance Changes in an Independent Cohort of CSF

[0125] To assess the consistently of these findings in the Emory ALS CSF, a TMT-MS dataset from a second independent ALS CSF proteomic dataset ALS CSF proteome was analyzed, which allowed comparison of the findings of this analysis of individual proteins as well as co-expressing modules in an independent cohort Hereafter the Oh et al. ALS dataset will be referred to as “Set 2”. The aim was to view these data collectively, (Set1 is TMT18 and Set 2 ALS is TMT 11), Set 1 proteomes were compared to Set 2 proteomes at both the protein and module level outlined in FIG. 5A. Similarity in proteins identified by each dataset as well as the structure of the network modules in Set 2 were compared.

[0126] The re-analysis of the independent Set 2 dataset differential abundance between ALS and control subjects closely resembled those presented elsewhere (FIG. 5B). Of the 2,105 and 1 ,831 proteins identified from Set 1 and Set 2 proteomes, respectively, 78.2 and 90.0% of those proteins were shared (FIG. 5C). The Emory ALS proteome included 458 proteins not found in the published Set 2 whereas the Oh dataset included 184 proteins not found in Emory. All modules from Emory ALS proteome were found to be preserved in Oh et al. ALS proteome (FIG. 5D). Modules associated with M1-Neuronal, M2-Complement activation, M3-Adaptive immune response, M4- Neuron development, M5-Extracellular matrix / Heparin binding, M6-Lysosomal / Vesicle, and M7- Cytoskeleton / Microglial had Zsummary values over 10 (i.e. q=1 x 10'23) corresponding to being well preserved. Modules associated with M9-Lysosomal, M10-Ubiquitination / Gluconeogenesis, M11-Postsynaptic membrane / signaling, and M12-Nervous system development had Zsummary values over 2 (q=0.05) but less than 10 indicating preserved. Module eigenproteins and synthetic module eigenproteins (Emory ALS module first principal component in Set 2) were concordant in 9 modules, with three modules showing increase in Set 2 but decrease in Set 1 (FIG. 5E). In all cases discordance was minimal. Several module eigenprotein, synthetic eigenproteincomparisons were of particular interest, including the modules that were significantly different across disease and control in Set 1 as well as an addition module with M4-Neuron development ontology that was not significantly different in Set 1 but was significantly different in Set 2 (FIG. 5F).

[0127] Synthetic eigenproteins were compared across Set 1 and Set 2 proteomes (FIG. 7). Synthetic eigenproteins associated with M1-Neuronal, M2-Complement activation, M4-Neuron development, M11-Postsynaptic membrane / signaling, and M12-Nervous system development were found to be significantly different across disease (ALS versus Control) for Oh et al. ALS proteome. While the datasets largely recapitulate, in distinct datasets from different centers, the lack of genomic traits in Set 2 prevents further inference.ALS-Specific Proteomic Changes Compared to Alzheimer’s Disease

[0128] To assess the specificity of the changes for ALS versus other neurodegenerative diseases, data from 149 controls and 149 AD samples in an additional Emory dataset were analyzed. Differentially expressed proteins were compared across Set 1 , Set 2, and Alzheimer’s Disease CSF datasets to determine which differentially expressed proteins are associated with neurodegenerative disease in general and which proteins were associated with ALS or AD (FIGs. 6A-6F). Three different datasets from varying TMT types and different centers (Emory and Johns Hopkins) (FIG. 6A) composed a total of 220 disease cases and 213 control individuals.

[0129] Across Emory sALS-Control and Emory AD-Control 1 ,130 proteins were concordant, and 739 proteins were discordant (FIG. 6B). The top 10 hub proteins were compared for significance across all three data sets with three found in common for all datasets and two specific to Emory ALS and Oh et al. ALS (FIG. 6C). Several proteins were found to be significantly different from controls in the same direction in all three proteomes including NEFL, NEFM, UCHL1, CHIT1 , and CHI3L1 were thus characterized as general neurodegeneration proteins (FIG. 6D). In total two proteins from module hubs were significantly different from controls in the same direction in Emory ALS and Oh et al. ALS proteomes and were either not significantly different from controls or not detected in the disease specificity proteome. In addition, 14 proteins from modules representing M5-Extracellular matrix / Heparin binding, M7-Cytoskeleton / Microglial, and M10- Ubiquitination / Gluconeogenesis varied significantly, in the same direction, in Emory ALS and Oh et al. ALS but did not vary significantly or varied significantly in the opposite direction in disease specificity. Of these proteins, 10 representative proteins are visualized (FIG. 6D). Similarly, several proteins associated with AD were assessed in all three datasets including SMOC1 , MDK,CTHRC1 , SP0N1, SP0CK2, and 0LFML3. Four general neurodegeneration proteins NEFL, NEFM, CHIT1 , and CHI3L1 were visualized to convey their significance across Emory ALS, Oh et al. ALS, and Emory AD; respectively (FIG. 6E). Four ALS-specific proteins IGF2, RARRES2, LGALS3, and LYZ were also visualized to demonstrate significance in Emory ALS and Oh et al. ALS proteomes but not Emory AD proteome (FIG. 6F). These differential abundances indicate that in addition to markers of neurodegeneration that are consistent across ALS and AD there are also differences associated with one disease or the other which may aid in elucidating disease mechanisms or targets for intervention.Example 3: Discussion for Diagnostic Applications

[0130] In this study, the CSF proteome of several groups of ALS patients defined by genetic status and clinical diagnosis was characterized in order to identify shared and unique biomarkers across each subgroup. By examining coordinated protein co-expression across genetic and sporadic forms of ALS, shared and group-specific changes linked to gene ontology and cell-type function in the proteome were identified. sALS, C9orf72 ALS, C9orf72 asymptomatic carriers, SOD1 ALS, and healthy controls were compared to identify biomarkers associated with each ALS type. This analysis validated previously identified ALS associated CSF biomarkers but importantly, identified novel ALS biomarkers specific to genetic status as well as the onset of clinical symptoms.

[0131] Similar to previous studies, a significant increase in the abundance of neurofilament proteins (e.g. NEFL, NEFM) and chitinase associated proteins (e.g. CHIT1 , CHI3L1 , CHI3L2) was found across all clinically diagnosed ALS cohorts. Increased abundance of neurofilament proteins in the CSF likely reflects the axonal degeneration of neuronal populations in the central nervous system (CNS) as these proteins are upregulated in the CSF of other neurodegenerative diseases including AD and frontotemporal lobar dementia (FTLD). The functional consequence of increased abundance of CHIT1 and chitosan-like proteins (e.g. CHI3L1 , CHI3L2) are unknown, these proteins have been shown to co-localize with markers of microglia and macrophages, thus are likely associated with microglial and / or macrophage activation. Increased abundance of other proteins associated with microglial activation was also observed, including GPNMB and LYZ. While increased GPNMB abundance has been previously reported in ALS CSF proteomes, the presence of LYZ has not been documented. However, both GPNMB and LYZ have been demonstrated to be upregulated at the transcript level in the spinal cord of ALS patients.

