WO2025226707A1 - Assay and kit for detecting and quantifying glycogen - Google Patents

Abstract

Described herein are kits and methods for quantifying glycogen in at least one sample. Disclosed methods involve detecting a presence of linear glucose chains in at least one sample and plurality of pre-applied glycogen standard spots; optionally generating a calibration curve based on the detection of linear glucose chains from the pre-applied glycogen standard spots; and quantifying an amount of glycogen in the at least one sample based on amounts of the linear glucose chains detected in the at least one sample and the pre-applied glycogen standard spots.

Description

ASSAY AND KIT FOR DETECTING AND QUANTIFYING GLYCOGEN

GOVERNMENT SUPPORT CLAUSE

[0001] This invention was made with government support under R01 AG066653 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0002] Glycogen is a central glucose storage molecule in humans. Glycogen metabolism can be dysregulated in disease, leading to accumulation of aberrant glycogen. Key to diagnosing and treating disorders related to glycogen is the ability to quantitatively and spatially measure glycogen in terms of both absolute quantity and structure. Examples of diseases of glycogen include classical Glycogen Storage Disorders (GSDs), which are characterized by accumulation of glycogen in specific organs and cellular compartments. For example, Pompe disease is an autosomal- recessive lysosomal storage disorder caused by alpha-1 ,4-glucosidase (GAA) enzyme deficiency. Prevalence ranges between 1 :40,000 and 1 :100,000. GAA dysfunction results in accumulation of large amounts of glycogen in skeletal and smooth muscle cells, hepatocytes, endothelial cells, and cells of the central nervous system, interfering with the cells' functioning. GAA activity usually less than 1 % is associated with infantile onset Pome disease (IOPD), and with cardiomyopathy, cardiorespiratory failure, and early death if enzyme replacement therapy (ERT) is not initiated. Partial reduction of GAA enzyme activity is associated with juvenile and adult late-onset Pompe disease (LOPD) onset, which is mainly characterized by progressive weakness of the limb girdle and axial muscles. In many instances, respiratory muscles are impaired early, and mechanical ventilatory support is indicated prior to wheelchair dependence in about 30% of LOPD patients. Affected respiratory muscles comprise the diaphragm in particular, but also the upper airway, and intercostal and abdominal muscles in severe disease, leading to recurrent pneumonia, respiratory acidosis, and other morbidities. BRIEF DESCRIPTION OF THE DRAWINGS

[0003] FIG. 1 . FIG. 1 A Schematic of MALDI-MSI workflow for in situ glycogen imaging and glycogen biodistribution by MALDI imaging (GlycoSense). FIG. 1 B Glycogen heatmap and distribution from brains of IOPD patient and normal specimen postmortem (3-month-old). FIG. 1 C biochemical characterization of brain glycogen from the IOPD brain and three additional age and sex-matched normal specimens. FIG. 1 D Glycogen heatmap and distribution in the quadricep muscle (Quad) of IOPD patient and normal specimen post-mortem (3-month-old). FIG. 1 E biochemical characterization of quad glycogen from the IOPD brain and three additional age and sex-matched normal specimens.

[0004] FIG. 2: FIG. 2A Schematics of the improved prototype liquid biopsy assay for the diagnosis of GSDs by GlycoSense. FIG. 2B. Glycogen standards used for direct quantification of available glycogen in patient blood. Each spot represent a specific glycogen concentration stated on the left in nanogram (ng) concentration. FIG. 2C Standard curve using purified human glycogen showing 10³ dynamic range of the assay. FIG. 2D. Schematics of isolation of peripheral blood mononuclear cells (PBMC) from IPO, LOPD, and normal patients fore th quantification of glycogen and diagnosis of GSDs. FIG. 2E. quantification of glycogen in two IOPD, 2 LOPD, and 4 normal volunteers after PBMC isolation from the blood by GlycoSense with proposed diagnostic cutoff for all GSDS.

