WO2022015810A2 - Biomarkers of trpml1 activation - Google Patents

Abstract

The present disclosure pertains to biomarkers of TRPML1 modulation and methods and kits using the same.

Description

BIOMARKERS OF TRPML1 ACTIVATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No.

63/053,416, filed July 17, 2020, which is incorporated herein in its entirety.

SEQUENCE LISTING

[0002] In accordance with 37 CFR 1.52(e)(5), a Sequence Listing in the form of an

ASCII text file (entitled “2013075-0044_SL.txt”, created on July 14, 2021, and 2,567 bytes in size) is incorporated herein by reference in its entirety.

BACKGROUND

[0003] The TRPML1 calcium channel resides within the limiting membrane of lysosomes and is ubiquitously expressed. Channel activity is required for lysosomal exocytosis, a process that is required for plasma membrane repair in the context, for example, of mechanical, chemical or toxin injury. Absence of a functional TRPMLl channel underlies the human lysosomal storage disease Mucolipidosis IV, which is characterized by cognitive deficits, retinal degeneration and delayed motor milestones. In the murine mcolnl (TRPMLl) knockout model, a prominent phenotype is muscular dystrophy. Pharmacological activation of the channel with TRPMLl agonists leads to (1) cytoplasmic to nuclear translocation of Transcription Factor EB (TFEB) and upregulation of expression of TFEB target genes involved in the autophagy pathway and lysosomal biogenesis, and (2) induction of lysosomal exocytosis.

[0004] There are no established target engagement or pharmacodynamic biomarkers for

TRPMLl agonists.

SUMMARY

[0005] It has recently been discovered that upregulation of autophagy is beneficial to patients suffering from a number of diseases and disorders. For example, it has been reported that inducing autophagy promotes clearance of hepatotoxic alpha- 1 -anti -trypsin (ATZ) in the liver. See Pastore, et al, EMBO Mol. Med. 5(3): 397-412 (Mar. 2013). Moreover, autophagy was recently found to be useful in the treatment of neurodegenerative disorders, cancer, and heart disease. See Pierzynowska, etal. , Metab. Brain Dis., 33(4); 989-1008 (2018) (discussing neurodegenerative disorders); Nelson & Shacka, Curr. Pathobiol. Rep., 1(4): 239-245 (2013) (discussing cancer); Sciaretta, et al. , Annual Review of Physiology, 80:1-26 (2018) (discussing heart disease); Maiuri & Kroemer, Cell Death & Differentiation, 26: 680-689 (2019) (discussing therapeutic applications of autophagy, generally).

[0006] The present disclosure provides, among other things, biomarkers of activation of autophagy regulators (e.g., TRPMLl). The present application encompasses the insight that certain cellular components are useful biomarkers of TRPMLl modulation (e.g., activation).

The present disclosure also provides certain technologies for use in medicine, and in particular for identifying, characterizing, and/or manufacturing certain agents and/or compositions that are useful in treating certain diseases, disorders or conditions.

[0007] In one aspect, the present disclosure provides methods comprising a step of: determining a level of one or more biomarkers in a sample from a subject, wherein one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, T -O-methyl guanosine, T -O-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

[0008] In some embodiments, a step of determining comprises determining the level of one or more biomarkers relative to a reference level of the one or more biomarkers.

[0009] In some embodiments, a subject is receiving or has received a therapy comprising a TRPMLl agonist.

[0010] In some embodiments, methods of the present disclosure further comprise a step of modifying a therapy after a step of determining. In some embodiments, a step of modifying a therapy comprises increasing frequency and/or dosage of a TRPMLl agonist. In some embodiments, a step of modifying a therapy comprises decreasing frequency and/or dosage of a TRPMLl agonist. In some embodiments, a step of modifying a therapy comprises ceasing administration of a TRPMLl agonist.

[0011] In some embodiments, methods of the present disclosure further comprise a step of continuing a therapy after a step of determining.

[0012] In some embodiments, a sample comprises serum, plasma, cerebrospinal fluid, urine, extracellular vesicles, exosomes, peripheral blood mononuclear cells, or biopsy specimens and one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine, monophosphate, uridine monophosphate, xanthosine monophosphate, T -O-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, 2’-0-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

[0013] In some embodiments, a sample comprises cells or tissues and one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, T -O-methyl guanosine, T -O-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

[0014] In some embodiments, methods of the present disclosure further comprise a step of lysing cells or performing a tissue biopsy and one or more biomarkers include one or more intracellular biomarkers. In some embodiments, one or more intracellular biomarkers comprise adenosine, cytidine, deoxyguanosine, guanine, guanosine, hypoxanthine, inosine, uridine, xanthosine, adenosine monophosphate, guanosine monophosphate, xanthosine monophosphate, cytidine monophosphate, inosine monophosphate or a combination thereof.

[0015] In another aspect, the present disclosure provides methods comprising a step of: administering a therapy comprising a TRPMLl agonist to a subject who has been determined to have a reduced level of one or more biomarkers relative to a reference level of one or more biomarkers, wherein one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, 2’-0-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

[0016] In another aspect, the present disclosure provides methods comprising a step of: characterizing a candidate therapy comprising a TRPMLl agonist by assessing its impact on one or more biomarkers in a subject to whom a candidate therapy is being or has been administered, wherein one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, T -O-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, 2’-0-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

[0017] In some embodiments, a subject is or comprises a model organism. In some embodiments, a subject is or comprises a cell culture. In some embodiments, a subject is or comprises a human. In some embodiments, a human is suffering from or susceptible to a muscular disease, a liver disease, a metabolic disease, an atherosclerotic disease, an inflammatory bowel disease, an atherosclerotic disease, a neurodegenerative disease, an infectious disease, an inflammatory disease, or an oncological disease.

[0018] In another aspect, the present disclosure provides kits comprising reagents to detect one or more biomarkers in a sample, wherein one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, T -O-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

[0019] In another aspect, the present disclosure provides methods comprising a step of: determining a level of one or more biomarkers in a sample from a subject, wherein one or more biomarkers comprise arachidonic acid, lysophosphatidylethanolamine (LPE) 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, lysophosphatidylcholine (LPC) 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4, or a combination thereof.

[0020] In some embodiments, a step of determining comprises determining a level of one or more biomarkers relative to a reference level of one or more biomarkers. [0021] In some embodiments, a subject is receiving or has received a therapy comprising a TRPML1 agonist.

[0022] In some embodiments, methods of the present disclosure further comprise a step of modifying a therapy after a step of determining. In some embodiments, a step of modifying a therapy comprises increasing the frequency and/or dosage of the TRPMLl agonist. In some embodiments, a step of modifying a therapy comprises decreasing the frequency and/or dosage of a TRPMLl agonist. In some embodiments, a step of modifying a therapy comprises ceasing administration of a TRPMLl agonist.

[0023] In some embodiments, methods of the present disclosure further comprise a step of continuing a therapy after a step of determining.

[0024] In some embodiments, a sample comprises serum, plasma, cerebrospinal fluid, urine, extracellular vesicles, exosomes, peripheral blood mononuclear cells, or biopsy specimens and one or more biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof. In some embodiments, a sample comprises cells and one or more biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2,

LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof.

[0025] In some embodiments, methods of the present disclosure further comprise a step of lysing cells or performing a tissue biopsy and one or more biomarkers include one or more intracellular biomarkers. In some embodiments, one or more intracellular biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof.

[0026] In another aspect, the present disclosure provides methods comprising a step of: administering a therapy comprising a TRPMLl agonist to a subject who has been determined to have a reduced level of one or more biomarkers relative to a reference level of one or more biomarkers, wherein one or more biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof. [0027] In another aspect, the present disclosure provides methods comprising a step of: characterizing a candidate therapy comprising a TRPMLl agonist by assessing its impact on one or more biomarkers in a subject to whom a candidate therapy is being or has been administered, wherein one or more biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof.

[0028] In some embodiments, a subject is or comprises a model organism. In some embodiments, a subject is or comprises a cell culture. In some embodiments, a subject is or comprises a human. In some embodiments, a human is suffering from or susceptible to a muscular disease, a liver disease, a metabolic disease, an atherosclerotic disease, an inflammatory bowel disease, an atherosclerotic disease, a neurodegenerative disease, or an oncological disease.

[0029] In another aspect, the present disclosure provides kits comprising reagents to detect one or more biomarkers in a sample, wherein one or more biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0,

LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof.

[0030] In another aspect, the present disclosure provides methods comprising a step of: determining a level of one or more biomarkers in a sample from a subject, wherein one or more biomarkers comprise acylcarnitine 14:0, acylcamitine 14:1, acylcarnitine 16:0, acylcarnitine 16:1, acylcamitine 18:0, acylcarnitine 18:1, acylcamitine 18:2, acylcarnitine 20:0, acylcarnitine 20:4, or a combination thereof.

[0031] In some embodiments, a step of determining comprises determining a level of one or more biomarkers relative to a reference level of one or more biomarkers.

[0032] In some embodiments, a subject is receiving or has received a therapy comprising a TRPMLl agonist.

[0033] In some embodiments, methods of the present disclosure further comprise a step of modifying a therapy after a step of determining. In some embodiments, a step of modifying a therapy comprises increasing frequency and/or dosage of a TRPMLl agonist. In some embodiments, a step of modifying the therapy comprises decreasing frequency and/or dosage of a TRPMLl agonist. In some embodiments, a step of modifying the therapy comprises ceasing administration of a TRPMLl agonist.

[0034] In some embodiments, methods of the present disclosure further comprise a step of continuing a therapy after a step of determining.

[0035] In some embodiments, a sample comprises serum, plasma, cerebrospinal fluid, urine, extracellular vesicles, exosomes, peripheral blood mononuclear cells, or biopsy specimens and one or more biomarkers comprise acylcamitine 14:0, acylcarnitine 14:1, acylcarnitine 16:0, acylcamitine 16:1, acylcarnitine 18:0, acylcamitine 18:1, acylcarnitine 18:2, acylcarnitine 20:0, acylcamitine 20:4, or a combination thereof.

[0036] In some embodiments, a sample comprises cells or tissues and one or more biomarkers comprise acylcarnitine 14:0, acylcamitine 14:1, acylcarnitine 16:0, acylcarnitine 16:1, acylcamitine 18:0, acylcarnitine 18:1, acylcamitine 18:2, acylcarnitine 20:0, acylcarnitine 20:4, or a combination thereof.

