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WO2025179146A1 - Compositions et procédés de visualisation de glycosylation - Google Patents

Compositions et procédés de visualisation de glycosylation

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Publication number
WO2025179146A1
WO2025179146A1 PCT/US2025/016798 US2025016798W WO2025179146A1 WO 2025179146 A1 WO2025179146 A1 WO 2025179146A1 US 2025016798 W US2025016798 W US 2025016798W WO 2025179146 A1 WO2025179146 A1 WO 2025179146A1
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Prior art keywords
oligonucleotide
sequence
bound
seq
sequence identity
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Inventor
Yu Xin Wang
Sara ANCEL
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Sanford Burnham Prebys Medical Discovery Institute
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Sanford Burnham Prebys Medical Discovery Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H10/00—ICT specially adapted for the handling or processing of patient-related medical or healthcare data
    • G16H10/40—ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H20/00—ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance
    • G16H20/10—ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance relating to drugs or medications, e.g. for ensuring correct administration to patients
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H30/00—ICT specially adapted for the handling or processing of medical images
    • G16H30/20—ICT specially adapted for the handling or processing of medical images for handling medical images, e.g. DICOM, HL7 or PACS
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H30/00—ICT specially adapted for the handling or processing of medical images
    • G16H30/40—ICT specially adapted for the handling or processing of medical images for processing medical images, e.g. editing
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/70—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for mining of medical data, e.g. analysing previous cases of other patients
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
    • G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/38—Post-translational modifications [PTMs] in chemical analysis of biological material addition of carbohydrates, e.g. glycosylation, glycation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders

Definitions

  • glycosylation is a highly conserved molecular process by which carbohydrate moieties are added to proteins or lipids in order to modulate their overall structure and function. These modifications are critical molecular determinants of cellular function and can play key roles in signaling, cellular adhesion, protein folding, trafficking, or as a biomarker of disease. Glycosylation is also spatially regulated and disparate glycosylation patterns are found in various locations within the cell. Therefore, there is a need for methods of analysis of glycosylation in situ. SEQUENCE LISTING [0004] The application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety.
  • a method of imaging a sample comprising: (i) contacting the sample with (a) a plurality of carbohydrate binding proteins, wherein each carbohydrate binding protein is covalently bound to an oligonucleotide; and (b) a subset of a plurality of fluorescently labeled nucleic acid probes, wherein each nucleic acid probe specifically binds to only one of the oligonucleotides of (i)(a), thereby generating a probe/carbohydrate binding protein complex; (ii) imaging the sample to detect a binding of each of the probe/carbohydrate binding protein complexes of step (i)(b); and (iii) removing the probes bound to the
  • the method further comprises repeating steps (i) to (iii) with a different subset of the plurality of fluorescently labeled nucleic acid probes of (i)(b) until all subsets of the plurality of fluorescently labeled nucleic acid probes have been utilized.
  • the plurality of carbohydrate binding proteins comprises a lectin.
  • the plurality of carbohydrate binding proteins comprises a mannose-binding protein.
  • the plurality of carbohydrate binding proteins comprises a catalytically inactivated enzyme.
  • the catalytically inactive enzyme is a glycosylase. [0011] In some embodiments, the catalytically inactive enzyme is a transferase. [0012] In some embodiments, the catalytically inactive enzyme is inactivated by one or more mutations. [0013] In some embodiments, the catalytically inactive enzyme is inactivated by truncation. [0014] In some embodiments, the plurality of carbohydrate binding proteins comprises about 5, about 10, about 15, about 20, about 25, about 30, about 35, or about 40 different carbohydrate binding proteins. [0015] In some embodiments, the plurality of carbohydrate binding proteins comprises two or more of the carbohydrate binding proteins listed in Table 1 or Table 3.
  • the plurality of carbohydrate binding proteins comprises a recombinant protein.
  • the carbohydrate binding protein is covalently bound to the oligonucleotide via a copper-free click chemistry reaction.
  • each carbohydrate binding protein of the plurality of carbohydrate binding proteins is covalently bound to from 1 to about 15 copies of the oligonucleotide.
  • the sample comprises a whole tissue.
  • the sample comprises a tissue section.
  • the sample is obtained from muscle, brain, a nerve, a sciatic nerve, spinal cord, kidney, liver, heart, lung, lymph node, spleen, or intestine tissue. [0022] In some embodiments, the sample is fixed. [0023] In some embodiments, the sample is not fixed. [0024] In some embodiments, step (iii) comprises contacting the sample with dimethyl sulfoxide (DMSO) or formamide.