[0132] Unique to this study is the assessment of asymptomatic C9orf72 repeat expansion carriers. There is a strong age-related penetrance associated with C9orf72 ALS / FTD thus, inclusion of asymptomatic C9orf72 carriers provides an early view into pre-symptomatic stages of disease. Notably, established biomarkers discussed above that are commonly associated with neurodegeneration (NEFL and NEFM) and microglial activation (CHIT1 , GPNMB, LYZ) were unchanged in asymptomatic C9orf72 carriers compared to controls. These data suggest that the observation of proteins associated with axonal degeneration and microglia activation in ALS cases are reflective of the presence of clinical symptoms. The increased abundance of protein markers thought to reflect neurodegeneration (NEFL and NEFM), only in the symptomatic C9orf72 carriers, provides internal validation of specificity of the proteomic findings. The discovery of differences in protein abundance between C9orf72 carriers with and without ALS is particularly important, as they may identify prognostic markers of disease (such as AKR1 B1 , TKT, CFL1 , CFL2, PPIA and FABP3). A shift to a pattern seen in symptomatic C9orf72 ALS could represent a precursor of imminent transition to symptomatic disease.

[0133] Interestingly, Lactate dehydrogenase A chain (LDHA), which was observed to be upregulated in the CSF of symptomatic ALS cases and has previously been shown to be increased in the CSF of other neurodegenerative diseases, including AD, is also significantly increased in the CSF of asymptomatic C9orf72 carriers. LDHA is a glycolytic protein involved in the concomitant interconversion of pyruvate with lactate and NADH with NAD+. The robust increase of LDHA abundance in the CSF of both asymptomatic C9orf72 carriers and symptomatic ALS patients suggest that shifts in energy metabolism from oxidative phosphorylation to glycolysis (i.e. The Warburg effect) is an early cellular change associated with ALS prior to clinical onset.

[0134] Importantly, several proteins were identified that were robustly increased in the CSF of asymptomatic C9orf72 carriers but not C9orf72 ALS patients including EMILIN1 and DNAJC3. Knockout experiments in mice and culture have shown that EM I LI N 1 plays a role in elastogenesis and maintenance of vascular cells. These findings were confirmed in bi-allelic, loss of function for EMILIN1 families, demonstrating that not only does EM I LI N 1 assist EFEMP in elastogenesis, but also affects lysyl oxidase by EFEMP2 to establish collagen crosslinks. Similarly, an investigation into siblings that demonstrated loss of DNAJC3 has been shown to result in diabetes mellitus as well as “multisystemic neurodegeneration.” DNAJ / HSP40 proteins are also known to regulate HSP70 co-chaperones through stimulation of ATP hydrolysis.

[0135] Differential changes of neurodegenerative proteins in the CSF proteomes stratified by disease subgroup were also observed. For example, decreased levels of some keratins were found, similar to observations in CSF of asymptomatic AD versus AD. Intermediate filaments (like keratins and neurofilaments) have previously been implicated in toxic dipeptide repeats associated with C9orf72-associated ALS, with repeats promoting a high-density network of intermediate filaments in the cytoplasm. This would suggest that dense networks of irregular intermediate filaments in the cytoplasm may have implications on phase separation of mutant TDP-43. Previous work has shown that keratins are significantly enriched in the cerebrovascular fraction of human brain and may be derived from smooth muscle cells, suggesting a role of cerebrovascular dysfunction in C9orf72-associated ALS potentially linked to TDP-43 pathobiology. In support of this hypothesis, keratins are not significantly decreased in abundance in SOD1 ALS, which does not have TDP-43 inclusions as a pathological hallmark.

[0136] Histone proteins (HIST1 H1B, HIST1 H4A, HIST1 H2AB, HIST1 H1 E, H2AFZ, and HIST2H2BF) were decreased in abundance relative to controls in genetic forms of ALS but not sporadic, which may provide insight to differences in disease mechanisms. It may be that decreased abundance of histone proteins in genetic forms of ALS may be due to decreased histone deacetylases, which were not detected in CSF in this study. There are 18 histone deacetylases (HDACs) in humans that deacetylate histone and non-histone proteins. Decreased histone deacetylation has been linked to neurodegeneration and HDAC abundance has downstream effects on HSP70 as well as TDP-43 deacetylation and aggregation. Mutations in FUS (including the 521C mutation) cause a preferential interaction between FUS and RBM45, rather than HDAC1 . Histone acetylation / deacetylaces may present several druggable targets.

[0137] Though TDP-43 pathology is a neuropathological hallmark of sporadic and C9orf72 associated ALS, TDP-43 was not identified in any CSF samples. Similarly, cryptic peptides that are associated with TDP-43 dysfunction or the inclusion of cryptic exons were also not identified. Markers of TDP-43 pathology are not expected in SOD1-related ALS, however the present systems biology approach identified a module (i.e., M8-lnflammatory response) as associated with SOD1 as well as other protein markers and modules. Total SOD1 protein was decreased in SOD1 ALS relative to controls and to the other patient groups. A recent study found that mutant SOD1 was 16-fold lower in concentration in CSF than wildtype SOD1 and that the turnover of mutant SOD1 was two times faster. In conjunction with the finding that Tofersen lowered SOD1 by 30%, these results demonstrate the need to better understand how these concentrations are allocated amongst the myriad known SOD1 mutations and peptides. These results thereforeindicate that the underlying pathogenic differences between different genetic forms of ALS is reflected in the CSF proteome and is consistent with differences observed in the neuropathology and clinical course of these subgroups.

[0138] There are several shortcomings to this study that should be addressed in future analyses. Additional representation in ALS cohorts is necessary to confirm that biomarkers (such as NEFL, CHIT1 , and recently elucidated novel biomarkers) are useful across diverse populations. Similarly, diverse cohorts may provide additional insight to ALS biology through a more complete view of how ALS affects patients. A larger sample size of genetic forms of ALS, the addition of asymptomatic cases, and the inclusion of other genetic forms of ALS (in particular cases associated with the RNA binding protein fused in sarcoma [FUS]) would address further unmet needs in future studies. In addition, the current dataset represents a snapshot of the biological processes at work in ALS and longitudinal datasets such as, as well as longitudinal data characterized with the depth provided with TMT-MS will help to better reveal these processes. The identification of additional ALS biomarkers will inform future efforts to identify disease onset and track ALS progression.

[0139] Numerous peptides were detected for each protein; these are shown in FIG. 2C for SOD1 and Table 1 for NEFL, NEFM, CHIT1 , CHI3L1, CHI3L2, and UCHLI. As expected, neurofilament proteins (NEFM and NEFL) were elevated in both sporadic and genetic ALS subtypes compared to controls, consistent with ongoing neurodegeneration. For instance, NEFL and NEFM levels were 2.79-fold and 2.21 -fold higher, respectively, in C9 ALS CSF compared to controls.