[0037] In some embodiments, methods of the present disclosure further comprise a step of lysing the cells or performing a tissue biopsy and one or more biomarkers include one or more intracellular biomarkers. In some embodiments, one or more intracellular biomarkers comprise acylcamitine 16:0, acylcarnitine 16:1, acylcamitine 18:1, or a combination thereof.

[0038] In another aspect, the present disclosure provides methods comprising a step of: administering a therapy comprising a TRPMLl agonist to a subject who has been determined to have a reduced level of one or more biomarkers relative to a reference level of one or more biomarkers, wherein one or more biomarkers comprise acylcamitine 14:0, acylcarnitine 14:1, acylcamitine 16:0, acylcarnitine 16:1, acylcamitine 18:0, acylcarnitine 18:1, acylcarnitine 18:2, acylcamitine 20:0, acylcarnitine 20:4, or a combination thereof.

[0039] In another aspect, the present disclosure provides methods comprising a step of: characterizing a candidate therapy comprising a TRPMLl agonist by assessing its impact on one or more biomarkers in a subject to whom a candidate therapy is being or has been administered, wherein one or more biomarkers comprise acylcamitine 14:0, acylcarnitine 14:1, acylcamitine 16:0, acylcamitine 16:1, acylcarnitine 18:0, acylcamitine 18:1, acylcarnitine 18:2, acylcarnitine 20:0, acylcamitine 20:4, or a combination thereof. [0040] In some embodiments, a subject is or comprises a model organism. In some embodiments, a subject is or comprises a cell culture. In some embodiments, a subject is or comprises a human. In some embodiments, a human is suffering from or susceptible to a muscular disease, a liver disease, a metabolic disease, an atherosclerotic disease, an inflammatory bowel disease, an atherosclerotic disease, a neurodegenerative disease, or an oncological disease.

[0041] In another aspect, the present disclosure provides kits comprising reagents to detect one or more biomarkers in a sample, wherein one or more biomarkers comprise acylcamitine 14:0, acylcarnitine 14:1, acylcamitine 16:0, acylcarnitine 16:1, acylcarnitine 18:0, acylcamitine 18:1, acylcarnitine 18:2, acylcamitine 20:0, acylcarnitine 20:4, or a combination thereof.

[0042] In another aspect, the present disclosure provides kits comprising reagents to detect one or more biomarkers in a sample, wherein the one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, T -O-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, arachidonic acid, lysophosphatidylethanolamine (LPE) 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, lysophosphatidylcholine (LPC) 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4, acylcarnitine 14:0, acylcarnitine 14:1, acylcarnitine 16:0, acylcamitine 16:1, acylcamitine 18:0, acylcarnitine 18:1, acylcamitine 18:2, acylcarnitine 20:0, acylcarnitine 20:4, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWING

[0043] The drawings are for illustration purposes only, not for limitation.

[0044] Figure 1 shows graphs illustrating release of nucleotide monophosphates into

THP-1 cell culture media after TRPML1 activation.

[0045] Figure 2 shows graphs illustrating release of nucleosides into THP-1 cell culture media after TRPML1 activation. [0046] Figure 3 shows a graph illustrating accumulation of hypoxanthine in THEM cells treated with TRPML1 activator.

[0047] Figure 4 shows graphs illustrating accumulation of T -O-methyl nucleosides in

PBMCs after TRPMLl activation.

[0048] Figures 5A and 5B show graphs illustrating accumulation of nucleosides in

PBMCs after TRPMLl activation.

[0049] Figures 6A and 6B show graphs illustrating accumulation of nucleotide monophosphates in peripheral blood mononuclear cells (PBMCs) after TRPMLl activation.

[0050] Figure 7 shows graphs illustrating release of arachidonic acid and lysophosphatidylethanolamine (18:1) into THP-1 cell culture media after TRPMLl activation.

[0051] Figure 8 shows a graph illustrating an increased arachidonic acid response in

THP-1 cells exposed to PLA2 inhibitors and a TRPMLl agonist.

[0052] Figure 9 shows graphs illustrating accumulation of acylcamitines in C2C12 myotubes after TRPMLl activation.

[0053] Figure 10 shows graphs illustrating accumulation of acylcamitines in C2C12 myotubes in a dose-dependent manner after TRPMLl activation.

[0054] Figure 11 shows a graph illustrating an increase in acylcamitines in C2C12 myoblasts after TRPMLl activation.

[0055] Figure 12 shows a graph illustrating an exemplary acylcarnitine 16:0 response in

THP-1 cells exposed to PLA2 inhibitors and a TRPMLl agonist.

[0056] Figure 13 shows graphs illustrating an increase in acylcamitines in mouse plasma after TRPMLl activation.

[0057] Figure 14 shows graphs illustrating an increase in acylcamitines in mouse muscle tissue after TRPMLl activation.

[0058] Figure 15 shows graphs illustrating an increase in acylcamitines in mouse heart tissue after TRPMLl activation. [0059] Figure 16 shows a western blot illustrating that an exemplary extracellular vesicle

(EV) precipitation methodology for EVs displays canonical protein markers. Whole cell lysate (WCL) served as a positive control for these EV markers.

[0060] Figure 17 shows a western blot illustrating an increase in canonical protein markers in extracellular vesicles (EVs) after TRPML1 activation. Whole cell lysate (WCL) served as a positive control for these EV markers.

[0061] Figures 18A and 18B show graphs illustrating an increase in extracellular vesicle

(EV) snoRNA after TRPML1 activation. This effect on EV snoRNA release was inhibited upon exposure of cells to excess TRPML1 inhibitor (Figure 18A) and was not the result of a false positive qPCR signal (Figure 18B).

DEFINITIONS

[0062] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

[0063] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0064] Agonist. As will be understood by those skilled in the art, the term “agonist” generally refers to an agent whose presence or level correlates with elevated level or activity of a target, as compared with that observed absent the agent (or with the agent at a different level). In some embodiments, an agonist is one whose presence or level correlates with a target level or activity that is comparable to or greater than a particular reference level or activity (e.g., that observed under appropriate reference conditions, such as presence of a known agonist, e.g., a positive control). In some embodiments, an agonist may be a direct agonist in that it exerts its influence directly on (e.g., interacts directly with) the target; in some embodiments, an agonist may be an indirect agonist in that it exerts its influence indirectly (e.g., by acting on, such as interacting with, a regulator of the target, or with some other component or entity.

[0065] Antagonist. As will be understood by those skilled in the art, the term

“antagonist” generally refers to an agent whose presence or level correlates with decreased level or activity of a target, as compared with that observed absent the agent (or with the agent at a different level). In some embodiments, an antagonist is one whose presence or level correlates with a target level or activity that is comparable to or less than a particular reference level or activity (e.g., that observed under appropriate reference conditions, such as presence of a known antagonist, e.g., a positive control). In some embodiments, an antagonist may be a direct antagonist in that it exerts its influence directly on (e.g., interacts directly with) the target; in some embodiments, an antagonist may be an indirect antagonist in that it exerts its influence indirectly (e.g., by acting on, such as interacting with, a regulator of the target, or with some other component or entity.

[0066] Biological sample. As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell- containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi- permeable membrane. Such a “processed sample” may comprise, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.

[0067] Biomarker. The term “biomarker” is used herein, consistent with its use in the art, to refer to a to an entity (or form thereof) whose presence, or level, correlates with a particular biological event or state of interest, so that it is considered to be a “marker” of that event or state. To give but a few examples, in some embodiments, a biomarker may be or comprise a marker for a particular disease state, or for likelihood that a particular disease, disorder or condition may develop, occur, or reoccur. In some embodiments, a biomarker may be or comprise a marker for a particular disease or therapeutic outcome, or likelihood thereof. Thus, in some embodiments, a biomarker is predictive, in some embodiments, a biomarker is prognostic, in some embodiments, a biomarker is diagnostic, of the relevant biological event or state of interest.

[0068] Dosing regimen or therapeutic regimen. Those skilled in the art will appreciate that the terms “dosing regimen” and “therapeutic regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0069] Patient or subject. As used herein, the term “patient” or “subject” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients or subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. In some embodiments, a patient or a subject is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient or subject displays one or more symptoms of a disorder or condition. In some embodiments, a patient or subject has been diagnosed with one or more disorders or conditions.

In some embodiments, a patient or a subject is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition.

[0070] Reference. As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

[0071] Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, and/or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid. In some embodiments, a biological fluid may be or comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.

[0072] Treat. As used herein, the terms “treat,” “treatment,” or “treating” refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

TRPML1 and Autophagy

[0073] Autophagy is a mechanism of the cell that degrades cytoplasmic material and organelles. There are multiple types of autophagy: (1) macroautophagy (generally referred to as autophagy); (2) microautophagy; and (3) chaperone-mediated autophagy. See Eskelinen & Saftig, Biochimica et Biophysica Acta - Mol. Cell Res., 1793(4):664-673 (2009). In macroautophagy, the autophagosome engulfs waste materials in the cytoplasm and fuses to the lysosome, where materials are delivered for degradation. The lysosome is as a subcellular organelle containing more than 50 soluble acid hydrolases useful for digesting cellular components. Fusion of the lysosome to the autophagosome is activated, in part, by release of ions through ion channels in the membrane of the lysosome, including Ca2+. See Cao, et al., J. Bio. Chem., 292(20)8424-8435 (2017).

[0074] Transient Receptor Potential Mucolipin-1 (also known as TRPMLl or MLl) is a

Ca2+ channel that is ubiquitously expressed in the lysosome and that regulates autophagy. See Wang, et al., PNAS, E1373-E1381 (March 2, 2015). In particular, TRPMLl is an inwardly rectifying current channel that transports cations from the lumen of the lysosome to the cytosol. See Di Paolda, et al., Cell Calcium 69:112-121 (2018). Release of Ca2+ from the lysosome via TRPMLl modulates transcription factor EB activity via local calcineurin activation, which ultimately induces autophagy and lysosomal biogenesis. See Medina, et al., Nat. Cell. Biol., 17(3):288-299 (2015).

