Tripeptidyl peptidase 1-related compositions and methods for the treatment and diagnosis of amyotrophic lateral sclerosis

Abstract

The present disclosure provides TPP1-related methods for the diagnosis and / or treatment of Amyotrophic Lateral Sclerosis (ALS). These methods can comprise the modulation of TPP1 to treat ALS and determining TPP1 expression, activity and genotypes to determine a subject's risk of ALS, responsiveness to an ALS treatment and whether they will benefit from treatment with a TPP1-modulating agent.

Description

TRIPEPTIDYL PEPTIDASE 1-RELATED COMPOSITIONS AND METHODS FOR THE TREATMENT AND DIAGNOSIS OF AMYOTROPHIC LATERAL SCLEROSISRELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Application No. 63 / 635,028, filed on April 17, 2024. The contents of the aforementioned patent application are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided electronically in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is “5PSC_001wo_SeqList”. The XML file is 2,349 bytes, created on March 31, 2025, and is being submitted electronically via USPTO Patent Center.BACKGROUND OF THE INVENTION

[0003] ALS, commonly known as Lou Gehrig's disease, is a progressive neurodegenerative disease that results in the degeneration of motor neurons in the brain and spinal cord. This degeneration leads to muscle weakness, atrophy, and eventually, respiratory failure. Annually, approximately 5,000 individuals in the United States are diagnosed with ALS. The disease presents mainly in two forms: sporadic ALS (SALS), which accounts for about 90% of cases, and familial - ALS (FALS), which is hereditary. Several genes have been implicated in ALS, but the exact mechanisms of motor neuron degeneration remain elusive. Current treatments are limited and focus mainly on symptomatic relief. Accordingly, there is a need in the art for improved compositions and methods for the treatment and diagnosis of ALS. The present disclosure addresses this need by providing Tripeptidyl peptidase l(TPPl)-related compositions and methods for the treatment and diagnosis of ALS. Tripeptidyl-peptidase 1 (TPP1), a protein encoded by the TPP1 gene, is a lysosomal enzyme involved in protein degradation. TPP1 plays a key role in breaking down proteolipids within the lysosome, a process essential for cellular maintenance and function. Mutations in TPP1 are linked to Neuronal Ceroid Lipofuscinosis 2 (NCL2), a form of Batten disease, which is a rare, fatal, autosomal recessive neurodegenerative disorder. It manifests in early childhood withsymptoms like seizures, motor and cognitive decline due to the accumulation of lipofuscin in cells.SUMMARY

[0004] The present disclosure provides methods of identifying the risk that a subject will develop ALS, the methods comprising: a) determining the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in a biological sample from the subject; b) comparing the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in the biological sample to a corresponding predetermined cutoff value; and c) determining that the subject is at low risk of developing ALS if the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in the biological sample is greater than or equal to the corresponding predetermined cutoff value, or determining that the subject is at a high risk of developing ALS if the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration is less than the corresponding predetermined cutoff value.

[0005] In some aspects, the corresponding predetermined cutoff value is determined through the analysis of the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in a training set, wherein the training set comprises the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration measured in biological samples from one or more training subjects, wherein the one or more training subjects comprise a plurality of training subjects that do not have ALS and a plurality of training subjects that have ALS.

[0006] In some aspects, the analysis comprises genome-wide association study (GWAS) analysis and / or mendelian randomization (MR) analysis.

[0007] The present disclosure provides methods of identifying the risk that a subject will develop ALS, the methods comprising: a) determining the TPP1 genotype of the subject; and b) determining the risk that the subject will develop ALS based on the TPP1 genotype.

[0008] In some aspects, identifying the TPP1 genotype of the subject comprises determining if the subject is major allele homozygous, minor allele heterozygous, or minor allele homozygous at a specific locus associated with a specific single nucleotides polymorphism (SNP). In some aspects, the specific locus is rs3758978, wherein major allele at rs3758978 is cytosine (C) and the minor allele is guanine (G), wherein subjects with a major allele homozygous or minor allele heterozygous rs3758978 genotype are identified as being at increased risk of ALS and subjects with a minor allele homozygous rs3758978 genotype areidentified as being at decreased risk of ALS. In some aspects, the specific locus is a locus that is in linkage disequilibrium with rs3758978.

[0009] The present disclosure provides methods of identifying a subject having ALS for treatment with a TPP1 -modulating agent, the method comprising: a) determining the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in a biological sample from the subject; b) comparing the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in the biological sample to a corresponding predetermined cutoff value; and c) identifying the subject for treatment with the TPP1 -modulating agent if the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration is less than or equal to the corresponding predetermined cutoff value.

[0010] The present disclosure provides methods of identifying a subject having ALS for treatment with a TPP1 -modulating agent, the methods comprising: a) determining the TPP1 genotype of the subject; and b) identifying whether the subject should be treated with the TPP1 -modulating agent based on the TPP1 genotype.

[0011] In some aspects, identifying the TPP1 genotype of the subject comprises determining if the subject is major allele homozygous, minor allele heterozygous, or minor allele homozygous at a specific locus associated with a specific single nucleotides polymorphism (SNP). In some aspects, the specific locus is rs3758978, wherein major allele at rs3758978 is cytosine (C) and the minor allele is guanine (G), wherein subjects with a major allele homozygous or minor allele heterozygous rs3758978 genotype are identified to be treated with a TPPl-modualting agent. In some aspects, the specific locus is a locus that is in linkage disequilibrium with rs3758978.

[0012] The present disclosure provides methods of determining the responsiveness of a subject to an ALS treatment, the methods comprising: a) at a first time point, determining the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in a biological sample from the subject; b) at a second time point, determining the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in a biological sample from the subject; c) comparing the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration at the first time point and the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration at the second time point; and d) identifying that the subject is responding to the ALS treatment when the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration at the second time point is greater than the TPP1 expression level, the TPP1 activity level, and / orthe TPP1 protein concentration at the first time point, or identifying that the subject is not responding to the ALS treatment when the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration at the second time point is the same or less than the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration at the first time point. In some aspects, the first time point is before the administration of the ALS treatment and the second time point is after the administration of at least one dose of the ALS treatment. In some aspects, the first time point is after the initial administration of the ALS treatment and the second time point is after one or more additional doses of the ALS treatment has been administered to the subject.

[0013] In some aspects, the methods further comprise hanging the dosage of the ALS treatment based on the change in the expression level and / or activity level of TPP1 between the first time point and the second time point.

[0014] In some aspects, a biological sample comprises blood, plasma, serum, urine, breast milk, cerebrospinal fluid, mucus, gastric juice, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, endolymph, perilymph, neuronal tissue or any combination thereof.

[0015] In some aspects, determining the TPP1 expression level comprises the use of PCR, high-throughput sequencing, next generation sequencing (NGS), Northern Blot, reverse transcription PCR (RT-PCR), real-time PCR (qPCR), quantitative PCR, qRT-PCR, flow cytometry, microarray analysis, digital droplet PCR, RNA sequencing, or any combination thereof.

[0016] In some aspects, determining TPP1 protein concentration comprises mass spectrometry, western blot, aptamer-based methods, enzyme-linked immunoabsorbent assays (ELISAs) measurements, proximity extension assays, or any combination thereof.

[0017] In some aspects, determining TPP1 activity level comprises the use of one or more enzymatic assays, activity -based protein profiling (ABPP), one or more functional assays, one or more biosensors, or any combination thereof.

[0018] The present disclosure provides methods of treating ALS in a subject, the methods comprising administering to a subject a TPP1 -modulating agent, wherein the TPP1- modulating agent is administered in one or more amounts sufficient to increase TPP1 activity, TPP1 expression, and / or TPP1 protein concentration in a plurality of cells in the subject such that the TPP1 activity, TPP1 expression, and / or TPP1 protein concentration in the plurality of cells is greater than a predetermined cutoff value.

[0019] In some aspects, a TPP1 -modulating agent comprises a gene therapy, a small molecule, an enzyme-replacement therapy, or any combination thereof.

[0020] In some aspects, a subject has been identified to have a TPP1 genotype that is associated with ALS.

[0021] In some aspects, the methods further comprise administering at least one additional therapy, preferably wherein the at least one additional therapy is selected from riluzole, edaravone, a neurotrophic factor, and an anti-inflammatory agent.

[0022] Any of the above aspects, or any other aspect described herein, can be combined with any other aspect described herein.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.” Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

[0024] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claim.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and further features will be more clearly appreciated from the following detailed description when taken in conjunction with the accompanying drawings.

[0026] FIG. l is a series of graphs that show colocalization of genetic associations with the risk of amyotrophic lateral sclerosis, circulating TPP1 levels, and cerebellum TPP1 expression levels. The cis-pQTL lead variant used as the genetic instrument is indicated. Genetic variants located in a ±500kb window centered around the genetic instrument are plotted with their significance in respective studies, and colored by the magnitude of correlation (linkage disequilibrium,LD r2) with the genetic instrument.