  • DMSO dimethyl sulfoxide
  • the plurality of carbohydrate binding proteins comprise two or more oligonucleotide-bound carbohydrate binding proteins selected from the group consisting of: i) HHA bound to an oligonucleotide having at least 80% sequence identity to a sequence of WSGR Docket No.: 42256-622.601 SEQ ID NO: 1; ii) CSA bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 2; iii) TKA bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 3; iv) MNAG bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 5; v) TL bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 6; vi) WFA bound to an oligonucleotide
  • a method of imaging a sample comprising: (i) contacting the sample with a plurality of different carbohydrate binding proteins, wherein each different carbohydrate binding protein of the plurality of different carbohydrate binding proteins is covalently bound to a barcode oligonucleotide of a plurality of barcode oligonucleotides, wherein, upon the contacting, a subset of the plurality of different carbohydrate binding proteins WSGR Docket No.: 42256-622.601 binds to the sample; (ii) contacting the sample with a plurality of fluorescently labeled first nucleic acid probes, wherein each fluorescently labeled first nucleic acid probe of the plurality of fluorescently labeled first nucleic acid probes binds to a first barcode oligonucleotide of the plurality of barcode oligonucleotides of step (i), thereby generating a first binary complex of the fluorescently labeled first
  • the method further comprises: (ii’) contacting the sample with a plurality of fluorescently labeled second nucleic acid probes, wherein each fluorescently labeled second nucleic acid probe of the plurality of fluorescently labeled second nucleic acid probes is configured to bind to a second barcode oligonucleotide of the plurality of barcode oligonucleotides of step (i), thereby generating a second binary complex of the fluorescently labeled second nucleic acid probe/second barcode oligonucleotide; (iii’) imaging the sample to detect a presence or absence of the second binary complex of step (ii’); and (iv’) removing the plurality of fluorescently labeled second nucleic acid probes from the sample, wherein the subset of the plurality of different carbohydrate binding proteins remain bound to the sample.
  • the method further comprises repeating steps (ii) to (iv) multiple times, each time with a plurality of fluorescently labeled additional nucleic acid probes, wherein each fluorescently labeled additional nucleic acid probe of the plurality of fluorescently labeled additional nucleic acid probes is configured to bind to an additional barcode oligonucleotide of the plurality of barcode oligonucleotides of step (i).
  • the imaging comprises measuring a staining intensity.
  • the plurality of different carbohydrate binding proteins comprises one or more lectins.
  • the plurality of different carbohydrate binding proteins comprise two or more oligonucleotide-bound carbohydrate binding proteins selected from the group consisting of: i) HHA bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 1; ii) CSA bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 2; iii) TKA bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 3; iv) MNAG bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 5; v) TL bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 6; vi) WFA bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 1; ii
  • composition comprising a plurality of carbohydrate binding proteins, wherein each carbohydrate binding protein is covalently bound to an oligonucleotide, and wherein the oligonucleotide comprises a sequence having at least 80% identity to a sequence set forth in any one of SEQ ID NOs: 1-55.
  • the oligonucleotide comprises a sequence set forth in any one of SEQ ID NOs: 1-55.
  • the plurality of carbohydrate binding proteins comprises a catalytically inactivated enzyme.
  • the catalytically inactive enzyme is a glycosylase.
  • the catalytically inactive enzyme is a transferase.
  • the catalytically inactive enzyme is inactivated by one or more mutations.
  • the catalytically inactive enzyme is inactivated by truncation.
  • the plurality of carbohydrate binding proteins comprises a carbohydrate binding protein listed in Table 1 or Table 3, or two or more of the carbohydrate binding proteins listed in Table 1 or Table 3.
  • the plurality of carbohydrate binding proteins comprises two or more oligonucleotide-bound carbohydrate binding proteins selected from the group consisting of: i) HHA bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 1; ii) CSA bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 2; iii) TKA bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 3; iv) MNAG bound to an oligonucleotide having at least 80% sequence identity to a sequence of SEQ ID NO: 5; v) TL bound to an oligonucleo
  • kits comprising: (iii) a plurality of carbohydrate binding proteins, wherein each carbohydrate binding protein is covalently bound to a different oligonucleotide; (ii) a plurality of fluorescently labeled nucleic acid probes, wherein each nucleic acid probe is configured to specifically bind to only one of the oligonucleotides of step (i); and (iii) a denaturing agent capable of removing a nucleic acid probe that is bound to an oligonucleotide-bound-carbohydrate binding protein.