[0075] TRPMLl channel activity is required for lysosomal exocytosis, a process that is required for plasma membrane repair in the context, for example, of mechanical, chemical or toxic injury. Absence of a functional TRPMLl channel underlies the human lysosomal storage disease Mucolipidosis IV, which is characterized by cognitive deficits, retinal degeneration and delayed motor milestones. In a murine mcolnl (TRPMLl) knockout model, a prominent phenotype is muscular dystrophy. Pharmacological activation of the TRPMLl channel with agonists leads to (1) cytoplasmic to nuclear translocation of Transcription Factor EB (TFEB) and upregulation of expression of TFEB target genes involved in the autophagy pathway and lysosomal biogenesis, and (2) induction of lysosomal exocytosis. [0076] It has recently been discovered that upregulation of autophagy is beneficial to patients suffering from a number of diseases and disorders. For example, it has been reported that inducing autophagy promotes clearance of hepatotoxic alpha- 1 -anti -trypsin (ATZ) in the liver. See Pastore, et ak, EMBO Mol. Med. 5(3): 397-412 (Mar. 2013). Moreover, autophagy was recently found to be useful in the treatment of neurodegenerative disorders, cancer, and heart disease. See Pierzynowska, et ak, Metab. Brain Dis., 33(4); 989-1008 (2018) (discussing neurodegenerative disorders); Nelson & Shacka, Curr. Pathobiok Rep., 1(4): 239-245 (2013) (discussing cancer); Sciaretta, et ak, Annual Review of Physiology, 80:1-26 (2018) (discussing heart disease); Maiuri & Kroemer, Cell Death & Differentiation, 26: 680-689 (2019) (discussing therapeutic applications of autophagy, generally). It is, therefore, desirable to assess one or more features of TRPMLl activity.

TRPML1 Modulators

[0077] In some embodiments, the present disclosure utilizes TRPML1 modulators of a chemical class selected from the group consisting of polypeptides, nucleic acids, lipids, carbohydrates, small molecules, metals, and combinations thereof. In some embodiments, TRPMLl modulators are small molecule compounds. In some embodiments, a TRPML1 modulator comprises ML-SA1, ML-SA3, ML-SA5, MK6-83, C8 (see WO 2018/005713), or C2 (see WO 2018/005713). In some embodiments, a TRPMLl modulator may show activity in one or more assays as described herein. In some embodiments, a small molecule compound is determined to be a TRPMLl modulator by showing activity in a TFEB assay wherein TFEB translocation is measured after wild-type and TRPMLl knock-out HeLa cells are treated with the small molecule compound. In some embodiments, a small molecule compound is determined to be a TRPMLl modulator by showing endogenous lysosomal calcium flux activity in an assay comprising Fluorescent Imaging Plate Reader (FLIPR) technology performed on wild-type and TRPMLl knock-out HeLa cells treated with the small molecule compound. In some embodiments, a small molecule compound is determined to be a TRPMLl modulator by showing exogenous calcium flux activity in an assay comprising Fluorescent Imaging Plate Reader (FLIPR) technology performed on a cell line that expresses tetracycline-inducible TRPMLl on the cell surface and has been treated with the small molecule compound. Biomarkers of TRPML1 Activation

[0078] Small molecule activation of the TRPMLl ion channel leads to rapid release of calcium and other divalent cations from the lysosome. Calcium is an important second messenger and enzymatic substrate that catalyzes downstream processes after TRPMLl activation and leads to the increase of target engagement biomarkers in vitro and in vivo. The calcium dependent phospholipase cPLA2a is involved in the production of arachidonic acid and acylcamitines following TRPMLl activation. The cPLA2a enzyme cleaves membrane- associated phosphatidylethanolamine and phosphatidylcholine to produce free fatty acids, predominantly arachidonate, and lysophospholipids. While free fatty acids are an enzymatic byproduct and precursors for biosynthesis of downstream metabolites, lysophospholipids generated in situ in cell membranes promote negative curvature, providing the membrane architecture necessary for lysosomal fusion and exocytosis. The final step in exocytosis involves the release of intra-lysosomal material, including nucleotides, nucleosides and nucleobases, which are products of RNA catabolism.

[0079] TRPMLl agonists also stimulate exocytosis from late endosome/lysosomes, which can lead to the release of small, non-coding RNAs into the extracellular space. The most well-studied of these small, non-coding RNAs are microRNAs (miRNAs), which are found in plasma in small membrane-bound vesicles or exosomes and/or bound to lipoproteins (Vladi Nat Cell Bio 20079:654; Vickers Nat Cell Bio 2011 13:423). The abundance of different miRNAs in plasma, serum, and urine tracks with a spectrum of neurological, cardiovascular, oncologic, metabolic, and hematological disorders, suggesting that miRNAs in the blood can serve as biomarkers for disease processes (Cortez Nat Rev Clin Oncol 2011 8:467). RNA-Seq has also revealed the presence of small nucleolar RNAs (snoRNAs) in the medium of cultured cells (Lefebvre Sci Reports 20166:27680; Kaur Sci Reports 2018 8:2577; Wei Nat Comm 2017 8:1145) and in human plasma exosomes (Valleron Blood 2012 120:3997; Huang BMC Genomics 2013 14:319).

[0080] SnoRNAs are noncoding RNAs that guide chemical modifications of structural

RNAs. While snoRNAs primarily localize in the nucleolus, where their canonical function is to target nascent ribosomal RNAs for 2’-0-methylation, recent studies have shown that snoRNAs can traffic out of the nucleus. SnoRNAs are present in exosomes produced in and released from late endosomes/multivesicular bodies (MVB) and are released extracellularly via MVB fusion with the plasma membrane (Leidal Nat Cell Bio 202022: 187). RNA-Seq data indicate that extracellular vesicles released from cells contain snoRNAs. Thus, snoRNAs, like miRNAs, have the potential to serve as circulating biomarkers.

[0081] Acylcarnitines represent a distal biomarker of TRPMLl activation. Calcium- dependent initiation of cPLA2a activity provides the fatty acid substrates required for acyl-CoA production and further metabolism into acylcarnitines by carnitine palmitoyltransferase I (CPT1). The appearance of increased acylcarnitines after TRPMLl activation in vitro and in vivo is potentially due to the inability of beta-oxidation to keep up with the transient burst of acylcarnitines following TRPMLl activation. The process of shuttling acylcarnitines across the mitochondrial membranes by CPT1 and CACT for metabolism into acyl-CoA and their use in beta-oxidation is rate limiting and may underlie to the transient increase in acylcarnitines following TRPMLl activation.

[0082] Suitable biomarkers for the present invention may include any substances (e.g., nucleic acid components or cell membrane components) that can be used as an indicator of activation of the TRPMLl channel. Typically, a suitable biomarker has a characteristic that can be objectively measured and evaluated as an indicator. In some embodiments, a suitable biomarker of the present disclosure is differentially expressed between samples from a subject treated with a TRPMLl modulator and samples from a subject treated with a control treatment.

In some embodiments, a suitable biomarker of the present disclosure is differentially expressed between samples before and after treatment with a TRPMLl modulator.

Nucleic Acid Components

[0083] In some embodiments of the present disclosure, suitable biomarkers comprise one or more nucleic acid components. In some embodiments of the present disclosure, suitable biomarkers comprise one or more nucleobases. In some embodiments, a nucleobase comprises adenine, guanine, cytosine, thymine or uracil. In some embodiments of the present disclosure, suitable biomarkers comprise one or more nucleosides. In some embodiments, a nucleoside comprises adenosine, guanosine, cytidine, uridine, inosine, 2’-0-methyl adenosine, 2’-0-methyl cytidine, T -O-methyl guanosine, T -O-methyl uridine, or deoxyguanosine. In some embodiments of the present disclosure, suitable biomarkers comprise one or more nucleotides.

In some embodiments, a nucleotide comprises adenosine monophosphate, guanosine monophosphate, cytidine monophosphate, uridine monophosphate, inosine monophosphate, or xanthosine monophosphate. In some embodiments of the present disclosure, one or more suitable biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, T -O-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, 2’-0-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, or a combination thereof. In some embodiments of the present disclosure, suitable biomarkers comprise one or more small, non-coding RNAs. In some embodiments, a small, non-coding RNA is or comprises a microRNA (miRNA). In some embodiments, a small, non-coding RNA is or comprises a small nucleolar RNA (snoRNA). In some embodiments, a species of snoRNA is selected from exemplary species of snoRNA disclosed in Bouchard-Bourelle, P., et al., (2020) snoDB: an interactive database of human snoRNA sequences, abundance and interactions. Nucleic Acids Res. 48, D220-D225, which is incorporated herein by reference in its entirety. In some embodiments, a species of snoRNA is or comprises U32a, U33, U34 or U35a.

[0084] In some embodiments of the present disclosure, provided methods comprise a step of determining a level of one or more biomarkers in a sample from a subject, wherein one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, T -O-methyl guanosine, T -O-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA or a combination thereof.

Membrane Components

[0085] In some embodiments of the present disclosure, suitable biomarkers comprise one or more cellular membrane components. In some embodiments, a membrane component comprises arachidonic acid, lysophosphatidylethanolamine or lysophosphatidylcholine. In some embodiments of the present disclosure, a suitable biomarker comprises arachidonic acid. In some embodiments of the present disclosure, a suitable biomarker comprises lysophosphatidylethanolamine (LPE). In some embodiments, LPE comprises LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, or LPE 20:4. The nomenclature for LPE corresponds to acyl chain length and number of double bonds. For example, LPE 16:1 contains a 16 carbon acyl chain with 1 double bond. In some embodiments of the present disclosure, a suitable biomarker comprises lysophosphatidylcholine (LPC). The nomenclature for LPC corresponds to acyl chain length and number of double bonds. For example, LPC 16:1 contains a 16 carbon acyl chain with 1 double bond. In some embodiments, LPC comprises LPC 16:0, LPC 16:1,

LPC 18:0, LPC 18:1, LPC 18:2, or LPC 20:4. In some embodiments of the present disclosure, provided methods comprise a step of determining a level of one or more biomarkers in a sample from a subject, wherein one or more biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4, or a combination thereof.

