Immuno-oncology gene panel compositions and methods of making and use thereof

Abstract

Described herein are gene panel compositions and methods of use thereof for spatial omic analysis in biological samples. The gene panels may be organized into distinct modules that target various aspects of immune response, tumor biology, and intercellular communication within the microenvironment. These methods involve spatial omic techniques, such as spatial transcriptomics, to localize gene expression across tissue sections, providing detailed insights into immune cell infiltration, immune checkpoint activation, and tumor-immune interactions. The disclosed compositions and methods are applicable for diagnostics, monitoring therapeutic responses, and guiding personalized treatment strategies. The modular design of the gene panels allows for customization based on specific research or clinical objectives.

Description

RES-PA52-PCTGENE PANEL COMPOSITIONS AND METHODS OF MAKING AND USE THEREOFBACKGROUND

[0001] Described herein are gene panel compositions for analyzing genes involved in immunooncology indications and related methods of analysis. Immuno-oncology has advanced cancer treatment by using the body’s immune system to target cancer cells. Treatments such as checkpoint inhibitors have provided new options for patients. However, the success of these treatments can vary significantly across patients due to differences in the genetic factors influencing immune system function and interactions with tumors. Current diagnostic approaches often focus on specific biomarkers, such as PD-1 or PD-L1, which do not provide a comprehensive view of immune activity in patients. As a result, improved methods are needed to assess immune system activity and tumor-immune interactions in a broader context. The present invention addresses these issues by providing a gene panel that includes multiple immune related pathways, allowing for a more complete assessment of a patient's immune status in relation to cancer and immunotherapy.SUMMARY

[0002] In various aspects the present disclosure provides a gene panel comprising a plurality of genes selected from the group consisting of tumor-related genes, immune function-related genes, T-cell related genes, Natural Killer (NK) cell related genes, Innate Lymphoid Cell (ILC) related genes, plasma cell (B-cell) related genes, monocyte and macrophage-related genes, dendritic cell (DC) related genes, granulocyte-related genes, vascular-related genes, and stromal-related genes. Such panels are useful for the analysis of immune responses and tumor-immune interactions in a human or other mammalian sample, wherein the expression, accumulation pattern or mutational status of said genes is used in spatial omic analysis to assess immune activity related to cancer, immune therapy response, chemotherapy, radiotherapy, inflammation, autoimmune disorder assessment, development, sample or patient stratification, drug test sample assessment or other analysis.

[0003] In some aspects, the spatial omic analysis comprises spatial transcriptomics, spatial proteomics, spatial metabolomics, or any combination thereof. In some aspects, the gene panel comprises at least one gene selected from any one of TABLE 1 - TABLE 10, or any combination thereof, alone or in combination with additional genes on any one of the tables or other non-listed genes. In some aspects, the gene panel comprises a gene panel module. In some aspects, the gene panel module comprises a gene panel module selected from TABLE 11.RES-PA52-PCT

[0004] In various aspects the present disclosure provides a method for assessing the immune status of a human subject with cancer, an autoimmune disorder, inflammation, or undergoing treatment so as to perturb immune status or cancer activity, comprising: obtaining a biological sample from the subject; analyzing the expression levels or mutational status of genes within the gene panel as described herein, comparing the expression levels or mutational status to reference data to assess immune cell infiltration, immune checkpoint activation, or tumor-immune interactions to predict the subject's response to immunotherapy.

[0005] In some aspects, the immunotherapy comprises a checkpoint inhibitor selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors. In some aspects, the biological sample comprises tumor tissue, blood, serum, plasma, or FFPE tissue samples.

[0006] In various aspects the present disclosure provides a diagnostic kit for use in immunooncology, comprising: the gene panel as described herein; reagents for isolating and amplifying nucleic acids from a biological sample; and instructions for analyzing the expression or mutational status of said genes using spatial omic techniques to assess immune responses and tumor-immune interactions in a human subject. Similarly, the present disclosure provides datasets and images, such as images presented on a screen or printed images, conveying information obtained through use of such kits or reagents, so as to present accumulation patters for one or more members of such panels, for example so as to obtain information relevant to immunotherapy, cancer progression or treatment, or other disorder or developmental stage progression disclosed or contemplated herein.

[0007] Similarly, the present disclosure provides methods of treatment evaluation or selection, comprising administering a therapeutic or therapy regimen to a subject such as a subject having or suspected of having cancer, evaluating accumulation levels of panel constituents in a tissue or other sample from the subject, and selecting a course of treatment informed by impact of the therapeutic or therapy regimen on subject accumulation levels. Evaluating in some cases comprises comparing to a reference or to a subject sample taken prior to initiation of the therapeutic treatment or therapy regimen. Selecting a course of treatment variously comprises selecting or excluding from consideration a therapeutic or therapy regimen, selecting or modulating a dosage amount or dosage frequency of a therapeutic or therapy regimen, or selecting or modulating a dosage amount or dosage frequency of a companion therapeutic or therapy regimen.RES-PA52-PCTINCORPORATION BY REFERENCE

[0008] 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.DETAILED DESCRIPTION

[0009] Described herein is the development and use of immuno-oncology gene panels for spatial omic analysis (e.g., spatial transcriptomics, spatial proteomics, spatial metabolomics, or any combination thereof) in biological samples. The gene panels described herein are designed to capture a comprehensive profile of immune and tumor microenvironment activity by comprising genes from various categories such as tumor related genes, immune function related genes, and genes specific to different immune cell types. These panels enable detailed analysis of immune responses, tumor-immune interactions, and other cellular dynamics within a spatially resolved context, providing valuable insights into cancer biology and responses to immunotherapies.Immuno-Oncology Spatial Omic Analysis

[0010] Spatial omic analysis (e.g., spatial transcriptomics, spatial proteomics, spatial metabolomics, or any combination thereof) enables the localization of molecular information within tissue sections, offering insights into the spatial heterogeneity of tumors and immune cell infiltration. In the context of immuno-oncology, spatial omic techniques allow for the measurement of gene expression, protein expression, and metabolic profiles related to immune checkpoints, immune activation, and tumor-immune interactions, which are important for understanding responses to immunotherapies.

[0011] In some embodiments, the gene panels described herein are designed for use in various sample types, including formalin-fixed, paraffin-embedded (FFPE) tissues, fresh-frozen tissue sections, or other relevant biological samples. The spatial resolution provided by these panels can reveal patterns of immune exclusion or infiltration that may correlate with a patient’s response to immunotherapies.

[0012] In some advantageous embodiments, the Spatial transcriptomics (or Spatial *omics) according to the present disclosure means any kind of analysis where data from the sample are derived in a spatial manner from in-situ samples of tissues or whole organisms. The in-situ sample may be a section of an organ or an organism. The in-situ sample may be not pretreated or pretreated in a way that is required for improving the result. Spatial*omics may include the detection of small molecules compounds of tissues or cells, proteins, DNA, and / or RNA. More preferentially, spatial*omics is restricted to proteins, DNA, and / or RNA. More preferentially,RES-PA52-PCT spatial*omics is restricted to DNA and / or RNA. Even more preferentially, spatial*omics is restricted to smFISH. Even more preferentially, spatial*omics is restricted to any kind of sequential smFISH. In particular, the spatial transcriptomics detecting comprises a multiplex method for detecting different analytes in a sample by sequential signal-encoding of said analytes as described in WO 2020 / 254519 Al, WO 2021 / 255244 and WO 2021 / 255263.

[0013] In one embodiment, the smFISH application is a technique known in this field as “Molecular Cartography, described for example in WO 2020 / 254519 Al, WO 2021 / 255244 and WO 2021 / 255263, the disclosures of which are each hereby incorporated by reference in their respective entireties.

[0014] The skilled person knows this method so that materials and methods for carrying it out are known as well. Briefly, Molecular Cartography is based on combinatorial single-molecule fluorescent in situ hybridization (smFISH). There are hybridized several tens of transcriptspecific probes per target RNA for analysis of up to 100 different types of RNA or even more per sample. Through a proprietary colorizing and de-colorizing chemistry during several imaging rounds, this technology accurately identifies individual transcripts by decoding the barcode that is specific to each transcript type. This innovative approach enables Molecular Cartography to offer unprecedented specificity and sensitivity with one spot corresponding to one transcript. The result is the visualization and identification of millions of individual transcripts per sample.Gene Panel Development

[0015] The gene panels described herein are developed to target a comprehensive set of genes relevant to immuno-oncology. These panels are intended to probe a wide range of immune responses, tumor biology, and the tumor microenvironment. In some embodiments, the panels may comprise genes associated with tumor related processes, immune function, and specific immune cell types, including T-cells, Natural Killer (NK) cells, Innate Lymphoid Cells (ILCs), plasma cells (B-cells), monocytes, macrophages, dendritic cells (DCs), granulocytes, vascular systems, and stromal components.

[0016] In some embodiments, combinations of these gene categories may be employed to create gene panels that are tailored to specific immuno-oncology indications, such as detecting immune cell infiltration, identifying immune checkpoint activation, or monitoring immune responses to tumor antigens.Tumor Related Genes

[0017] The gene panels described herein may comprise tumor related genes to provide insights into the genetic landscape of the tumor, such as mutations, gene expression changes, and otherRES-PA52-PCT alterations that contribute to tumor progression or therapy resistance. These genes may comprise oncogenes, tumor suppressor genes, and genes involved in cell proliferation, survival, and immune evasion. By comprising tumor related genes, the panels can help identify mechanisms of immune escape or tumor-induced suppression of immune responses, which may be valuable for selecting patients for immune checkpoint inhibitors or other immunotherapies. In some embodiments, the gene panels as described herein may comprise genes selected from TABLE 1.TABLE 1 - Tumor Related GenesRES-PA52-PCTImmune Function Related Genes

[0018] The gene panels described herein may comprise immune function related genes to provide a broad overview of the immune system’s activity within the tumor microenvironment. These genes may encode cytokines, chemokines, and other signaling molecules that regulate immune responses. By analyzing these immune function related genes, the panels can assess immune cell recruitment, inflammatory signals, and the overall immune landscape. This analysis may detect signatures of immune activation or suppression, which may predict responses to immunotherapies. In some embodiments, the gene panels as described herein may comprise genes selected from TABLE 2.TABLE 2 - Immune Function Related GenesRES-PA52-PCTRES-PA52-PCTRES-PA52-PCTRES-PA52-PCTT-Cell Related Genes