  • the plurality of carbohydrate binding proteins comprise two or more oligonucleotide-bound carbohydrate binding proteins selected from the group consisting of: i) HHA bound to an oligonucleotide having a sequence of SEQ ID NO: 1; ii) CSA bound to an oligonucleotide having a sequence of SEQ ID NO: 2; iii) TKA bound to an oligonucleotide having a sequence of SEQ ID NO: 3; iv) MNAG bound to an oligonucleotide having a sequence of SEQ ID NO: 5; v) TL bound to an oligonucleotide having a sequence of SEQ ID NO: 6; vi) WFA bound to an oligonucleotide having a sequence of SEQ ID NO: 7; vii) ECA bound to an oligonucleotide having a sequence of SEQ ID NO: 8; viii) LEA bound to an an oligonucleotide
  • the oligonucleotide comprises sequence having at least 80% identity to a sequence set forth in any one of SEQ ID NOs: 1-55. [0070] In some embodiments, the oligonucleotide comprises sequence set forth in any one of SEQ ID NOs: 1-55. [0071] In some embodiments, the carbohydrate binding protein is selected from the carbohydrate binding proteins listed in Table 1 or Table 3.
  • a computer implemented method for analyzing a sample comprising: (i) receiving input comprising images of the sample, wherein the images of the sample comprise images generated by the method of any one of the preceding embodiments; and (ii) generating, using a machine learning algorithm, an output comprising a quantitative or qualitative value of functional or structural features of the sample.
  • a method of detecting a tissue pathology comprising: (i) obtaining a tissue sample; and (ii) applying the method of any one of the preceding embodiments to the tissue sample.
  • the tissue sample comprises cancer cells.
  • the tissue sample comprises cells comprising a disorder of glycosylation.
  • the tissue sample comprises a glycosylation pattern that is different from a tissue sample that does not comprise cancer cells or cells comprising a disorder of glycosylation.
  • the tissue sample comprises a lower or higher expression level of the fluorescently labeled nucleic acid probes of any one of the preceding embodiments.
  • the disorder of glycosylation comprises Saul-Wilson Syndrome (SWS) or GNE myopathy.
  • the cancer is pancreatic ductal adenocarcinoma (PDAC).
  • a method of characterizing a cancer in a subject comprising: (i) obtaining a tissue sample from the subject, wherein the tissue sample comprises a cancer tissue; and (ii) applying the method of any one of the preceding embodiments to the tissue sample.
  • the cancer is pancreatic ductal adenocarcinoma (PDAC).
  • the characterizing comprises identifying a border between the cancer tissue and a healthy tissue in the tissue sample.
  • the border is identified by detecting a lower or higher expression level of the fluorescently labeled nucleic acid probes of any one of the preceding embodiments by the cancer tissue compared to the healthy tissue.
  • the characterizing comprises determining a size of a tumor. [0085] In some embodiments, the characterizing comprises determining progression of the tumor. [0086] In some embodiments, the progression is measured by comparing results obtained from a first tissue sample to results obtained from a second tissue sample, wherein the second tissue sample is obtained at least one week later than the first tissue sample. INCORPORATION BY REFERENCE [0087] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
  • FIGs.1A-1C depict results measuring conjugation of dibenzocyclooctyne (DBCO) to IgG antibodies.
  • FIG.1A depicts number of DBCO molecules per antibody when reacted with various concentrations of DBCO.
  • FIG.1B depicts fluorescence microscopy images detecting antibodies conjugated with an optimal concentration of DBCO (middle), the limit of detection (left), or at saturation (right).
  • FIG.1C is a quantification of the fluorescence intensity across the stained tissue.
  • FIG.2 depicts results measuring number of DBCO molecules per carbohydrate binding protein Wheat Germ Agglutinin (WGA) when reacted with various concentrations of DBCO.
  • FIG.3A is a fluorescence microscopy image of a skeletal muscle sample obtained using oligonucleotide-conjugated carbohydrate binding proteins bound to fluorescent oligonucleotide probes.
  • FIG.3B is a fluorescence microscopy image of a skeletal muscle sample obtained using oligonucleotide-conjugated antibodies bound to fluorescent oligonucleotide probes.
  • FIGs.4A-4E are fluorescence microscopy images detecting staining with carbohydrate binding proteins in a variety of cellular locations in a skeletal muscle sample.