[0027] FIG. 2 is a graph showing the association between normalized cerebrospinal fluid TPP1 levels and age and sex. Effects of age and sex on normalized cerebrospinal fluid TPP1 levels were estimated using multiple linear regression based on 188 samples, adjusted for Alzheimer’s disease status. Each dot represents one sample. Red and blue lines indicate the fitted linear relationships between TPP1 levels and age in female control subjects and male control subjects, respectively.DETAILED DESCRIPTION

[0028] The present disclosure provides, inter alia, TPP1 -related compositions and methods for the treatment and diagnosis of ALS. For example, the present disclosure provides compositions and methods directed to the modulation of TPP1 activity, TPP1 expression, and / or TPP1 protein concentration (e.g. through the modulation of the expression of the TPP1 gene) in at least one cell in the subject for the treatment of ALS. In another example, the present disclosure provides compositions and methods for determining TPP1 expression levels, TPP1 genotypes, and / or TPP1 protein concentration. The compositions and methods described herein are based on the surprising and unexpected finding, described in the experimental examples that TPP1 plays a significant role in the development of ALS in a subject. More, specifically, Applicants have unexpectedly found that lower expression levels and / or activity levels of TPP1 are linked to an increased risk of ALS in a subject.

[0029] Methods of Treatment

[0030] The present disclosure provides a method of treating ALS in a subject in need thereof, wherein the treatment comprises modulating TPP1 activity, TPP1 expression, and / or TPP1 protein concentration in at least one cell in the subject. In some aspects, the treatment comprises modulating the TPP1 expression in at least one cell in the subject. In some aspects,the treatment comprises modulating TPP1 activity in at least one cell in the subject. In some aspects, the treatment comprises modulating the TPP1 protein concentration in at least one cell in the subject.

[0031] The present disclosure provides a method of treating ALS in a subject, the method comprising administering to the subject an agent that modulates TPP1 activity, TPP1 expression, and / or TPP1 protein concentration in at least one cell in the subject. Such agents are also referred to herein as “TPP1 -modulating agents”. Accordingly, the present disclosure provides a method of treating ALS in a subject, the method comprising administering to the subject a TPP1 -modulating agent, wherein the TPP1 -modulating agent modulates TPP1 expressionin at least one cell in the subject. The present disclosure provides a method of treating ALS in a subject, the method comprising administering to the subject a TPP1- modulating agent, wherein the TPP1 -modulating agent modulates TPP1 activity in at least one cell in the subject. The present disclosure provides a method of treating ALS in a subject, the method comprising administering to the subject a TPP1 -modulating agent, wherein the TPP1 -modulating agent modulates TPP1 protein concentration in at least one cell in the subject. In some aspects, the at least one cell comprises a population of neuronal cells in the subject.

[0032] In some aspects, modulating TPP1 expression comprises increasing the expression level of TPP1 by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99%, or at least about 100%, or at least about 125%, or at least about 150%, or at least about 175%, or at least about 200%, or at least about 225%, or at least about 250%, or at least about 275%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about 1000% in at least one cell in a subject. In some aspects, the increase in TPP1 expression is compared to the TPP1 expression prior to the administration of the TPP1 -modulating agent.

[0033] In some aspects, modulating TPP1 expression comprises increasing the expression level of TPP1 in at least one cell in a subject such that the expression level of TPP1 is equal to or greater than a predetermined cutoff value.

[0034] In some aspects, modulating TPP1 activity comprises increasing the activity level of TPP1 by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about99%, or at least about 100%, or at least about 125%, or at least about 150%, or at least about 175%, or at least about 200%, or at least about 225%, or at least about 250%, or at least about275%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about450%, or at least about 500%, or at least about 550%, or at least about 600%, or at least about650%, or at least about 700%, or at least about 750%, or at least about 800%, or at least about850%, or at least about 900%, or at least about 950%, or at least about 1000% in at least one cell in a subject. In some aspects, the increase in TPP1 activity is compared to the TPP1 activity prior to the administration of the TPP1 -modulating agent.

[0035] In some aspects, modulating TPP1 activity comprises increasing the activity level of TPP1 in at least one cell in a subject such that the activity level of TPP1 is equal to or greater than a predetermined cutoff value.

[0036] In some aspects, modulating TPP1 protein concentration comprises increasing the concentration of TPP1 protein by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about95%, or at least about 99%, or at least about 100%, or at least about 125%, or at least about 150%, or at least about 175%, or at least about 200%, or at least about 225%, or at least about250%, or at least about 275%, or at least about 300%, or at least about 350%, or at least about400%, or at least about 450%, or at least about 500%, or at least about 550%, or at least about600%, or at least about 650%, or at least about 700%, or at least about 750%, or at least about800%, or at least about 850%, or at least about 900%, or at least about 950%, or at least about1000% in at least one cell in a subject. In some aspects, the increase in TPP1 protein concentration is compared to the TPP1 protein concentration prior to the administration of the TPP1 -modulating agent.

[0037] In some aspects, modulating TPP1 protein concentration comprises increasing the TPP1 protein concentration in at least one cell in a subject such that the TPP1 protein concentration is equal to or greater than a predetermined cutoff value.

[0038] In some aspects, the predetermined cutoff values described above can be determined through the analysis of the TPP1 activity, TPP1 expression, and / or TPP1 protein concentration in a training set comprising the TPP1 activity, TPP1 expression, and / or TPP1 protein concentration measured in biological samples from one or more training subjects. In some aspects, the one or more training subjects comprise a plurality of training subjects that do not have ALS and a plurality of training subjects that have ALS. In some aspects, the predetermined cutoff values described above can be determined by a method comprising the generation of a polygenic risk score (PRS) for ALS, wherein the generation of the PRS is accomplished by analyzing genetic variations in one or more training subjects. The generation of a PRS for ALS may also be accomplished using data from large-scale genomewide association studies of ALS. In some aspects, the one or more training subjects comprise a plurality of training subjects that do not have ALS and a plurality of training subjects that have ALS. In some aspects, the PRS analysis and the TPPl-related analysis can be combined to provide the predetermined cutoff values described above.

[0039] In some aspects, the TPP1 -modulating agents are administered to the subject in a therapeutically effective amount. The term “therapeutically effective amount” of a TPP1- modulating agent is used in the broadest sense to refer to a nontoxic but sufficient amount of the agent to provide the desired effect or benefit. For example, a therapeutically effective amount of a TPP1 -modulating agent may be the amount needed to increase the activity level of TPP1 in at least one cell in a subject such that the activity level of TPP1 is equal to or greater than a predetermined cutoff value, or the amount needed to increase TPP1 expression in at least one cell in a subject such that the expression level of TPP1 is equal to or greater than a predetermined cutoff value, or the amount needed to increase the TPP1 protein concentration in at least one cell in a subject such that the TPP1 protein concentration is equal to or greater than a predetermined cutoff value.

[0040] The term “TPP1 -modulating agent,” as used herein, refers a molecule that has a desired biological effect, i.e. the modulation of TPP1. TPP1 -modulating agents include, but are not limited to: i) proteinaceous molecules, comprising, but not limited to, peptides, polypeptides, proteins, antibodies or antigen-binding fragments thereof, post-translationally modified proteins, antibodies, small molecules, inorganic or organic compounds; ii) nucleic acid molecules comprising, but not limited to, polynucleotides, double-stranded or single-stranded DNA, or double-stranded or single-stranded RNA (e.g., antisense, RNAi, siRNA, etc.), aptamers, as well as triple helix nucleic acid molecules; iii) gene therapy compositions, including, but not limited to viral vectors (e.g. adeno-associated viral (AAV) vectors) and genome editing modalities e.g. CRISPR-based gene editing systems); and iv) small molecules, including, but not limited to organic or inorganic compounds that are less than 1000 daltons; TPP1 -modulating agents can be derived or obtained from any known organism (comprising, but not limited to, animals (e.g., mammals (human and non-human mammals)), plants, bacteria, fungi, and protista, or viruses) or from a library of synthetic molecules.

[0041] In some aspects, the TPP1 -modulating agent comprises a TPP1 -enzyme replacement therapy. Accordingly, the present disclosure provides methods of treating ALS in a subject, the methods comprising administering to the subject a TPPl-enzyme replacement therapy. In some aspects, the TPPl-enzyme replacement therapy comprises a recombinant protein that recapitulates the activity of TPP1. As would be appreciated by the skilled artisan, TPPl- enzyme replacement therapies include, but are not limited to cerliponase alfa (marketed as BRINEURA®). TPPl-enzyme replacement therapies are described in further detail in US Patent No. 8029781, which is incorporated herein by reference in its entirety.