[0019] T-cell related genes play a central role in analyzing immune responses in cancer. The gene panels described herein may comprise genes involved in T-cell activation, differentiation, proliferation, and immune checkpoint pathways, such as PD-1, CTLA-4, and LAG-3. These genes can assess the presence and activity of tumor-infiltrating lymphocytes (TILs) and provide insight into whether T-cells are in an activated or exhausted state. The analysis of T-cell related genes may help predict patient responses to T-cell-based immunotherapies, including checkpoint inhibitors or adoptive cell therapies. In some embodiments, the gene panels as described herein may comprise genes selected from TABLE 3.TABLE 3 - T-Cell Related GenesRES-PA52-PCTRES-PA52-PCTNatural Killer (NK) Cell and Innate Lymphoid Cell (ILC) Related Genes

[0020] The gene panels described herein may comprise Natural Killer (NK) cell-related genes and genes related to Innate Lymphoid Cells (ILCs) to evaluate the presence, activity, and contribution of these cells within the tumor microenvironment. NK cells are part of the innate immune response against tumors, and the selected genes may involve NK cell activation receptors, cytokine production, and cytotoxicity mechanisms. By analyzing NK cell-related genes, the panels can assess their role in tumor elimination and identify potential evasion mechanisms, such as the downregulation of NK cell ligands. Similarly, the panels may comprise genes related to ILCs, which are involved in early immune responses and tissue regulation. These genes may target ILC subsets, such as ILC1, ILC2, and ILC3, each with distinct roles in cytokine production and immune regulation. By analyzing ILC-related genes, the panels can provide insights into early immune dynamics within the tumor microenvironment, particularly regarding inflammation and immune modulation. Together, the NK and ILC gene analyses offer a comprehensive view of innate immune responses to tumor development and immune evasion strategies. In some embodiments, the gene panels as described herein may comprise genes selected from TABLE 4.TABLE 4 - Natural Killer (NK) Cell and Innate Lymphoid Cell (ILC) Related GenesPlasma Cell (B-Cell) Related Genes

[0021] Plasma cells (e.g., B-Cells) are important for antibody production and antigen presentation. The gene panels described herein may comprise genes involved in B-cell receptor signaling, antibody production, and plasma cell differentiation. By probing these genes, the panels can identify humoral immune responses within the tumor microenvironment, which may contribute to anti-tumor immunity or immune evasion, depending on the context. This analysisRES-PA52-PCT may evaluate the role of B-cells in immuno-oncology therapies. In some embodiments, the gene panels as described herein may comprise genes selected from TABLE 5.TABLE 5 - Plasma Cell (B-Cell) Related GenesMonocyte and Macrophage Related Genes

[0022] Monocytes and macrophages play key roles in regulating immune responses and maintaining tissue homeostasis in tumors. The gene panels described herein may comprise genes related to macrophage polarization, cytokine production, and phagocytic activity. By analyzing these genes, the panels can reveal whether tumor-associated macrophages (TAMs) are contributing to an immune-suppressive environment or promoting tumor elimination. The identification of macrophage related signatures can help target therapies aimed at reprogramming macrophages for anti-tumor activity. In some embodiments, the gene panels as described herein may comprise genes selected from TABLE 6.TABLE 6 - Monocyte and Macrophage Related GenesRES-PA52-PCTDendritic Cell (DC) Related Genes

[0023] Dendritic cells are essential for antigen presentation and the initiation of T-cell responses. The gene panels described herein may comprise genes involved in dendritic cell maturation, antigen presentation, and cytokine production. By probing these genes, the panels can assess the functionality of dendritic cells in presenting tumor antigens and stimulating antitumor immune responses. This analysis may be used for therapies that seek to enhance dendritic cell function, such as cancer vaccines. In some embodiments, the gene panels as described herein may comprise genes selected from TABLE 7.TABLE 7 - Dendritic Cell (DC) Related GenesGranulocyte Related Genes

[0024] The gene panels described herein may comprise genes related to granulocytes, such as neutrophils, eosinophils, and basophils, which are involved in inflammation and immune responses. These genes may provide insights into how granulocyte activity contributes to the tumor immune environment, either by promoting inflammation or aiding anti-tumor responses. Granulocyte related genes can be valuable for identifying inflammatory signatures that may influence therapeutic decisions, including those related to targeting specific cytokines. In some embodiments, the gene panels as described herein may comprise genes selected from TABLE 8.TABLE 8 - Granulocyte Related GenesRES-PA52-PCTVascular Related Genes

[0025] The gene panels described herein may comprise vascular related genes to assess the role of the tumor vasculature in regulating immune cell infiltration and tumor growth. These genes may be involved in angiogenesis, endothelial function, and vascular permeability. By analyzing vascular related genes, the panels can provide insight into how the tumor vasculature affects immune cell access to the tumor, which may be important for the efficacy of immunotherapies targeting immune cell infiltration or angiogenesis. In some embodiments, the gene panels as described herein may comprise genes selected from TABLE 9.TABLE 9 - Vascular Related GenesStromal Related Genes

[0026] The tumor stroma, which consists of fibroblasts, extracellular matrix components, and other non-immune cells, plays a role in shaping immune responses within the tumor microenvironment. The gene panels described herein may comprise stromal related genes that regulate matrix remodeling, fibroblast activity, and immune modulation. These genes may reveal whether the tumor stroma is creating an immune-suppressive environment or facilitating immune cell infiltration, which could inform therapeutic strategies targeting the tumor stroma.RES-PA52-PCTIn some embodiments, the gene panels as described herein may comprise genes selected fromTABLE 10.TABLE 10 - Stromal Related GenesGene Panel Modules

[0027] The gene panels described herein may be organized into multiple modules, each tailored to assess distinct aspects of immune function, tumor biology, and intercellular communication within the tumor microenvironment. The number of genes in each module may vary based on the intended use, but exemplary sizes are provided for illustration. In some embodiments, a gene panel module may comprise genes selected from any one of TABLE 1 - TABLE 10, or any combination thereof, alone or in combination with additional genes on any one of the tables or other non-listed genes. In some embodiments, a gene panel module may comprise at least 10 genes, at least 20 genes, at least 30 genes, at least 40 genes, at least 50 genes, at least 60 genes, at least 100 genes, at least 125 genes, or at least 150 genes. In some embodiments a gene panel as described herein may comprise 1 gene panel module, 2 gene panel modules, 3 gene panel modules, 4 gene panel modules, 5 gene panel modules, or greater than 5 gene panel modules. Some gene panel modules comprise representatives from at least, no more than, about or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or all Tables, alone or in combination with other genes, proteins or other analytes. Assaying for a ‘gene’ in a panel or selected from a table may variously comprise assaying for transcript accumulation level or location, or both level and location associated with that gene, or of protein accumulation level or location, or both level and location associated with that gene, or both transcript and protein levels, alone or in combination with other analyteRES-PA52-PCT markers. Such markers may comprise subject genome encoded analytes or natively produced analytes, or xenic markers such as bacterial or eukaryotic markers.

[0028] A gene panel module as described herein may be a core gene panel module. In some embodiments, a core module may comprise at least 150 genes (e.g., 165 genes, or for example, at least, at most, about or exactly 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, or more than 200). This module is in some cases designed to provide a comprehensive overview of immune activity across various cell types. In some cases, the core module may comprise a set number of genes focused on broad immune functions, including genes responsible for cytokine signaling, immune cell activation, and regulatory pathways that control immune surveillance. By analyzing this core set of genes, researchers can gather a general profile of the immune environment, covering multiple immune cell types such as T-cells, B-cells, macrophages, and dendritic cells. The 165 genes, for example, included in this module are selected to provide a baseline immune profile that is relevant across multiple types of cancer, allowing for broad diagnostic and therapeutic applications. Alternately, some panels are selected or designed to focus upon single cell types or a subset of cell types, such as associated with a particular tissue or sample.

[0029] A gene panel module as described herein may be a tumor gene panel module. In some embodiments, a tumor module may comprise at least 30 genes (e.g., 35 genes, or for example at least, at most, about or no more than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 100) and focus on tumor-specific biology, including oncogenesis, cell proliferation, immune evasion, and resistance to apoptosis. In some embodiments, this module comprises genes that are frequently mutated in cancer or are known to play critical roles in tumor progression. By analyzing a limited but targeted set of genes, this module provides detailed insights into how tumors evade immune detection or manipulate surrounding tissues to support growth. This 35- gene module allows for a focused investigation into tumor-specific pathways, potentially identifying actionable targets for immuno-oncology treatments. Alternately or in combination, some panels further comprise unrelated, reference or control panel members.

[0030] A gene panel module as described herein may be a cell-cell communication module. In some embodiments, a cell-cell communication module may comprise at least 100 genes (or for example, at least, at most, about or exactly 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, or more than 200), and may be designed to study how cells interact within the tumor microenvironment, particularly focusing on communication between tumor cells and immune cells. In some embodiments, this module comprises genes involved in cell signaling pathways, such as those controlling cytokine production, chemokine interactions, and cell adhesion molecules. ByRES-PA52-PCT focusing on intercellular signaling, this module helps reveal how tumor cells modulate immune responses, either by recruiting immune-suppressive cells or by evading immune detection. The 100 genes in this module are selected to cover a wide range of signaling pathways that influence immune cell migration, activation, and suppression, providing insights into the dynamics of cell- to-cell interactions in the tumor microenvironment.

[0031] Exemplary gene panel modules are provided in TABLE 11.TABLE 11 - Exemplary Gene Panel ModulesRES-PA52-PCTRES-PA52-PCTRES-PA52-PCT

[0032] In some embodiments, the number of genes in each module may be adjusted based on the depth of analysis required. For instance, if more detailed profiling of immune responses is needed, the core module may be expanded to comprise additional genes related to specific immune cell subsets, while the tumor module may be adjusted to include more genes involved in immune evasion strategies. Similarly, the cell-cell communication module may be modified to focus on specific interactions between tumor cells and distinct immune cell populations, such as regulatory T-cells or tumor-associated macrophages.

[0033] This modular approach allows for flexibility in designing gene panels that are tailored to specific research or clinical applications, offering both broad and targeted insights into the complex dynamics of tumor-immune interactions.RES-PA52-PCTGene Panel Modifications and Concentrations

[0034] The gene panels described herein may be modified to comprise additional genes or remove genes based on new discoveries in immuno-oncology. The flexibility of the panels allows for adaptation to emerging biomarkers, therapeutic targets, and patient-specific needs. For example, if new immune checkpoint pathways are discovered, the gene panels may be updated to comprise relevant genes, or panels may be customized for specific tumor types or treatment regimens.