  • FIG.4A depicts nuclear staining
  • FIG.4B depicts peri-nuclear staining
  • FIG.4C depicts cytoplasmic staining
  • FIG.4D depicts cell membrane staining
  • FIG.4E depicts extracellular matrix (ECM) staining.
  • ECM extracellular matrix
  • FIGs.5A-5B are fluorescence microscopy image of a skeletal muscle sample obtained using carbohydrate binding proteins specific for certain cell types or tissue structures.
  • FIG.5A depicts muscle fiber (LCA) and NMJ (VVL) staining.
  • FIG.5B depicts staining of vessels (DSL), muscle spindles (PHAE), and motor nerves (ECL).
  • FIG.6 is a schematic diagram depicting a process by which a glycan profile is obtained for a skeletal muscle tissue sample and the resulting images are used to train an artificial intelligence algorithm to analyze structural features of the tissue.
  • FIGs.7A-7C depict examples of data output from an analysis performed by an artificial intelligence algorithm analyzing various parameters of skeletal muscle structure and function.
  • FIG.7A depicts the capillary count
  • FIG.7B depicts the myofiber count
  • FIG.7C depicts the myofiber size.
  • FIGs.8A-8C are fluorescence microscopy images measuring glycan profiles in healthy (FIG.8A), denervated (FIG.8B), and regenerating (FIG.8C) skeletal muscle.
  • FIG.9 is a schematic diagram depicting method of obtaining a glycan profile by staining a sample with a variety of carbohydrate binding proteins conjugated to oligonucleotides and obtaining serial images by staining with oligonucleotides complementary to a subset of the bound oligonucleotide-conjugated carbohydrate binding proteins.
  • the resultant images can be used to resolve distributions of individual glycan moieties.
  • FIG.10A is a schematic diagram depicting a method of obtaining a glycan profile by staining a sample with a glycosylation landscape analysis by probe hybridization (GLYPH) panel of barcoded carbohydrate binding proteins including lectins, antibodies, and peptides.
  • Carbohydrate binding proteins are conjugated with oligonucleotide barcodes using CLICK- chemistry and rendered via annealing with fluorescent cDNA probes. After imaging, probes are denatured and washed before the next cycle of imaging. Images are processed and aligned to generate GLYPH maps.
  • FIG.10B is a schematic diagram depicting a computational analysis of the profile obtained in FIG.10A by signal decomposition of mixed signals using known binding kinetics of carbohydrate binding proteins into individual glycan moieties.
  • Cells and tissue structures are segmented, glycan signals are quantified for each cell, and analyzed for spatial relationships and enriched interactions.
  • FIG.11 depicts fluorescence microscopy images detecting glycosylation patterns of induced pluripotent stem cell (iPSC)-derived osteo-chondral organoids comparing organoids obtained from healthy iPSCs to those obtained from iPSCs carrying Saul-Wilson Syndrome mutations.
  • iPSC induced pluripotent stem cell
  • FIG.12 depicts a fluorescence microscopy image detecting glycosylation patterns of pancreatic ductal adenocarcinoma tissue samples comparing healthy tissue with tumor tissue.
  • FIG.13 depicts fluorescence microscopy images detecting glycosylation patterns of human muscle tissue comparing control samples to samples obtained from patients with GNE myopathy. DETAILED DESCRIPTION [00104] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
  • Co-detection by indexing generally uses deoxyribonucleic acid (DNA)- conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes to simultaneously visualize many markers in situ.
  • CODEX enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in developmental biology, disease and therapeutic design.
  • Glycosylation landscape analysis by probe hybridization generally uses DNA-conjugated carbohydrate binding proteins and the cyclic addition and removal of complementary fluorescently labeled DNA probes to simultaneously visualize many markers in situ.
  • GLYPH enables an unbiased, high resolution spatial analysis of patterns of glycosylation and may depict how these patterns contribute to biological functions and cellular responses in healthy or diseased tissues.
  • the present disclosure allows for multiplexed spatial glycan profiling using DNA barcoding and iterative rendering to visualize and study glycosylation in situ. The results may provide combinatorial glycan profiles for specific cell types and/or tissue structures that can provide insights into disease, aging, and development.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.
  • the term “about” or “approximately” may mean within an acceptable error range for the particular value, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value may be assumed.
  • DNA barcoding generally refer to a unique molecular tag/label that identifies the carbohydrate binding proteins to which the unique molecular tag/label is covalently bound.
  • the length of the barcode can be from about 10 nucleic acids to about 20 nucleic acids, from about 20 nucleic acids to about 30 nucleic acids, or from about 30 nucleic acids to about 40 nucleic acids.