[0042] In some aspects, the TPP1 -modulating agent comprises a nucleic acid molecule comprising a nucleic acid sequence encoding for TPP1, or a portion thereof. In some aspects, the TPP1 -modulating agent comprises a nucleic acid molecule comprising a nucleic acid sequence encoding for a protein that exhibits the same biological activity as TPP1 (i.e. the cleavage of N-terminal tripeptides from substrates within the lysosome). In some aspects, the nucleic acid molecule can be a DNA molecule, a cDNA molecule, an RNA molecule, or any combination thereof. In some aspects, the nucleic acid molecule can comprise one or more chemical modifications.

[0043] In some aspects, the TPP1 -modulating agent comprises a TPPl-gene therapy composition. Accordingly, the present disclosure provides methods of treating ALS in a subject, the methods comprising administering to the subject a TPPl-gene therapy. As would be appreciated by the skilled artisan, TPPl-gene therapies include, but are not limited to, LX1004, (an rh.10 AAV vector comprising CLN2 cDNA), RGX-181 (an AAV9 vector comprising a TPP1 -encoding sequence).

[0044] In some aspects, a TPP1 -modulating agent can be administered in combination with one or more additional therapies.

[0045] In some aspects, an additional therapy can be a therapy known in the art for the treatment of ALS and / or the treatment of symptoms of ALS.

[0046] In some aspects, an additional agent can be riluzole.

[0047] In some aspects, an additional agent can be edaravone.

[0048] In some aspects, an additional agent can be tofersen.

[0049] In some aspects, an additional agent can be mecobalamin.

[0050] In some aspects, an additional agent can be a neurotrophic factor.

[0051] In some aspects, an additional agent can be an anti-inflammatory agent.

[0052] In some aspects, the at least one additional therapy and the TPPl-modualting agent can be administered in temporal proximity. As used herein, the term “temporal proximity” refers to that administration of one therapeutic agent (e.g., one or more pluralities of therapeutic cells of the present disclosure) occurs within a time period before or after the administration of another therapeutic agent (e.g., radiation therapy), such that the therapeutic effect of the one therapeutic agent overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, the therapeutic effect of the one therapeutic agent completely overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, “temporal proximity” means that administration of one therapeutic agent occurs within a time period before or after the administration of another therapeutic agent, such that there is a synergistic effect between the one therapeutic agent and the other therapeutic agent.“Temporal proximity” may vary according to various factors, including but not limited to, the age, gender, weight, genetic background, medical condition, disease history, and treatment history of the subject to which the therapeutic agents are to be administered; the disease or condition to be treated or ameliorated; the therapeutic outcome to be achieved; the dosage, dosing frequency, and dosing duration of the therapeutic agents; the pharmacokinetics and pharmacodynamics of the therapeutic agents; and the route(s) through which the therapeutic agents are administered. In some embodiments, “temporal proximity” means within 15 minutes, within 30 minutes, within an hour, within two hours, within four hours, within six hours, within eight hours, within 12 hours, within 18 hours, within 24 hours, within 36 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within a week, within 2 weeks, within 3 weeks, within 4 weeks, with 6 weeks, or within 8 weeks. In some embodiments, multiple administration of one therapeutic agent can occur in temporal proximity to a single administration of another therapeutic agent. In some embodiments, temporal proximity may change during a treatment cycle or within a dosing regimen.

[0053] TPP1 -modulating agents can be administered via a variety of routes, including, but not limited to, oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous,intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, or any combination thereof.

[0054] Methods of Identifying Subjects for Treatment with a TPP1 -modulating agent

[0055] The present disclosure provides methods of identifying subjects having ALS as candidates for treatment with a TPP1 -modulating agent based on TPP1 expression in the subject, the TPP1 protein concentration in the subject, the activity of TPP1 in the subject, and / or the TPP1 genotype of the subject. That is, the methods herein can be used to screen for subjects having ALS who would most likely benefit from treatment with a TPP1- modulating agent. In a non-limiting example, such methods could be used to identify subjects for inclusion in clinical trials.

[0056] Accordingly, the present disclosure provides methods of identifying a subject having ALS for treatment with a TPP1 -modulating agent, the methods comprising: a) determining the expression level of TPP1 in a biological sample from the subject; b) comparing the expression level of TPP1 in the biological sample to a predetermined cutoff value; and c) identifying the subject for treatment with the TPP1 -modulating agent if the expression level is less than or equal to the predetermined cutoff value.

[0057] The present disclosure provides methods of identifying a subject having ALS for treatment with a TPP1 -modulating agent, the methods comprising: a) determining the activity level of TPP1 in a biological sample from the subject; b) comparing the activity level of TPP1 in the biological sample to a predetermined cutoff value; and c) identifying the subject for treatment with the TPP1 -modulating agent if the activity level is less than or equal to the predetermined cutoff value.

[0058] The present disclosure provides methods of identifying a subject having ALS for treatment with a TPP1 -modulating agent, the methods comprising: a) determining the TPP1 protein concentration in a biological sample from the subject; b) comparing the TPP1 protein concentration in the biological sample to a predetermined cutoff value; and c) identifying the subject for treatment with the TPP1 -modulating agent if the TPP1 protein concentration level is less than or equal to the predetermined cutoff value.

[0059] The present disclosure provides methods of identifying a subject having ALS for treatment with a TPP1 -modulating agent, the methods comprising: a) determining the TPP1 genotype of the subject; and b) identifying whether the subject should be treated with the TPP1 -modulating agent based on the TPP1 genotype.

[0060] In some aspects, identifying the TPP1 genotype of the subject comprises determining if the subject is major allele homozygous, minor allele heterozygous, or minor allelehomozygous at a specific single nucleotides polymorphism (SNP). In some aspects, the SNP is rs3758978. The major allele at this SNP locus is cytosine (C) and the minor allele is guanine (G). As described in the experimental examples herein, Applicants have identified that subjects with a TPP1 major allele (C) have increased risk of developing ALS, and therefore will likely benefit from treatment with a TPP1 -modulating agent. Accordingly, in the methods of the present disclosure, subjects with a major allele homozygous or minor allele heterozygous rs3758978 genotype are identified to likely benefit from treatment with a TPP1 -modulating agent, while subjects with a minor allele homozygous rs3758978 genotype are identified as not likely to benefit from treatment with a TPP1 -modulating agent. In some aspects, the SNP is an SNP in linkage disequilibrium with rs3758978.

[0061] Accordingly, the present disclosure provides methods of identifying a subject having ALS for treatment with a TPP1 -modulating agent, the methods comprising: a) determining if the subject is major allele homozygous, minor allele heterozygous, or minor allele homozygous at locus rs3758978, wherein the major allele is cytosine (C) and the minor allele is guanine (G); and b) identifying the subject for treatment with the TPP-modulating agent when the subject is identified as major allele homozygous or minor allele heterozygous at locus rs3758978.

[0062] In some aspects, identifying the TPP1 genotype comprises determining if the subject has a genetic variant encoding a predicted loss-of-function TPP1 polypeptide. The genetic variant can be anyone of a missense mutation, a nonsynonymous mutation, an insertion of one or more nucleotides, a deletion of one or more nucleotides, an inversion and a deletioninsertion within a TPP1 -encoding sequence in the genome. In the methods of the present disclosure, subjects who are identified as having a genetic variant encoding a predicted loss- of-function TPP1 polypeptide will be identified as likely to benefit from treatment with a TPP1 -modulating agent, and therefore should be identified for treatment with the TPP1- modulating agent.

[0063] In some aspects, identifying the TPP1 genotype comprises determining if the subject has a genetic variant in a non-TPPl-enocding sequence in the genome that results in decreased expression and / or activity of TPP1. In a non-limiting example, such genetic variants include those within 1 megabases (Mb) of the transcription start site of the TPP1 gene. In the methods of the present disclosure, subjects who are identified as having a genetic variant in a non-TPPl-enocding sequence in the genome that results in decreased expression and / or activity of TPP1 will be identified as likely to benefit from treatment with a TPP1-modulating agent, and therefore should be identified for treatment with the TPP1 -modulating agent.

[0064] In some aspects of the preceding methods, identifying the subject for treatment with the TPP1 -modulating agent comprises identifying them for inclusion in a clinical trial for a TPP1 -modulating agent. In some aspects, identifying the subject for treatment with the TPP1- modulating agent comprises identifying that the subject will likely benefit from treatment with the TPP1 -modulating agent.

[0065] Methods of Identifying ALS Risk

[0066] The present disclosure provides a methods of identifying the risk that a subject will develop ALS, wherein the risk is determined based on the expression of TPP1 in the subject, the activity of TPP1 in the subject, the TPP1 protein concentration, and / or the TPP1 genotype of the subject.