[0035] The concentration of targeting probes across the gene panels may be adjusted to achieve the desired specificity and sensitivity. In some embodiments, the gene panels may be constructed with consistent probe concentrations across all genes to provide an equal representation of each gene. In other embodiments, probe concentrations may vary, with highly expressed genes targeted by fewer probes, while low-abundance transcripts or genes of particular interest may be targeted by higher concentrations of probes. This approach allows for a more tailored analysis based on the expected expression levels of the genes in different tissue types or disease states.Methods of Measuring an Analyte in Cells within a Sample

[0036] Methods of the present disclosure include detecting and / or measuring an analyte in a sample (e.g., a gene comprised by a gene panel of the current disclosure). In an embodiment of the disclosure the sample is a biological sample, preferably comprising biological tissue, further preferably comprising biological cells. A biological sample may be derived from an organ, organoids, cell cultures, stem cells, cell suspensions, primary cells, samples infected by viruses, bacteria, or fungi, eukaryotic or prokaryotic samples, smears, disease samples, a tissue section. Methods of the present disclosure include detecting an analyte by spatial *omics (e.g., spatial transcriptomics, spatial genomics, spatial proteomics, spatial metabolomics, etc.). In particular, the spatial transcriptomics detecting may in some cases comprise a multiplex method for detecting different analytes in a sample by sequential signal-encoding of said analytes as described in WO 2020 / 254519 Al, published December 24, 2020, WO 2021 / 255244, published December 23, 2021 and WO 2021 / 255263, published February 3, 2022, each of which is hereby incorporated by reference in its entirety. Other approaches, such as sequence or positional barcode based approaches are also contemplated herein, and methods using panels herein are in some cases not limited to hybridization or fluorescence based panel detection.

[0037] Hybridization and fluorescence detection based methods are in some cases particularly qualified to encode, identify, detect, count, or quantify analytes or single analytes molecules in a biological sample, such as a sample which contains nucleic acids as said analytes. It isRES-PA52-PCT understood that the biological sample may be in a form as it is in its natural environment (e.g., liquid, semi-liquid, solid, etc.), or processed environment, e.g., as a dried film on the surface of a device which may be re-liquefied before the method is carried out. Fixing the sample (e.g., biological tissue or biological cells) has the advantage that the analytes to be encoded, e.g., the nucleic acids, are immobilized and cannot escape. In doing so, the analytes then prepared for a better detection or encoding by the method according to the disclosure.

[0038] In yet a further embodiment within the set of analyte-specific probes the individual analyte-specific probes comprise binding elements (SI, S2, S3, S4, S5) which specifically interact with different sub-structures of one of the analytes to be encoded. By this measure the method becomes even more robust and reliable because the signal intensity obtained at the end of the method or a cycle, respectively, is increased. It is understood that the individual probes of a set while binding to the same analyte differ in their binding position or binding site at or on the analyte. The binding elements SI, S2, S3, S4, S5 etc. of the first, second, third fourth, fifth etc. analyte-specific probes therefore bind to or at a different position which, however, may or may not overlap.

[0039] In an embodiment, the present disclosure pertains to kit for multiplex analyte encoding, comprising:(A) at least twenty (20) different sets of analyte-specific probes for encoding of at least 20 different analytes, each set of analyte-specific probes interacting with a different analyte, wherein if the analyte is a nucleic acid each set of analyte-specific probes comprises at least five (5) analyte-specific probes which specifically interact with different sub-structures of the same analyte, each analyte-specific probe comprising:(aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded, and(bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), wherein the analyte-specific probes of a particular set of analyte-specific probes differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T), wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and(B) at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte each decoding oligonucleotide comprises:RES-PA52-PCT(aa) an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and(bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the identifier connect element (t); and(C) a set of signal oligonucleotides, each signal oligonucleotide comprising:(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and(bb) a signal element.

[0040] A multiplex method or assay allows for the simultaneously or sequential measurement of multiple analytes. According to the present disclosure it may be used to determine simultaneously or sequential the presence or absence of a plurality of predetermined (known) analytes like genomic as well as extra-genomic (e.g., mRNA) nucleic acid target sequences in a sample. An analyte may be “predetermined” in that its sequence is known to design a probe that binds to the that target.

[0041] In some embodiments according to the present disclosure at least 20, in particular at least 25, in particular at least 30 different analytes are detected and / or quantified in a sample in parallel. For example, there may be at least 5, at least 10, at least 20, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, or at least 30,000 distinguishable analyte-specific probes that are applied to a sample, e.g., simultaneously or sequentially. In some embodiments, there may be at least 100 analyte-specific probe sets (100 distinguishable analyte probes). In some embodiments, there may be at least 300 analytespecific probe sets (100 distinguishable analyte probes).

[0042] In some embodiments for the multiplexing twenty (20) or more different sets of analytespecific probes for encoding of at least 20 different analytes or more are required, in particular more than 50, more than 100 or more than 200. In the multiplexing methods of the present disclosure, in particular at least 20 different groups of analytes (e.g., mRNA molecules).

[0043] In some embodiments, at least 4 rounds to collect information for identification of the analyte are carried out, wherein multiple readout increases the accuracy of identification and avoids false positives. The unique tag (e.g., a unique identifier sequence) can be identified by various techniques, including hybridization, e.g., with labeled probes, directly or indirectly or byRES-PA52-PCT sequencing (by synthesis, ligation). In particular, the identity of the tag can be encoded with one single signal (binary code), two or more signals, wherein the signal can be a fluorescent label (e.g., attached to an oligonucleotide).

[0044] In some embodiments, a kit does not comprise sets of analyte-specific probes as defined under item A).

[0045] Preferably, if the analyte in the kits or methods according to the present disclosure is a nucleic acid, each set of analyte-specific probes comprises at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 different analyte-specific probes which specifically interact with different sub-structures of the same analyte. Nucleic acid analytes may include DNA molecules (e.g., genomic DNA, nuclear DNA, circular DNA, mitochondrial DNA, viral DNA, bacterial DNA, extra- or intracellular DNA) and / or RNA molecules (e.g., mRNA, hnRNA, miRNA, viral RNA, bacterial RNA, extra- or intracellular RNA, circular mRNA, tRNA, siRNA, snRNA, or rRNA).

[0046] In some embodiments according to the present disclosure the kit comprises at least two different sets of signal oligonucleotides, wherein a set of signal oligonucleotides comprises a different signal element and a different connector element (C) than a second set of signal oligonucleotides. For example, a first set of signal oligonucleotides may comprise signal oligonucleotides that comprise a different signal elements and a different connector element (C) than signal oligonucleotides comprised by a second set of signal oligonucleotides.

[0047] In particular, the kit may comprise at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0048] In some embodiments the kit comprises at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and wherein the decoding oligonucleotides of the different sets for at least one analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0049] In some embodiments, in a given cycle, the number of different sets of decoding oligonucleotides (wherein each of the sets differ in at least the translator element (c)) per analyteRES-PA52-PCT corresponds to the number of different sets of signal oligonucleotides comprising different connector elements (C). However, the decoding oligonucleotides in a particular set of decoding oligonucleotides may interact with identical identifier elements (T) which are unique to a particular analyte. In some embodiments, sets of decoding oligonucleotides for the different analytes may comprise a common translator element(s) (c). For example, a first set of decoding oligonucleotides for a first analyte may have the same translator element (c) as a second set of decoding oligonucleotides for a second analyte.

[0050] In another aspect, the present disclosure is generally directed to methods including acts of exposing a sample to a plurality of analyte-specific probes; for each of the analyte-specific probes, determining binding of the analyte-specific probes within the sample; creating code words based on the binding of the analyte-specific probes, the decoding oligonucleotides and the signal oligonucleotides; and for at least some of the code words, matching the code word to a valid code word. In certain embodiments, this pattern of binding or hybridization of the analytespecific probes, the decoding oligonucleotides and the signal oligonucleotides may be converted into a “code word.” For example, for instance, the code words may be “101” and “110” for a first analyte and a second analyte, respectively, where a value of 1 represents binding and a value of 0 represents no binding of decoding oligonucleotides and / or the binding of signal oligonucleotides without and / or quenched signal element. The analyte in the detection round / cycle is therefore not detectable during imaging.

[0051] To create such a zero (0) in a code word for an individual analyte the kit may comprise: (D) at least a set of non-signal decoding oligonucleotides for binding to a particular identifier element (T) of analyte-specific probes, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interacting with the same different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a unique identifier sequence and does not comprise a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0052] To create such a zero (0) in a code word for an individual analyte the kit may comprise: (D) at least a set of non-signal decoding oligonucleotides for binding to a particular identifier element (T) of analyte-specific probes, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interacting with the same different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a unique identifier sequence and comprise a translator element that does not interact / bind to a signal oligonucleotide due to an instable binding sequence and / or due to the translator element isRES-PA52-PCT to short (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0053] In some embodiments, the kit comprises: (D) at least two (2) different sets of non-signal decoding oligonucleotides for binding to at least two different identifier elements (T) of analytespecific probes, each set of non-signal decoding oligonucleotides interacting with a different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a unique identifier sequence and does not comprise a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0054] In some embodiments, the different sets of non-signal decoding oligonucleotides may be comprised in a pre-mixture of different sets of non-signal decoding oligonucleotides or exist separately.

[0055] Furthermore, in some embodiments the kit may comprise: (E) a set of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

[0056] In some embodiments, the kit comprises: (E) at least two sets of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

[0057] In some embodiments, the different sets of non-signal oligonucleotides may be comprised in a pre-mixture of different sets of non-signal oligonucleotides or exist separately.

[0058] Further, in some embodiments the decoding oligonucleotides in a particular set of decoding oligonucleotides interacts with the same identifier elements (T) which are unique to a particular analyte.

[0059] In some embodiments, the different sets of decoding oligonucleotides may be comprised in a pre-mixture of different sets of decoding oligonucleotides or exist separately. In some embodiments, the different sets of analyte-specific probes may be comprised in a pre-mixture of different sets of analyte-specific probes or exist separately. In some embodiments, the different sets of signal oligonucleotides may be comprised in a pre-mixture of different sets of signal oligonucleotides or exist separately.