  • the nucleic acid sequence of the barcode oligonucleotide can be determined, thereby identifying the carbohydrate binding proteins to which the barcode oligonucleotide is covalently bound.
  • the present disclosure provides a method of imaging a sample.
  • the method comprises contacting the sample with (i) a plurality of carbohydrate- binding proteins, wherein each carbohydrate-binding protein is covalently bound to an oligonucleotide and (ii) a subset of a corresponding plurality of fluorescently labeled nucleic acid probes, wherein each nucleic acid probe specifically binds to only one of the oligonucleotides covalently bound to each carbohydrate-binding protein, thereby generating a probe/carbohydrate-binding protein complex.
  • the plurality of carbohydrate-binding proteins binds or couples to the sample.
  • the plurality of carbohydrate-binding proteins is coupled to a WSGR Docket No.: 42256-622.601 plurality of oligonucleotides.
  • a carbohydrate-binding protein of the plurality of carbohydrate-binding proteins is coupled to an oligonucleotide of the plurality of oligonucleotides.
  • a carbohydrate-binding protein of the plurality of carbohydrate-binding proteins is covalently bound to an oligonucleotide of the plurality of oligonucleotides.
  • a first subset of the plurality of carbohydrate-binding protein is coupled to a first subset of the plurality of oligonucleotides.
  • a second subset of the plurality of carbohydrate-binding proteins is coupled to a second subset of the plurality of oligonucleotides.
  • the first subset and the second subset of the plurality of carbohydrate-binding proteins are different.
  • the first subset and the second subset of the plurality of carbohydrate-binding proteins are same.
  • the first subset and the second subset of the plurality of oligonucleotides are same.
  • a nucleic acid probe specifically binds to an oligonucleotide that is covalently bound to a carbohydrate-binding protein, thereby generating a probe/carbohydrate-binding protein complex (or probe/protein complex).
  • the method comprises contacting the sample with a subset of fluorescently labeled nucleic acid probes. [00116] In some embodiments, the method further comprises imaging the sample to detect the binding of each of the probe/carbohydrate-binding protein complexes. In some embodiments, the method further comprises removing the probes bound to the carbohydrate-binding protein.
  • the catalytically inactivated enzyme is a transferase. In some embodiments, the catalytically inactivated enzyme is a lipase. In some embodiments, the catalytically inactivated enzyme is a polymerase. In some embodiments, the catalytically inactivated enzyme is a ligase. In some embodiments, the catalytically inactivated enzyme is a protease. In some embodiments, the catalytically inactivated enzyme is a hydrolase. In some embodiments, the catalytically inactivated enzyme is an oxidase. In some embodiments, the catalytically inactivated enzyme is a reductase.
  • the catalytically inactivated enzyme is an isomerase. In some embodiments, the catalytically inactivated enzyme is inactivated by one or more mutations, comprising one or more of: point mutations (e.g., missense, nonsense, silent), insertion/deletion mutations, and/or frameshift mutations. In some embodiments, the catalytically inactivated enzyme is inactivated by truncation.
  • the plurality of carbohydrate binding proteins comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 different carbohydrate binding proteins.
  • the plurality of carbohydrate binding proteins comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 different carbohydrate binding proteins.
  • the plurality of carbohydrate binding proteins comprises a carbohydrate binding protein listed in Table 1 or Table 3, or two or more of the carbohydrate binding proteins listed in Table 1 or in Table 3.
  • the carbohydrate binding protein comprises a carbohydrate binding protein with an amino acid or nucleic acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to a sequence of a carbohydrate binding protein of Table 1 or Table 3.
  • the carbohydrate binding protein comprises a recombinant protein.
  • the sample comprises a whole tissue. In some embodiments, the sample comprises at least a portion of a tissue. In some embodiments, the sample comprises a tissue section. In some embodiments, the tissue is obtained from muscle. In some embodiments, the sample is obtained from skeletal muscle. In some embodiments, the sample is obtained from smooth muscle. In some embodiments, the sample is obtained from cardiac muscle. In some embodiments, the sample is obtained from brain. In some embodiments, the sample is obtained from a nerve. In some embodiments, the tissue is obtained from a sciatic nerve. In some embodiments, the sample is obtained from spinal cord. In some embodiments, the sample is obtained from kidney. In some embodiments, the sample is obtained from liver. In some embodiments, the sample is obtained from lung.