[0067] Accordingly, the present disclosure provides methods of identifying the risk that a subject will develop ALS, the methods comprising: a) determining the expression level of TPP1 in a biological sample from the subject; b) comparing the expression level of TPP1 in the biological sample to a predetermined cutoff value; and c) determining that the subject is at low risk of developing ALS if the expression level of TPP1 in the biological sample is greater than or equal to the predetermined cutoff value, or determining that the subject is at a high risk of developing ALS if the expression level of TPP1 is less than the predetermined cutoff value.

[0068] The present disclosure provides methods of identifying the risk that a subject will develop ALS, the methods comprising: a) determining the activity level of TPP1 in a biological sample from the subject; b) comparing the activity level of TPP1 in the biological sample to a predetermined cutoff value; and c) determining that the subject is at low risk of developing ALS if the activity level of TPP1 in the biological sample is greater than or equal to the predetermined cutoff value, or determining that the subject is at a high risk of developing ALS if the activity level of TPP1 is less than the predetermined cutoff value.

[0069] The present disclosure provides methods of identifying the risk that a subject will develop ALS, the methods comprising: a) determining the TPP1 protein concentration in a biological sample from the subject; b) comparing the TPP1 protein concentration in the biological sample to a predetermined cutoff value; and c) determining that the subject is at low risk of developing ALS if the TPP1 protein concentration in the biological sample is greater than or equal to the predetermined cutoff value, or determining that the subject is at ahigh risk of developing ALS if the TPP1 protein concentration is less than the predetermined cutoff value.

[0070] In some aspects, the predetermined cutoff values described above can be determined through the analysis of the TPP1 activity, TPP1 expression, and / or TPP1 protein concentration in a training set comprising the TPP1 activity, TPP1 expression, and / or TPP1 protein concentration measured in biological samples from one or more training subjects. In some aspects, the one or more training subjects comprise a plurality of training subjects that do not have ALS and a plurality of training subjects that have ALS. In some aspects, the predetermined cutoff values described above can be determined by a method comprising the generation of a polygenic risk score (PRS) for ALS, wherein the generation of the PRS is accomplished by analyzing genetic variations in one or more training subjects. The generation of a PRS for ALS may also be accomplished using data from large-scale genomewide association studies of ALS. In some aspects, the one or more training subjects comprise a plurality of training subjects that do not have ALS and a plurality of training subjects that have ALS. In some aspects, the PRS analysis and the TPPl-related analysis can be combined to provide the predetermined cutoff values described above.

[0071] The present disclosure provides methods of identifying the risk that a subject will develop ALS, the methods comprising: a) determining the TPP1 genotype of the subject; and b) determining the risk that the subject will develop ALS based on the TPP1 genotype.

[0072] In some aspects, TPP1 genotypes that are indicative of high risk or low risk of ALS can be identified through the analysis of TPP1 genotypes in a training set comprising the TPP1 genotypes from one or more training subjects. In some aspects, the one or more training subjects comprise a plurality of training subjects that do not have ALS and a plurality of training subjects that have ALS.

[0073] In some aspects, identifying the TPP1 genotype of the subject comprises determining if the subject is major allele homozygous, minor allele heterozygous, or minor allele homozygous at a specific locus associated with a specific single nucleotides polymorphism (SNP).

[0074] In some aspects, the SNP is rs3758978. The major allele at this SNP locus is cytosine (C) and the minor allele is guanine (G). As described in the experimental examples herein, Applicants have identified that subjects with a TPP1 major allele (C) have increased risk of developing ALS. Accordingly, subjects with a major allele homozygous or minor allele heterozygous rs3758978 genotype are at increased risk of ALS while subjects with a minorallele homozygous rs3758978 genotype are at decreased risk of ALS. In some aspects, the SNP is an SNP in linkage disequilibrium with rs3758978.

[0075] Accordingly, the present disclosure provides methods of identifying if a subject is at increased risk of developing ALS, the methods comprising: a) determining if the subject is major allele homozygous, minor allele heterozygous, or minor allele homozygous at locus rs3758978, wherein the major allele is cytosine (C) and the minor allele is guanine (G); and b) identifying that the subject is at increased risk of ALS when the subject is identified as major allele homozygous or minor allele heterozygous at locus rs3758978, or identifying that the subject is at decreased risk of ALS when the subject is identified as minor allele homozygous at locus rs3758978.

[0076] In some aspects, identifying the TPP1 genotype comprises determining if the subject has a genetic variant encoding a predicted loss-of-function TPP1 polypeptide. The genetic variant can be anyone of a missense mutation, a nonsynonymous mutation, an insertion of one or more nucleotides, a deletion of one or more nucleotides, an inversion and a deletioninsertion within a TPP1 -encoding sequence in the genome. In the methods of the present disclosure, subjects who are identified as having a genetic variant encoding a predicted loss- of-function TPP1 polypeptide will be identified as having an increased risk of developing ALS as compared to subjects that do not have such a genetic variant.

[0077] In some aspects, identifying the TPP1 genotype comprises determining if the subject has a genetic variant in a non-TPPl-enocding sequence in the genome that results in decreased expression and / or activity of TPP1. In a non-limiting example, such genetic variants include those within 1 megabases (Mb) of the transcription start site of the TPP1 gene. In the methods of the present disclosure, subjects who are identified as having a genetic variant in a non-TPPl-enocding sequence in the genome that results in decreased expression and / or activity of TPP1 will be identified as having an increased risk of developing ALS as compared to subjects that do not have such a genetic variant.

[0078] In some aspects, the methods described above can further comprise administering to a subject identified as being at high or increased risk for ALS one or more ALS therapies, including, but not limited to, therapies comprising the TPP1 -modulating agents described herein.

[0079] Methods of Determining Initial Dosages of ALS Treatments

[0080] The present disclosure provides methods of determining the initial dosage of an ALS treatment to administer to a subject based on the expression of TPP1 in the subject, theactivity of TPP1 in the subject, the TPP1 protein concentration in the subject, and / or the TPP1 genotype of the subject.

[0081] Accordingly, the present disclosure provides methods of determining the initial dosage of an ALS treatment to administer to a subject, the methods comprising: a) determining the expression level of TPP1 in a biological sample from the subject; b) comparing the expression level of TPP1 in the biological sample to a predetermined cutoff value; and c) determining that the subject should receive an initial dosage of a first amount of the ALS treatment if the expression level of TPP1 in the biological sample is greater than or equal to the predetermined cutoff value, or determining that the subject should receive an initial dosage of a second amount if the expression level of TPP1 is less than the predetermined cutoff value, wherein the second amount is greater than the first amount.

[0082] The present disclosure provides methods of determining the initial dosage of an ALS treatment to administer to a subject, the methods comprising: a) determining the activity level of TPP1 in a biological sample from the subject; b) comparing the activity level of TPP1 in the biological sample to a predetermined cutoff value; and c) determining that the subject should receive an initial dosage of a first amount of the ALS treatment if the activity level of TPP1 in the biological sample is greater than or equal to the predetermined cutoff value, or determining that the subject should receive an initial dosage of a second amount if the activity level of TPP1 is less than the predetermined cutoff value, wherein the second amount is greater than the first amount.

[0083] The present disclosure provides methods of determining the initial dosage of an ALS treatment to administer to a subject, the methods comprising: a) determining the TPP1 protein concentration in a biological sample from the subject; b) comparing the TPP1 protein concentration in the biological sample to a predetermined cutoff value; and c) determining that the subject should receive an initial dosage of a first amount of the ALS treatment if the TPP1 protein concentration in the biological sample is greater than or equal to the predetermined cutoff value, or determining that the subject should receive an initial dosage of a second amount if the TPP1 protein concentration is less than the predetermined cutoff value, wherein the second amount is greater than the first amount.

[0084] In some aspects, the predetermined cutoff values described above can be determined through the analysis of the TPP1 activity, TPP1 expression, and / or TPP1 protein concentration in a training set comprising the TPP1 activity, TPP1 expression, and / or TPP1 protein concentration measured in biological samples from one or more training subjects. In some aspects, the one or more training subjects comprise a plurality of training subjects thatdo not have ALS and a plurality of training subjects that have ALS. In some aspects, the predetermined cutoff values described above can be determined by a method comprising the generation of a polygenic risk score (PRS) for ALS, wherein the generation of the PRS is accomplished by analyzing genetic variations in one or more training subjects. The generation of a PRS for ALS may also be accomplished using data from large-scale genomewide association studies of ALS. In some aspects, the one or more training subjects comprise a plurality of training subjects that do not have ALS and a plurality of training subjects that have ALS. In some aspects, the PRS analysis and the TPPl-related analysis can be combined to provide the predetermined cutoff values described above.

[0085] The present disclosure provides methods of determining the initial dosage of an ALS treatment to administer to a subject, the methods comprising: a) determining the TPP1 genotype of the subject; and b) determining the initial dosage of the ALS treatment that the subject should receive based on the TPP1 genotype.