[0005] FIG. 3 a, Scanned image showing the location of spotted glycogen standards (0.06, 0.18, 0.55, 1.6, and 5 ng) next to a tissue section on a microscope slide, b, Mass spectra of different glycogen concentrations spotted on the slide, indicating the glucose polymer 7 m/z peaks corresponding to varying amounts of glycogen, c, XY plots showing the relationship between glycogen concentration (ng) and relative intensity per pixel for each spotted standard, d, Log-transformed plots of relative intensity versus glycogen concentration for the standards for different glucose chain length indicated above. The linear regression lines indicate the strong correlation used for glycogen quantification. R² values for each plot are shown, line equations are displayed on top. e, Absolute quantification of glycogen in four human lung adenocarcinoma (LUAD) and one lung squamous cell carcinoma (LUSC) tissue sections (n = 3 ROIs of 500 pixels/tissue; mean +/- s.e.m. p-values indicated; one-way ANOVA adjusted for Tukey’s multiple comparisons).

DETAILED DESCRIPTION

[0006] Disclosed herein are embodiments that improve upon the GlycoSense platform shown in FIG. 1 (also described in US Pat. Pub 20220229026) and relate to clinical assays useful for assessing glycogen storage diseases (GSDs) by detecting glycogen levels in liquid biopsy samples as well as tissue biopsies, and surgical specimens. The previously devised GlycoSense assay involves adding an isoamylase to a sample, wherein the isoamylase cleaving glucose chains from glycogen; applying a matrix- assisted laser desorption ionization (MALDI) ionization matrix to the sample; and analyzing the samples using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In an embodiment provided herein, the biopsy sample (e.g. blood/RBC/PBMC/tissue) is directly spotted onto a microscope slide next to specifically- designed glycogen-based standards. Once fixed on the slide, the samples can be processed by standard MALDI glycogen protocols such as those described in FIG. 1 . The assay embodiments described herein have successfully been implemented to obtain glycogen quantitation from five Pompe patients and five healthy volunteers with exceptional results. The assay embodiments described herein can distinguish the difference between healthy volunteers, IOPD as well as LOPD with clear nonintercepting values. While the data presented herein relates to Pompe disease, those skilled in the art will appreciate that the embodiments disclosed herein can be used to detect aberrant glycogen levels and/or to diagnose subjects with aberrant glycogen storage conditions, which would include classic GSDs, as well as other diseases of excess glycogen, or conditions that involve reduced glycogen.

[0007] In another embodiment, provided is a method that involves detecting a presence of linear glucose chains in at least one sample and plurality of pre-applied glycogen standard spots; optionally generating a calibration curve based on the detection of linear glucose chains from the pre-applied glycogen standard spots; and quantifying an amount of glycogen in the at least one sample based on amounts of the linear glucose chains detected in the at least one sample and the pre-applied glycogen standard spots.

Definitions

[0008] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, including the methods and materials are described below.

[0009] Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a liquid” includes a plurality of liquids, and so forth.

[0010] The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0011] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0012] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration, percentage, or the like is meant to encompass variations of in some embodiments ±50%, in some embodiments ±40%, in some embodiments ±30%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1 %, in some embodiments ±0.5%, and in some embodiments ±0.1 % from the specified amount, as such variations are appropriate to perform the disclosed method.

[0013] As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11 , 12, 13, and 14 are also disclosed.

[0014] As used herein, nomenclature for compounds, including organic compounds, can be given using common names, IIIPAC, IlIBMB, or CAS recommendations for nomenclature. When one or more stereochemical features are present, Cahn-lngold- Prelog rules for stereochemistry can be employed to designate stereochemical priority, E1 Z specification, and the like. One of skill in the art can readily ascertain the structure of a compound if given a name, either by systemic reduction of the compound structure using naming conventions, or by commercially available software, such as CHEMDRAW™ (Cambridgesoft Corporation, U.S.A.).

[0015] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0016] The term “subject” as used herein refers to an animal. Typical examples of an animal include but are not limited to mammals. In specific embodiments, the subject is a human, dog, cat, cow, horse, pig, goat, sheep, rat, mouse, guinea pig, or a nonhuman primate.

[0017] The term “effective amount” refers to an amount of a therapeutic agent that is sufficient to exert a physiological effect in the subject.