[0140] The present investigation identified protein biomarkers that may distinguish the transition from asymptomatic to symptomatic phase and differences between genetic and sporadic forms that indicate a difference in mechanisms of disease. External and disease specific datasets were incorporated to validate the present proteome and distinguish ALS specific pathways. Future analyses should utilize these differences to determine their applicability to diverse ALS cohorts and identify whether these biomarkers are consistent longitudinally.Example 4: Results for Therapeutic ApplicationsFluid biomarkers are variable in c9ALS ASO treated patients

[0141] The present study includes eight c9ALS patients treated with BIIB078 ASO; four cases had matched CSF and post-mortem tissue, two cases had only post-mortem tissue, and two cases had only CSF. Drug-naive c9ALS (n=31) and non-ALS cases (n=32) were included in theanalyses as controls. Demographic, dosing, and clinical characteristics of BIIB078 treated cases are summarized in Table 3. For the six cases with longitudinal CSF collected at each dosing interval, the abundance of soluble poly(GP), a pharmacodynamic biomarker of target engagement for G4C2 targeting therapeutic strategies including ASOs, and neurofilament light chain (NfL), a prognostic biomarker of ALS (34) and a potential biomarker of therapeutic response (FIGs. BABB) were measured. In five of six cases, a reduction in poly(GP) was observed after BIIB078 treatment (avg. = -37.2%), with considerable variation in the relative abundance of poly(GP) at each timepoint (FIG. 8A). Only one case, Case #6, had a robust, monotonic reduction in poly(GP) abundance in the CSF (-77.4%). Near-contemporaneously recorded measurements of neurological function (ALSFRS-R) showed a strong decline in scores, indicating disease progression, during BIIB078 treatment (FIGs. 8A, 8C). Measurements of NfL abundance in CSF collected immediately prior to administrating the first BIIB078 dose and following the last BIIB078 dose (FIG. 8D) also demonstrated variability among cases. Four cases showed an increase in NfL and two cases a decrease in NfL (FIG. 8D).Intrathecally delivered BIIB078 is broadly distributed throughout the CNS parenchyma

[0142] The observation that soluble poly(GP) was decreased in the CSF of some Bl I B078- treated c9ALS patients suggests that BIIB078 is internalized into the CNS parenchyma. Thus, using postmortem tissues from c9ALS patients treated with BIIB078, it was investigated whether BIIB078 was detectable in the CNS parenchyma. Immunohistochemical (IHC) analysis using an established polyclonal antibody that recognizes phosphorothioate (PS) linkages, a commonly used backbone modification to improve pharmacological properties of ASOs including BIIB078, revealed positive staining in the spinal cord of BIIB078-treated c9ALS cases (FIG. 9A). ASO-PS staining was more robust and widely distributed in the spinal cord of cases with a shorter interval between last BIIB078 dose and autopsy. Positive ASO-PS staining was observed throughout thelumbar spinal cord including the grey matter, white matter tracts, and neuropil. Based on morphological assessment of cell types, ASO-PS staining was predominately detected in vascular and glial cells; notably, IHC detection was absent from motor neurons of the ventral horn.

[0143] To validate and extend these IHC findings, an orthogonal RNA in situ hybridization (ISH) approach was employed to assess ASO distribution. A custom miRNAscope (ACDBio) probe was designed to specifically detect the BIIB078 sequence. Compared to ASO-PS IHC staining, miRNAscope-based ISH revealed a more robust ASO distribution throughout the spinal cord, and the ASO was detected in all cases irrespective of dose or interval between last dose and autopsy (FIG. 9A). As with ASO-PS staining, however, there was a noticeable reduction in BIIB078 detection as the interval between last dose and autopsy increased. BIIB078 was observed in both white matter tracts of the dorsal, ventral, and lateral columns as well as the gray matter dorsal and ventral horns. Internalization of BIIB078 was detected in both the cytoplasm and nucleus of all cell types in the spinal cord including motor neurons. Given the increased sensitivity and specificity of the BIIB078-targeting miRNAscope ISH probe, this approach was used to investigate BIIB078 distribution throughout the CNS. Broad distribution of BIIB078 was observed throughout subarachnoid brain regions including the motor, frontal, and cerebellar cortices (FIGs. 9B-9D) as well as periventricular regions including the hippocampus, basal ganglia, and medulla (FIGs. 9E- 9G). As observed in the spinal cord, there was a reduction in BIIB078 signal across brain regions in cases with a longer interval between last dose and autopsy. In the motor and frontal cortex, BIIB078 was most strongly localized to the arachnoid and pial lining along sulco-gyral patterns, but also BIIB078 penetrated both superficial and deep cortical laminae including the internal pyramidal (layer 5) and multiform (layer 6) layers; it was less abundant in white matter (FIGs. 9B- 9C). In cases with a shorter interval between last dose and autopsy (e.g. Case #1; 17 days), BIIB078 was detectable in both glial and neuronal cells as well as the neuropil. In cases with a longer interval between last dose and autopsy (e.g. Case #6; 455 days), BIIB078 was noticeably less abundant throughout cortical layers, but strong BIIB078 ISH signal was observed in cells surrounding Virchow-Robin spaces. BIIB078 was abundant in the granule, Purkinje, and molecular layers of the cerebellum (FIG. 9D). In periventricular regions robust BIIB078 signal was observed in regions abutting the respective ventricular space (FIGs. 9E-9G).

[0144] To further quantify BIIB078 distribution in CNS tissues, a plate-based oligonucleotide electro-chemiluminescent (POE) immunoassay was adapted to measure the concentration of BIIB078 ASO in tissue. With this method BIIB078 was detected in the spinal cord and motor cortex of Bl I B078- treated c9ALS cases but not drug-naive c9ALS or control cases (FIGs. 10A-10B).BIIB078 concentrations in motor and frontal cortex, cerebellum, hippocampus, caudate, and medulla were normalized to the concentration of BIIB078 in the spinal cord for each case. It was found that periventricular regions had reduced BIIB078 ASO concentrations whereas subarachnoid regions had considerable variation in relative BIIB078 ASO concentration (FIG. 10C). Correlation of BIIB078 concentration with several study dosing parameters revealed a positive correlation with the dose administered (mg) at last visit and a negative correlation with interval between last dose and autopsy in several brain regions (FIG. 10D). Together, these data provided compelling evidence that intrathecally administered BIIB078 ASO results in broad distribution throughout the CNS, internalizes in both superficial and internal cell populations, and persists in CNS parenchyma for extended time periods.C9orf72 transcripts are moderately reduced in BIIB078 ASO-treated cases