[0086] In some aspects, identifying the TPP1 genotype of the subject comprises determining if the subject is major allele homozygous, minor allele heterozygous, or minor allele homozygous at a specific locus associated with a specific single nucleotides polymorphism (SNP).

[0087] In some aspects, determining the TPP1 genotype comprises determining if the subject is major allele homozygous, minor allele heterozygous, or minor allele homozygous at locus rs3758978, wherein the major allele is cytosine (C) and the minor allele is guanine (G). Subjects who are major allele homozygous should receive an initial dosage of a first amount, subjects who are minor allele heterozygous should receive an initial dosage of a second amount, and subjects who are minor allele homozygous should receive an initial dosage of a third amount, wherein the first amount is greater than the second amount, and the second amount is greater than the third amount.

[0088] Accordingly, the present disclosure provides methods of determining the initial dosage of an ALS treatment to administer to a subject, the methods comprising: a) determining if the subject is major allele homozygous, minor allele heterozygous, or minor allele homozygous at locus rs3758978, wherein the major allele is cytosine (C) and the minor allele is guanine (G); and b) determining that: i) the subject should receive an initial dosage of a first amount if the subject is major allele homozygous; ii) the subject should receive an initial dosage of a second amount if the subject is minor allele heterozygous; and iii) the subject should receive an initial dosage of a third amount if the subject is minor allelehomozygous, wherein the first amount is greater than the second amount, and the second amount is greater than the third amount.

[0089] In some aspects, identifying the TPP1 genotype comprises determining if the subject has a genetic variant encoding a predicted loss-of-function TPP1 polypeptide. The genetic variant can be anyone of a missense mutation, a nonsynonymous mutation, an insertion of one or more nucleotides, a deletion of one or more nucleotides, an inversion and a deletioninsertion within a TPP1 -encoding sequence in the genome. In the methods of the present disclosure, subjects who are identified as having a genetic variant encoding a predicted loss- of-function TPP1 polypeptide will be identified as requiring an increased initial dose of an ALS treatment as compared to subjects who do not have such a genetic variant.

[0090] In some aspects, identifying the TPP1 genotype comprises determining if the subject has a genetic variant in a non-TPPl-enocding sequence in the genome that results in decreased expression and / or activity of the TPP1. In a non-limiting example, such genetic variants include those within 1 megabases (Mb) of the transcription start site of the TPP1 gene. In the methods of the present disclosure, subjects who are identified as having a genetic variant in a non-TPPl-enocding sequence in the genome that results in decreased expression and / or activity of the TPP1 will be identified as requiring an increased initial dose of an ALS treatment as compared to subjects that do not have such a genetic variant.

[0091] The methods described above for the determination of initial dosages can be applied to any ALS treatment known in the art. The methods described above for the determination of initial dosages can also be applied to the TPP1 -modulating agent therapies described herein.

[0092] Methods of Determining Responsiveness to ALS Treatments and Modifying Dosages

[0093] The present disclosure provides methods of determining the responsiveness of a subject to an ALS treatment based on changes in the expression of TPP1 in the subject, the TPP1 protein concentration in the subject, and / or the activity of TPP1 in the subject after receiving the treatment. That is, subjects who exhibit an increase in the expression of TPP1, an increase in the TPP! Protein concentration and / or an increase in the activity of TPP1 after the administration of an ALS treatment are identified as responding to the treatment, while subjects that exhibit no change in the expression of TPP1, no change in TPP1 protein concentration, and / or no chance in the activity of TPP1 after the administration of the ALS treatment are identified as not responding to the treatment.

[0094] Accordingly, the present disclosure provides methods of determining the responsiveness of a subject to an ALS treatment, the methods comprising: a) at a first time point, determining the expression level of TPP1 in a biological sample from the subject; b) ata second time point, determining the expression level of TPP1 in a biological sample from the subject; c) comparing the expression level of TPP1 at the first time point and the expression level of TPP1 at the second time point; and d) identifying that the subject is responding to the ALS treatment when the expression level of TPP1 at the second time point is greater than the expression level of TPP1 at the first time point, or identifying that the subject is not responding to the ALS treatment when the expression level of TPP1 at the second time point is the same or less than the expression level of TPP1 at the first time point.

[0095] In some aspects of the preceding method, a subject is identified as being responsive to the ALS treatment only if the increase in TPP1 expression from the first time point to the second time point is greater than or equal to a predetermined cutoff (e.g. a predetermined cutoff percentage). In some aspects, a predetermined cutoff percentage can be about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 90%, or about 95%, or at least 100% increase in the expression of TPPP

[0096] Accordingly, the present disclosure provides methods of determining the responsiveness of a subject to an ALS treatment, the methods comprising: a) at a first time point, determining the activity level of TPP1 in a biological sample from the subject; b) at a second time point, determining the activity level of TPP1 in a biological sample from the subject; c) comparing the activity level of TPP1 at the first time point and the activity level of TPP1 at the second time point; and d) identifying that the subject is responding to the ALS treatment when the activity level of TPP1 at the second time point is greater than the activity level of TPP1 at the first time point, or identifying that the subject is not responding to the ALS treatment when the activity level of TPP1 at the second time point is the same or less than the activity level of TPP1 at the first time point.

[0097] In some aspects of the preceding method, a subject is identified as being responsive to the ALS treatment only if the increase in TPP1 activity from the first time point to the second time point is greater than or equal to a predetermined cutoff (e.g. a predetermined cutoff percentage). In some aspects, a predetermined cutoff percentage can be about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 90%, or about 95%, or at least 100% increase in the activity of TPP1.

[0098] Accordingly, the present disclosure provides methods of determining the responsiveness of a subject to an ALS treatment, the methods comprising: a) at a first time point, determining the TPP1 protein concentration in a biological sample from the subject; b) at a second time point, determining the TPP1 protein concentration in a biological sample from the subject; c) comparing the TPP1 protein concentration at the first time point and the TPP1 protein concentration at the second time point; and d) identifying that the subject is responding to the ALS treatment when the TPP1 protein concentration at the second time point is greater than the TPP1 protein concentration at the first time point, or identifying that the subject is not responding to the ALS treatment when the TPP1 protein concentration at the second time point is the same or less than the TPP1 protein concentration at the first time point.

[0099] In some aspects of the preceding method, a subject is identified as being responsive to the ALS treatment only if the increase in TPP1 protein concentration from the first time point to the second time point is greater than or equal to a predetermined cutoff (e.g. a predetermined cutoff percentage). In some aspects, a predetermined cutoff percentage can be about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 90%, or about 95%, or at least 100% increase in the TPP1 protein concentration.

[0100] In some aspects of the preceding methods, the first time point can be before the administration of the ALS treatment and the second time point can be after the administration of at least one dose of the ALS treatment. In some aspects of the preceding methods, the first time point can be after the initial administration of the ALS treatment and the second time point can be after one or more additional doses of the ALS treatment is administered to the subject.

[0101] In some aspects, the predetermined cutoff values described above can be determined through the analysis of the temporal changes in TPP1 activity, TPP1 protein concentration, and / or TPP1 expression in a training set comprising the TPP1 expression level, the TPP1 protein concentration level, and / or TPP1 activity level measured in sets of biological samples collected from one or more training subjects who had been administered the ALS treatment. In some aspects, the one or more training subjects comprise a plurality of training subjects responded to the ALS treatment and a plurality of training subjects that did not respond to the ALS treatment.

[0102] The methods described herein for determining the responsiveness of a subject to an ALS therapy can be further extrapolated to methods of modifying the dosage of an ALS therapy. That is, subjects who are identified as responding to a particular ALS therapy may be given additional and / or increased amounts of the ALS therapy since they are responsive. Subjects who are identified as not responding to a particular ALS therapy may be discontinued on that particular ALS therapy and switched to a different ALS therapy. Alternatively, subjects who are identified as not responding to a particular ALS treatment may be administered an increased amount of that ALS treatment to determine if they will respond to a higher dose. Additionally, in the methods described above, the percentage change between the first and second time points can be used to inform a modification to the dose of ALS therapy administered to the subject. In a non-limiting example, a subject may exhibit an increase in TPP1 activity following the administration of a first dose of an ALS therapy, but that increase is not sufficient for a therapeutic effect. In this case, the amount of ALS therapy that is administered in the next dose can be increased to effectuate a larger increase in TPP1 activity.

[0103] The methods described above for the determination of a subject’s response to an ALS therapy can be applied to any ALS treatment known in the art, including the TPP1- modulating agent therapies described herein.

[0104] Kits

[0105] The present disclosure provides kits for use in the methods described herein.

[0106] Accordingly, the present disclosure provides kits comprising at least one TPP1- modulating agent described herein.