[0060] As mentioned above the analyte to be encoded may be a nucleic acid (e.g., DNA, PNA, RNA, or mixtures thereof). In some embodiments the analyte to be encoded may be RNA (e.g.,RES-PA52-PCT mRNA, hnRNA, miRNA, viral RNA, bacterial RNA, extra- or intracellular RNA, circular mRNA, tRNA, siRNA, snRNA, rRNA, or any combination thereof). In some embodiments the analyte to be encoded may be mRNA.

[0061] The present disclosure pertains to a multiplex method for detecting different analytes in a sample by sequential signal-encoding of said analytes, comprising the steps of(A) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding of at least 20 different analytes, each set of analyte-specific probes interacting with a different analyte, wherein if the analyte is a nucleic acid each set of analytespecific probes comprises at least five (5) analyte-specific probes which specifically interact with different sub-structures of the same analyte, each analyte-specific probe comprising:(aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded, and(bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), wherein the analyte-specific probes of a particular set of analyte-specific probes differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T), wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and(B) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte each decoding oligonucleotide comprises:(aa) an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and(bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the first connect element (t); and(C) contacting the sample with at least a set of signal oligonucleotides, each signal oligonucleotide comprising:RES-PA52-PCT(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and(bb) a signal element,(D) detecting the signal caused by the signal element;(E) selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining the specific binding of the analyte-specific probes to the analytes to be encoded;(F) performing at least three (3) further cycles comprising steps B) to E) to generate an encoding scheme with a code word per analyte, wherein in particular the last cycle may stop with step (D).

[0062] As mentioned above, the method according to the present disclosure comprises selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining the specific binding of the analyte-specific probes to the analyte to be encoded. All steps are performed sequentially. However, some steps may be performed simultaneously or sequential, the contacting steps A) to C), in particular B) and C).

[0063] By this measure the requirements for another round / cycle of binding further decoding oligonucleotides to the same analyte-specific probes are established, thus finally resulting in a code, or encoding scheme comprising more than one signal. This step is realized by applying conditions and factors well known to the skilled person, e.g., pH, temperature, salt conditions, oligonucleotide concentration, polymers etc.

[0064] In another embodiment of the present disclosure, the method may comprise repeating steps (B)-(E) at least three times to generate an encoding scheme. With this measure a code of four signals in case of four cycles / rounds which are carried out by the user, where 'n' is an integer representing the number of rounds. The encoding capacity of the method according to the disclosure is herewith increased depending on the nature of the analyte and the needs of the operator. In an embodiment of the disclosure said encoding scheme is predetermined and allocated to the analyte to be encoded.

[0065] However, this measure enables a precise experimental set-up by providing the appropriate sequential order of the employed decoding and signal oligonucleotides and, therefore, allows the correct allocation of a specific analyte to a respective encoding scheme. The decoding oligonucleotides which are used in repeated steps (B)-(D2) may comprise a translator element (c2) which is identical with the translator element (cl) of the decoding oligonucleotides used in previous steps (B)-(E). In another embodiment of the disclosure decoding oligonucleotides are used in repeated steps (B)-(E) comprising a translator elementRES-PA52-PCT(c2) which differs from the translator element (cl) of the decoding oligonucleotides used in previous steps (B)-(E). It is understood that the decoding elements may or may not be changed from round to round, i.e., in the second round (B)-(E) comprising the translator element c2, in the third round (B)-(E) comprising the translator element c3, in the fourth round (B)-(E) comprising the translator element c4 etc., wherein 'n' is an integer representing the number of rounds.

[0066] The signal oligonucleotides which are used in repeated steps (B)-(E) may comprise a signal element which is identical with the signal element of the decoding oligonucleotides used in previous steps (B)-(E). In a further embodiment of the disclosure signal oligonucleotides are used in repeated steps (B)-(E) comprising a signal element which differs from the signal element of the decoding oligonucleotides used in previous steps (B)-(E). In some embodiments no-signal oligonucleotides and / or no-signal decoding oligonucleotides for an individual analyte are used, resulting to the value 0 in the code word for this cycle / position. In some embodiments in a repeated cycle no decoding oligonucleotides for an individual analyte is contacted with the sample resulting also to the value 0 in the code word for this cycle / position.

[0067] By this measure each round the same or a different signal is provided resulting in an encoding scheme characterized by a signal sequence consisting of numerous different signals. This measure allows the creation of a unique code or code word which differs from all other code words of the encoding scheme. In another embodiment of the disclosure, the binding element (S) of the analyte-specific probe comprises a nucleic acid comprising a nucleotide sequence allowing a specific binding to the analyte to be encoded, preferably a specific hybridization to the analyte to be encoded.

[0068] In some embodiments, all steps are automated, in particular wherein steps B) to F) are automated, in particular by using a robotic system and / or an optical multiplexing system according to the present disclosure. In some examples, the steps may be performed in a fluidic system.

[0069] As mentioned above, with the methods according to the present disclosure an encoding scheme with a code word per analyte is generated. Therefore, each analyte may be associated with a specific code word, wherein said code word comprise a number of positions, and wherein each position corresponds to one cycle resulting in a plurality of distinguishable encoding schemes with the plurality of code words. In particular, said encoding scheme may be predetermined and allocated to the analyte to be encoded.

[0070] In some embodiments, the code words obtained for the individual analytes in the performed cycles comprise the detected signals and additionally at least one element corresponding to no detected signal like 0,1 or 0,1,2 etc.. No signal is detected for at least oneRES-PA52-PCT analyte within at least one cycle if using a non-signal probe or a non-signal decoding oligonucleotide, or if in one cycle no decoding oligonucleotide is contacted with the corresponding identifier sequence comprised on analyte-specific probe interacting with the corresponding analyte in the sample. In this cycle the position has the value zero (0).

[0071] In some embodiments, at least for one individual analyte a position of the code word is zero (0). In particular, the code word zero (0) is generated by using no decoding oligonucleotides having an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of a corresponding analyte-specific probe for an individual analyte. As mentioned above, in some embodiments, if at least for one individual analyte a position of the code word is zero (0) in this cycle no corresponding decoding oligonucleotides having an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of a corresponding analyte-specific probe for an individual analyte are used.

[0072] Furthermore, in some embodiments the sample is contacted with at least two different sets of signal oligonucleotides, wherein the signal oligonucleotides in each set comprise a different signal element and comprise a different connector element (C).

[0073] In more embodiments, the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0074] In more embodiments, the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides comprised in these different sets comprise the same identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and wherein the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide; wherein only one set of decoding oligonucleotides per analyte is used per cycle, and / or wherein different sets of decoding oligonucleotides are used in different cycles in combination with the corresponding set of signal oligonucleotides in the same cycle.RES-PA52-PCT

[0075] In some embodiments, the number of different sets of decoding oligonucleotides per analyte comprising different translator elements (c) corresponds to the number of different sets of signal oligonucleotides comprising different connector elements (C). All sets of decoding oligonucleotides for the different analytes may comprise the same type(s) of translator element(s) (c).

[0076] In some embodiments of the method according to the present disclosure, the sample is contacted with at least a set of non-signal decoding oligonucleotides for binding to a particular identifier element (T) of analyte-specific probes, wherein the decoding oligonucleotides in the same set of non-signal decoding oligonucleotides interacting with the same different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a unique identifier sequence, and does not comprise a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0077] As mentioned above, the sample may be contacted with at least two (2) different sets of non-signal decoding oligonucleotides for binding to at least two different identifier elements (T) of analyte-specific probes, each set of non-signal decoding oligonucleotides interacting with a different identifier element (T), wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a unique identifier sequence, and does not comprise a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

[0078] In some embodiments of the method according to the present disclosure, the different sets of non-signal decoding oligonucleotides may be comprised in a pre-mixture of different sets of non-signal decoding oligonucleotides or exist separately.

[0079] Furthermore, in some embodiments of the method according to the present disclosure, the sample is contacted with a set of non-signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

[0080] In further embodiments, the sample may be contacted with: at least two sets of non- signal oligonucleotides, each non-signal oligonucleotide comprising: (aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and (bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.RES-PA52-PCT

[0081] As mentioned above, the different sets of non-signal oligonucleotides may be comprised in a pre-mixture of different sets of non-signal oligonucleotides or exist separately.

[0082] In further embodiments, the decoding oligonucleotides in a particular set of decoding oligonucleotides interacts with identical identifier elements (T) which are unique to a particular analyte.

[0083] As mentioned above, the different sets of decoding oligonucleotides may be comprised in a pre-mixture of different sets of decoding oligonucleotides or exist separately as well as the different sets of analyte-specific probes may be comprised in a pre-mixture of different sets of analyte-specific probes or exist separately as well the different sets of signal oligonucleotides may be comprised in a pre-mixture of different sets of signal oligonucleotides or exist separately.

[0084] In some embodiments of the method according to the present disclosure, the binding element (S) comprise a nucleic acid comprising a nucleotide sequence allowing a specific binding to the analyte to be encoded, preferably a specific hybridization to the analyte to be encoded.

[0085] In some embodiments of the method according to the present disclosure, after step A) and before step B) the non-bound analyte-specific probes may be removed, in particular by washing, further after step B) and before step C) the non-bound decoding oligonucleotides may be removed, in particular by washing further, after step C) and before step D) the non-bound signal oligonucleotides may be removed, in particular by washing.

[0086] In some embodiments of the method according to the present disclosure, the analyte specific probes may be incubated with the sample, thereby allowing a specific binding of the analyte specific probes to the analytes to be encoded, further the decoding oligonucleotides may be incubated with the sample, thereby allowing a specific hybridization of the decoding oligonucleotides to identifier elements (T) of the respective analyte-specific probes, further the signal oligonucleotides may be incubated with the sample, thereby allowing a specific hybridization of the signal oligonucleotides to translator elements (T) of the respective decoding oligonucleotides.

[0087] As mentioned above, the analyte to be encoded may be a nucleic acid (e.g., DNA, PNA, RNA, or combinations thereof). In some embodiments, the analyte to be encoded may be a RNA (e.g., mRNA, hnRNA, miRNA, viral RNA, bacterial RNA, extra- or intracellular RNA, circular mRNA, tRNA, siRNA, snRNA, or rRNA).

[0088] By this measure the method is further developed to such an extent that the encoded analytes can be detected by any means which is adapted to visualize the signal element.RES-PA52-PCTExamples of detectable physical features include e.g., light, chemical reactions, molecular mass, radioactivity, etc.

[0089] In some embodiments, the signal caused by the signal element, therefore in particular the binding of the signal oligonucleotides to the decoding oligonucleotides, interacting with the corresponding analyte probes, bound to the respective analyte is determined by: imaging at least a portion of the sample; and / or using an optical imaging technique; and / or using a fluorescence imaging technique; and / or multi-color fluorescence imaging technique; and / or super-resolution fluorescence imaging technique.