[0145] Three predominant transcript variants are produced by C9orf72. The G4C2 repeat expansion is positioned between two alternative transcription start sites demarcated by exon 1a and exon 1b. Transcript variant 1 (V1 ; NM_14005.6) and variant 3 (V3; NM_001256054.2) are transcribed from exon 1a and the G4C2 repeat is positioned in the first intron; variant 2 (V2; NM_018325.5), the most abundant C9orf72 transcript, is transcribed from the downstream exon 1b. BIIB078 ASO was designed to selectively target intronic G4C2 repeat containing transcript variants, V1 and V3. Therefore, it was next determined if C9orf72 transcripts were reduced in Bl I B078- treated c9ALS cases. Quantitative PCR (qPCR) was next performed using Taqman primer sets designed to detect total C9orf72, C9orf72 V1 , or C9orf72 V3 in the spinal cord of control, drug-naive c9ALS, and BIIB078-treated c9ALS cases (FIG. 11 A). No difference was observed in total C9orf72 expression among control, drug-naive c9ALS, and BIIB078-treated c9ALS cases, although there was a trend towards lower expression in both drug-naive and Bl I B078- treated c9ALS cases compared to controls. Additionally, differences in C9orf72 V1 and V3 expression between drug-naive c9ALS and control cases were not found, but there was a reduction in C9orf72 V3 expression in the BIIB078-treated cases. Notably, three BIIB078-treated c9ALS cases had the lowest C9orf72 V1 and V3 expression of all cases measured (FIG. 11 A) and these three cases had the highest concentration of BIIB078 ASO and the shortest interval between last dose and autopsy (17, 27, 92 days). C9orf72 V1 and V3, but not total C9orf72 expression in the treated cases, correlated with BIIB078 concentration in the spinal cord (FIG. 11B). Similarly, there was a moderate correlation between C9orf72 V3 but not total C9orf72 expression in the motor cortex of BIIB078-treated c9ALS cases compared to drug-naive c9ALS and non-ALS controls. To directly assess intronic G4C2-containing RNA transcript abundance, acustom Nanostring probe designed to recognize intron 1 upstream of the G4C2 repeat was used. An increase in intronic G4C2-containing transcripts in drug-naive c9ALS cases was observed compared to non-ALS controls, and there was a trend towards lower intronic G4C2-containing transcript expression in Bl I B078- treated cases. A custom Nanostring probe was also used to assess the C4G2-containing antisense transcripts. An increase in antisense expression in c9ALS cases was observed compared to non-ALS controls but no difference between drug-naive and Bl I B078- treated c9ALS cases. These results revealed that C9orf72 transcripts V1 and V3 are decreased in some BIIB078-treated c9ALS cases compared to drug-naive c9ALS cases and demonstrate target engagement of BIIB078 in cases with higher levels of BIIB078 or short intervals between dose and autopsy.Dipeptide repeat proteins persist throughout the CNS in BIIB078 ASO-treated c9ALS patients

[0146] To determine whether BIIB078 treatment reduced DPRs, it was assessed whether poly(GP) and poly(GA) were present in several disease-relevant CNS brain regions. Poly(GP) and poly(GA) inclusions were detected in the spinal cord, motor cortex, and frontal cortex of both drug-naive and BIIB078-treated c9ALS cases (FIGs. 12A-12B). In the spinal cord, poly(GP) and poly(GA) immunostaining revealed perinuclear, cytoplasmic inclusions in spinal motor neurons of the ventral horn, though rare inclusions in neurons of the dorsal horn as well as glial cells were also observed. In the motor and frontal cortices, poly(GP) and poly(GA) inclusions were predominately detected in layer 2 and layer 5; occasional diffuse poly(GP)-immunopositive neurons were detected. Using established DPR immunoassays, poly(GP) and poly(GA) abundance were quantified between drug-naive and BIIB078-treated c9ALS cases (Fig 5C, D). The abundance of poly(GP) in BIIB078-treated c9ALS cases was indistinguishable from drug- naive c9ALS cases in all regions tested except for spinal cord where poly(GP) abundance in the spinal cord of BIIB078-treated c9ALS cases was lower compared to drug-naive c9ALS cases (FIG. 12C). Poly(GA) abundance in the motor and frontal cortices of BIIB078-treated cases was indistinguishable from drug-naTve c9ALS cases (FIG. 12D); poly(GA) was not detectable by immunoassay in the spinal cord of c9ALS cases. Additionally, dual IHC staining demonstrated that poly(GA) inclusions persist in cells with internalized BIIB078 ASO (FIG. 12E). These findings provide evidence that BIIB078 ASO treatment does not result in robust reduction of DPRs.Abundant TDP-43 pathology is observed in BIIB078-treated c9ALS patients

[0147] The mislocalization, hyperphosphorylation and aggregation of the RNA binding protein, TDP-43, is a characteristic pathology of several neurodegenerative disease including ALS andfrontotemporal lobar degeneration (FTLD) with TDP-43 pathology. Thus, it was assessed whether TDP-43 pathology was different in BIIB078-treated c9ALS cases compared to drug-naTve c9ALS cases. Quantitative immunoassays were performed to measure the abundance of phosphorylated TDP-43 (pTDP-43) in brain regions associated with neurodegeneration in C9ALS / FTD cases including the spinal cord, motor cortex, and frontal cortex. Phosphorylated TDP-43 was absent in controls and present in c9ALS cases treated or not with BIIB078, but no difference was observed in pTDP-43 abundance between drug-naive and BIIB078-treated cases (FIG. 13A). To support these findings, truncated STMN2 was also quantified (tSTMN2) expression, a surrogate measure for loss of nuclear TDP-43 function, in these same brain regions. It was found that tSTMN2 was detectable in both c9ALS groups compared to controls, but tSTMN2 expression was not different between drug-naive and BIIB078-treated cases. These data demonstrate that BIIB078 treatment did not reduce TDP-43 pathology.Example 5: Discussion for Therapeutic Applications

[0148] Herein it is demonstrated that intrathecal administration of BIIB078 results in widespread distribution of this ASO throughout the CNS. Importantly, BIIB078 was detected in the spinal cord and motor cortex, regions primarily affected in ALS. The distribution of BIIB078 is dependent on CSF circulation, and thus BIIB078 was also detected in subarachnoid and periventricular brain regions. Given that the primary site of CSF secretion is in the choroid plexus of the ventricles where it ultimately flows out into the subarachnoid space, it was interesting to note that BIIB078 was detectable in periventricular brain regions even in cases with short intervals between dosing and autopsy (e.g. Case #1), suggesting that intrathecally administered ASOs can diffuse against outward CSF flow into the ventricles. BIIB078 was detectable in spinal cord and several brain regions in all cases, even though the time interval between last dose and autopsy varied (17 - 455 days), demonstrating that BIIB078 persists in tissue long after cessation of dosing. The described quantitative approach to measuring BIIB078 in tissue also allowed for correlation of ASO abundance with various dosing parameters. As expected, it was found that ASO abundance in tissue was most strongly correlated with dose level (mg) and the time interval between last dose and autopsy.

[0149] As a marker of target engagement, DPR abundance was measured in tissue and CSF of Bl I B078- treated patients. In 5 of the 6 cases with available CSF, CSF poly(GP) was lower at the final measurement when compared to the baseline measurement, indicating that BIIB078 successfully targeted the G4C2 repeat (FIG. 8B). In these 5 cases, poly(GP) abundancefluctuated above or below baseline by as much as -26%, and this variability in poly(GP) was even seen in c9ALS cases treated with placebo prior to transitioning to active drug during the open label extension (Cases #4 and #8). It is important to note that time intervals between poly(GP) measurements were relatively short, ranging from every 2 weeks to 4 weeks, which may have contributed to the observed fluctuations in poly(GP) abundance. In comparison to the findings in CSF, the abundance of poly(GP) and poly(GA) in CNS tissues were not robustly different between Bl I B078- treated and drug-naive c9ALS cases. Only in the spinal cord did poly(GP) show reduced abundance compared to the drug-naive c9ALS group, which may be related to proximity of dosing to spinal cord tissue. This discrepancy between CSF and tissue measurements implies a less than direct relationship between this fluid biomarker of target engagement and global CNS pathology.