[0107] The present disclosure also provides kits comprising at least one reagent that allows for the determination of the activity of TPP1, the expression of TPP1, the TPP1 protein concentration, and / or TPP1 genotype in a subject. For example, the present disclosure provides kits comprising one or more nucleic acid primers for use in sequencing the TPP1 locus in a biological sample from a subject, one or more nucleic acid primers for use in quantitative PCR to assess TPP1 expression levels in a biological sample from a subject, and / or one or more antibodies to assess TPP1 protein concentration in a biological sample from the subject.

[0108] The kits of the present disclosure can further comprise instructions for performing one or more of the methods described herein.

[0109] General Methods and Definitions

[0110] As would be appreciated by the skilled artisan, the protein Tripeptidyl Peptidase 1 (TTP1) can also be referred to as lysosomal pepstatin-insensitive protease or CLN2. As used herein, TTP1 can refer to any of the isoforms of the protein known in the art. SEQ ID NO: 1 shows an exemplary TTP1 sequence:MGLQACLLGLFALILSGKCSYSPEPDQRRTLPPGWVSLGRADPEEELS LTFALRQQNVERLSELVQAVSDPSSPQYGKYLTLENVADLVRPSPLTL HTVQKWLLAAGAQKCHSVITQDFLTCWLSIRQAELLLPGAEFHHYVG GPTETHVVRSPHPYQLPQALAPHVDFVGGLHRFPPTSSLRQRPEPQVT GTVGLHLGVTPSVIRKRYNLTSQDVGSGTSNNSQACAQFLEQYFHDS DLAQFMRLFGGNFAHQASVARVVGQQGRGRAGIEASLDVQYLMSAG ANISTWVYSSPGRHEGQEPFLQWLMLLSNESALPHVHTVSYGDDEDS LSSAYIQRVNTELMKAAARGLTLLFASGDSGAGCWSVSGRHQFRPTFP ASSPYVTTVGGTSFQEPFLITNEIVDYISGGGFSNVFPRPSYQEEAVTKF LSSSPHLPPSSYFNASGRAYPDVAALSDGYWVVSNRVPIPWVSGTSAS TPVFGGILSLINEHRILSGRPPLGFLNPRLYQQHGAGLFDVTRGCHESC LDEEVEGQGFCSGPGWDPVTGWGTPNFPALLKTLLNP (SEQ ID NO: 1)

[0111] In the methods of the present disclosure, determining the expression of TPP1, the activity of TPP1, the TPP1 protein concentration and / or a TPP1 genotype can be accomplished using any suitable known in the art.

[0112] For example, determining the expression of TPP1 can comprise PCR, high- throughput sequencing, next generation sequencing, Northern Blot, reverse transcription PCR (RT-PCR), real-time PCR (qPCR), quantitative PCR, qRT-PCR, flow cytometry, microarray analysis, digital droplet PCR, RNA sequencing, or any combination thereof. In another example, determining a TPP1 genotype can comprise next generation sequencing or microarray analysis. In another example, determining TPP1 protein concentration can comprise mass spectrometry, western blot, aptamer-based methods, enzyme-linked immunoabsorbent assays (ELISAs) measurements, proximity extension assays, or any other assay known in the art for the determination of protein concentration. In another example, determining TPP1 activity can comprise performing one or more enzymatic assays (e.g. assays that directly measure the enzyme activity of TPP1 by monitoring the conversion of substrates to products), activity-based protein profiling (e.g. techniques that employs probes that bind to and measure the activity of TPP1 in its active states), one or more functional assay (e.g. assays that are designed to assess the outcomes of a protein’s activity, for example, the extent of phosphorylation by a kinase), one or more biosensors (e.g. sensors that detect changes in biological activity through variations in electrical signals or fluorescence, or any other assay / method that is known in the art for the determination of TPP1 activity.

[0113] As used herein, the term “loss-of-function polypeptide” (e.g. a loss-of-function TPP1 polypeptide) refers to a polypeptide that contains at least one mutation e.g. missense mutations, nonsense mutations, frameshift mutations, and splice site mutations, insertions, deletions, and translocations) such that the biological activity, protein level, and / or expression of the polypeptide is decreased as compared to the same polypeptide without the at least one mutation. In some aspects, the decrease is complete, i.e. the loss-of-function polypeptide exhibits no relevant biological activity and / or expression, or the decrease can be partial, i.e. the loss-of-function polypeptide exhibits some relevant biological activity and / or expression, but less than that of the same polypeptide without the at least one mutation.

[0114] The term "biological sample" as used herein refers to any sample of biological origin potentially containing one or more biomarkers. A biological sample can comprise blood, plasma, serum, urine, breast milk, cerebrospinal fluid, mucus, gastric juice, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, endolymph, perilymph or any combination thereof. A biological sample can be a blood sample. A biological sample can be a cerebrospinal fluid sample.

[0115] As described herein, TPP1 genotypes that are indicative of high risk or low risk of ALS can be identified through the analysis of TPP1 genotypes in a training set comprising the TPP1 genotypes from one or more training subjects. This analysis can comprise genomewide association study (GWAS) analysis and / or mendelian randomization (MR) analysis.

[0116] The terms “subject” and “patient” are used interchangeably herein. In some embodiments, the subject treated in accordance with the methods described herein is a human patient. In some aspects, the subject is male. In some aspects, the subject is female.

[0117] As used herein, the term “portion” when used in reference to a polypeptide or a peptide refers to a fragment of the polypeptide or peptide. In some embodiments, a “portion” of a polypeptide or peptide retains at least one function and / or activity of the full-length polypeptide or peptide from which it was derived. For example, in some embodiments, if a full-length polypeptide binds a given ligand, a portion of that full-length polypeptide also binds to the same ligand.

[0118] The terms “protein” and “polypeptide” are used interchangeably herein.

[0119] As known in the art, “nucleic acid molecule,” “polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deox ribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylatednucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0120] A "gene," "polynucleotide," "coding region," "sequence," "nucleic acid sequence," "segment," "fragment," “genotype,” or "transgene" that "encodes" a particular protein, is a section of a nucleic acid molecule that is transcribed and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences. The coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence.

[0121] The term "cell" is herein used in its broadest sense in the art and refers to a living body that is a structural unit of tissue of a multicellular organism, is surrounded by a membrane structure that isolates it from the outside, has the capability of self-replicating, and has genetic information and a mechanism for expressing it.

[0122] Experimental Examples

[0123] Methods

[0124] Genome-wide association studies of circulating protein levels

[0125] GWAS analysis of the circulating levels of 4,907 proteins were conducted in the deCODE study, based on 35,559 Icelanders (see Ferkingstad et al. Large-scale integration of the plasma proteome with genetics and disease. Nat Genet. 2021;53(12): 1712-1721. doi: 10.1038 / s41588-021-00978-w). Circulating protein levels were measured using the SomaLogic SomaScan assay v.4. Details of this study have been described previously. Conditional analyses were performed to identify conditionally independent lead variants with a p-value < 0.05 / 27,200,000 = 1.8xl0'9, which represented the Bonferroni-corrected significance threshold accounting for 27.2 million variants tested throughout the genome for each protein. Cis-pQTL lead variants were defined for each protein as conditionally independent lead variants located within 1 Mb of the transcription start site of the proteincoding gene.

[0126] Genome-wide association studies of cerebrospinal fluid protein levels

[0127] GWAS analysis of the cerebrospinal fluid (CSF) levels of TPP1 was undertaken in a consortium of cohorts comprised of 3,506 samples (see Western D et al. Proteogenomic analysis of human cerebrospinal fluid identifies neurologically relevant regulation and implicates causal proteins for Alzheimer’s disease. Nat Genet. 2024;56(12):2672-2684). Circulating levels of TPP1 were measured using the Soma logic SomaScan 7k assay. Cis-pQTL lead variants for TPP1 that were conditionally independent and located within 1Mb of the transcription start site of TPP1 were identified.

[0128] Genome-wide association study of amyotrophic lateral sclerosis

[0129] Applicants obtained the summary statistics from the largest ALS GWAS meta-analysis, encompassing 117 cohorts (see van Rheenen W et al. Common and rare varsriant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology. Nat Genet. 2021;53(12): 1636-1648. doi: 10.1038 / s41588-021- 00973-1). The European ancestry-specific analysis of this study included a total of 27,205 patients with ALS and 110,881 control subjects. The participants were not selected for a family history of ALS. There was no known overlap between the participants of the deCODE study and participants of this meta-analysis.