Acyl-CoAs

[0086] In some embodiments of the present disclosure, suitable biomarkers comprise one or more fatty acyl CoAs. The nomenclature for acyl CoAs correspond to acyl chain length and number of double bonds. For example, acyl CoA16:l contains a 16 carbon acyl chain with 1 double bond. In some embodiments, one or more acyl CoAs comprise acyl CoA14:0, acyl CoA14:l, acyl CoA16:0, acyl CoA16:l, acyl CoA18:0, acyl CoA18:l, acyl CoA18:2, acyl CoA20:0, acyl CoA20:4, or a combination thereof. In some embodiments of the present disclosure, provided methods comprise a step of determining a level of one or more biomarkers in a sample from a subject, wherein one or more biomarkers comprise acyl CoA14:0, acyl CoA14:l, acyl CoA16:0, acyl CoA16:l, acyl CoA18:0, acyl CoA18:l, acyl CoA18:2, acyl CoA20:0, acyl CoA20:4, or a combination thereof.

Acylcarnitines

[0087] In some embodiments of the present disclosure, suitable biomarkers comprise one or more acylcarnitines. The nomenclature for acylcarnitines correspond to acyl chain length and number of double bonds. For example, acylcarnitine 16:1 contains a 16 carbon acyl chain with 1 double bond. In some embodiments, one or more acylcarnitines comprise acylcarnitine 14:0, acylcarnitine 14:1, acylcarnitine 16:0, acylcarnitine 16:1, acylcarnitine 18:0, acylcarnitine 18:1, acylcarnitine 18:2, acylcarnitine 20:0, acylcarnitine 20:4, or a combination thereof. In some embodiments of the present disclosure, provided methods comprise a step of determining a level of one or more biomarkers in a sample from a subject, wherein one or more biomarkers comprise acylcarnitine 14:0, acylcarnitine 14:1, acylcarnitine 16:0, acylcarnitine 16:1, acylcarnitine 18:0, acylcarnitine 18:1, acylcarnitine 18:2, acylcarnitine 20:0, acylcarnitine 20:4, or a combination thereof.

Uses

[0088] In some embodiments of the present disclosure, biomarkers provided herein permit assessment of one or more features of TRPMLl activity. In some embodiments, such assessment may be useful in assessing and/or monitoring patient population(s) (e.g., to assess suitability for, likely responsiveness to, and/or progress of therapy, for example with TRPMLl agonist therapy). In some embodiments, such assessment may be useful in identifying (e.g., screening) and/or characterizing one or more TRPMLl modulators and/or modulating treatments (e.g., agonists/agonistic treatments). In some embodiments, such assessment may be useful in patient stratification.

[0089] In some embodiments of the present disclosure, provided methods comprise a step of determining a level of one or more biomarkers in a sample from a subject, wherein one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, T -O-methyl guanosine, T -O-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4, acylcarnitine 14:0, acylcarnitine 14:1, acylcarnitine 16:0, acylcarnitine 16:1, acylcarnitine 18:0, acylcarnitine 18:1, acylcarnitine 18:2, acylcarnitine 20:0, acylcarnitine 20:4, or a combination thereof. In some embodiments, the step of determining comprises determining the level of one or more biomarkers relative to a reference level of the one or more biomarkers. Monitor therapy

[0090] In some embodiments of the present disclosure, provided methods comprise monitoring a therapy a subject is receiving or has received. In some embodiments, the subject is receiving or has received a therapy comprising a TRPMLl modulator (e.g., agonist). In some embodiments, provided methods further comprise a step of modifying a therapy after the step of determining a level of one or more biomarkers in a sample from a subject. In some embodiments, a step of modifying a therapy comprises increasing frequency and/or magnitude of dosing of a TRPMLl modulator (e.g., agonist). In some embodiments, a step of modifying a therapy comprises decreasing frequency and/or magnitude of dosing of a TRPMLl modulator (e.g., agonist). In some embodiments, a step of modifying a therapy comprises ceasing administration of a TRPMLl modulator (e.g., agonist). In some embodiments, provided methods further comprise a step of continuing the therapy after the step of determining a level of one or more biomarkers in a sample from a subject.

[0091] In some embodiments of the present disclosure, provided methods comprise a step of administering a therapy comprising a TRPMLl modulator (e.g., agonist) to a subject who has been determined to have a reduced level of one or more biomarkers relative to a reference level of the one or more biomarkers. In some embodiments of the present disclosure, provided methods comprise a step of characterizing a candidate therapy comprising a TRPMLl modulator (e.g., agonist) by assessing its impact on one or more biomarkers in a subject to whom the candidate therapy is being or has been administered. In some embodiments, a subject is or comprises a model organism. Examples of model organisms include, but are not limited to, rodents, dogs, non-human primates and humans. In some embodiments, a subject is or comprises a human. In some embodiments, a subject is or comprises a cell culture.

[0092] In some embodiments, a human is suffering from or susceptible to a muscular disease. In some embodiments, a muscular disease is a muscular dystrophy. In some embodiments, a muscular dystrophy is or comprises limb girdle muscular dystrophy type 1 A

(myofibrillar myopathy), IB (Emery-Dreifuss musclar dystrophy), 1C (rippling muscle disease),

ID (D1 DNAJB6-related LGMD), IE (myofibrillar myopathy), IF (D2 TNP03-related LGMD), 1G (D3 HNRNPDL-related LGMD), 1H, 1L (D4 calpain3 -related LGMD), 2A (R1 calpain3- related LGMD), 2B (R2 dysferlin-related LGMD), 2C (R5 g-sarcoglycan-related LGMD), 2D (R3 a-sarcoglycan-related LGMD), 2E (R4 b-sarcoglycan-related LGMD), 2F (R6 d- sarcoglycan-related LGMD), 2G (R7 telethonin-related LGMD), 2H (R8 TRIM 32-related LGMD), 21 (R9 FKRP-related LGMD), 2J (RIO titin-related LGMD), 2K (R11 POMT1 -related LGMD), 2L (R12 anoctamin 5-related LGMD), 2M (R13 Fukutin-related LGMD), 2N (R14 POMT2-related LGMD), 20 (R15 POMGnTl -related LGMD), 2P (R16 a -dystrogly can-related LGMD), 2Q (R17 plectin-related LGMD), 2R (myofibrillar myopathy), 2S (R18 TRAPPC11- r elated LGMD), 2T (R19 GMPPB-related LGMD), 2U (R20 ISPD-related LGMD), 2V (Pompe disease), 2W (PINCH-2-related myopathy), 2X (BVES-related myopathy), or 2Y (TORIAIPI- related myopathy). In some embodiments, a human is suffering from or susceptible to a liver disease (e.g., alpha-1 antitrypsin deficiency), a metabolic disease (e.g., NAFLD, NASH, lysosomal storage diseases, and adenosine deaminase deficiency), an atherosclerotic disease (e.g., coronary artery disease, stroke, peripheral vascular disease, and age-related macular degeneration), an inflammatory bowel disease (e.g., Crohn’s disease and ulcerative colitis), a neurodegenerative disease (e.g., polyglutamine expansion disorders (Huntington disease and spinocerebellar ataxias SCA1, SCA2, SCA3, SCA6, SCA7 and SCA17), disorders of protein aggregation (e.g., tauopathies, such as Alzheimer disease, Frontotemporal dementia, progressive supranuclear palsy, Niemann-Pick type Cl and C2 diseases), Parkinson’s disease and Lewy Body dementia), an infectious disease (e.g., H. pylori-induced gastritis and peptic ulcer disease, tuberculosis, salmonellosis, and listeriosis), an inflammatory disorder (e.g., Crohn’s disease, ulcerative colitis, asthma, peptic ulcer disease, autoimmune disorders (e.g., systemic lupus erythematosis and rheumatoid arthritis), periodontitis, hepatitis, and tuberculosis), or an oncological disease (e.g., blood disorders (e.g., leukemias, myelomas, and lymphomas) and solid tumors (e.g. breast cancers, colon cancers, pancreatic cancers, liver cancers, lung cancers, osteosarcomas, rhabdosarcomas, head and neck cancers, and brain cancers).

Biological sample

[0093] Methods of the present disclosure may be applied to any type of biological samples allowing one or more biomarkers of the present disclosure to be assayed. Examples of suitable biological samples include, but are not limited to, serum, plasma, cerebrospinal fluid, urine, extracellular vesicles, exosomes, circulating blood cells (e.g., peripheral blood mononuclear cells), and biopsy specimens. In some embodiments, extracellular vesicles are derived from blood. In some embodiments, extracellular vesicles are derived from cerebrospinal fluid. In some embodiments, exosomes are derived from blood. In some embodiments, exosomes are derived from cerebrospinal fluid. In some embodiments, a sample comprises cells or tissues. In some embodiments, provided methods further comprises a step of lysing cells or performing a tissue biopsy and one or more biomarkers include one or more intracellular biomarkers. Biological samples suitable for the present disclosure may be fresh or frozen samples collected from a subject, or archival samples with known diagnosis, treatment and/or outcome history. Biological samples may be collected by any invasive or non-invasive means, such as, for example, by drawing CSF or blood from a subject, or using fine needle aspiration or needle biopsy, or by surgical biopsy. In some embodiments, biological samples may be used without or with limited processing of the sample.

Assays

[0094] In some embodiments, the present disclosure provides certain biological and/or chemical assays (e.g., that facilitate and/or permit assessment of one or more feature(s) of TRMPL1 expression and/or activity, and/or of impact of TRPMLl modulator(s) on such expression and/or activity. Examples of suitable assays include, but are not limited to, UV spectroscopy, imaging with fluorescent dyes (e.g., DAPI), polyclonal antibodies (e.g., against arachidonic acid, LPE or LPC), antibody-based assays (e.g., an enzyme-linked immunosorbent assay (ELISA)), mass spectrometry, liquid chromatography tandem mass spectrometry (LC- MS/MS), colorimetric kits, and fluorometric kits. Alternatively or additionally, the present disclosure provides technologies for identifying and/or characterizing one or more aspects of biological pathway(s) (e.g., autophagy pathway(s)) involving TRMPL1, and thus permits identification and/or characterization of additional useful targets within such pathway(s) and/or of modulator(s) that impact such pathway(s) (whether or not targeting TRPMLl itself).