[0150] The detection and internalization of BIIB078 ASO in the CNS parenchyma coupled with an overall, but variable, reduction in CSF poly(GP) abundance suggests target engagement of BIIB078 with G4C2 repeat-containing transcripts. This was explicitly evaluated by measuring G4C2 repeat-containing transcript expression and translated DPR abundance. In the spinal cord or motor cortex, a difference in the expression of either total or variant-specific C9orf72 transcripts was not observed between BIIB078-treated and drug-naive c9ALS cases; however, three Bl I B078- treated c9ALS cases had considerably low C9orf72 transcript V1 and V3 expression across all samples measured. A strong correlation was observed between C9orf72 transcript V1 and V3 expression and BIIB078 concentration. Of note, these three cases had the highest concentration of BIIB078 and the shortest interval between last dose and autopsy.

[0151] While these findings, in aggregate, demonstrate broad distribution of BIIB078 in the CNS of c9ALS patients, and provide some evidence of target engagement, it is unclear why there was not a more consistent and robust reduction in targeting G4C2 repeat-containing transcripts. Since the three cases (Cases #1-3) with lowest C9orf72 V1 and V3 expression were the three BIIB078- treated cases with highest ASO concentrations, it is possible that a critical BIIB078 concentration is needed for robust target engagement. Countering this hypothesis is the fact that a similar trend between C9orf72 V3 expression and BIIB078 concentration was observed in the motor cortex of the same three patients, even though the motor cortex had significantly lower BIIB078 concentration as compared to spinal cord. Moreover, the fact that BIIB078 is robustly detected in tissue by probe-based hybridization assays (e.g. ISH) suggests that the BIIB078 ASO was not degraded by RNase H and is in excess in tissue. Interestingly, these three cases also had the shortest interval between last dose and autopsy, which raises the possibility that internalizedBIIB078 can initially engage with G4C2 repeat-containing transcripts but, perhaps due to nonspecific interactions with other proteins, are prevented from further engaging its respective target.

[0152] Although clinical-pathological correlations in this study were limited by the small number of patients, there were no observable correlations between CSF poly(GP) and either ALSFRS-R or CSF neurofilament proteins, data that are consistent with prior findings. As an example, one patient (Case #6) who showed the most pronounced monotonic reduction in poly(GP) in response to BIIB078 treatment (77.6% reduction) did not show a parallel response to change in ALSFRS- R and also showed an increase in CSF neurofilament. Despite a remarkable reduction of poly(GP) in the CSF, Case #6 had the lowest BIIB078 concentration at autopsy, though it is important to consider that the long interval between last CSF poly(GP) measurement and poly(GP) measurement at autopsy in this case could contribute to this discrepancy. The other cases are less informative, but again suggest that robust delivery of BIIB078 to CNS tissue and reduction of CSF poly(GP) does not correlate with a reduction of DPRs in tissue nor with a positive clinical response.

[0153] An important limitation of this study is that C9orf72 expression and DPR abundance were not able to be assessed before and after BIIB078 treatment, as only post-mortem tissue was available. However, the diverse sample cohort collected for this study with respect to different dosing schemes, total dose administered, and time interval between last dose and autopsy, provided a broader picture of BIIB078 distribution and pathological efficacy throughout the CNS. Predicated on preclinical findings that ASOs robustly mitigate G4C2 sense strand gain-of-function toxicity in animal models coupled with the failure of BIIB078 to provide a clinical benefit in a phase 1 clinical trial (30), it is tempting to conclude that G4C2 sense strand gain-of-function mechanisms do not underly neurodegeneration in C9ALS / FTD; however, this data suggests such a conclusion may be premature. Ultimately, this study resolves important knowledge gaps in understanding of BIIB078 distribution and efficacy in patient tissues and provides evidence that despite broad BIIB078 ASO distribution throughout the CNS, G4C2 expansion pathology was not significantly altered. Thus, strategies to silence the G4C2 encoding sense strand remain a potential therapeutic option.