[0018] The term "sample" as used herein includes any biological specimen obtained from a patient. Samples include, without limitation, whole blood, plasma, serum, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), cord blood, ductal lavage fluid, nipple aspirate, lymph, bone marrow aspirate, saliva, urine, stool (i.e., feces), sputum, bronchial lavage fluid, tears, fine needle aspirate, any other bodily fluid, a tissue such as a biopsy of a tumor (e.g., needle biopsy) or a lymph node, and cellular extracts thereof. In some embodiments, the sample is whole blood or a fractional component thereof such as plasma, serum, or a cell pellet.

[0019] As used herein, the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the severity, duration and/or progression of a disease or disorder such as a GSD, or one or more symptoms thereof resulting, from the administration of one or more therapies.

[0020] The term “therapy” refers to the standard of care needed to treat a specific disease or disorder. In a typical example, therapy involves the act of administering to a subject a therapeutic agent(s) in an effective amount.

[0021] As used herein, the term "diagnostic threshold" refers to an amount of glycogen in a sample from a subject that is at least 30, 40 or 50% above or below the amount of glycogen in a sample from a control subject (i.e. healthy subject). In a specific example, the diagnostic threshold pertains to glycogen levels 50-300% higher than control subjects. In another embodiment, the diagnostic threshold level pertains to glycogen levels at least 300% higher than control subjects. The diagnostic threshold can be established empirically by analyzing samples from healthy patients and from patients known to have a glycogen storage disease.

[0022] The term glycogen storage disease therapy (GSD) therapy as used herein refers to a therapy for treating GSD, and in particular glycogen storage disease type II. Examples of therapeutic agents used in GSD therapy include, but are not limited to, enzymes (enzyme replacement therapy or ERT), gene therapy constructs or vectors, small molecules, oligonucleotide inhibitor molecules (e.g. siRNA, shRNA, antisense molecules), and small molecules. Delivery of enzymes for ERT can involve packaged or unpackaged enzymes. Packaged enzymes include, but are not limited to, modified cells (e.g. B-cells) engineered to express enzymes, enzyme-containing microvesicles (e.g. exosomes), enzyme-containing liposomes, or nanoparticle associated enzymes.

[0023] Unless explicitly stated otherwise, as used herein, the term “glycogen” refers to both normal glycogen and diseased glycogen, which is also called polyglucans or polyglucosan bodies.

Assay and Kit Reagents

[0024] Assay and kit embodiments described herein utilize or include, respectively, certain enzymes for cleaving glycogen in a sample. In a specific example, a glycogen cleaving enzyme pertains to an isomylase. The glucose chains may be cleaved from the glycogen using any suitable isoamylase. In some embodiments, the isoamylase includes any suitable enzyme isoamylase that specifically cleaves glucose chains at only the a1 ,-6 linkages.

[0025] In some embodiments, the method includes dual imaging of glycogen and N- glycans. In such embodiments, the method also includes releasing N-linked glycans with any suitable enzyme, such as, but not limited to, peptide-N-glycosidase F (PNGase F).

[0026] In a specific embodiment, Isoamylase is isolated from bacteria (such as Pseudomonas sp, or other suitable bacteria sources). In an alternative embodiment, an enzyme from another source with glycogen 6-a-D-glucanohydrolase activity is utilized. Another enzyme, such as PNGase F, can be included as secondary enzyme control.

[0027] One of the improvements presented by the embodiments here is the implementation of specific glycogen-based standards that enable the ability to produce a calibration curve for more accurate quantification of glycogen in a sample. The standards used in the kit may include purified glycogen, long chain polysaccharides (glucose 7-12 length polymers), or starch. In a specific example, isotopically labeled glycogen is utilized for improved quantitative accuracy and linear range. 13C-glycogen as well as 12C-glycogen. 13C-glycogen is produced by feeding 13C-gluocse to bacteria or mice and purified per the method that we developed (Sun RC et al, Nature communications, (2017) 8(1 ), 1646). In typically embodiments, concentrations of 1 picogram to 1 milligram will be used as a linear range for glycogen quantitation. Other glycogen standards can include, rabbit, bovine, and oyster glycogen, as well as linear polysaccharide with 5-10 glucose unit long.