[0090] Methods as disclosed herein may also comprise additional methods for detection of an additional analyte. In some embodiments, an additional analyte is detected that is a protein or peptide. In some embodiments, 10 protein targets, 20 protein targets, 30 protein targets, or 40 protein targets are detected in the sample in addition to another analyte (e.g., a nucleic acid). In some embodiments, various antibody staining techniques may be employed to detect and visualize specific proteins or antigens within a sample. These techniques include direct and indirect staining, wherein antibodies either directly bind to antigens or use secondary antibodies for signal amplification. In some embodiments, fluorescent antibody staining is used. Multiplex staining allows for the simultaneous detection of multiple antigens. In some embodiments, the sample will be treated with a blocking agent (e.g., salmon sperm, BSA, goat serum) prior to analyte detection.

[0091] Described herein are methods and compositions for multi-modal analysis, combining spatial transcriptomics with protein assays and histological staining techniques, such as hematoxylin and eosin (H&E) staining, for comprehensive analysis of biological samples. In some embodiments, spatial transcriptomics is used to map gene expression across tissue sections, providing insights into the spatial distribution of RNA molecules within the sample. This transcriptomic data may be further enhanced by combining it with protein detection assays, such as immunohistochemistry or multiplex protein staining, to quantify the expression and localization of key protein markers.

[0092] In some embodiments, histological staining techniques, such as H&E staining, are employed alongside spatial transcriptomics and protein assays. H&E staining offers a structural overview of the tissue morphology, highlighting the cellular architecture, tumor boundaries, and stromal components. This allows for the spatial transcriptomic and protein data to be interpreted in the context of the tissue’s histological structure, ensuring that gene and protein expression is linked to specific regions of interest, such as tumor margins or immune cell infiltrates.

[0093] By integrating spatial transcriptomics, protein assays, and histological staining, this multi-modal approach offers a comprehensive view of the tissue microenvironment. ThisRES-PA52-PCT enables researchers and clinicians to correlate gene expression, protein expression, and tissue architecture, providing a more detailed understanding of cellular interactions, tumor-immune dynamics, and the impact of therapeutic interventions. In some embodiments, these methods are applied for diagnostic purposes, treatment monitoring, and to inform therapeutic strategies in oncology and other diseases.

[0094] Alternate detection approaches are also consistent with the disclosure of the panels and approaches herein. For example, some methods comprise or employ fluorescence in situ hybridization (FISH) using fluorophore labeled gene, transcript, or antibody probes, such as traditional FISH, merFISH, seqFISH or seqFISH+. Alternately, one may obtain positional information relevant to panels disclosed or contemplated herein via deposition of samples onto positionally encoded barcode oligo arrays and tagging of sample nucleic acids therein, or similarly tagging barcoded antibodies hybridized thereto. Positionally tagged nucleic acids or other tags may then be packaged as libraries and subjected to traditional sequencing, for example sequencing by synthesis, sequencing by binding, other sequencing by extension, or long read sequencing, for example. Positional information may then be deconvoluted to generate positional information in these embodiments.

[0095] The kits and method according to the present disclosure may be used ideally for in vitro methods for diagnosis, monitor, assay for, characterize, assess the status of, assess impact of a treatment or assess progression of a disease, such as a disease selected from the group comprising cancer, neuronal diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases due to a viral or bacterial infection, skin diseases, skeletal muscle diseases, dental diseases, and prenatal diseases.

[0096] Further, the kits and method according to the present disclosure may be used also ideally for in vitro methods for diagnosis of a disease in a subject individual, or nonhuman mammalian, animal or plant subject selected from the group comprising: diseases caused by biotic stress, preferably by infectious and / or parasitic origin, or diseases caused by abiotic stress, preferably caused by nutritional deficiencies and / or unfavorable environment, genetic disorder, impact of a therapeutic treatment, impact of a chemical treatment such as a fertilizer, nutrient treatment or supplement, or other condition, treatment or effect.

[0097] Further, the kits and method according to the present disclosure may be used also ideally for in vitro methods for screening, identifying and / or testing a substance and / or drug comprising: (a) contacting a test sample comprising a sample with a substance and / or drug, (b) detecting different analytes in a sample by sequential signal-encoding of said analytes with a method according to the present disclosure.RES-PA52-PCT

[0098] An optical multiplexing system suitable for the method according to the present disclosure, comprising at least: a reaction vessel for containing the kits or part of the kits according to the present disclosure; a detection unit comprising a microscope, in particular a fluorescence microscope; a camera; a liquid handling device.

[0099] In some embodiments, optical multiplexing system may comprise further a heat and cooling device and / or a robotic system.

[0100] In some embodiments, the method according to the present disclosure encodes a nucleic acid analyte, such as an mRNA, e.g., such an mRNA coding for a particular protein.

[0101] In some embodiments, the method described herein is used for specific detection of many different analytes in parallel. The technology allows to distinguish a higher number of analytes than different signals are available. The process includes at least four consecutive rounds of specific binding, signal detection and selective denaturation (if a next round is required), eventually producing a signal code. To decouple the dependency between the analyte specific binding and the oligonucleotides providing the detectable signal, a so called “decoding”-oligonucleotide is introduced. The decoding oligonucleotide transcribes the information of the analyte specific probe set to the signal oligonucleotides.

[0102] In a specific embodiment, the method may comprise the steps of (1) providing one or more analyte specific probe sets, the set of analyte specific probes consist of one or more different probes, each differing in the binding moiety that specifically interacts with the analyte, all probes of a single probe set are tethered to a sequence element (unique identifier), that is unique to a single probe set and allows the specific hybridization of a decoding oligonucleotide, (2) specific binding of the probe sets to their target binding sites of the analyte, (3) eliminating non-bound probes (e.g. by a wash step), (4) providing a mixture of decoding oligonucleotides that specifically hybridize to the unique identifier sequences of the probe sets, the decoding oligonucleotides comprise of at least two sequence elements, a first element that is complementary to the unique identifier sequences of the corresponding probe set and a second sequence element (translator element) that provides a sequence for the specific hybridization of a signal oligonucleotide, the translator element defines the type of signal that is recruited to the decoding oligonucleotide, (5) specific hybridization of the decoding oligonucleotides to the unique identifier sequences provided by the bound probe sets, (6) eliminating non-bound decoding oligonucleotides (e.g. by washing step), (7) providing a mixture of signal oligonucleotides, comprising of a signal that can be detected and a nucleic acid sequence that specifically hybridizes to the translator element of one of the decoding oligonucleotides used in the former hybridization step, (8) specific hybridization of the signal oligonucleotides, (9) eliminating non-bound signal oligonucleotides, (10) detection of the signals, (11) selectiveRES-PA52-PCT release of decoding oligonucleotides and signal oligonucleotides while the binding of specific probe sets to the analyte is almost or completely unaffected, (12) eliminating released decoding oligonucleotide and signal oligonucleotides (e.g., by a washing step) while the binding of specific probes sets to the analytes is almost or completely unaffected , repeating the steps 4 to 12 at least three times until the detection of a sufficient number of signals to generate an encoding scheme for each different analyte of interest.

[0103] It is to be understood that the before-mentioned features and those to be mentioned in the following cannot only be used in the combination indicated in the respective case, but also in other combinations or in an isolated manner without departing from the scope of the disclosure.

[0104] The method disclosed herein is used for specific detection of many different analytes in parallel. The technology allows distinguishing a higher number of analytes than different signals are available. The process preferably includes at least two consecutive rounds of specific binding, signal detection and selective denaturation (if a next round is required), eventually producing a signal code. To decouple the dependency between the analyte specific binding and the oligonucleotides providing the detectable signal, a so called “decoding” oligonucleotide is introduced. The decoding oligonucleotide transcribes the information of the analyte specific probe set to the signal oligonucleotides.

[0105] The present disclosure pertains further to methods of detecting an analyte, comprising: attaching a plurality of analyte-specific probes to the analyte, wherein the analyte-specific probes independently attach to the analyte and wherein the analyte-specific probes share a common identifier segment (T); annealing a plurality of first decoding oligonucleotides to the analyte-specific probes, wherein the first decoding oligonucleotides share a first common region that is reverse complementary to the common identifier segment and a second common region; annealing a first signal oligonucleotide to at least one of the pluralities of first decoding oligonucleotides such that an oligo tethered to the first signal oligonucleotide is reverse complementary to the second common region; detecting the first signal oligonucleotide; removing the plurality of first decoding oligonucleotides; annealing a plurality of second decoding oligonucleotides to the analyte-specific probes, wherein the second decoding oligonucleotides share a first common region that is reverse complementary to the common identifier segment and a second decoding oligonucleotides second common region that differs from the second common region of the first decoding; annealing a second signal oligonucleotide to at least one of the pluralities of second decoding oligonucleotides such that an oligo tethered to the signal oligonucleotide is reverse complementary to the second decoding oligonucleotide second common region; and detecting the second signal oligonucleotide.RES-PA52-PCT

[0106] In the above-mentioned embodiment a second aliquot of a plurality of first decoding oligonucleotides is annealed to the analyte-specific probes. Furthermore, a first aliquot of a plurality of first decoding oligonucleotides is annealed to the analyte-specific probes.

[0107] In some embodiments, no second signal oligonucleotide to the at least one of the pluralities of first decoding oligonucleotides is annealed. No third signal oligonucleotide to the at least one of the pluralities of first decoding oligonucleotides is annealed.

[0108] In some further embodiments, the present disclosure pertains to a method of assigning an analyte to a position in an image, comprising assigning a fluorescence pattern to the analyte, observing the fluorescence pattern at the position in the image, and assigning the analyte to the position, in particular wherein observing the fluorescence pattern comprises repeating steps of labeling the position using a fluorophore tagged oligo drawn from a re-accessible pool, performing a single excitation at the position in the image, and contacting the analyte to a denaturant, in particular wherein observing the fluorescence pattern comprises repeating steps of labeling the position using a fluorophore tag-recruiting bridging oligo drawn from a re- accessible pool, performing a single excitation at the position in the image, and contacting the analyte to a denaturant.