  • removing the probes bound to the carbohydrate binding proteins or the oligonucleotides comprises contacting the sample with an acidic solution such as, but not limited to, hydrochloric acid or acetic acid. In some embodiments, removing the probes bound to the carbohydrate binding proteins or the oligonucleotides comprises contacting the sample with a basic solution, such as, but not limited to, sodium hydroxide, calcium hydroxide, or potassium hydroxide. [00126] Also provided herein is a method of imaging a sample. In some embodiments, the method comprises contacting the sample with a plurality of different carbohydrate binding proteins.
  • this binding generates a first binary complex of the fluorescently labeled first nucleic acid probe/first barcode oligonucleotide.
  • the method further comprises imaging the sample to detect a presence or absence of the first binary complex.
  • the method further comprises removing the plurality of fluorescently labeled first nucleic acid probes from the sample, wherein the subset of the plurality of different carbohydrate binding proteins remain bound to the sample. [00127] In some embodiments, the method further comprises contacting the sample with a plurality of fluorescently labeled second nucleic acid probes.
  • the method further comprises removing the plurality of fluorescently labeled second nucleic acid probes from the sample, wherein the subset of the plurality of different carbohydrate binding proteins remain bound to the sample.
  • the method comprises repeating contacting the sample with a plurality of fluorescently labeled nucleic acid probes.
  • the method further comprises imaging the sample to detect a presence or absence of the binary complex.
  • the method further comprises removing the plurality of fluorescently labeled nucleic acid probes from the sample.
  • the subset of the plurality of different carbohydrate binding proteins remain bound to the sample multiple times, each time with a plurality of fluorescently labeled additional nucleic acid probes.
  • each fluorescently labeled additional nucleic acid probe of the plurality of fluorescently labeled additional nucleic acid probes is configured to bind to an additional barcode oligonucleotide of the plurality of barcode oligonucleotides.
  • imaging comprises measuring a staining intensity.
  • the staining intensity can be used to quantify the relative number of carbohydrate binding proteins bound to an area of the sample.
  • the plurality of different carbohydrate binding proteins comprises one or more lectins.
  • the plurality of different carbohydrate binding proteins comprises one or more mannose-binding proteins.
  • the oligonucleotide (or barcode oligonucleotide) comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, or 100% identity to a sequence set forth in any one of SEQ ID NOs: 1-55 as shown in Table 2.
  • each of the carbohydrate binding proteins are conjugated to an oligonucleotide with at least 60%, at least 70%, at least 80%, at least 90%, or 100% sequence identity to any one of oligonucleotides as shown in Table 2.
  • each of the carbohydrate binding proteins are conjugated to an oligonucleotide as shown in Table 3.
  • the catalytically inactivated enzyme is inactivated by truncation.
  • the plurality of carbohydrate binding proteins comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 different carbohydrate binding proteins.
  • the method further comprises contacting the DBCO-labeled carbohydrate binding protein with an oligonucleotide conjugated to an azide moiety.
  • the oligonucleotide comprises a sequence with at least 60%, at least 70%, at least 80%, at least 90%, or 100% identity to a sequence set forth in any one of SEQ ID NOs: 1-55.
  • the oligonucleotide comprises a sequence set forth in any one of SEQ ID NOs: 1-55.
  • the carbohydrate binding protein is selected from the carbohydrate binding proteins listed in Table 1 or Table 3.
  • the method comprises receiving input comprising images of a sample, wherein the images of a sample comprise the images generated by the methods of imaging disclosed herein. In some embodiments, the method further comprises generating, e.g., using a machine learning algorithm, an output comprising a quantitative or qualitative value of functional or structural features of the sample.
  • the sample comprises a whole tissue. In some embodiments, the sample comprises a tissue section. In some embodiments, the tissue is obtained from muscle. In some embodiments, the sample is obtained from skeletal muscle. In some embodiments, the sample is obtained from smooth muscle.
  • the sample is obtained from cardiac muscle. In some embodiments, the sample is obtained from brain. In some embodiments, the sample is obtained from a nerve. In some embodiments, the tissue is obtained from a sciatic nerve. In some embodiments, the sample is obtained from spinal cord. In some embodiments, the sample is obtained from kidney. In some embodiments, the sample is obtained from liver. In some embodiments, the sample is obtained from lung. In some embodiments, the sample is obtained from lymph node. In some embodiments, the sample is obtained from spleen. In some embodiments, the sample is obtained from intestine. In some embodiments, the sample is obtained from skin. In some embodiments, the sample is obtained from eye. In some embodiments, the sample is obtained from bone.