[0130] Mendelian randomization

[0131] Applicants identified candidate circulating proteins whose protein-coding genes are located within 1 Mb of any variants that had a p-value < LOxlO'5in the ALS GWAS dataset. Applicants performed MR analyses utilizing the cis-pQTL lead variants as genetic instruments. If a cis-pQTL lead variant was not available in the ALS GWAS summary statistics, a proxy was identified as the genetic instrument using the LDlink R package (see Machiela MJ and Chanock SJ. LDlink: a web-based application for exploring population-specific haplotype structure and linking correlated alleles of possible functional variants. Bioinforma Oxf Engl. 2015;31(21):3555-3557. doi: 10.1093 / bioinformatics / btv402). The proxy chosen included those in high linkage disequilibrium (LD; r2 > 0.8) with the cis-pQTL lead variant based on the LD reference panel consisting of non-Finnish European ancestry populations in the 1000 Genomes Project (see 1000 Genomes Project Consortium, Auton A, Brooks LD, et al. A global reference for human genetic variation. Nature. 2015;526(7571):68-74. doi: 10.1038 / naturel5393). GWAS summary statistics for genetic instruments were harmonized with forward strand alleles inferred using allele frequency information. Palindromic variants with a minor allele frequency > 0.42 were discarded to avoid strand mismatches.

[0132] Wald ratio estimates were derived for proteins with only one genetic instrument, while inverse variance weighted estimates were derived for proteins with two or more genetic instruments. Associations with a Benjamini -Hochberg-corrected p-value (false discovery rate; FDR) < 0.05, accounting for the number of proteins tested, were considered significant.

[0133] For significant associations where the circulating protein levels were instrumented using three or more genetic instruments, Applicants conducted sensitivity analyses using the weighted median, penalized weighted median, and weighted mode methods (see Burgess S et al. A review of instrumental variable estimators for Mendelian randomization. Stat Methods Med Res. 2017;26(5):2333-2355. doi:10.1177 / 0962280215597579; see also Bowden J et al. A framework for the investigation of pleiotropy in two-sample summary data Mendelian randomization. Stat Med. 2017;36(l 1): 1783-1802. doi: 10.1002 / sim.7221; see Bowden J et al. Consistent Estimation in Mendelian Randomization with Some Invalid Instruments Using a Weighted Median Estimator. Genet Epidemiol. 2016;40(4):304-314. doi: 10.1002 / gepi.21965). These different methods were examined to determine if they yielded estimates with a consistent effect direction and magnitude. The mendelian randomization (MR)-Egger regression was implemented, wherein a significant MR-Egger intercept (p-value < 0.05) would indicate a risk of directional horizontal pleiotropy (see Bowden J et al. Mendelian randomization with invalid instruments: effect estimation and bias detection through Egger regression. Int J Epidemiol. 2015;44(2):512-525. doi: 10.1093 / ije / dyv080). Furthermore, the F-statistic for each test was calculated, where an F-statistic > 10 would indicate a low risk of weak instrument bias (see Burgess S, Thompson SG, CRP CHD Genetics Collaboration. Avoiding bias from weak instruments in Mendelian randomization studies. Int J Epidemiol. 2011;40(3):755-764. doi: 10.1093 / ije / dyr036). MR analyses were conducted using the TwoSampleMR R package version 0.5.6 (see Hemani G, Zheng J, Elsworth B, et al. The MR-Base platform supports systematic causal inference across the human phenome. eLife. 2018;7:e34408. doi: 10.7554 / eLife.34408).

[0134] Colocalization assessment

[0135] For significant circulating protein-ALS associations, colocalization analyses was performed using PWCoCo to guard against potential confounding due to LD (see Zheng J, Haberland V, Baird D, et al. Phenome-wide Mendelian randomization mapping the influence of the plasma proteome on complex diseases. Nat Genet. 2020;52(10): 1122-1131. doi: 10.1038 / s41588-020-0682-6; see also Zuber V, Grinberg NF, Gill D, et al. Combining evidence from Mendelian randomization and colocalization: Review and comparison of approaches. Am J Hum Genet. 2022;109(5):767-782. doi: 10.1016 / j.ajhg.2022.04.001).Applicants utilized GWAS summary statistics of all variants located within 500 kb of the genetic instruments and an LD reference panel based on 5,000 randomly selected unrelated European ancestry individuals from the UK Biobank to infer the colocalization probability for each protein- ALS association. Default priors of PWCoCo were used, i.e. pi (prior probability of the exposure having a causal variant) = l.OxlO'4, p2 (prior probability of the outcome having a causal variant) = l.OxlO'4, and pn (prior probability of the exposure and the outcome sharing the same causal variant) = l.OxlO'5. A colocalization probability > 80% was considered strong evidence of colocalization.

[0136] Horizontal pleiotropy assessment

[0137] For significant circulating protein-ALS associations supported by strong colocalization evidence, Applicants further examined whether the genetic instruments have been associated with circulating levels of other proteins. Each genetic instrument was queried in the Open Targets Genetics database (see Mountjoy E, Schmidt EM, Carmona M, et al. An open approach to systematically prioritize causal variants and genes at all published human GWAS trait- associated loci. Nat Genet. 2021;53(l 1): 1527-1533. doi: 10.1038 / s41588-021-00945-5) and pQTL evidence scores were obtained that link each variant to its neighboring genes. A nonzero pQTL evidence score linking a genetic instrument to a neighboring gene other than the protein-coding gene would indicate a risk of horizontal pleiotropy. Protein-ALS associations that were unlikely to be biased by such horizontal pleiotropy were prioritized.

[0138] Gene expression in brain tissues

[0139] Applicants next examined whether the expression of the prioritized protein-coding genes in brain tissues was also associated with the risk of ALS. Genetic associations with mRNA levels were obtained from the cis-expression quantitative trait loci (cis-eQTL) analyses from the Genotype-Tissue Expression (GTEx) database version 8 (see THE GTEX CONSORTIUM. The GTEx Consortium atlas of genetic regulatory effects across human tissues. Science. 2020;369(6509): 1318-1330. doi: 10.1126 / science.aazl776). MR analyses were performed using the cis-eQTL lead variants as genetic instruments. Colocalization analyses were performed using PWCoCo with default priors as described above.

[0140] Protein measurement in cerebrospinal fluid

[0141] Finally, Applicants investigated whether the prioritized proteins have been measured in cerebrospinal fluid (CSF). CSF protein profiles were obtained from a recent multicenter cohort study of Alzheimer’s disease (see Bader JM, Geyer PE, Muller JB, et al. Proteome profiling in cerebrospinal fluid reveals novel biomarkers of Alzheimer's disease. Mol Syst Biol. 2020;16(6):e9356. doi:10.15252 / msb.20199356). CSF protein levels underwent rank-based inverse normaltransformation. The association between the normalized CSF protein levels and age and sex was assessed using multiple linear regression, adjusted for Alzheimer’s disease status.

[0142] Results

[0143] After data harmonization, 126 circulating proteins had coding genes located within 1 Mb of loci demonstrating suggestive associations with the risk of ALS (p-value < l.OxlO'5in the ALS GWAS). The genetic instruments of these proteins are presented in Table 1.

[0144] MR analyses identified nine circulating protein-ALS associations that had an FDR < 0.05 (Table 2). The minimal F-statistic of these nine tests was 43.5, suggesting a low risk of weak instrument bias. Three or more genetic instruments were used for assessing the associations between circulating levels of TPP1, SHBG, and TXNDC15 and the risk of ALS. Highly consistent MR estimates were obtained in sensitivity analyses using alternative methods (Table 3). No evidence of directional horizontal pleiotropy was identified (Table 3).

[0145] Two of these nine significant circulating protein-ALS associations were supported by strong colocalization evidence (Table 4). Specifically, a one standard deviation increase in genetically predicted circulating levels of TPP1 was associated with an odds ratio (OR) of 0.76 for ALS (95% CL 0.65-0.88; p-value = 4.0xl0'4; FDR = 7.3xl0'3; colocalization probability = 93.5%; FIG. 1). Meanwhile, a one standard deviation increase in genetically predicted circulating levels of SHBG was associated with an OR of 1.19 for ALS (95% CL 1.07-1.33; p- value = 1.2xl0'3; FDR = 1.7xl0'2; colocalization probability = 87.2%). However, the genetic instruments for circulating SHBG levels have been associated with the circulating levels of other proteins, including TNFSF12 and ATP1B2, whose coding genes are close to SHBG (Table 5). In contrast, the TPP1-ALS association was unlikely to be biased by such horizontal pleiotropy (Table 5).

[0146] TPP1 expression was profiled in multiple tissues available in the GTEx database. Amongst the brain tissues, significant genetic associations with TPP1 expression were only detected in the cerebellum based on 209 individuals. MR analyses based on cis-eQTL associations showed that a one standard deviation increase in genetically predicted cerebellum TPP1 expression levels was associated with an OR of 0.81 for ALS (95% CL 0.72-0.90; p- value = 1.5xl0'4; Table 6). The genetic associations with cerebellum 7PP1 expression levels and circulating plasma TPP1 levels had a high colocalization probability of 90.3%, while the genetic associations with cerebellum TPP1 expression levels and the risk of ALS had a moderate colocalization probability of 72.7% (FIG. 1).