[0109] In some further embodiments, the present disclosure pertains to a composition comprising a cell having nucleic acids distributed therein, wherein a first nucleic acid is tagged by a first plurality of probes that target adjacent segments of the first nucleic acid and that share a common first tether segment; a second nucleic acid is tagged by a second plurality of probes that target adjacent segments of the second nucleic acid and that share a common second tether segment; and a third nucleic acid is tagged by a third plurality of probes that target adjacent segments of the third nucleic acid and that share a common third tether segment; a first adapter population comprising molecules having a first tether reverse complementary region and a first fluorophore adapter tether; a second adapter population comprising molecules having a second tether reverse complementary region and a second fluorophore adapter tether; a third adapter population comprising molecules having a third tether reverse complementary region and a first fluorophore adapter tether; a population of first fluorophores having a first tether reverse complementary region; and a population of second fluorophores having a second tether reverse complementary region.

[0110] In some further embodiments, the present disclosure pertains to a method of assigning coded fluorescence patterns to a plurality of target analytes in a cell, comprising subjecting the cell to a plurality of detection rounds, each detection round comprising contacting the cell to representatives of the same at least two populations of tagged fluorescence moieties, and removing the fluorescent moieties after a single excitation event, wherein the number of patternsRES-PA52-PCT detectable increases exponentially with the number of detection rounds, wherein the fluorescence moieties are not tagged with nucleic acid tags that are specific to the target nucleic acids, and wherein separate aliquots of common tagged fluorescence moieties are used across multiple detection rounds.[OHl] With the above notified methods a high degree of decoding efficiency may be achieved. For example, a total decoding efficiency of at least 30% may be achieved, or alternately at least, at most, about or exactly 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or greater.

[0112] In some further embodiments, the present disclosure pertains to a method of assigning coded fluorescence patterns to a plurality of target analytes in a cell, comprising: contacting a target to a bipartite labeling probe, the bipartite labeling probe comprising a target-specific moiety and a fluorophore-specifying moiety; contacting the bipartite labeling probe to a first aliquot of a fluorophore reservoir comprising no more than two populations of fluorophores; replacing the fluorophore specifying moiety in the bipartite probe, and contacting the bipartite labeling probe to a second aliquot of the fluorophore reservoir comprising the same no more than two populations.

[0113] In some embodiments of the above notified method, replacing the fluorophore specifying moiety in the bipartite probe comprises denaturing a binding between a target-specific moiety and a fluorophore-specifying moiety after subjecting the bipartite labeling probe bound to a fluorophore of the fluorophore to excitation energy. Replacing the fluorophore specifying moiety in the bipartite probe comprises drawing from one of no more than two fluorophore specifying moiety reservoirs.

[0114] In some further embodiments, the present disclosure pertains to a method of detecting an analyte, comprising: attaching a plurality of probes to the analyte, in particular a nucleic acid, wherein the probes independently attach / anneal to the analyte and wherein the probes share a common identifier segment; annealing a plurality of first adapter segments to the probes, wherein the first adapter segments share a first common region that is reverse complementary to the common identifier segment and a second common region, in particular configured to accommodate a single reporter / selected from no more than two reporter categories; annealing a first reporter to at least one of the plurality of first adapter segments such that an oligo tethered to the first reporter is reverse complementary to the second common region; detecting the first reporter; removing the plurality of first adapter segments, in particular without annealing a second reporter to the at least one of the pluralities of first adapter segments; annealing a plurality of second adapter segments to the probes, wherein the second adapter segments share a first common region that is reverse complementary to the common identifier segment and a second adapter second common region that differs from the second common region of the firstRES-PA52-PCT adapter segments, in particular configured to accommodate a single reporter / selected from no more than two reporter categories; annealing a second reporter to at least one of the pluralities of second adapter segments such that an oligo tethered to the second reporter is reverse complementary to the second adapter second common region; and detecting the second reporter, in particular without annealing a third reporter to the at least one of the pluralities of first adapter segments.

[0115] The analyte or target may be a nucleic acid, e.g., DNA or RNA, and the probe set comprises oligonucleotides that are partially or completely complementary to the whole sequence or a subsequence of the analyte (e.g., a nucleic acid sequence) to be detected. The nucleic acid sequence specific oligonucleotide probe sets comprising analyte-specific probes (1) including a binding element (S) that specifically hybridizes to the target nucleic acid sequence to be detected, and an identifier element (T) comprising a nucleotide sequence which is unique to said set of analyte-specific probes (unique identifier sequence).

[0116] In some embodiments, at least one analyte is a first nucleic acid and at least a second analyte is a second nucleic acid and at least the first probe set binds to the first nucleic acid sequence and at least the second probe set binds specifically to the second nucleic acid analyte. Other combinations are possible as well.

[0117] An embodiment of the general method of the present disclosure may be as follows. Step 1 : applying the at least 20 analyte- or target-specific probe sets. The target nucleic acid sequence is incubated with a probe set consisting of oligonucleotides with sequences complementary to the target nucleic acid. In this example, a probe set of 5 different probes is shown, each comprising a sequence element complementary to an individual subsequence of the target nucleic acid sequence (SI to S5). In this example, the regions do not overlap. Each of the oligonucleotides targeting the same nucleic acid sequence comprises the identifier element or unique identifier sequence (T), respectively. Step 2: Hybridization of the probe set. The probe set is hybridized to the target nucleic acid sequence under conditions allowing a specific hybridization. After the incubation, the probes are hybridized to their corresponding target sequences and provide the identifier element (T) for the next steps. Step 3 : Eliminating nonbound probes. After hybridization, the unbound oligonucleotides are eliminated, e.g., by washing steps. Step 4: Applying the decoding oligonucleotides. The decoding oligonucleotides consisting of at least two sequence elements (t) and (c) are applied. While sequence element (t) is complementary to the unique identifier sequence (T), the sequence element (c) provides a region for the subsequent hybridization of signal oligonucleotides (translator element). Step 5: Hybridization of decoding oligonucleotides. The decoding oligonucleotides are hybridized with the unique identifier sequences of the probes (T) via their complementary first sequenceRES-PA52-PCT elements (t). After incubation, the decoding oligonucleotides provide the translator sequence element (c) for a subsequent hybridization step. Step 6: Eliminating the excess of decoding oligonucleotides. After hybridization, the unbound decoding oligonucleotides are eliminated, e.g., by washing steps. Step 7: Applying the signal oligonucleotide. The signal oligonucleotides are applied. The signal oligonucleotides comprise at least one second connector element (C) that is essentially complementary to the translator sequence element (c) and at least one signal element that provides a detectable signal (F). Step 8: Hybridization of the signal oligonucleotides. The signal oligonucleotides are hybridized via the complementary sequence connector element (C) to the translator element (c) of decoding oligonucleotide. After incubation, the signal oligonucleotides are hybridized to their corresponding decoding oligonucleotides and provide a signal (F) that can be detected. Step 9: Eliminating the excess of signal oligonucleotides. After hybridization, the unbound signal oligonucleotides are eliminated, e.g., by washing steps. Step 10: Signal detection. The signals provided by the signal oligonucleotides are detected. The following steps (steps 11 and 12) may be unnecessary for the last detection round or may be necessary (e.g., if further measurements are performed on the sample). Step 11 : Selective denaturation. The hybridization between the unique identifier sequence (T) and the first sequence element (t) of the decoding oligonucleotides is dissolved. The destabilization can be achieved via different mechanisms well known to the trained person like for example: increased temperature, denaturing agents, etc. The target- or analyte-specific probes are not affected by this step. Step 12: Eliminating the denatured decoding oligonucleotides. The denatured decoding oligonucleotides and signal oligonucleotides are eliminated (e.g., by washing steps) leaving the specific probe sets with free unique identifier sequences, reusable in a next round of hybridization and detection (steps 4 to 10). This detection cycle (steps 4 to 12) is repeated at least four times until the planed encoding scheme is completed.

[0118] The “analyte-specific probe” (e.g., “probe”) comprises at least two elements, the so- called binding element (S) which specifically interacts with (e.g., is essentially complementary to, binds to, hybridizes to, or a combination thereof) an analyte, and a so-called identifier element (T) comprising a “unique identifier sequence”. The binding element (S) may be a nucleic acid such as a hybridization sequence or an aptamer.

[0119] The “unique identifier sequence” as comprised by the analyte-specific probe is unique in its sequence compared to other unique identifiers. “Unique” in this context means that it specifically identifies only one analyte, e.g., a nucleic acid, or, alternatively, it specifically identifies only a group of analytes, e.g., a group of nucleic acids, independently whether the group of analytes comprises a gene family or not. Therefore, the analyte or a group of analytesRES-PA52-PCT to be encoded by this unique identifier can be distinguished from all other analytes or groups of analytes that are to be encoded based on the unique identifier sequence of the identifier element (T). Or, in other words, there is only one “unique identifier sequence” for a particular analyte or a group of analytes, but not more than one, e.g., not even two. Due to the uniqueness of the unique identifier sequence the identifier element (T) hybridizes to one type of decoding oligonucleotides during a cycle. The length of the unique identifier sequence is within the range 8-60 nt, 12-40 nt, or 14-20 nt, which may depend on the number of analytes encoded in parallel and the stability of interaction needed. A unique identifier may be a sequence element of the analyte-specific probe, attached directly or by a linker, a covalent bond or high affinity binding modes, e.g., antibody-antigen interaction, streptavidin-biotin interaction etc. It is understood that the term “analyte specific probe set” includes a plurality of analyte specific probes which may differ in their binding elements (S) in a way that each probe binds to the same analyte but possibly to different parts thereof, for instance to different (e.g., neighboring) or overlapping sections of the nucleotide sequence comprised by the nucleic acid molecule to be encoded. However, each of the plurality of the probes comprises the same identifier element (T). For example, an analyte specific probe set may comprise a plurality of analyte specific probes that each independently comprise a different binding element (S) and a common identifier element (T). The different binding elements (S) comprised by the plurality of analyte specific probes in an analyte specific probe set may bind different regions (e.g., different sequences) comprised by an analyte (e.g., a nucleic acid).

[0120] A “bipartite labeling probe” comprises a binding sequence capable of hybridizing the analyte and a binding probe sequence capable of binding a detectable signal molecule like a fluorophore or a nucleic acid sequence comprising a fluorophore.

[0121] A “decoding oligonucleotide” or an “adapter” or a / adapter segment” comprises at least two sequence elements, one sequence element that can specifically bind to a unique identifier sequence, referred to as an “identifier connector element” (t) or “first connector element” (t), and a second sequence element specifically binding to a signal oligonucleotide, referred to as “translator element” (c). The length of the sequence elements is within the range 8-60 nt, 12-40 nt, 14-20 nt, which may be dependent on the number of analytes to be encoded in parallel, the stability of interaction needed, the number of different signal oligonucleotides used, or a combination thereof. The length of the two sequence elements may or may not be the same.