  • the sample is obtained from a tumor. In some embodiments, the sample is fixed. In some embodiments, the sample is not fixed.
  • WSGR Docket No.: 42256-622.601 Methods of Detecting a Tissue Pathology Also provided herein is a method of detecting a tissue pathology. In some embodiments, the method comprises obtaining a tissue sample. In some embodiments, the method further comprises applying the method of any of the foregoing embodiments to the tissue sample. In some embodiments, the tissue sample comprises cancer cells. In some embodiments, the tissue sample comprises cells comprising a disorder of glycosylation.
  • the tissue sample comprising cancer cells or cells comprising a disorder of glycosylation comprises a glycosylation pattern that is different from a tissue sample that does not comprise cancer cells or cells comprising a disorder of glycosylation.
  • the tissue sample comprising cancer cells or cells comprising a disorder of glycosylation comprises a lower or higher expression level of the fluorescently labeled nucleic acid probes of any of the foregoing embodiments.
  • the disorder of glycosylation comprises Saul-Wilson Syndrome (SWS) or GNE myopathy.
  • the cancer is pancreatic ductal adenocarcinoma (PDAC).
  • the method comprises obtaining a tissue sample from the subject.
  • the tissue sample comprises a cancer tissue.
  • the method comprises applying the method of any of the foregoing embodiments to the tissue sample.
  • the cancer is pancreatic ductal adenocarcinoma (PDAC).
  • characterizing comprises identifying a border between the cancer tissue and healthy tissue in the tissue sample.
  • the border is identified by detecting a lower or higher expression level of the fluorescently labeled nucleic acid probes of any one of the preceding embodiments by the cancer tissue compared to the healthy tissue.
  • characterizing comprise determining the size of the tumor. In some embodiments, characterizing comprises determining progression of the tumor. In some embodiments, progression is measured by comparing the results obtained from a first tissue sample to the results obtained from a second tissue sample. In some embodiments, the second tissue sample is obtained at least one day, at least one week, at least 2 weeks, at least one month, at least 3 months, at least 6 months, at least one year, at least two years, at least five years later than the first tissue sample.
  • Diseases and Tissues [00143] The methods and compositions disclosed herein may be used in the diagnosis or monitoring of a disease affecting a tissue. In some embodiments, the is a disease characterized by tissue specific increased or decreased expression of a carbohydrate.
  • the disease is aging. In some embodiments the disease is a degenerative disease. In some embodiments, the disease is muscular dystrophy. In some embodiments, the tissue is muscle. In some embodiments, the tissue is skeletal muscle. In some embodiments, the tissue is smooth muscle. In some embodiments, the tissue is cardiac muscle. In some embodiments, the tissue is brain. In some embodiments, the tissue is a nerve. In some embodiments, the tissue is a sciatic nerve. In some embodiments, the tissue is spinal cord. In some embodiments, the tissue is kidney. In some embodiments, the tissue is liver. In some embodiments, the tissue is lung. In some embodiments, the tissue is lymph node. In some embodiments, the tissue is spleen.
  • the protein was concentrated.
  • a 50 kDa Molecular Weight Cutoff (MWCO) filter, for IgG antibodies, or 10 kDa MWCO filter for lectins was blocked with 400 ⁇ L of 0.05% Tween in WSGR Docket No.: 42256-622.601 PBS and centrifuged at 12000xg for 8 mins.
  • Protein content was measured from a master tube via Nanodrop by measuring absorbance at 280 nm.
  • 50 ⁇ g of protein either glycan- binding proteins or IgG antibodies
  • the column was centrifuged at 12000xg for 8 mins and the flow through was discarded.
  • the protein was resuspended in 100 ⁇ L of PBS.
  • DBCO dibenzocylooctyne
  • 0.25 ⁇ L (12.5 nmols) of 50 mM Sulfo DBCO-PEG4-TFP was added to the column and allowed to react for 4 hrs at room temperature.
  • the column was washed with 400 ⁇ L of PBS and centrifuged at 12000xg for 8 mins.
  • 100 ⁇ L of PBS was added and protein absorbance was measured using a Nanodrop spectrophotometer.
  • the rate of DBCO addition was calculated to confirm that between 3 and 10 DBCO molecules were added to each protein molecule.
  • the optimal level of conjugation for both antibodies and carbohydrate binding proteins was estimated by establishing the dynamic range of fluorescence after conjugation with increasing amounts of DBCO, as shown in FIGs. 1A-1C and FIG.2.400 ⁇ L of PBS was added to the column and centrifuged at 12000xg for 8 min.