[0147] In cis-pQTL MR analyses to test the causal effect of decreased CSF levels of TPP1, results were observed that were directionally consistent with the plasma cis-pQTL MRfindings. Specifically, a one standard deviation decrease in CSF TPP1 levels was associated with an increase in the odds of ALS, however, the 95% confidence intervals included the null (p-value = 0.20) and there was little evidence of colocalization (12% using pair-wise conditional and colocalization (PWCoco) as a method to assess colocalization; see). While directionally consistent with the plasma MR findings, these results could be under-powered and this may have been the reason why the 95% confidence intervals included the null.

[0148] The association of TPP1 levels in CSF with Alzheimer’s disease. Based on 188 individuals (85 patients with Alzheimer’s disease and 103 control subjects) with measured CSF TPP1 levels, suggestive observational associations between CSF TPP1 levels and age and sex were identified (FIG. 2 and Table 7). Specifically, a one-year increase in age was associated with a 0.018 standard deviation decrease in CSF TPP1 levels (95% CI: 0.006-0.029; 4.0xl0'3). Additionally, CSF TPP1 levels in males were, on average, 0.362 standard deviations lower than those in females (95% CI: 0.083-0.641; p-value = 1.2xl0'2). CSF TPP1 levels were not associated with Alzheimer’s disease status.

[0149] Without wishing to be bound by theory, the results described in this example demonstrate that subjects having decreased expression and / or activity of TPP1 results in a higher risk of ALS. Accordingly, the levels of expression and activity of TPP1, as well as the identification of specific TPP1 genotypes, can be used to determine a subject’s risk for ALS, as well as indicate whether they are a potential candidate for treatment with a TPP1 -modulating agent.Table 1. Association results for all instruments, “exp” is “exposure”; “oc” is “outcome”Table 2. Primary Mendelian randomization results. Beta represents the log-OR for amyotrophic lateral sclerosis per 1 SD increase in genetically predicted circulating protein levels.

[0150] Table 3. Mendelian randomization results using alternative methods. Beta represents the log-OR for amyotrophic lateral sclerosis per 1 SD increase in genetically predicted circulating protein levels.Table 4. Colocalization results for significant associations between circulating proteins and amyotrophic lateral sclerosis.Table 5. Open Targets Genetics pQTL evidence score. A non-zero pQTL evidence score indicates association between the variant and the circulating levels of the corresponding protein.Table 6. Mendelian randomization and colocalization results based on cerebellum cis-eQTL of TPP1.Table 7. Association between cerebrospinal fluid TPP1 levels and age and sex. Association test was performed using multiple linear regression.

Claims

What is claimed is:

1. A method of identifying the risk that a subject will develop ALS, the method comprising: a) determining the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in a biological sample from the subject; b) comparing the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in the biological sample to a corresponding predetermined cutoff value; and c) determining that the subject is at low risk of developing ALS if the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in the biological sample is greater than or equal to the corresponding predetermined cutoff value, or determining that the subject is at a high risk of developing ALS if the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration is less than the corresponding predetermined cutoff value.

2. The method of claim 1, wherein the corresponding predetermined cutoff value is determined through the analysis of the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in a training set, wherein the training set comprises the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration measured in biological samples from one or more training subjects, wherein the one or more training subjects comprise a plurality of training subjects that do not have ALS and a plurality of training subjects that have ALS.

3. The method of claim 2, wherein the analysis comprises genome-wide association study (GWAS) analysis and / or mendelian randomization (MR) analysis.

4. A method of identifying the risk that a subject will develop ALS, the method comprising: a) determining the TPP1 genotype of the subject; and b) determining the risk that the subject will develop ALS based on the TPP1 genotype.

5. The method of claim 4, wherein the identifying the TPP1 genotype of the subject comprises determining if the subject is major allele homozygous, minor allele heterozygous, or minor allele homozygous at a specific locus associated with a specific single nucleotides polymorphism (SNP).

6. The method of claim 5, wherein the specific locus is rs3758978, wherein major allele at rs3758978 is cytosine (C) and the minor allele is guanine (G), wherein subjects with a major allele homozygous or minor allele heterozygous rs3758978 genotype are identified as being at increased risk of ALS and subjects with a minor allele homozygous rs3758978 genotype are identified as being at decreased risk of ALS.

7. The method of claim 5, wherein the specific locus is a locus that is in linkage disequilibrium with rs3758978.

8. A method of identifying a subject having ALS for treatment with a TPP1 -modulating agent, the method comprising: a) determining the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in a biological sample from the subject; b) comparing the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in the biological sample to a corresponding predetermined cutoff value; and c) identifying the subject for treatment with the TPP1 -modulating agent if the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration is less than or equal to the corresponding predetermined cutoff value.

9. A method of identifying a subject having ALS for treatment with a TPP1 -modulating agent, the method comprising: a) determining the TPP1 genotype of the subject; and b) identifying whether the subject should be treated with the TPP1 -modulating agent based on the TPP1 genotype.

10. The method of claim 9, wherein the identifying the TPP1 genotype of the subject comprises determining if the subject is major allele homozygous, minor allele heterozygous,or minor allele homozygous at a specific locus associated with a specific single nucleotides polymorphism (SNP).

11. The method of claim 9, wherein the specific locus is rs3758978, wherein major allele at rs3758978 is cytosine (C) and the minor allele is guanine (G), wherein subjects with a major allele homozygous or minor allele heterozygous rs3758978 genotype are identified to be treated with a TPPl-modualting agent.

12. The method of claim 9, wherein the specific locus is a locus that is in linkage disequilibrium with rs3758978.

13. A method of determining the responsiveness of a subject to an ALS treatment, the method comprising: a) at a first time point, determining the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in a biological sample from the subject; b) at a second time point, determining the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration in a biological sample from the subject; c) comparing the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration at the first time point and the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration at the second time point; and d) identifying that the subject is responding to the ALS treatment when the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration at the second time point is greater than the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration at the first time point, or identifying that the subject is not responding to the ALS treatment when the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration at the second time point is the same or less than the TPP1 expression level, the TPP1 activity level, and / or the TPP1 protein concentration at the first time point.

14. The method of claim 13, wherein first time point is before the administration of the ALS treatment and the second time point is after the administration of at least one dose of the ALS treatment.

15. The method of claim 13, wherein the first time point is after the initial administration of the ALS treatment and the second time point is after one or more additional doses of the ALS treatment has been administered to the subject.

16. The method of any one of claims 13-15, further comprising changing the dosage of the ALS treatment based on the change in the expression level and / or activity level of TPP1 between the first time point and the second time point.

17. The method of any one of the preceding claims, wherein the biological sample comprises blood, plasma, serum, urine, breast milk, cerebrospinal fluid, mucus, gastric juice, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, endolymph, perilymph, neuronal tissue, or any combination thereof, preferably wherein the biological sample comprises plasma or cerebrospinal fluid.

18. The method of any one of the preceding claims, wherein determining the TPP1 expression level comprises the use of PCR, high-throughput sequencing, next generation sequencing (NGS), Northern Blot, reverse transcription PCR (RT-PCR), real-time PCR (qPCR), quantitative PCR, qRT-PCR, flow cytometry, microarray analysis, digital droplet PCR, RNA sequencing, or any combination thereof.

19. The method of any one of the preceding claims, wherein determining TPP1 protein concentration comprises mass spectrometry, western blot, aptamer-based methods, enzyme- linked immunoabsorbent assays (ELISAs) measurements, proximity extension assays, or any combination thereof.

20. The method of any one of the preceding claims, wherein determining TPP1 activity level comprises the use of one or more enzymatic assays, activity-based protein profiling (ABPP), one or more functional assays, one or more biosensors, or any combination thereof.

21. A method of treating ALS in a subject, the method comprising administering to a subject a TPP1 -modulating agent, wherein the TPP1 -modulating agent is administered in one or more amounts sufficient to increase TPP1 activity, TPP1 expression, and / or TPP1 protein concentration in a pluralityof cells in the subject such that the TPP1 activity, TPP1 expression, and / or TPP1 protein concentration in the plurality of cells is greater than a predetermined cutoff value.

22. The method of claim 21, wherein the TPP1 -modulating agent comprises a gene therapy, a small molecule, an enzyme-replacement therapy, or any combination thereof.

23. The method of claim 21 or claim 22, wherein the subject has been identified to have a TPP1 genotype that is associated with ALS.

24. The method of any one of claims 21-23, wherein the method further comprises administering at least one additional therapy, preferably wherein the at least one additional therapy is selected from riluzole, edaravone, a neurotrophic factor, and an anti-inflammatory agent.

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