[0154] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the abovedescribed embodiment(s) without departing substantially from the spirit and principles of thedisclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.REFERENCES1. Adamo CS, et al. EMI LIN 1 deficiency causes arterial tortuosity with osteopenia and connects impaired elastogenesis with defective collagen fibrillogenesis. The American Journal of Human Genetics. 2022;109(12):2230-52.2. Akcimen, F et al., Amyotrophic lateral sclerosis: translating genetic discoveries into therapies. Nature Reviews Genetics 24, 642-658 (2023).3. Bacioglu M, et al. Neurofilament Light Chain in Blood and CSF as Marker of Disease Progression in Mouse Models and in Neurodegenerative Diseases. Neuron. 2016;91(1):56- 66.4. Balendra, R et al, C9orf72-mediated ALS and FTD: multiple pathways to disease. Nat Rev Neurol 14, 544-558 (2018).5. Benatar, M et al, Neurofilament light chain in drug development for amyotrophic lateral sclerosis: a critical appraisal. Brain 146, 2711-2716 (2022).6. Bennett, CF et al, Pharmacology of Antisense Drugs. Annual Review of Pharmacology and Toxicology 57, 81-105 (2017).7. Bennett, CF, Therapeutic Antisense Oligonucleotides Are Coming of Age. Annual Review of Medicine 70, 307-321 (2019).8. Bereman MS, et al. Machine Learning Reveals Protein Signatures in CSF and Plasma Fluids of Clinical Value for ALS. Scientific Reports. 2018;8(1).9. Boeynaems, S et al., Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics. Mol Cell 65, 1044-1055.e1045 (2017).10. Bothwell, SW et al, Cerebrospinal fluid dynamics and intracranial pressure elevation in neurological diseases. Fluids and Barriers of the CNS 16, 9 (2019).11. Brown A-L, et al. TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A. Nature. 2022;603(7899):131-7.12. Brown RH, et al. Amyotrophic Lateral Sclerosis. New England Journal of Medicine. 2017;377(2):162-72.Bruneteau G, et al. Muscle histone deacetylase 4 upregulation in amyotrophic lateral sclerosis: potential role in reinnervation ability and disease progression. Brain. 2013;136(8):2359-68. Calaway R, et al. Package ‘doParallel1. 1.0.14 ed. CRAN2018. p. Provides a parallel backend for the %dopar% function using the parallel package. Cohen TJ, et al. An acetylation switch controls TDP-43 function and aggregation propensity. Nature Communications. 2015;6(1):5845. Cooper-Knock, J et al., Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions. Brain 137, 2040-2051 (2014). Dammer EB, et al. Batch correction and harmonization of -Omics datasets with a tunable median polish of ratio. Frontiers in Systems Biology. 2023;3. Dammer EB, et al. Multi-platform proteomic analysis of Alzheimer’s disease cerebrospinal fluid and plasma reveals network biomarkers associated with proteostasis and the matrisome. Alzheimer's Research & Therapy. 2022;14(1). Dammer EB, et al. Proteomic Network Analysis of Alzheimer’s Disease Cerebrospinal Fluid Reveals Alterations Associated withAPOE£4 Genotype and Atomoxetine Treatment. 2023. De Geus MB, et al. Mass spectrometry in cerebrospinal fluid uncovers association of glycolysis biomarkers with Alzheimer’s disease in a large clinical sample. Scientific Reports. 2023;13(1). Dejesus-Hernandez M, et al. Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS. Neuron. 2011 ;72(2):245- 56. Donnelly, CJ et al., RNA toxicity from the ALS / FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 80, 415-428 (2013). Gendron, TF et al., Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in C9FTD / ALS. Acta Neuropathol 126, 829-844 (2013). Gendron, TF et al., Phosphorylated neurofilament heavy chain: A biomarker of survival for C9ORF72-associated amyotrophic lateral 5 sclerosis. Ann Neurol 82, 139-146 (2017).Gendron, TF et al., Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72- associated amyotrophic lateral sclerosis. Sci Transl Med 9, eaai7866 (2017). Gong J, et al. RBM45 competes with HDAC1 for binding to FUS in response to DNA damage. Nucleic Acids Research. 2017;45(22): 12862-76. Guo Q, et al. Global analysis of the heparin-enriched plasma proteome captures matrisome- associated proteins in Alzheimer’s disease. 2023. Han Q, et al. Gcn5- and Elp3-induced histone H3 acetylation regulates hsp70 gene transcription in yeast. Biochemical Journal. 2008;409(3):779-88. Hardiman O, et al. Amyotrophic lateral sclerosis. Nature Reviews Disease Primers. 2017;3(1):17071. Higginbotham L, et al. Integrated proteomics reveals brain-based cerebrospinal fluid biomarkers in asymptomatic and symptomatic Alzheimer’s disease. Science Advances. 2020;6(43):eaaz9360. Higginbotham L, et al. Unbiased classification of the elderly human brain proteome resolves distinct clinical and pathophysiological subtypes of cognitive impairment. Neurobiology of Disease. 2023; 186: 106286. Hu, J et al, Engineering Duplex RNAs for Challenging Targets: Recognition of GGGGCC / CCCCGG Repeats at the ALS / FTD C9orf72 Locus. Chem Biol 22, 1505-1511 (2015). Hu, J et al, Recognition of c9orf72 Mutant RNA by Single-Stranded Silencing RNAs. Nucleic Acid Ther 27, 87-94 (2017). Humphrey J, et al. Integrative transcriptomic analysis of the amyotrophic lateral sclerosis spinal cord implicates glial activation and suggests new risk genes. Nature Neuroscience. 2023;26(1):150-62. Irwin KE, et al. A fluid biomarker reveals loss of TDP-43 splicing repression in presymptomatic ALS-FTD. Nature Medicine. 2024. Jiang, J et al., Gain of Toxicity from ALS / FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs. Neuron 90, 535-550 (2016).Johnson ECB, et al. Large-scale deep multi-layer analysis of Alzheimer’s disease brain reveals strong proteomic disease-related changes not observed at the RNA level. Nature Neuroscience. 2022;25(2):213-25. Johnson ECB, et al. Large-scale proteomic analysis of Alzheimer’s disease brain and cerebrospinal fluid reveals early changes in energy metabolism associated with microglia and astrocyte activation. Nature Medicine. 2020;26(5):769-80. Kall L, et al. Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nature Methods. 2007;4(11):923-5. Kaufman L, et al. Finding groups in data : an introduction to cluster analysis. Hoboken, N.J.: Wiley; 2005. xiv, 342 p. p. Klim, JR et al., ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair. Nat Neurosci 22, 167-179 (2019). Klingl YE, et al. Opportunities for histone deacetylase inhibition in amyotrophic lateral sclerosis. British Journal of Pharmacology. 2021 ;178(6):1353-72. Kong AT, et al. MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics. Nature Methods. 2017;14(5):513-20. Krishnan, G et al., Poly(GR) and poly(GA) in cerebrospinal fluid as potential biomarkers for C9ORF72-ALS / FTD. Nat Commun 13, 2799 (2022). Lagier-Tourenne, C et al., Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration. Proc Natl Acad Sci U S A 110, E4530-4539 (2013). Landqvist Waldo M, et al. Cerebrospinal fluid neurofilament light chain protein levels in subtypes of frontotemporal dementia. BMC Neurology. 2013;13(1):54. Langfelder P, et al. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008;9(1):559. Lee, KH et al., C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles. Cell 167, 774-788. e717 (2016). Lee, YB et al., Hexanucleotide repeats in ALS / FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic. Cell Rep 5, 1178-1186 (2013). Lin Y, et al. Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers. Cell. 2016;167(3):789-802.e12.Liu, Y et al., Variant-selective stereopure oligonucleotides protect against pathologies associated with C9orf72- repeat expansion in preclinical models. Nat Commun 12, 847 (2021). Lu, CH et al., Neurofilament light chain. Neurology 84, 2247-2257 (2015). Ly CV, et al. Protein kinetics of superoxide dismutase-1 in familial and sporadic amyotrophic lateral sclerosis. Annals of Clinical and Translational Neurology. 