  • Example 2 Tissue Staining with Oligonucleotide-Conjugated Carbohydrate Binding Proteins [00149] The oligonucleotide-conjugated carbohydrate binding proteins generated as in Example 1 were used in imaging samples to assess the capability of the carbohydrate binding proteins to identify a glycan profile in the sample.
  • the panel comprised 11 different oligonucleotide- conjugated carbohydrate binding proteins. Muscle tissue was obtained from mice and fixed.
  • Staining reagents were prepared by mixing oligonucleotide-conjugated antibodies or carbohydrate binding proteins. As negative controls, non-fluorescent complementary DNA oligonucleotides of each barcode at 500 ⁇ M, a combination of IgG molecules from mouse, rat and rabbit at 10 WSGR Docket No.: 42256-622.601 ⁇ g/mL, and salmon sperm DNA were also prepared. The tissue slides were incubated with the staining reagents for 3 hr at room temperature or overnight at 4 oC.
  • Tissue slides were then imaged using an iterative rendering process by serially applying a subset of fluorescently tagged oligonucleotides complementary to a subset of the oligonucleotides bound to the carbohydrate binding proteins or antibodies, capturing an image, denaturing the oligonucleotide binding, washing away the subset of fluorescently tagged oligonucleotides complementary with DMSO, applying a new subset of fluorescently tagged oligonucleotides, and repeating this process until all carbohydrate binding proteins or antibodies have been captured.
  • FIG.9 and FIG.10A Images obtained using carbohydrate binding proteins (FIG.3A), demonstrated staining patterns illustrating glycan expression profiles in the skeletal muscle tissue. Images obtained using antibodies (FIG.3B) identified specific cell types in the tissue. [00151] Additionally, carbohydrate binding protein binding profiles were obtained in various subcellular locations including the nucleus, the perinuclear region, the cytoplasm, the cell membrane, and the extracellular matrix (FIGs.4A-4E). FIG.4A depicts nuclear staining; FIG.
  • FIG.4A depicts muscle fiber (LCA) and NMJ (VVL) staining.
  • FIG.5B depicts staining of vessels (DSL), muscle spindles (PHAE), and motor nerves (ECL).
  • DSL blood pressure
  • PHAE muscle spindles
  • ECL motor nerves
  • FIG.7A depicts the capillary count
  • FIG.7B depicts the myofiber count
  • FIG.7C depicts the myofiber size.
  • the AI model was trained to distinguish healthy (FIG.8A) from denervated (FIG.8B) or regenerating (FIG. 8C) cells.
  • Quantified glycan-binding staining can be calculated based on cell type and its localization within the tissue, or the presence of nearby cell types or tissue features. Moreover, comparisons of cell type or tissue structures can be made to identify changes glycosylation across disease states (healthy vs. injured, aging, or degenerating) or correlated to tissue function (i.e. contraction strength of skeletal muscle, insulin secretion of pancreas, aggressiveness of tumor growth) or in response to a drug treatment (e.g., tumor cells in response to a chemotherapy, the location of CAR-T cells within a tumor, restoration of the Dystroglycan complex by a gene therapy).
  • tissue function i.e. contraction strength of skeletal muscle, insulin secretion of pancreas, aggressiveness of tumor growth
  • a drug treatment e.g., tumor cells in response to a chemotherapy, the location of CAR-T cells within a tumor, restoration of the Dystroglycan complex by a gene therapy.
  • FIG.10B An exemplary workflow of this analysis is shown in FIG.10B.
  • Example 5 Glycosylation Patterns of Abnormal Human Skeletal Development
  • iPSCs healthy human induced pluripotent stem cells
  • COG4 mutated COG4
  • SWS is a congenital disorder of glycosylation characterized by short stature (dwarfism) and skeletal abnormalities.
  • Control and SWS organoids were cultured for 21 days, and the glycosylation patterns of the organoids were assayed using the methods detailed in Examples 1 and 2.

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Abstract

Selon certains aspects, l'invention concerne des compositions et des procédés d'imagerie d'un échantillon en obtenant un profil de glucides présents dans l'échantillon. L'invention concerne également un kit d'imagerie d'un échantillon pour obtenir un profil de glucides présents dans l'échantillon. Des images obtenues peuvent être utilisées dans un procédé mis en œuvre par ordinateur divulgué ici pour analyser un échantillon.
PCT/US2025/016798 2024-02-23 2025-02-21 Compositions et procédés de visualisation de glycosylation Pending WO2025179146A1 (fr)

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