[0028] Standards and enzymes are typically included in a kit as lyophilized powder that can be resuspended in ultra-pure water or saline buffer provided with the diagnostic test. In a specific embodiment, glycogen standards are wet-printed directly onto Mass- spec suitable slides using a reagent printer and can included as ready to use slides as part of the kit. Volumes will be on the 10 nL to 1 ml_ scale, and will be precisely spatially located on the slides using automated liquid dispensing methods.

[0029] The kit may also comprise components for isolation of peripheral blood mononuclear cells (PBMC) from a fresh whole blood sample. PBMC will provide improved sensitivity to detect mild/single mutation(s) in GSDs and whole blood will be used to detect severe or loss of function mutations

[0030] The kit embodiments provided herein may include Mass-spec suitable slides. These include charged microscope slides, coated slides, or preferably intelli-slides (Bruker). In other embodiments, liquid sample slides are included, which have liquid sample spots pre-allocated using hydrophobic barrier material to contain liquid samples in place.

[0031] In another aspect, the present invention provides a method for quantifying glycogen in a sample (e.g. at least one population of cells), the method comprising the steps of: applying at least one population of cells to a surface of a substrate; optionally, fixing and rinsing the at least one population of cells; spraying the substrate with an enzyme solution; and scanning the substrate by mass spectrometry to detect and quantify the presence of linear glucose chains. In one embodiment, the substrate is a glass or plastic microscope slide or multiwell plate, and which has been treated to include different concentrations of glycogen for use a standards. In one embodiment, the substrate surface may include one or more of: an indium tin oxide coating, a gelatin coating, a collagen coating, a poly-1 -lysine coating, a poly-ornithine coating, an extracellular matrix coating, a protein coating, and surface ionization. In one embodiment, the enzymatic releasing solution comprises isoamylase.

[0032] In certain examples, the mass spectrometry utilized in the methods/assay is selected from the group consisting of: matrix-assisted laser desorption/ionization imaging Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, scanning microprobe MALDI (SMALDI) mass spectrometry, infrared matrix assisted laser desorption electrospray ionization (MALD-ESI) mass spectrometry, surface-assisted laser desorption/ionization (SALDI) mass spectrometry, desorption electrospray ionization (DESI) mass spectrometry, secondary ion mass spectrometry (SIMS) mass spectrometry, and easy ambient sonic spray ionization (EASI) mass spectrometry. In one embodiment, the scanning step is preceded by a step of spraying the substrate with a MALDI matrix material. In one embodiment, the MALDI matrix solution is selected from the group consisting of: 2,5-dihydroxybenzoic acid (DHB), a- cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), 1 ,5-diaminonaphthalene (DAP), and 9-aminoacridine (AAD).

[0033] In a specific assay embodiment, the assay includes a method with the following steps:

1 . Isolating PBMC from whole blood.

2. Spotting PBMC on pre-supplied slides with glycogen standards already printed on the slide. The PBMC may be spotted on designated spots on the slide (created by hydrophobic barrier), and can be 1 sample or up to 96 samples. (1 -100 designated spots for receiving 1 -100 different samples)

3. Optionally, dissolving enzyme provided in a kit in water.

4. Applying enzyme to spots using an enzyme dry sprayer (e.g. HTX enzyme dry sprayer), this sprayer assists in even application of enzymes to all samples. 5. Incubating slide with sample spots and enzymes.

6. Optionally, storing slides with enzymes in desiccator until ready to use. At this stage, slides can be safely store up to 3 days.

7. Spray MALDI-matrix (e.g., CHCA, DHB, SA, AAD, or DAP) onto slide using a dry sprayer (e.g. HTX dry sprayer.

8. Analyzing slide using MALDI.

[0034] PBMCs from normal control subjects may be utilized as a control. In a specific example, the glycogen amount is calculated based on glucose polymer 3-20 and quantified by glycogen standards. In a specific example, a normal cut off (diagnostic threshold) is produced by comparing the quantitative amount of glycogen in normal control patients to those with aberrant glycogen accumulation.