Identify and/or characterize TRPML1 modulators

[0095] The present disclosure also provides methods and assays for assessing (e.g., identifying and/or characterizing) agents and/or other compositions that modulate TRPMLl activity. Provided methods and assays may be useful for identifying TRPMLl modulators. In some instances, a TRPMLl modulator is expected to affect the health state of a subject exposed to it (e.g., a beneficial or adverse effect). For example, provided methods allow for the evaluation of agents and/or compositions intended for use in humans (e.g., a drug candidate).

[0096] In some embodiments, methods and assays of the present disclosure can be used to screen candidate agents. Exemplary candidate agents include, but are not limited to, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or combination or complex thereof.

[0097] In some embodiments, methods and assays of the present disclosure can be used to characterize agents. In some embodiments, a characterized agent has already been established as having activity as a TRPML1 modulator. In some embodiments, a characterized agent was not previously established as having activity as a TRPML1 modulator. In some embodiments, methods and assays of the present disclosure can be used to characterize compositions comprising agents. In some embodiments, methods and assays of the present disclosure can be used to characterize compositions used to deliver agents.

[0098] In some embodiments, methods and assays of the present disclosure can be used to monitor manufacturing of agents and/or other compositions that modulate TRPML1 activity. In some embodiments, methods and assays of the present disclosure can be used to monitor manufacturing of a drug product that modulates TRPML1 activity.

[0099] In some embodiments, methods and assays of the present disclosure can be used to assess characteristics and/or quality of a batch of an agent and/or other composition that modulate TRPML1 activity (i.e., a release test). In some embodiments, methods and assays of the present disclosure can be used to assess characteristics and/or quality of a batch of a drug product that modulates TRPML1 activity

Kits

[0100] The present disclosure further provides kits comprising various reagents and materials useful for carrying out methods according to the present disclosure. The diagnosis/characterization/monitoring procedures described herein may be performed by diagnostic laboratories, experimental laboratories, or practitioners. The invention provides kits that can be used in these different settings. [0101] For example, materials and reagents for characterizing biological samples, measuring biomarker levels, diagnosing a disease, disorder, or condition in a subject, identifying subtypes, characterizing severity, staging a disease, and/or monitoring treatment response in a subject according to methods of the present disclosure may be assembled together in a kit. In some embodiments, a kit of the present disclosure comprises at least one or more reagents that specifically detects expression levels of one or more biomarkers and instructions for using the kit according to a method of the present disclosure.

[0102] Kits or other articles of manufacture of the present disclosure may include one or more containers to hold various reagents. Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), and ampules. A container may be formed from a variety of materials such as glass or plastic.

[0103] In some embodiments, kits of the present disclosure may include suitable control levels or control samples for determining control levels as described herein. In some embodiments, kits of the present disclosure may include instructions for using the kit according to one or more methods of the present disclosure and may comprise instructions for processing a biological sample obtained from a subject and/or for performing the test, instructions for interpreting the results as well as a notice in the form prescribed by a governmental agency (e.g., FDA) regulating the manufacture, use or sale of pharmaceuticals or biological products.

[0104] In some embodiments, kits of the present disclosure comprise reagents to detect one or more biomarkers in a sample, wherein the one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, T -O-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4, acylcarnitine 14:0, acylcamitine 14:1, acylcamitine 16:0, acylcarnitine 16:1, acylcamitine 18:0, acylcarnitine 18:1, acylcarnitine 18:2, acylcamitine 20:0, acylcarnitine 20:4, or a combination thereof. [0105] Other features of certain embodiments will become apparent in the course of the following descriptions of exemplary embodiments, which are given for illustration and are not intended to be limiting thereof.

EXAMPLES

[0106] The following examples are provided so as to describe to the skilled artisan how to make and use methods and compositions described herein, and are not intended to limit the scope of the present disclosure.

[0107] In an effort to discover biomarkers associated with TRPML1 activation, THP-1 human monocyte cells and peripheral blood mononuclear cells (PBMCs) were exposed to a series of experimental conditions and cellular-associated metabolites and metabolites released into the cell culture media were examined using both targeted and unbiased metabolomics workflows.

Example 1 - Targeted Metabolomics

[0108] Targeted metabolomics was used to quantify 280 components of the well- characterized metabolome in each cell type, including organic acids, amino acids, nucleotides and pentose phosphate cycle components. Criteria for hit calling included a fold change > 2 and a p-value < 0.05. Matrices examined were THP-1 and PBMC cells and cell culture media.

Methods

Cell culture media preparation

[0109] 800pL of ice-cold methanol was added to 100pL of cell culture media and the mixture was vortexed for 1 minute and incubated at 4°C for 30 minutes. The mixture was centrifuged to pellet proteins and the supernatant was aliquoted into a new 1.5mL Eppendorf tube. The sample was dried completely under vacuum and the pellet was solubilized in 100pL of LC/MS grade water.

Treatment of cells [0110] THP-1 cells were grown in RPMI media with 10% fetal bovine serum. Treatment conditions included a one hour exposure to the TRPMLl activator Agonist 2 or the mock treatment (binds TRPMLl with weak affinity) compound Agonist 3. Both compounds were used at a 1 OmM concentration.

Cell preparation

[0111] The cell plate was placed on ice immediately following supernatant removal and lmL of 80:20 ice cold methanol: water was added to the well containing cells. The cell layer was scraped and the plate was incubated at 4°C for 30 minutes. The cell slurry was then aliquoted into a 1.5mL Eppendorf tube and briefly vortexed. The cell slurry was then centrifuged and the supernatant was aliquoted into a new 1.5mL Eppendorf tube. The sample was dried completely under vacuum and the pellet was solubilized in 100pL of LC/MS grade water.

Chromatographic method

[0112] A Waters Xbridge Amide 2.5pm, 4.6x100mm column was used at a temperature of 30°C, a flow rate of 350pL/minute and with an injection volume of 10pL for gradient elution. Mobile phase component A was 95:5 water: acetonitrile with 20mM ammonium acetate and 20mM ammonium hydroxide at pH 9.0 and component B was 100% acetonitrile. Table 1 shows the gradient used and the mobile phase was diverted to waste for minutes 0-1 and for minutes 30- 32.

Table 1

Mass spectrometry (MS) Parameters

[0113] Targeted metabolomics was performed using an AB Sciex 6500+ operating in

MRM mode with polarity switching tuned for Q1 and Q3 transitions for each targeted metabolite. Table 2 shows the MS source parameters. Table 2

Results

[0114] After TRPMLl activation with Agonist 2, THP-1 cells released a series of nucleotide monophosphates and nucleosides into the cell culture media that were discovered using targeted metabolomics. As shown in Figure 1 and Figure 2, these metabolites include adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, xanthosine monophosphate, uridine monophosphate, adenosine, cytidine, guanosine, inosine and uridine. The increased release of these nucleotide monophosphates and nucleosides was blocked by a 30 minute pre-treatment with 50mM mucolipin specific inhibitor 3 (ML-SI3), a TRPMLl inhibitor. The increased release of these nucleotide monophosphates and nucleosides was also not seen in the cell culture media of THP-1 cells that were treated with mock treatment (binds to TRPMLl with weak affinity) Agonist 3, or with negative controls dimethylsulfoxide (DMSO) and THP-1 cell culture media (RPMI). An accumulation of hypoxanthine within THP-1 cells was also observed after TRPMLl activation (Figure 3).

[0115] Additionally, after TRPMLl activation an accumulation of nucleosides and nucleotide monophosphates within PBMCs was observed. As shown in Figures 4, 5A, 5B, 6A and 6B, these metabolites include 2’-0-methylcytidine, 2’-0-methylguanosine, 2’-0- methyluridine, adenosine, guanine, guanosine, hypoxanthine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, xanthosine monophosphate. The accumulation of these nucleosides and nucleotide monophosphates within PBMCs was blocked by pre-treatment with ML-SI3, a TRPMLl inhibitor. The accumulation of these nucleosides and nucleotide monophosphates was also not seen in the PBMCs that were treated with mock treatment Agonist 3, or with negative control dimethylsulfoxide (DMSO). The release of metabolites into cell culture media upon TRPMLl activation in PBMCs was not observed.

Example 2 - Unbiased Metabolomics

[0116] Unbiased metabolomics was used to broadly observe all ionizable components in

THEM cell culture media only. Again, criteria for hit calling included a fold change > 2 and a p- value < 0.05.

Methods

Cell culture media preparation

[0117] 800pL of ice-cold methanol was added to 100pL of cell culture media and the mixture was vortexed for 1 minute and incubated at 4°C for 30 minutes. The mixture was centrifuged to pellet proteins and the supernatant was aliquoted into a new 1.5mL Eppendorf tube. The sample was dried completely under vacuum and the pellet was solubilized in 100pL of LC/MS grade water.

Treatment of cells

[0118] THP-1 cells were grown in RPMI media with 10% fetal bovine serum. Treatment conditions included a one hour exposure to the TRPML1 activator Agonist 2or the mock treatment (binds TRPML1 with weak affinity) compound Agonist 3. Both compounds were used at a 1 OmM concentration. Additional conditions including a 30 minute pre-treatment with 50mM ML-SI3 (mucolipin specific inhibitor 3, a TRPML1 inhibitor) plus IOmM C8 (a TRPML1 activator) and a 30 minute pre-treatment with DMSO or 10mM PLA2 inhibitor were also used. Inhibitors tested include CAY10502 (cPLA2a inhibitor), arachidonyl trifluoroketone (ATK, dual cPLA2a and iPLA2 inhibitor), bromoenol lactone (BEL, iPLA2 inhibitor) and LY315920 (sPLA2 inhibitor). After treatment, the cells and media were immediately prepared for liquid chromatography/mass spectrometry (LC/MS) analysis.

Cell preparation

[0119] The cell plate was placed on ice immediately following supernatant removal and lmL of 80:20 ice cold methanol: water was added to the well containing cells. The cell layer was scraped and the plate was incubated at 4°C for 30 minutes. The cell slurry was then aliquoted into a 1.5mL Eppendorf tube and briefly vortexed. The cell slurry was then centrifuged and the supernatant was aliquoted into a new 1.5mL Eppendorf tube. The sample was dried completely under vacuum and the pellet was solubilized in 100pL of LC/MS grade water.