[0122] A “signal oligonucleotide” or a “reporter” as used herein comprises at least two elements, a so-called “translator connector element” (C) or "second connector element" (C) having a nucleotide sequence specifically hybridizable to at least a section of the nucleotide sequence of the translator element (c) of the decoding oligonucleotide, and a “signal element”RES-PA52-PCT which provides a detectable signal. This element can either actively generate a detectable signal or provide such a signal via manipulation, e.g., fluorescent excitation. Typical signal elements are, for example, enzymes that catalyze a detectable reaction, fluorophores, radioactive elements, or dyes.

[0123] A “set” refers to a plurality of moieties or subjects, e.g., analyte-specific probes or decoding oligonucleotides, whether the individual members of said plurality are identical or different from each other. In an analyte specific probe set, the analyte specific probes are identical in the identifier element (T) but may comprise a different binding element (S) for specifically interacting with the same analyte but for specifically interacting with different substructures of the same analyte to be encoded.

[0124] An “analyte specific probe set” refers to a plurality of moieties or subjects, e.g., analytespecific probes that are different from each other and bind to independent regions of the analyte. A single analyte specific probe set is further characterized by the same unique identifier.

[0125] A “decoding oligonucleotide set” refers to a plurality of decoding oligonucleotides specific for a certain unique identifier needed to realize the encoding independent of the length of the code word. Each and all of the decoding oligonucleotides included in a “decoding oligonucleotide set” bind to the same unique identifier element (T) of the analyte-specific probe.

[0126] In this disclosure “simultaneously detecting” means that all probes detecting the analyte may be added in one detection round. For example, genomic as well as mRNA may be detected in one detection round. In this disclosure “sequential detecting” means that the probes detecting the analyte may be added sequentially, e.g., probe set 1 for detecting analyte 1 is added first, then, optionally after a washing step, probe set 2 for detecting analyte 2 is added second, etc. For example, first, genomic nucleic acid elements may be detected in round 1, then followed by the detection of nucleic acid elements of mRNA in round 2, optionally, with a washing step in between round 1 and round 2. In some embodiments both “simultaneously detecting” as well as “sequential detecting” may be performed, e.g., detecting simultaneously analyte 1 and 2 with a first and second set of probes, the, optionally performing a washing step, and then detecting simultaneously analyte 3 and 4 with a third and fourth set of probes.

[0127] In certain embodiments, this pattern of binding or hybridization of the decoding oligonucleotides may be converted into a “code word.” For example, the code words could be also "101" and "110" for an analyte, where a value of 1 represents binding and a value of 0 represents no binding. The code words may also have longer lengths in other embodiments. A code word can be directly related to a specific unique identifier sequence of an analyte-specific probe. Accordingly, different analyte-specific probe may match certain code words, which can then be used to identify the different analytes of the analyte-specific probe based on the bindingRES-PA52-PCT patterns of the decoding oligonucleotide. However, if no binding is evident, then the code word would be "000" in this example.

[0128] The values in each code word can also be assigned in different fashions in some embodiments. For example, a value of 0 could represent binding while a value of 1 represents no binding. Similarly, a value of 1 could represent binding of a secondary nucleic acid probe with one type of signaling entity while a value of 0 could represent binding of a secondary nucleic acid probe with another type of distinguishable signaling entity. These signaling entities could be distinguished, for example, via different colors of fluorescence. In some cases, values in code words need not be confined to 0 and 1. The values could also be drawn from larger alphabets, such as ternary (e.g., 0, 1, and 2) or quaternary (e.g., 0, 1, 2, and 3) systems. Each different value could, for example, be represented by a different distinguishable signaling entity, including (in some cases) one value that may be represented by the absence of signal.

[0129] The code words for each analyte may be assigned sequentially or may be assigned at random. For instance, a first analyte may be assigned to 101, while a second nucleic acid target may be assigned to 110. In addition, in some embodiments, the code words may be assigned using an error-detection system or an error- correcting system, such as a Hamming system, a Golay code, or an extended Hamming system (or a SECDED system, i.e., single error correction, double error detection). Such systems can be used to identify where errors have occurred, and in some cases, such systems can also be used to correct the errors and determine what the correct code word should have been. For example, a code word such as 001 may be detected as invalid and corrected using such a system to 101, e.g., if 001 is not previously assigned to a different target sequence. A variety of different error-correcting codes can be used, many of which have previously been developed for use within the computer industry; however, such error-correcting systems have not typically been used within biological systems. Additional examples of such error-correcting codes are discussed in more detail below.

[0130] In the “incubation” steps as understood herein the respective moi eties or subjects such as probes or oligonucleotide, are brought into contact with each other under conditions well known to the skilled person allowing a specific binding or hybridization reaction, e.g., pH, temperature, salt conditions etc. Such steps may, therefore, be preferably carried out in a liquid environment such as a buffer system which is well known in the art.

[0131] The “removing” steps according to the disclosure may include the washing away of the moi eties or subjects to be removed such as the probes or oligonucleotides by certain conditions, e.g., pH, temperature, salt conditions etc., as known in the art.

[0132] It is understood that in an embodiment of the method according to the present disclosure a plurality of analytes can be encoded in parallel. This requires the use of different sets ofRES-PA52-PCT analyte-specific probes in step (1). The analyte-specific probes of a particular set differ from the analyte-specific probes of another set. This means that the analyte-specific probes of set 1 bind to analyte 1, the analyte-specific probes of set 2 binds to analyte 2, the analyte-specific probes of set 3 binds to analyte 3, etc. In this embodiment also the use of different sets of decoding oligonucleotides is required in the methods according to the present disclosure.

[0133] The decoding oligonucleotides of a particular set differ from the decoding oligonucleotides of another set. This means, the decoding oligonucleotides of set 1 bind to the analyte-specific probes of above set 1 of analyte-specific probes, the decoding oligonucleotides of set 2 binds to the analyte-specific probes of above set 2 of analyte-specific probes, the decoding oligonucleotides of set 3 binds to the analyte-specific probes of above set 3 of analytespecific probes, etc.

[0134] In this embodiment where a plurality of analytes is to be encoded in parallel the different sets of analyte-specific probes may be provided as a premixture of different sets of analytespecific probes and / or the different sets of decoding oligonucleotides may be provided as a premixture of different sets of decoding oligonucleotides. Each mixture may be contained in a single vial. Alternatively, the different sets of analyte-specific probes and / or the different sets of decoding oligonucleotides may be provided in steps singularly.

[0135] A “kit” is a combination of individual elements useful for carrying out the use and / or method of the disclosure, wherein the elements are optimized for use together in the methods. The kits may also contain additional reagents, chemicals, buffers, reaction vials etc. which may be useful for carrying out the method according to the disclosure. Such kits unify all essential elements required to work the method according to the disclosure, thus minimizing the risk of errors. Therefore, such kits also allow semi-skilled laboratory staff to perform the method according to the present disclosure.

[0136] The term “quencher” or “quencher dye” or “quencher molecule” refers to a dye or an equivalent molecule, such as nucleoside guanosine (G) or 2'-deoxyguanosine (dG), which is capable of reducing the fluorescence of a fluorescent reporter dye or donor dye. A quencher dye may be a fluorescent dye or non-fluore scent dye. When the quencher is a fluorescent dye, its fluorescence wavelength is typically substantially different from that of the reporter dye and the quencher fluorescence is usually not monitored during an assay. Some embodiments of the present disclosure disclose signal oligonucleotides comprising a quencher and / or a quencher in combination with a signal element, and therefore the signal oligonucleotides is not detectable during imaging.

[0137] A “sample” as referred to herein is a composition in liquid or solid form suspected of comprising the analytes to be encoded. A sample described herein may be a biological sample.RES-PA52-PCTA sample may comprise biological tissue, biological cells, and / or extracts and / or part of cells, or any combination thereof. The sample may comprise eukaryotic or procaryotic cells. The sample may derive from any kind of animal (including Homo, rat, mouse, mammalia, birds, fish, insects, worms), plant, or fungi. In some embodiments, the sample may comprise a mammalian cell. In some embodiments, the sample may comprise a human cell. The sample can be selected from any organ, any tissue, any kind of culture, any specimen taken for diagnostic purposes (e.g. smear, liquid biopsy, tissue biopsy etc.). The sample may comprise DNA and / or RNA. Samples may taken from alive or dead organisms. Cells may not be complete and samples may contain only partial cells. The sample may be frozen, fixed or embedded. In some embodiments, the biological tissue, biological cells, extracts and / or part of cells are fixed. In some embodiments, the analytes are fixed in a permeabilized sample, such as a cell-containing sample.

[0138] The term “cell” as used herein is the smallest unit of life and comprises a number (for example, at least one, at least two, at least 5, at least 10, at least 20) genome elements that can be differentiated. The cell may be dead or alive. The cell may be eukaryotic or prokaryotic. The cell may not be complete and contain only a part of the cell (e.g., due to preparation or fixation of the sample or cell).

[0139] The term “genome” as used herein is a complete identity or a part of this. It can be DNA or RNA.

[0140] The term “transcription” is used herein for a process during which one strand of the genome sequence of the genome element is copied into a complementary RNA (e.g., an mRNA) strand. These single-stranded copies may be independent molecules (not covalently connected to another molecule) but may be connected for a certain time to the genome element by a non- covalently binding (e.g., by hydrogen bonding). The connection can be stabilized by a fixation method (e.g., methanol, formalin etc.).

[0141] The term “biological sample” is defined as a material that is derived from an organism and at least contains detectable nucleic acids, cells or parts of cells. These cells may originate from the same or different organs or even organisms.

[0142] The term “tissue” is used herein for any kind of a sample material that is formed by a certain number of cells of the same or different type with a meaningful structural relationship (or the lack thereof), and thus does comprise genome elements. The term “tissue section” is used herein for a thin section of a tissue favorably done by a cryotome or a microtome.