2023; 10(6): 1012-24. Magistretti J, et al. A Cellular Perspective on Brain Energy Metabolism and Functional Imaging. Neuron. 2015;86(4):883-901. Maienschein-Cline M, et al. ARTS: automated randomization of multiple traits for study design. Bioinformatics. 2014;30(11):1637-9. Majounie E, et al. Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study. The Lancet Neurology. 2012;11(4):323-30. Melamed, Z et al., Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration. Nat Neurosci 22, 180-190 (2019). Milioto, C et al., PolyGR and polyPR knock-in mice 5 reveal a conserved neuroprotective extracellular matrix signature in C9orf72 ALS / FTD neurons. Nature Neuroscience 27, 643- 655 (2024). Miller TM, et al. Trial of Antisense Oligonucleotide Tofersen for SOD1 ALS. New England Journal of Medicine. 2022;387(12):1099-110. Modeste ES, et al. Quantitative proteomics of cerebrospinal fluid from African Americans and Caucasians reveals shared and divergent changes in Alzheimer’s disease. Molecular Neurodegeneration. 2023;18(1). Mori, K et al., The C9orf72 GGGGCC repeat is translated into aggregating dipepti derepeat proteins in FTLD / ALS. Science 339, 1335-1338 (2013). Murphy NA, et al. Age-related penetrance of the C9orf72 repeat expansion. Scientific Reports. 2017;7(1). Neumann, M et al., Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130-133 (2006).Oeckl P, et al. Proteomics in cerebrospinal fluid and spinal cord suggests UCHL1 , MAP2 and GPNMB as biomarkers and underpins importance of transcriptional pathways in amyotrophic lateral sclerosis. Acta Neuropathologica. 2020; 139(1): 119-34. Oh S, et al. Discovery of Biomarkers for Amyotrophic Lateral Sclerosis from Human Cerebrospinal Fluid Using Mass-Spectrometry-Based Proteomics. Biomedicines. 2023; 11 (5): 1250. Ping L, et al. Global quantitative analysis of the human brain proteome in Alzheimer’s and Parkinson’s Disease. Scientific Data. 2018;5(1):180036. Pottier, C et al, Genetics of FTLD: overview and what else we can expect from genetic studies. Journal of Neurochemistry 138, 32-53 (2016). Qiu X-B, et al. The diversity of the DnaJ / Hsp40 family, the crucial partners for Hsp70 chaperones. Cellular and Molecular Life Sciences. 2006;63(22):2560-70. Rayaprolu S, et al. Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau. Neuropsychopharmacology. 2021 ;46(1):98-115. Reimand J, et al. Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap. Nature Protocols. 2019;14(2):482-517. Renton E, et al. A Hexanucleotide Repeat Expansion in C9ORF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD. Neuron. 2011 ;72(2):257-68. Rosen DR, et al. Mutations in Cu / Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993;362(6415):59-62. Rothstein, JD et al., G(2)C(4) targeting antisense oligonucleotides potently mitigate TDP-43 dysfunction in human C9orf72 ALS / FTD induced pluripotent stem cell derived neurons. Acta Neuropathol 147, 1 (2023). Rouaux C. Critical loss of CBP / p300 histone acetylase activity by caspase-6 during neurodegeneration. The EMBO Journal. 2003;22(24):6537-49. Sareen, D et al., Targeting RNA Foci in iPSC-Derived Motor Neurons from ALS Patients with a C9ORF72Repeat Expansion. Sci Transl Med 5, 208ra149 (2013). Seto E, et al. Erasers of Histone Acetylation: The Histone Deacetylase Enzymes. Cold Spring Harbor Perspectives in Biology. 2014;6(4):a018713-a. Seyfried NT, et al. A Multi-network Approach Identifies Protein-Specific Co-expression in Asymptomatic and Symptomatic Alzheimer’s Disease. Cell Systems. 2017;4(1):60-72.e4.Shiota T, et al. C9orf72-Derived Proline:Arginine Poly-Dipeptides Modulate Cytoskeleton and Mechanical Stress Response. Frontiers in Cell and Developmental Biology. 2022;10. Steinacker P, et al. Neurofilaments in the diagnosis of motoneuron diseases: a prospective study on 455 patients. Journal of Neurology, Neurosurgery & Psychiatry. 2016;87(1):12-20. Synofzik M, et al. Absence of BiP Co-chaperone DNAJC3 Causes Diabetes Mellitus and Multisystemic Neurodegeneration. The American Journal of Human Genetics. 2014;95(6):689-97. Thayer, MB et al., POE Immunoassay: Plate-based oligonucleotide electrochemiluminescent immunoassay for the quantification of nucleic acids in biological matrices. Scientific Reports 10, 10425 (2020). Thompson AG, et al. Cerebrospinal fluid macrophage biomarkers in amyotrophic lateral sclerosis. Annals of Neurology. 2018;83(2):258-68. Thompson AG, et al. Network Analysis of the CSF Proteome Characterizes Convergent Pathways of Cellular Dysfunction in ALS. Frontiers in Neuroscience. 2021 ; 15. Tran, H et al., Suppression of mutant C9orf72 expression by a potent mixed backbone antisense oligonucleotide. Nat Med 28, 117-124 (2022). Umoh ME, et al. A proteomic network approach across the ALS‐FTD disease spectrum resolves clinical phenotypes and genetic vulnerability in human brain. EM BO Molecular Medicine. 2018;10(1):48-62. Valle C, et al. Tissue-specific deregulation of selected HDACs characterizes ALS progression in mouse models: pharmacological characterization of SIRT1 and SIRT2 pathways. Cell Death & Disease. 2014;5(6):e1296-e. Valvona CJ, et al. The Regulation and Function of Lactate Dehydrogenase A: Therapeutic Potential in Brain Tumor. Brain Pathology. 2016;26(1):3-17. van den Berg, LH et al., in ENCALS. (Edinburgh, 2022). Vu L, et al. Cross-sectional and longitudinal measures of chitinase proteins in amyotrophic lateral sclerosis and expression of CHI3L1 in activated astrocytes. Journal of Neurology, Neurosurgery & Psychiatry. 2020;91(4):350-8. Vu L, et al. Proteomics and mathematical modeling of longitudinal <scp>CSF< / scp> differentiates fast versus slow <scp>ALS< / scp> progression. Annals of Clinical and Translational Neurology. 2023;10(11):2025-42.Warburg O, et al. THE METABOLISM OF TUMORS IN THE BODY. Journal of General Physiology. 1927;8(6):519-30. Warburg O. The Metabolism of Carcinoma Cellsl . The Journal of Cancer Research. 1925;9(1):148-63. Weydt P, et al. Neurofilament levels as biomarkers in asymptomatic and symptomatic familial amyotrophic lateral sclerosis. Annals of Neurology. 2016;79(1):152-8. Wickham H, et al. Package ‘ggplot2’. Create elegant data visualisations using the grammar of graphics. 2 ed. CRAN2016. Winiarska A, et al. Decreased Levels of Histidine-Rich Glycoprotein in Advanced Lung Cancer: Association with Prothrombotic Alterations. Disease Markers. 2019;2019:8170759. Wojtas AM, et al. Proteomic Changes in the Human Cerebrovasculature in Alzheimer’s Disease and Related Tauopathies Linked to Peripheral Biomarkers in Plasma and Cerebrospinal Fluid. 2024. Yang KL, et al. MSBooster: improving peptide identification rates using deep learning-based features. Nature Communications. 2023; 14(1). Yu F, et al. Fast Quantitative Analysis of timsTOF PASEF Data with MSFragger and lonQuant. Molecular & Cellular Proteomics. 2020; 19(9): 1575-85. Zanetti M, et al. EMILIN-1 Deficiency Induces Elastogenesis and Vascular Cell Defects. Molecular and Cellular Biology. 2004;24(2):638-50. . Zetterberg H, et al. Association of Cerebrospinal Fluid Neurofilament Light Concentration With Alzheimer Disease Progression. JAMA Neurology. 2016;73(1):60-7. . Zhang, K et al., The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525, 56-61 (2015). . Zhang, YJ et al., Aggregation-prone C9FTD / ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress. Acta Neuropathol 128, 505-524 (2014). . Zhang, YJ et al., C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins. Nat Neurosci 19, 668-677 (2016). . Zhang, YJ et al., Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity. Science 363, (2019). . Zhang, YJ et al., Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis. Nature Medicine 24, 1136-1142 (2018).- SO -106. Zum T et al., RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc Natl Acad Sci U S A 110, E4968-4977 (2013).