Chromatographic method 1

[0120] A Waters Xbridge Amide 2.5pm, 4.6x100mm column was used at a temperature of 30°C, a flow rate of 350pL/minute and with an injection volume of 10pL for gradient elution. Mobile phase component A was 95:5 water: acetonitrile with 20mM ammonium acetate and 20mM ammonium hydroxide at pH 9.0 and component B was 100% acetonitrile. Table 3 shows the gradient used and the mobile phase was diverted to waste for minutes 0-1 and for minutes 30- 32.

Table 3

Chromatographic method 2

[0121] A Waters Acquity BEH Cl 8, 1.7 pm, 2.1 x 100mm column was used at a temperature of 45°C, a flow rate of 300pL/minute and with an injection volume of lOpL for gradient elution. Mobile phase component A was 0.1% formic acid in water and component B was 0.1% formic acid in 4: 1 methanol: acetonitrile. Table 4 shows the gradient used and the mobile phase was diverted to waste for minutes 0-2 and for minutes 22.1-24.

Table 4

Mass spectrometry (MS) Parameters

[0122] Unbiased metabolomics was performed on a Thermo QExactive operating in full scan mode with both positive and negative ionization. Table 5 shows the MS source settings and full scan MS settings.

Table 5

Results

[0123] Unbiased metabolomics was used to broadly survey metabolites released into cell culture media by THP-1 cells after TRPMLl activation. As shown in Figure 7, arachidonic acid and lysophophatidylethanolamine (18:1) (18:1 lysoPE) released into the cell culture media was observed. The increased release of arachidonic acid and 18:1 lysoPE was blocked by pre treatment with ML-SI3, a TRPMLl inhibitor. The increased release was also not seen in the cell culture media of THP-1 cells that were treated with mock treatment Agonist 3, or with negative controls dimethylsulfoxide (DMSO) and THP-1 cell culture media (RPMI).

[0124] As expected, the cells pretreated with DMSO for 30 minutes followed by

TRPMLl activation by C8 demonstrated robust arachidonic acid release. As shown in Figure 8, pre-treatment of THP-1 cells with the cPLA2a inhibitor CAY10502 blocked production of arachidonic acid in response to treatment with the TRPMLl activator C8, while pre-treatment with other PLA2 inhibitors did not reduce the effect of TRPMLl activation, which points to cPLA2a as playing a role in calcium-driven exocytosis in THP-1 cells. Example 3 - Acylcarnitines are biomarkers of TRPML1 target engagement

[0125] In an effort to discover biomarkers associated with TRPML1 activation, a C2C12 mouse myoblast cell line was exposed to a series of experimental conditions and cellular associated metabolites and metabolites released into the cell culture media were examined using targeted metabolomics. THP-1 human monocyte cells were used to investigate acylcarnitine accumulation after TRPMLl activation.

[0126] Targeted metabolomics was used to quantify 243 components of the well- characterized metabolome, including organic acids, amino acids, nucleotides and pentose phosphate cycle components. Criteria for hit calling included a fold change > 2 and a p-value < 0.05.

[0127] Following the initial observation of accumulated acylcarnitines in C2C12 myotubes after TRPMLl activation, the abundance of acylcarnitines in C57BL/10ScSn- Dmd^mdx/J (mdx) mice that were dosed with 5 or 30 mg/kg via intraperitoneal injection was examined. The abundance of acylcarnitines in these mice was compared to vehicle control treated mice.

Methods

Cell culture media preparation

[0128] 800pL of ice-cold methanol was added to 100pL of cell culture media and the mixture was vortexed for 1 minute and incubated at 4°C for 30 minutes. The mixture was centrifuged to pellet proteins and the supernatant was aliquoted into a new 1.5mL Eppendorf tube. The sample was dried completely under vacuum and the pellet was solubilized in 100pL of LC/MS grade water.

Treatment of C2C12 cells

[0129] C2C12 cells were grown in DMEM media with 10% fetal bovine serum.

Treatment conditions included a one hour exposure to the TRPMLl activator C8 or the mock treatment (binds TRPMLl with weak affinity) compound Agonist 3. Both compounds were used at a 1 OmM concentration. An additional condition including a 30 minute pre-treatment with 50mM B11 (a TRPMLl inhibitor) plus IOmM C8 (a TRPMLl activator) was also used. Some THP-1 cells were also pre-treated with DMSO or a PLA2 inhibitor for 30 minutes before treatment with IOmM C8. PLA2 inhibitors included: CAY10502 (a cPLA2a inhibitor) BEL (an iPLA2 inhibitor), and LY315920 (an sPLA2 inhibitor). Some C2C12 myoblasts were also pre treated with DMSO or the CPT1 inhibitor etomoxir for 30 minutes. After treatment, the cells and media were immediately prepared for liquid chromatography/mass spectrometry (LC/MS) analysis.

Treatment of THP-1 cells

[0130] THP-1 human monocyte cells were grown in RPMI media supplemented with

10% FBS and pretreated for 30 minutes with DMSO or IOmM PLA2 inhibitor. Inhibitors tested included CAY10502 (cPLA2a inhibitor), bromoenol lactone (BEL, iPLA2 inhibitor) and LY315920 (sPLA2 inhibitor). After 30 minutes, the cells were challenged with 10mM C8 for 30 minutes and intracellular acylcamitine abundance from each treatment condition was measured by LC-MS/MS.

Cell preparation

[0131] The cell plate was placed on ice immediately following supernatant removal and lmL of 80:20 ice cold methanol: water was added to the well containing cells. The cell layer was scraped and the plate was incubated at 4°C for 30 minutes. The cell slurry was aliquoted into a 1.5mL Eppendorf tube and briefly vortexed. The cell slurry was centrifuged and the supernatant was aliquoted into a new 1.5mL Eppendorf tube. The sample was dried completely under vacuum and the pellet was solubilized in 100 pL of LC/MS grade water.

Extraction of acylcarnitines from mouse plasma

[0132] 10pL of plasma was aliquoted into a 1 5mL Eppendorf tube and 90pL of methanol spiked with lOOnM acylcamitine 14:0 d-9 was added to the sample. The sample was vortexed for 5 minutes at room temperature and then centrifuged for 10 minutes at 10,000 rpm at 4°C. 50pL of supernatant was aliquoted into an LC/MS compatible vessel for analysis.

Extraction of acylcarnitines from solid tissue

[0133] A minimum of 30mg and a maximum of lOOmg of tissue was added to a 2mL

Eppendorf tube and three 3mm tungsten carbide beads were added to each tube. A lOx volume of 10% ethanol in water was added to each sample and the samples were homogenized on a Qiagen TissueLyzer II for 20 minutes. 100 pL of tissue homogenate was aliquoted into a new 1 5mL tube and the rest of the sample was saved at -80°C for future analysis. 900pL of ice-cold methanol spiked with lOOnM acylcamitine 14:0 d-9 was added to the sample. The sample was vortexed for 5 minutes at room temperature and then centrifuged for 10 minutes at 10,000 rpm at 4°C. 50pL of supernatant was aliquoted into an LC/MS compatible vessel for analysis.

Chromatographic Method 1

[0134] A first chromatographic method was used for targeted metabolomics and discovery of an acylcamitine phenotype in C2C12 cells. A Waters Xbridge Amide 2.5pm, 4.6x100mm column was used at a temperature of 30°C, a flow rate of 350pL/minute and with an injection volume of 10pL for gradient elution. Mobile phase component A was 95:5 water: acetonitrile with 20mM ammonium acetate and 20mM ammonium hydroxide at pH 9.0 and component B was 100% acetonitrile. Table 6 shows the gradient used and the mobile phase was diverted to waste for minutes 0-1 and for minutes 30-32.

Table 6

Chromatographic Method 2

[0135] A second chromatographic method was used for targeted quantitation of acylcamitines in plasma, heart and muscle tissue. A Phenomenex Synergi Fusion RP-80, 2 x 50mm, 4pm column was used at a temperature of 45 °C, a flow rate of 600pL/minute and with an injection volume of lpL for gradient elution. Mobile phase component A was 0.1% formic acid in water and component B was 0.1% formic acid in acetonitrile. Table 7 shows the gradient used and the mobile phase was diverted to waste for minutes 0-1.5 and for minutes 7-10.

Table 7

Mass spectrometry (MS) Method 1 Parameters

[0136] Targeted metabolomics was performed using an AB Sciex 6500+ operating in

MRM mode with polarity switching tuned for Q1 and Q3 transitions for each targeted metabolite. Table 8 shows the MS source parameters.

Table 8

Mass spectrometry (MS) Method 2 Parameters

[0137] Targeted metabolomics was performed using an AB Sciex 6500+ operating in

MRM mode with polarity switching tuned for Q1 and Q3 transitions for each targeted metabolite. Table 9 shows the MS source parameters.

Table 9

Table 10 - Mass transitions to detect acylcarnitines

Results

Accumulated acylcarnitines in C2C12 cells discovered through targeted metabolomics

[0138] After a one-hour incubation of C2C12 mouse myoblast cells with the TRPMLl activator C8, an increase in 3 species of acylcamitine (16:0, 16:1 and 18:1) was observed (Figure 9). This same effect was not observed in C2C12 cells that had been treated with DMSO or in C2C12 cells that treated with the mock treatment Agonist 3 (Figure 9). Additionally, cells exposed to excess TRPMLl inhibitor B11 in the presence of C8 did not produce increased acylcarnitines (Figure 9). This observation suggests that the acylcamitine phenotype is due to the TRPML activity and not from an off-target effect.

[0139] When the TRPMLl activator C8 was added to wild-type C2C12 myoblasts, a dose-dependent increase in acylcamitine production with measurable ECso was observed (Figure 10). However, this response was blocked in TRPMLl knock-out (KO) C2C12 myoblasts to near baseline level (Figure 10), providing further evidence that TRPMLl is required for increased acylcamitine production. The WT DMSO results and KO DMSO results appear as single data points and are overlaid by the lowest concentration WT data point in each dose response graph. [0140] Additionally, as shown in Figure 11, inhibition of the enzyme CPT1, a mitochondrial enzyme involved in production of acylcamitines, with etomoxir in C2C12 myoblasts prior to TRPMLl activation with C8 lead to a 50% reduction in acylcamitine production (e.g., AC 16:0) relative to control.