[0035] The MALDI ionization matrix may include any suitable ionization matrix that is compatible with MALDI. Suitable ionization matrixes include, but are not limited to, CHCA or DHB for detection of N-linked glycans by MALDI-MSL The enzyme(s) and MALDI matrix may be applied simultaneously or sequentially using any suitable method of application, such as, but not limited to, uniform application using a high velocity dryspraying robot.

[0036] In a particular embodiment, provided is a unique slide onto which the glycogen standards are directly printed. The slides with printed glycogen standards may also include areas to guide spotting of liquid samples of PBMC.

[0037] The kits can be promoted, distributed or sold as units for performing the methods described herein. Additionally, the kits can contain a package insert describing the kit and methods for its use. A kit for glycogen detection and quantification may include at least one substrate, each substrate having a surface spotted with a plurality of glycogen standards; at least one isoamylase solution; and at least one MALDI matrix material.

GSD Therapy

[0038] Upon the determination that a subject exhibits indicators of a glycogen storage disease, such as quantifying glycogen in a biological subject, a GSD therapy may be administered to a subject. Alternatively, GlycoSense can be used to detect response to therapy, i.e after administration of GSD therapy, glycoSense is used to define reduce or lack of reduction in glycogen and can be used to titrate therapeutic concentration to become effective dose or dosing. There are currently approved enzymes for administration as ERT for treating Pompe disease which include alglucosidase alfa (Sanofi) and Avagluoscidase alfa (Sanofi). Other examples of ERT agents include PGN004 (Pharming), Enzyme-AMFA (NanoMedSyn), OXY2810 (Oxyrane), JR-162 (JCR Pharma), Repleva GAA (Eleva), Algugosidase alfa biosimilar (UGA Biopharma), rhGAA (Huons), tobrhGAA (JME Group), EVT-GAA (Denali), M-021 (M6P), and GAA ERT.

[0039] Examples of Gene therapy approaches for GSD therapy include ACTUS-101 (AskBio, Bayer), SPK-3006 (Spark & Roche), AT845 (Astellas Gene Therapies), rAAV9- DES-hGAA (UF/Lacerta), Sel-339 (Selecta), hGAAAAV (Amicus), AVR-RD-03 (Avrobio), AAV Gene Tx (Sarepta/Lacerta), AAVB1 -GAA (Univ. Mass), AAV Gene Tx (Abeona), CRISPR-/Cas9-AAV (Rgeneron), FTX-PD01 , CAN202 (CANbridge), and AAV9 GYS1 Gene (Genethon).

[0040] Small Molecule drug approaches for GSD Therapy include MZE001 (Maze), CX-1739 (RespireRx), and GYS1 Modulator (Icagen/Ligand). Oligonucleotide approaches include GYS1 antisense (lonis), siRNA (Avidity and ABX1100 (Aro).

Examples of other GSD therapies are described in U.S. Patents 7,056,712; 9,050,333; 9,370,556; 9,644,216; 9,850,474; 10,647,969; 11 ,060,110; 11 ,214,782; 11 ,332,760; and 11 ,535,870, and US20160184410A1 , which are incorporated by reference.

GSD Diagnosis

[0041] According to another embodiment, disclosed is a method comprising applying at least one sample from the subject to a surface of a substrate, the substrate comprising a plurality of pre-applied glycogen standard spots; optionally, fixing and rinsing the sample; applying an isoamylase solution to the substrate; and scanning the substrate by mass spectrometry; detecting a presence of linear glucose chains in the at least one sample and the plurality of pre-applied glycogen standard spots; optionally generating a calibration curve based on the detection of linear glucose chains from the pre-applied glycogen standard spots; quantifying an amount of glycogen in the at least one sample based on amounts of the linear glucose chains detected in the at least one sample and the pre-applied glycogen standard spots; diagnosing the subject as having a glycogen storage disease if the amount of glycogen in the at least one sample is at least 50% higher than glycogen levels in a control subject, or above a diagnostic threshold; and administering a glycogen storage disease therapy to the subject. In a specific embodiment, the at least one sample of the subject comprises PBMCs.

[0042] According to another embodiment, provided is a method comprising quantifying an amount of glycogen in at least one PBMC containing sample from a subject using a slide or kit as described herein; diagnosing the subject as having a glycogen storage disease if an amount of glycogen in the at least one PBMC containing sample is above a diagnostic threshold level; and administering a glycogen storage disease therapy to the subject.