Claims

CLAIMSWhat is claimed is:

1. A method of diagnosing a neurodegenerative disease associated with loss of motor neurons comprising providing a sample from a patient, measuring one or more proteins in the sample, thereby providing measured relative quantities of each of the one or more proteins, comparing the measured relative quantities of the one or more proteins to reference relative quantities of the one or more proteins associated with a specific neurodegenerative disease associated with loss of motor neurons, and diagnosing the patient with a specific neurodegenerative disease associated with loss of motor neurons when the measured relative quantities of the one or more proteins are substantially similar to reference relative quantities of the one or more proteins associated with a specific neurodegenerative disease associated with loss of motor neurons.

2. The method of claim 1 , wherein the sample is from cerebrospinal fluid (CSF).

3. The method of claim 1 wherein the neurodegenerative disease associated with loss of motor neurons comprises Amyotrophic Lateral Sclerosis (ALS) and where the measured relative quantities of the one or more proteins correlate with disease onset, progression, or both disease onset and progression in an ALS subgroup.

4. The method of claim 3, wherein the ALS subgroup is associated with an inherited mutation.

5. The method of claim 4, wherein the inherited mutation is a c9orf72 hexanucleotide repeat.

6. The method of claim 5, wherein the patient is a symptomatic carrier or asymptomatic carrier of the c9orf72 hexanucleotide repeat.

7. The method of claim 4, wherein the inherited mutation is a superoxide dismutase 1 gene (SOD1) mutation.

8. The method of claim 3, wherein the ALS subgroup is sporadic ALS.

9. The method of claim 1 , wherein the one or more proteins comprise neurofilament medium protein (NEFM) and neurofilament light chain protein (NEFL).

10. The method of claim 1, wherein the one or more proteins comprise chitinase 1 protein (CHIT1) and chitinase-3 like-protein-1 (CHI3L1).

11. The method of claim 1 , wherein the one or more proteins comprise superoxide dismutase protein (SOD1).

12. The method of claim 11 , wherein a low abundance of the SOD1 protein indicates an A5T mutation is present in the SOD1 protein.

13. The method of claim 1 , wherein the one or more proteins comprise ALS-specific proteins, wherein the ALS-specific proteins comprise insulin-like growth factor 2 (IGF2), retinoic acid receptor responder 2 (RARRES2), galactose-specific lectin 3 (LGALS3), and lysozyme (LYZ).

14. The method of claim 1, wherein the one or more proteins comprise Glycoprotein nonmetastatic melanoma protein B (GPNMB).

15. The method of claim 1 , wherein the one or more proteins comprise aldo-keto reductase family 1 , member B1 (AKR1 B1), Transketolase (TKT), cofilin 1 (CFL1), cofilin 2 (CFL2), peptidylprolyl isomerase A (PPIA), and fatty-acid-binding protein 3 (FABP3).

16. The method of claim 1 , wherein the one or more proteins comprise lactate dehydrogenase A (LDHA).

17. The method of claim 1 , wherein the one or more proteins comprise elastin microfibril interfacer 1 (EMILIN1) and DnaJ heat shock protein family (Hsp40) member C3 (DNAJC3).

18. The method of claim 1 , wherein the one or more proteins comprise Histone H1.5 protein (HIST1 H1 B), Histone H4 (HIST1 H4A), Histone H2A (HIST1 H2AB), Histone H1.4 (HIST1H1 E), Histone H2A.Z (H2AFZ), and Histone H2B type 2-F HIST2H2BF.

19. The methods of any of one claims 1-18, wherein measuring the one or more proteins in the sample providing measured relative quantities of each protein comprises one or more of:(a) contacting the sample with an enzyme capable of C-terminal cleavage of lysine, arginine, or both; and(b) contacting the sample with an enzyme capable of N-terminal cleavage of lysine, arginine, or both; wherein performing (a), (b), or both (a) and (b) provides cleaved proteins; analyzing the cleaved proteins by mass spectrometry, wherein mass spectrometry generates fragmentation spectra from peptides; and analyzing cleavage of the cleaved proteins.

20. The method of claim 19, wherein analyzing cleavage of the cleaved proteins comprises mass selection of one or more target peptide ions, fragmentation of the one or more target peptide ions, and mass-to-charge (m / z) analysis of the one or more target peptide ions.

21. The method of claim 19, further comprising searching fragmentation spectra of the cleaved proteins against genomic or metagenomic databases to deduce amino acid sequences of the cleaved proteins, thereby providing positive identifications and measured relative quantities for the one or more proteins in the sample.22 A method for treating ALS in a subject, the method comprising administering a pharmaceutical composition comprising at least one antisense oligonucleotide (ASO) to the subject, wherein the ASO selectively binds to a target nucleotide sequence comprising one or more G4C2 repeats.

23. The method of claim 22, wherein the subject is a human.

24. The method of claim 22, wherein the ALS comprises c9ALS and the ASO comprises BIIB078.

25. The method of claim 22, wherein the pharmaceutical composition is administered intrathecally.

26. The method of claim 22, wherein performing the method reduces poly(GP) in cerebrospinal fluid (CSF) of the subject by at least about 50%.

27. The method of claim 26, wherein performing the method reduces poly(GP) in cerebrospinal fluid (CSF) of the subject by at least about 75%.

28. The method of claim 22, wherein the method is performed once.

29. The method of claim 22, wherein the method is performed 2 or more times, and wherein each dose of the pharmaceutical composition is separated by a time interval.

30. The method of claim 29, wherein the time interval comprises from about 2 weeks to about 4 weeks.

31. The method of claim 22, wherein the pharmaceutical composition comprises from about 10 mg to about 90 mg of the ASO.

32. The method of claim 22, wherein the ASO localizes to a CNS region affected by ALS.

33. The method of claim 32, wherein the CNS region comprises the spinal cord, the motor cortex, the subarachnoid brain region, or the periventricular brain region.

34. The method of claim 32, wherein the ASO persists in the CNS region for from at least 10 days to at least 500 days.

35. The method of claim 22, wherein performing the method reduces at least one C9orf72 transcript in the CSF of the subject.

36. The method of claim 35, wherein the at least one C9orf72 transcript comprises one or more G4C2 repeats.

37. The method of claim 35, wherein the at least one C9orf72 transcript comprises variant 1 , variant 3, or both.

38. The method of claim 22, further comprising administering at least one additional ALS treatment to the subject.

39. The method of claim 38, wherein the at least one additional ALS treatment comprises administration of a second pharmaceutical composition, stem cell therapy, gene therapy, an immunomodulatory therapy, or any combination thereof.

40. The method of claim 39, wherein the second pharmaceutical composition comprises riluzole, edaravone, sodium phenylbutyrate / taurursodiol, tofersen, or any combination thereof.

41. The method of claim 22, wherein performing the method reduces at least one symptom of ALS.

42. The method of claim 41 , wherein the at least one symptom comprises muscle weakness, fatigue, pain, loss of fine motor function, slurred speech, difficulty swallowing, muscle cramps, muscle atrophy, shortness of breath, coughing, speech impairment, cognitive changes, emotional changes, excessive salivation, difficulty controlling bowel movements, tripping, falling, difficulty walking, difficulty standing, or any combination thereof.

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