Accumulation of acylcarnitines in THP-1 cells after TRPML1 activation

[0141] As shown in Figure 12, pre-treatment of THP-1 cells with the cPLA2a inhibitor

CAY10502 also blocked production of acylcamitine 16:0 in response to treatment with the TRPMLl activator C8, while pre-treatment with other PLA2 inhibitors (BEL, LY315920) did not reduce the effect of TRPMLl activation. The calcium dependent phospholipase cPLA2a is involved in the production of arachidonic acid and acylcamitines following TRPMLl activation. The cPLA2a enzyme cleaves membrane-associated phosphatidylethanolamine and phosphatidylcholine to produce free fatty acids, predominantly arachidonate, and lysophospholipids.

Accumulation of acylcarnitines in mouse tissue after TRPMLl activation

[0142] Acylcamitine abundance was examined in C57BL/10ScSn-Dmd^mdx/J (mdx) mice one-hour after a single intraperitoneal injection of the TRPMLl activator Agonist 1. The 3 cohorts of mice examined included a vehicle control group and treatment groups that received either 5 mg/kg or 30 mg/kg of Agonist 1. Tissues profiled for AC abundance included plasma, muscle and heart. Accumulation of acylcamitine species 16:1, 18:1 and 18:2 was detected in mouse plasma after treatment with Agonist 1 (Figure 13), while accumulation of acylcamitine species 14:0, 16:0, 16:1, 18:0, 18:1, and 20:0 was detected in mouse muscle tissue after treatment with Agonist 1 (Figure 14) and accumulation of acylcamitine species 14:0, 16:0, 18:0, 20:0, and 20:4 was detected in mouse heart tissue after treatment with Agonist 1 (Figure 15).

Example 4 - Small noncoding RNAs are biomarkers of TRPMLl target engagement

[0143] Since late endosome/lysosomes contain snoRNAs and TRPMLl agonists can increase late endosome/lysosome fusion with the plasma membrane, whether snoRNAs could be detected in extracellular medium in response to TRPMLl activation and thus serve as potential target engagement biomarkers was examined.

Methods

Cell culture conditioned media preparation

[0144] THP-1 cells were centrifuged and washed twice in phosphate buffered saline and the cells were then re-suspended in cell culture media containing 10% extracellular (EV)- depleted fetal bovine serum and plated. Forty-eight hours later, conditioned media was collected and centrifuged at 330 x g for 10 minutes to pellet cells. Supernatant was transferred to a new 50mL tube and centrifuged at 3,200 x g for 15 minutes to pellet cellular debris and then the supernatant was transferred to a new 50mL tube.

Extracellular vesicle (EV) precipitation

[0145] 0.5x volumes Total Exosome Isolation Reagent (Life Technologies, Catalogue

#4478359) was added to conditioned media and samples were then vortexed for 15 seconds and incubated at 4°C for 16 hours. Samples were then centrifuged at 3,200 x g for 1 hour to pellet EVs. Supernatant was aspirated and discarded and the EV pellets were subjected to protein and RNA analysis.

Protein preparation and western blot

[0146] An appropriate volume of 2x Laemmli reducing sample buffer was added to the

EV pellets and the samples were vortexed for 10 seconds. The samples were then incubated at 42°C for 5 minutes, transferred to a 1.5 mL micro centrifuge tube and then incubated at 95°C for 5 minutes. The samples were then centrifuged for 30 seconds at 16,000 x g loaded into a 4-15% tris-glycine midi gel and electrophoresed for 1.5 hours at 150 volts. Samples were transferred onto 0.2 pm nitrocellulose membranes for 10 minutes at 25 volts. Membranes were blocked in 5% non-fat milk for 30 minutes at room temperature and were then incubated with indicated primary antibodies in 5% non-fat milk at 4°C for 16 hours. Membrane were then washed 3 times for 12 minutes in Tris-buffered saline with tween (TBST) and then incubated at room temperature for 1 hour with appropriate fluorescent secondary antibodies. Finally, membranes were washed 3 times for 12 minutes in TBST, then washed 1 time for 5 minutes in phosphate- buffered saline and then imaged with a BioRad ChemiDoc MP. RNA preparation and RT-qPCR

[0147] 200 pL of ice-cold Exosome Resuspension Buffer (Life Technologies, Catalogue

#4478545) was added to EV pellets and samples were incubated at for 5 minutes at room temperature. One volume of 2X Denaturing Solution (Life Technologies, Catalogue #4478545) was added to each sample and vortexed for 15 seconds and samples were then incubated at 4°C for 5 minutes. Synthetic C. elegans miR-39 (Qiagen, Catalogue #219610) was added to each sample (0.066 fmol) and small RNA was extracted using Total Exosome RNA & Protein Isolation Kit (Life Technologies, Catalogue #4478545) according to manufacturer’s protocol with Appendix A - Enriching for small RNAs. Stem-loop core snoRNA-specific and C. elegans miR-39-specific reverse transcription primers were combined (0.5 mM final concentration of each primer) in DNAse- and RNAse-free water.

Table 11

[0148] Fourteen pL of extracted RNA was combined with 2 pL snoRNA-specific reverse transcription primers, 2 pL DNAse- and RNAse-free water, and 2 pL dNTPs (10 mM). For a negative control using 01igo(dT)-mediated reverse transcription for mRNA, 14 pL of extracted RNA was combined with 2 pL 01igo(dT)2o (50 mM), 2 pL DNAse- and RNAse-free water, and 2 pL dNTPs (10 mM). Samples were incubated at 65 °C for 5 minutes to denature RNA and were then placed on ice for 2 minutes to allow primers to anneal to RNA templates. Twenty pL of Reverse Transcription mix (Life Technologies, Catalogue #18080-051) comprising lOx RT Buffer, 25 mM MgCh, 0.1 M DTT, 40 U /pL RNaseOUT, and 200 U /pL SS III RT according to manufacturer’s specifications was then added to each sample.

[0149] Samples were incubated under the following conditions for cDNA generation by reverse transcription: 25°C for 10 minutes, 50°C for 30 minutes and 85°C for 5 minutes and were then centrifuged and place on ice. Two pL of E. coli RNase H (2 U/pL) was added to each sample to degrade residual RNA, samples were incubated at 37°C for 30 minutes and cDNA samples were then placed on ice. Forward qPCR primers (5 pM) for snoRNAs and C. elegans miR-39 and a universal reverse primer against stem-loop core of reverse transcription primers were prepared in DNAse- and RNAse-free water.

Table 12

[0150] Additionally, a qPCR master mix comprising appropriate volumes of 5 pM forward primer, 5 pM reverse primer, water and 2x SYBR Green was prepared. 6.5 pL of qPCR master mix and 3.5 pL of cDNA was added to each well of a qPCR optical reaction plate and the qPCR plate was incubated under the following conditions for quantitative assessment of cDNA abundance: one cycle at 95 °C for 10 minutes and 40 cycles of 95 °C for 15 seconds and 60°C for 60 seconds. Levels of cDNA qPCR amplification (Ct) was first normalized to C. elegans miR- 39 spike-in and fold change was computed using AACT method relative to the DMSO sample. Results

Extracellular vesicle (EV) precipitation methodology is sufficient to enrich for EVs displaying canonical protein markers

[0151] As shown in Figure 16, the exemplary EV precipitation methodology described herein did not capture cellular contaminants from the Golgi Apparatus (GM130), Endoplasmic Reticulum (Calnexin), or Autophagic Vesicles (LC3). However, the methodology did enrich for canonical EV markers (Flotillin-1, TSG101, CD63). Ionomycin treatment had no effect on capture of EV or contaminant markers and non-conditioned media alone did not contain EV or contaminant markers. Ionomycin shuttles extracellular calcium into cells and is a separate calcium source from TRPMLl -mediated calcium release from lysosomes, which drives exocytosis. Therefore, the lack of an effect on EVs after treatment with ionomycin suggests that lysosomal calcium release, and not extracellular calcium influx, is the specific stimulus for snoRNA EV release. These results suggest that the precipitation methodology for EV capture generated relatively pure EV species.

TRPMLl activation increases the abundance of canonical EV protein markers

[0152] TRPMLl activation in THP-1 cells with C8 resulted in increased EV markers

(Flotillin-1, TSG101, CD63), but did not induce the release of cellular contaminant markers (GM130, Calnexin, LC3) (Figure 17). This suggests that the elevated detection of EV markers upon TRPMLl activation is not due to cell death and release of cellular components into the conditioned media. Importantly, cells exposed to excess TRPMLl inhibitor B11 in the presence of C8 did not exhibit increased EV markers. This observation suggests that TRPMLl activation specifically induces the release of EVs from THP-1 cells and does not represent an off-target effect of C8.

TRPMLl activation increases the abundance of EV snoRNA-U32a. U33. U34 and U35a

[0153] TRPMLl activation in THP-1 cells with C8 resulted in increased detection of snoRNAs U32a, U33, U34 and U35a (18-144 fold) relative to DMSO treated cells (Figure 18A). Importantly, this effect on EV snoRNA release by C8 was inhibited upon exposure of cells to excess TRPMLl inhibitor B 11. This observation suggests that TRPML1 activation specifically induces the release of these snoRNAs within secreted EVs. Additionally, as shown in Figure 18B, a negative control using Oligo(dT) as a reverse transcription primer resulted in no amplification of these snoRNAs during qPCR, suggesting that there is not a false positive qPCR signal from mRNA species or genomic DNA with similar sequences to these snoRNAs or contaminants in the reagents used.

EQUIVALENTS

[0154] It is to be appreciated by those skilled in the art that various alterations, modifications, and improvements to the present disclosure will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of the present disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawing are by way of example only and any invention described in the present disclosure if further described in detail by the claims that follow.

[0155] Those skilled in the art will appreciate typical standards of deviation or error attributable to values obtained in assays or other processes as described herein. The publications, websites and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference in their entireties.

Claims

1. A method comprising a step of: determining a level of one or more biomarkers in a sample from a subject, wherein one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, T -O-methyl guanosine, T -O-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

2. The method of claim 1, wherein the step of determining comprises determining the level of one or more biomarkers relative to a reference level of the one or more biomarkers.

3. The method of claim 1, wherein the subject is receiving or has received a therapy comprising a TRPML1 agonist.

4. The method of claim 3, further comprising a step of modifying the therapy after the step of determining.

5. The method of claim 4, wherein the step of modifying the therapy comprises increasing frequency and/or dosage of the TRPML1 agonist.