Examples

Glycogen MALDI MSI in FFPE tissues

[0043] Formalin-fixed paraffin-embedded tissues were sectioned (4 pm) and mounted on slides for MALDI imaging as described in Stanback, A. E. et al. Regional N-glycan and lipid analysis from tissues using MALDI-mass spectrometry imaging. STAR Protoc. 2, 100304 (2021 ). Slides were heated (60 °C, 1 h), deparaffinized in xylene and rehydrated in graded ethanol and water. Antigen retrieval was performed using citraconic anhydride buffer (pH 3) in a vegetable steamer (30 min). After cooling, buffer was gradually replaced with water, and slides were desiccated before enzymatic digestion. For absolute glycogen quantitation, serial dilutions of rabbit liver glycogen (0.05-0.00061 mg ml^-1) were spotted onto slides using a mosquito low-volume pipettor. FIG. 3A shows one example of spotted standards that have been applied to slided onto which tissue is fixed. Enzyme-coated slides were prepared using the HTX spray station with isoamylase (3 units per slide), PNGase F (20 mg per slide) or both, followed by incubation (37 °C, 2 h) and matrix application. MALDI MSI was performed using a Waters SynaptG2-Xs, with laser (1 ,000 Hz, 200 a.u., 50-pm spot), mass range (500- 3,500 m/z) and ion mobility. Mass drift correction and signal enhancement were applied using high-definition imaging software <Stanbeck et aL, supra), leveraging matrix and glycan peaks for calibration.

Multiplexed imaging of metabolome and glycogen from fresh frozen specimens

[0044] Mouse lungs were resected and snap frozen in liquid nitrogen. Fresh frozen lungs were cut at 10 pm onto microscope slides without optimal cutting temperature compound. Tissue sections were dried in a desiccator immediately to terminate metabolism. Slides were then sprayed with an N-(1 -naphthyl) ethylenediamine dihydrochloride/70% methanol matrix at 7 mg ml^-1 via an HTX M5 Sprayer. Slides were then run on Bruker timsTOF flex with a laser power operating at 10,000 Hz and 60% laser energy, with 300 laser shots per pixel, and raster and laser spot size of 50 pm to image for small molecules and lipids (Clarke, H. A. et al. Spatial metabolome lipidome and glycome from a single brain section. Preprint at bioRxiv). Mass acquisition was set from 50 to 2,000 m/z in negative mode. The matrix was then stripped off using ice-cold 100% methanol for 5 min, and the tissue was fixed overnight in 10% NBF. The next morning, slides were switched from NBF to 70% ethanol, and remained for 2 h before desiccating. The slides were then prepared according to our N-glycan and glycogen imaging using enzyme assist release of N-linked glycans and glycogen for MALDI ionization.

[0045] Fixed tissue sections were prepared using the HTX spray station (HTX) was used to coat the slide with a 0.2 ml aqueous solution of either isoamylase (3 units per slide), PNGase F (20 mg total per slide) or both. The spray nozzle was heated to 45 °C, and the spray velocity was 900 m min^-1. After enzyme application, slides were incubated at 37 °C for 2 h in a humidified chamber, then dried in a desiccator before matrix application with 0.04 g a-cyano-4-hydroxycinnamic acid matrix (CHCA) in 5.7 ml 50% acetonitrile/50% water and 5.7 pl trifluoroacetic acid) with the HTX sprayer. At the end of the sample preparation, tissue sections were run using the Bruker timsTOF flex with a laser power operating at 10,000 Hz and 60% laser energy, with 300 laser shots per pixel, and raster and laser spot size of 50 pm for the integration of metabolome and glycome using x,y laser coordinates produced from the MALDI imaging files. Mass acquisition was set from 500 to 4,000 m/z in positive mode. Samples were analysed using the SCiLS labs software (Bruker) using the manufacturer-recommended settings, and pixel data were exported using the SCiLS API and plotted using graphing software Prism.