[0143] An “analyte” according to the disclosure may be any molecule (e.g., a biomolecule) of interest. Sometimes herein the term “analyte” is replaced by “target.” In some embodiments an analyte may comprise a biomolecule (e.g., a protein, a nucleic acid, a biomolecule, a lipid, or any combination thereof). In some embodiments, an analyte may be a nucleic acid (e.g., DNA,RES-PA52-PCTPNA, LNA, RNA, or any combination thereof). In some embodiments, an analyte may be a DNA molecule (e.g., genomic DNA, nuclear DNA, circular DNA, mitochondrial DNA, viral DNA, bacterial DNA, extra- or intracellular DNA, or any combination thereof). In some embodiments, an analyte may be an RNA molecule (e.g., mRNA, hnRNA, miRNA, viral RNA, bacterial RNA, extra- or intracellular RNA, circular mRNA, tRNA, siRNA, snRNA, rRNA, or any combination thereof). In some embodiments, an analyte may be an mRNA (e.g., a transcript). In some embodiments, there may be multiple analytes, e.g., at least two individual nucleic acids.

[0144] In some embodiments, an analyte may be a “coding sequence”, “encoding sequence”, “structural nucleotide sequence”, or “structural nucleic acid molecule” which refers to a nucleotide sequence that is translated into a polypeptide, e.g., via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'- terminus. A coding sequence can include, but is not limited to, genomic DNA, cDNA, EST, and recombinant nucleotide sequences.

[0145] As used in the present disclosure, “cell”, “cell line”, and “cell culture” can be used interchangeably and all such designations include progeny. Thus, the words “transformants” or “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progenies may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that has the same functionality as screened for in the originally transformed cell are included.

[0146] An “encoding scheme” may describe a set of code words that are associated with the analytes to be detected. Each code word refers to one of the analytes and can be distinguished from all other code words. A code word hereby is a sequence of signs provided by the detection cycles of the method. A sign within a code word is a detectable signal or the absence of a signal. A code word does not need to comprise of all different signals used in the method. The number of signs in a code word is defined by the number of detection cycles.

[0147] An “oligonucleotide” as used herein, refers to a nucleic acid molecule, such as DNA, PNA, LNA or RNA. The length of the oligonucleotides may be within the range of 10-10,000 nucleotides (nt), 10-15,000 nt, 10-10,000 nt, 10-5,000 nt, 10-2,000 nt, 10-1,500 nt, 10-1,000 nt, or 10-800 nt. In some embodiments, the length of oligonucleotides may be within the range of 100-1,500 nt, 100-1,200 nt, 100-1,000 nt, or 100-800 nts. In some embodiments, the length of oligonucleotides may be within the range of 400-1,500 nt, 400-1,200 nt, 400-1,000 nt, or 400- 800 nts. The nucleic acid molecule can be fully or partially single-stranded. The oligonucleotides may be linear or may comprise hairpin or loop structures. The oligonucleotidesRES-PA52-PCT may comprise modifications such as biotin, labeling moieties, blocking moieties, or other modifications.

[0148] “Essentially complementary” means, when referring to two nucleotide sequences, that both sequences can specifically hybridize to each other under stringent conditions, thereby forming a hybrid nucleic acid molecule with a sense and an antisense strand connected to each other via hydrogen bonds (Watson-and-Crick base pairs). “Essentially complementary” includes not only perfect base-pairing along the entire strands, e.g., perfect complementary sequences but also imperfect complementary sequences which, however, still have the capability to hybridize to each other under stringent conditions. Among experts it is well accepted that an “essentially complementary” sequence has at least 88% sequence identity to a fully or perfectly complementary sequence.

[0149] As disclosed herein, percent sequence identity (e.g., “percent identity,” “percent sequence identity,” or “sequence identity”) describes the similarity between two or more sequences (e.g., a nucleic acid sequence or an amino acid sequence). Sequence similarity calculations may be performed using the BLAST algorithm for sequence alignment, which is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). Percent sequence identity compares a given sequence to a claimed or described sequence after alignment of the given sequence to be compared (the “Compared Sequence”) with the described or claimed sequence (the “Reference Sequence”). The percent identity is then determined according to the following formula: percent identity = 100*(l -(C / R)) wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence, wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference, and (iv) the alignment has to start at position 1 of the aligned sequences; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.

[0150] As used herein, the term “about” in the context of a number in some cases refers to a range spanning 10% below to 10% above that number. In the context of a range, the term refers to an expanded range spanning 10% blow the listed lower limit to 10% above the listed upper limit. In cases where the number is small and cannot be subdivided, the term in some cases refers to a range spanning one unit below to one unit above the number referred to.RES-PA52-PCT

[0151] If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity, then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the herein above calculated percent identity is less than the specified percent identity.EXAMPLES

[0152] The invention is further illustrated by the following non-limiting examples.EXAMPLE 1 - Tumor Analysis Using Immuno-Oncology Gene Panel

[0153] This example describes how spatial transcriptomics is used to analyze a tumor biopsy with an immuno-oncology gene panel to assess immune responses and the tumor microenvironment. A gene panel comprising a core immune module, a tumor-specific module, and a cell-cell communication module is applied to the spatial transcriptomic analysis of the tissue sections. The spatial transcriptomics analysis of the core module reveals localized areas of high immune checkpoint gene expression (e.g., PD-1, CTLA-4), indicating regions of immune suppression within the tumor. The tumor module identifies regions with high oncogene expression, indicating areas of active tumor proliferation. The cell-cell communication module shows spatial patterns of increased expression of cytokines and adhesion molecules involved in immune evasion and angiogenesis. This spatially resolved data supports therapeutic decisions for combining immune checkpoint inhibitors and anti -angiogenic therapies.EXAMPLE 2 - Diagnostic Monitoring with Spatial Transcriptomics and ImmunoOncology Gene Panel

[0154] This example describes the use of spatial transcriptomics with an immuno-oncology gene panel to monitor a cancer patient’s response to immunotherapy. A biopsy sample is analyzed at baseline and during therapy using spatial transcriptomics to map gene expression across the tumor microenvironment. The baseline analysis reveals spatially distinct regions of immune cell infiltration and immune checkpoint activation, suggesting localized immune suppression. After several weeks of immunotherapy, spatial transcriptomics shows a reduction in immune checkpoint gene expression and an increase in effector T-cell markers, indicating active antitumor immune responses in specific regions of the tumor. This spatially resolved gene expression data helps clinicians confirm treatment efficacy and guide adjustments in the therapeutic strategy based on localized immune responses.RES-PA52-PCTEXAMPLE 3 - Spatial vs Quantitative Assay outcomes for an Immuno-Oncology Gene Panel

[0155] This example illustrates the value of spatial transcriptomics with an immuno-oncology gene panel to monitor a cancer patient’s response to immunotherapy.

[0156] A first patient is subjected to an immunotherapy treatment. A sample is taken and bulk transcripts are obtained. Quantitative analysis indicates that immune-oncology transcript levels in the panel in bulk do not change in response to the treatment, and the treatment is discontinued.

[0157] A second patient is subjected to the same immunotherapy treatment. A sample is taken and subjected to spatial omic analysis consistent with the disclosure herein. Spatial analysis indicates that immune-oncology transcript levels in the panel in bulk do not change in response to the treatment, but that transcript levels within a small sub-population of cells in the sample are substantially impacted. The treatment is continued and the subject is observed to exhibit immunotherapy triggered remission.EXAMPLE 4 - Individual vs Panel Assay outcomes for an Immuno-Oncology Gene Assay

[0158] This example illustrates the value of spatial transcriptomics with an immuno-oncology gene panel rather than individual genes to monitor immune responses.

[0159] A first subject is given a cancer immunotherapy and an immunotherapy response marker is assayed to determine the efficacy of the immunotherapy to elicit an immune response.

[0160] Expression of the gene is found not to vary, and the therapy is not continued.

[0161] A second subject is given the same cancer immunotherapy and an immunotherapy response marker is assayed using a panel comprising at least one gene from multiple tables herein to determine the efficacy of the immunotherapy to elicit an immune response.

[0162] The gene assayed in the first subject is found not to vary, nor do other genes in the table of the gene assayed in the first subject. Genes from other tables in the panel are observed to vary, indicating that an immune response is elicited in response to the therapy through a pathway not involving the gene of the first subject assay. The treatment is continued and the subject is observed to exhibit immunotherapy triggered remission.

[0163] While preferred embodiments of the present invention 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 practicingRES-PA52-PCT the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

RES-PA52-PCTCLAIMSWHAT IS CLAIMED IS:

1. A gene panel comprising a plurality of genes selected from the group consisting of tumor-related genes, immune function-related genes, T-cell related genes, Natural Killer (NK) cell related genes, Innate Lymphoid Cell (ILC) related genes, plasma cell (B-cell) related genes, monocyte and macrophage-related genes, dendritic cell (DC) related genes, granulocyte-related genes, vascular-related genes, and stromal-related genes, for the analysis of immune responses and tumor-immune interactions in a human sample, wherein the expression or mutational status of said genes is used in spatial omic analysis to assess immune activity related to cancer and immune therapy response.

2. The gene panel of claim 1, wherein the spatial omic analysis comprises spatial transcriptomics, spatial proteomics, spatial metabolomics, or any combination thereof.

3. The gene panel of claim 1 or claim 2, wherein the gene panel comprises at least one gene selected from any one of TABLE 1 - TABLE 10, or any combination thereof.

4. The gene panel of any one of claims 1-3, wherein the gene panel comprises a gene panel module.

5. The gene panel of claim 4, wherein the gene panel module comprises a gene panel module selected from TABLE 11.

6. A method for assessing the immune status of a human subject with cancer, comprising: obtaining a biological sample from the subject; analyzing the expression levels or mutational status of genes within the gene panel of any one of claims 1-5, comparing the expression levels or mutational status to reference data to assess immune cell infiltration, immune checkpoint activation, or tumor-immune interactions to predict the subject's response to immunotherapy.

7. The method of claim 6, wherein the immunotherapy comprises a checkpoint inhibitor selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors.

8. The method of claim 6 or 7, wherein the biological sample comprises tumor tissue, blood, serum, plasma, or FFPE tissue samples.RES-PA52-PCT9. A diagnostic kit for use in immuno-oncology, comprising: the gene panel of any one of claims 1-5; reagents for isolating and amplifying nucleic acids from a biological sample; and instructions for analyzing the expression or mutational status of said genes using spatial omic techniques to assess immune responses and tumor-immune interactions in a human subject.

10. An image of a subject sample, said image assayed using a panel of any one of claims 1-5, to indicate subject gene activity for members of the panel.

11. The image of claim 10, wherein the gene activity comprises localized transcript accumulation levels.

12. The image of claim 10, wherein the gene activity comprises localized protein accumulation levels.

13. The image of claim 10, wherein the image comprises non-panel analyte accumulation levels.

Images