6. The method of claim 4, wherein the step of modifying the therapy comprises decreasing frequency and/or dosage of the TRPML1 agonist.

7. The method of claim 4, wherein the step of modifying the therapy comprises ceasing administration of the TRPML1 agonist.

8. The method of claim 3, further comprising a step of continuing the therapy after the step of determining.

9. The method of any one of claims 1-8, wherein the sample comprises serum, plasma, cerebrospinal fluid, urine, extracellular vesicles, exosomes, peripheral blood mononuclear cells, or biopsy specimens and the one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine, monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, 2’-0-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

10. The method of any one of claims 1-8, wherein the sample comprises cells or tissues and the one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, T -O-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, 2’-0-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

11. The method of claim 10, wherein the method further comprises a step of lysing the cells or performing a tissue biopsy and the one or more biomarkers include one or more intracellular biomarkers.

12. The method of claim 11, wherein the one or more intracellular biomarkers comprise adenosine, cytidine, deoxyguanosine, guanine, guanosine, hypoxanthine, inosine, uridine, xanthosine, adenosine monophosphate, guanosine monophosphate, xanthosine monophosphate, cytidine monophosphate, inosine monophosphate or a combination thereof.

13. A method comprising a step of: administering a therapy comprising a TRPML1 agonist to a subject who has been determined to have a reduced level of one or more biomarkers relative to a reference level of the one or more biomarkers, wherein the one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, 2’-0- methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

14. A method comprising a step of: characterizing a candidate therapy comprising a TRPMLl agonist by assessing its impact on one or more biomarkers in a subject to whom the candidate therapy is being or has been administered, wherein the one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, 2’-0-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, 2’-0-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

15. The method of claim 14, wherein the subject is or comprises a model organism.

16. The method of claim 14, wherein the subject is or comprises a cell culture.

17. The method of claim 15, wherein the subject is or comprises a human.

18. The method of claim 17, wherein the human is suffering from or susceptible to a muscular disease, a liver disease, a metabolic disease, an atherosclerotic disease, an inflammatory bowel disease, an atherosclerotic disease, a neurodegenerative disease, an infectious disease, an inflammatory disease, or an oncological disease.

19. A kit comprising reagents to detect one or more biomarkers in a sample, wherein the one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, T -O-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, 2’-0-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, or a combination thereof.

20. A method comprising a step of: determining a level of one or more biomarkers in a sample from a subject, wherein one or more biomarkers comprise arachidonic acid, lysophosphatidylethanolamine (LPE) 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, lysophosphatidylcholine (LPC) 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4, or a combination thereof

21. The method of claim 20, wherein the step of determining comprises determining the level of one or more biomarkers relative to a reference level of the one or more biomarkers.

22. The method of claim 20, wherein the subject is receiving or has received a therapy comprising a TRPML1 agonist.

23. The method of claim 22, further comprising a step of modifying the therapy after the step of determining.

24. The method of claim 23, wherein the step of modifying the therapy comprises increasing the frequency and/or dosage of the TRPML1 agonist.

25. The method of claim 23, wherein the step of modifying the therapy comprises decreasing the frequency and/or dosage of the TRPML1 agonist.

26. The method of claim 23, wherein the step of modifying the therapy comprises ceasing administration of the TRPML1 agonist.

27. The method of claim 22, further comprising a step of continuing the therapy after the step of determining.

28. The method of any one of claims 20-27, wherein the sample comprises serum, plasma, cerebrospinal fluid, urine, extracellular vesicles, exosomes, peripheral blood mononuclear cells, or biopsy specimens and the one or more biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof.

29. The method of any one of claims 20-27, wherein the sample comprises cells and the one or more biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof.

30. The method of claim 29, wherein the method further comprises a step of lysing the cells or performing a tissue biopsy and the one or more biomarkers include one or more intracellular biomarkers.

31. The method of claim 30, wherein the one or more intracellular biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof.

32. A method comprising a step of: administering a therapy comprising a TRPML1 agonist to a subject who has been determined to have a reduced level of one or more biomarkers relative to a reference level of the one or more biomarkers, wherein the one or more biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof.

33. A method comprising a step of: characterizing a candidate therapy comprising a TRPML1 agonist by assessing its impact on one or more biomarkers in a subject to whom the candidate therapy is being or has been administered, wherein the one or more biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof.

34. The method of claim 33, wherein the subject is or comprises a model organism.

35. The method of claim 33, wherein the subject is or comprises a cell culture.

36. The method of claim 34, wherein the subject is or comprises a human.

37. The method of claim 36, wherein the human is suffering from or susceptible to a muscular disease, a liver disease, a metabolic disease, an atherosclerotic disease, an inflammatory bowel disease, an atherosclerotic disease, a neurodegenerative disease, or an oncological disease.

38. A kit comprising reagents to detect one or more biomarkers in a sample, wherein the one or more biomarkers comprise arachidonic acid, LPE 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, LPC 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4 or a combination thereof.

39. A method comprising a step of: determining a level of one or more biomarkers in a sample from a subject, wherein the one or more biomarkers comprise acylcarnitine 14:0, acylcamitine 14:1, acylcarnitine 16:0, acylcamitine 16:1, acylcarnitine 18:0, acylcamitine 18:1, acylcarnitine 18:2, acylcarnitine 20:0, acylcamitine 20:4, or a combination thereof.

40. The method of claim 39, wherein the step of determining comprises determining the level of one or more biomarkers relative to a reference level of the one or more biomarkers.

41. The method of claim 39, wherein the subject is receiving or has received a therapy comprising a TRPML1 agonist.

42. The method of claim 41, further comprising a step of modifying the therapy after the step of determining.

43. The method of claim 42, wherein the step of modifying the therapy comprises increasing frequency and/or dosage of the TRPML1 agonist.

44. The method of claim 42, wherein the step of modifying the therapy comprises decreasing frequency and/or dosage of the TRPML1 agonist.

45. The method of claim 42, wherein the step of modifying the therapy comprises ceasing administration of the TRPML1 agonist.

46. The method of claim 41, further comprising a step of continuing the therapy after the step of determining.

47. The method of any one of claims 39-46, wherein the sample comprises serum, plasma, cerebrospinal fluid, urine, extracellular vesicles, exosomes, peripheral blood mononuclear cells, or biopsy specimens and the one or more biomarkers comprise acylcarnitine 14:0, acylcamitine 14:1, acylcamitine 16:0, acylcarnitine 16:1, acylcamitine 18:0, acylcarnitine 18:1, acylcarnitine 18:2, acylcamitine 20:0, acylcarnitine 20:4, or a combination thereof.

48. The method of any one of claims 39-46, wherein the sample comprises cells or tissues and the one or more biomarkers comprise acylcarnitine 14:0, acylcamitine 14:1, acylcamitine 16:0, acylcamitine 16:1, acylcarnitine 18:0, acylcamitine 18:1, acylcarnitine 18:2, acylcarnitine 20:0, acylcamitine 20:4, or a combination thereof.

49. The method of claim 48, wherein the method further comprises a step of lysing the cells or performing a tissue biopsy and the one or more biomarkers include one or more intracellular biomarkers.

50. The method of claim 49, wherein the one or more intracellular biomarkers comprise acylcamitine 16:0, acylcarnitine 16:1, acylcamitine 18:1, or a combination thereof.

51. A method comprising a step of: administering a therapy comprising a TRPML1 agonist to a subject who has been determined to have a reduced level of one or more biomarkers relative to a reference level of the one or more biomarkers, wherein the one or more biomarkers comprise acylcamitine 14:0, acylcamitine 14:1, acylcamitine 16:0, acylcamitine 16:1, acylcamitine 18:0, acylcamitine 18:1, acylcamitine 18:2, acylcamitine 20:0, acylcamitine 20:4, or a combination thereof.

52. A method comprising a step of: characterizing a candidate therapy comprising a TRPMLl agonist by assessing its impact on one or more biomarkers in a subject to whom the candidate therapy is being or has been administered, wherein the one or more biomarkers comprise acylcamitine 14:0, acylcamitine 14:1, acylcamitine 16:0, acylcamitine 16:1, acylcamitine 18:0, acylcamitine 18:1, acylcamitine 18:2, acylcamitine 20:0, acylcamitine 20:4, or a combination thereof.

53. The method of claim 52, wherein the subject is or comprises a model organism.

54. The method of claim 52, wherein the subject is or comprises a cell culture.

55. The method of claim 53, wherein the subject is or comprises a human.

56. The method of claim 55, wherein the human is suffering from or susceptible to a muscular disease, a liver disease, a metabolic disease, an atherosclerotic disease, an inflammatory bowel disease, an atherosclerotic disease, a neurodegenerative disease, or an oncological disease.

57. A kit comprising reagents to detect one or more biomarkers in a sample, wherein the one or more biomarkers comprise acylcamitine 14:0, acylcamitine 14:1, acylcamitine 16:0, acylcamitine 16:1, acylcamitine 18:0, acylcamitine 18:1, acylcamitine 18:2, acylcamitine 20:0, acylcamitine 20:4, or a combination thereof.

58. A kit comprising reagents to detect one or more biomarkers in a sample, wherein the one or more biomarkers comprise adenosine, cytidine, cytosine, guanosine, inosine, uridine, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, inosine monophosphate, uridine monophosphate, xanthosine monophosphate, T -O-methyl adenosine, 2’-0-methyl cytidine, 2’-0-methyl guanosine, 2’-0-methyl uridine, deoxyguanosine, guanine, hypoxanthine, xanthosine, miRNA, snoRNA, arachidonic acid, lysophosphatidylethanolamine (LPE) 16:0, LPE 16:1, LPE 18:0, LPE 18:1, LPE 18:2, LPE 20:4, lysophosphatidylcholine (LPC) 16:0, LPC 16:1, LPC 18:0, LPC 18:1, LPC 18:2, LPC 20:4, acylcarnitine 14:0, acylcamitine 14:1, acylcarnitine 16:0, acylcarnitine 16:1, acylcarnitine 18:0, acylcarnitine 18:1, acylcarnitine 18:2, acylcarnitine 20:0, acylcamitine 20:4, or a combination thereof.