Claims

CLAIMS What is claimed is:

1 . A kit for glycogen detection and quantification in a biological sample, the kit comprising: at least one substrate, each substrate having a surface onto which a plurality of glycogen standard spots are disposed; at least one isoamylase solution; and, optionally, at least one MALDI matrix material.

2. The kit of claim 1 , wherein the substrate is a glass or plastic microscope slide or multiwell plate onto which the plurality of glycogen standard spots are printed.

3. The kit of claims 1 or 2, wherein the plurality of glycogen standard spots comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different spots with varying concentrations of glycogen.

4. The kit of any of claims 1 -3, wherein the substrate comprises charged microscope slides, coated slides or liquid sample slides.

5. The kit of any of claims 1 -4, wherein the MALDI matrix solution comprises 2,5- dihydroxybenzoic acid (DHB), a-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), 1 ,5-diaminonaphthalene (DAP), and/or 9-aminoacridine (AAD).

6. The kit of any of claims 1 -5, wherein plurality of glycogen standard spots are dried.

7. The kit of any of claims 1 -5, wherein the plurality of glycogen standard spots are liquid spots contained by a hydrophobic barrier material.

8. A method for quantifying glycogen in at least one sample, the method comprising the steps of: applying at least one sample to a surface of a substrate, the substrate comprising a plurality of pre-applied glycogen standard spots; optionally, fixing and rinsing the sample; applying an enzyme solution to the substrate; scanning the substrate by mass spectrometry; detecting a presence of linear glucose chains in the at least one sample and the plurality of pre-applied glycogen standard spots; optionally generating a calibration curve based on the detection of linear glucose chains from the pre-applied glycogen standard spots; and quantifying an amount of glycogen in the at least one sample based on amounts of the linear glucose chains detected in the at least one sample and the pre-applied glycogen standard spots.

9. The method of claim 8, wherein the enzyme solution comprises isoamylase.

10. The method of claim 9, wherein the isoamylase is from a bacteria source.

11 . The method of any of claims 8-10, wherein the substrate is a glass or plastic microscope slide or multiwell plate onto which the plurality of pre-applied glycogen standard spots are printed onto the substrate.

12. The method of any of claims 8-11 , wherein the mass spectrometry comprises matrix-assisted laser desorption ionization mass spectrometry imaging.

13. The method of any of claims 8-12, wherein the at least one sample comprises peripheral blood mononuclear cells (PBMCs) or a tissue biopsy.

14. The method of any of claims 8-13, wherein applying comprises spraying.

15. A method comprising applying at least one sample from the subject to a surface of a substrate, the substrate comprising a plurality of pre-applied glycogen standard spots; optionally, fixing and rinsing the sample; applying an isoamylase solution to the substrate; and scanning the substrate by mass spectrometry; detecting a presence of linear glucose chains in the at least one sample and the plurality of pre-applied glycogen standard spots; optionally generating a calibration curve based on the detection of linear glucose chains from the pre-applied glycogen standard spots; quantifying an amount of glycogen in the at least one sample based on amounts of the linear glucose chains detected in the at least one sample and the pre-applied glycogen standard spots; diagnosing the subject as having a glycogen storage disease if the amount of glycogen in the at least one sample is at least 50% higher than glycogen levels in a control subject, or above a diagnostic threshold; and administering a glycogen storage disease therapy to the subject.

16. The method of claim 15, wherein the at least one sample comprises PBMCs.

17. A method comprising

Quantifying an amount of glycogen in at least one PBMC containing sample from a subject using the kit of claim 8;

Diagnosing the subject as having a glycogen storage disease if an amount of glycogen in the at least one PBMC containing sample is above a diagnostic threshold level; and

Administering a glycogen storage disease therapy to the subject.

18. The method of any of claims 15-17, wherein the glycogen storage disease therapy comprises administration of an effective amount of enzyme replacement therapy, gene therapy, a small molecule, or oligonucleotide inhibitor. 1

19. The method of any of claims 15-18, wherein the disease is one of aberrant excess or deficient glycogen, wherein, optionally, the GSD is Pompe disease.

20. The method of claim 19, wherein the Pompe disease is late onset form (LOPD) or Infantile onset form (IOPD).