Regulating mucus production

Abstract

Provided herein are ex vivo methods of engineering or culturing a submucosal gland (SMG) organoid, comprising isolating submucosal gland basal cells and / or myoepithelial cells (e.g., human cells), and / or submucosal gland stem and / or progenitor cells (e.g., human cells), and culturing such cells with an agonist or ligand of ERBB (e.g., in a three- dimensional matrix). Also provided herein are SMG organoid engineered or cultured ex vivo. Also provided herein are methods of identifying an agent that reduces airway mucus production using an ex vivo SMG organoid described herein. Also provided herein is a method of treating a hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands, in a subject, comprising administering to the subject an agent that inhibits ERBB.

Description

UCH-39925 REGULATING MUCUS PRODUCTION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. Provisional Patent ApplicationNo.63 / 650,618, filed May 22, 2024, which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] Abnormal mucus production (e.g., caused by hypersecretory disorders) raisesthe risk of infection and death for patients suffering from a host of diseases and conditions, including cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), emphysema, bronchitis, primary dyskinesia (PCD), microbial infections (including viral, bacterial, and fungal infections), and asthma. Notably, there are many patients worldwide suffering from such diseases and conditions. For example, more than 100,000 children and adults worldwide are living with cystic fibrosis, and more than 15 million people in the United States have COPD. COPD is the sixth leading cause of death in the United States, according to the Centers for Disease Control (CDC). Accordingly, there is an urgent, unmet clinical need to address abnormal mucus production to reduce the risk of infection and death, and to improve both longevity and quality of life for patients. SUMMARY

[0003] Cystic fibrosis, asthma, and chronic bronchitis are all hypersecretory disordersof the airways characterized by increased secretions from the submucosal glands (SMGs). These glands, which are contiguous with the lumen surface of human cartilaginous airways, produce and secrete mucus and antimicrobial enzymes to protect the lungs from environmental insults. However, the full complement of SMG cellular identities remains dubious, in part due to the unique complexity of airway SMGs in humans. Further understanding of the cellular lineages and niche interactions that govern SMG stem / progenitor behavior is instrumental for development of therapeutic strategies to address disorders afflicting the airways.

[0004] Provided herein are methods of ex vivo culturing and manipulation ofsubmucosal gland (SMG) organoids, and in vivo methods of treating diseases and disorders associated with aberrant submucosal gland activity, including hyperplasia, hypertrophy, and excessive mucus production. In some aspects, the disclosure provides a method of engineering a submucosal gland (SMG) organoid ex vivo, the method comprising:UCH-39925 (i) obtaining a population of cells from the proximal airway tissue of a subject; (ii) isolating from the obtained population of cells (a) submucosal gland basal cells and / or myoepithelial cells, and / or (b) submucosal gland stem and / or progenitor cells; (iii) culturing the isolated cells in an expansion media comprising one or more growth agents promoting branching and / or budding morphogenesis; and (iv) culturing the isolated cells in a differentiation media comprising one or more agonists or ligands of ERBB; thereby engineering an SMG organoid ex vivo.

[0005] In some embodiments, the isolating in step (ii) comprises isolating stem and / orprogenitor cells that express on the cell surface TROP2, NGFR, EGFR and / or ITGA2.

[0006] In some embodiments, the one or more growth agents promoting branchingand / or budding morphogenesis comprise HBEGF and / or Heregulinβ-1 (NRG1); optionally wherein the expansion media comprises (a) HBEGF at a concentration of 10-1000 ng / mL or about 100 ng / mL and (b) NRG1 at a concentration of 10-1000 ng / mL or about 100 ng / mL.

[0007] In some embodiments, the one or more agonists or ligands of ERBB compriseEGF; optionally wherein the differentiation media comprises EGF at a concentration of 10- 1000 ng / mL or about 100 ng / mL. In some embodiments, the ERBB is ERBB3.

[0008] In some embodiments, the culturing in the differentiation media in step (iv) isfor at least 5 or 7 days, is for between 5 and 14 days, or is for between 5 and 21 days.

[0009] In some embodiments, the expansion media further comprises amphiregulin(AREG) and / or epiregulin (EREG). In some embodiments, the culturing in the expansion media in step (iii) is for at least 5 or 7 days, or is between 5 and 14 days, optionally without cell passaging.

[0010] In some embodiments, the culturing in step (iii) and / or (iv) is in a three-dimensional matrix. In some embodiments, the three-dimensional matrix is a hydrogel (e.g., Matrigel or Culturex).

[0011] In some embodiments, the proximal airway tissue is derived from theintercartilaginous zone tracheae or bronchi. In some embodiments, the proximal airway tissue comprises submucosal gland duct basal cells and / or myoepithelial cells.

[0012] In some embodiments, the isolating submucosal gland stem and / or progenitorcells in step (ii) comprises mechanically manipulating the proximal airway tissue to deplete surface epithelium, exposing the depleted tissue to one or more enzymes to digestUCH-39925 extracellular matrix, and straining the exposed tissue with one or more markers needed to isolate the submucosal gland stem and / or progenitor cells.

[0013] In some embodiments, the isolating submucosal gland stem and / or progenitorcells in step (ii) comprises fluorescence-activated cell sorting (FACS). In some embodiments, the submucosal gland stem and / or progenitor cells are TROP2+, NGFR+, EGFR+, and ITGA2+. In some embodiments, the SMG organoid express mucin-5B (MUC5B), lysozyme (LYZ), and / or lactoferrin (LTF), and / or wherein the SMG organoid comprises branching and / or budding morphogenetic characteristics of in vivo SMG.

[0014] In some embodiments, the subject is human. In some embodiments, the SMGorganoid exhibits hypertrophy, hyperplasia, and / or increased mucus production, optionally wherein the SMG organoid exhibits physical and / or functional characteristics of a hypersecretory airway disorder or disease.

[0015] In some embodiments, the disclosure provides an SMG organoid engineeredusing a method disclosed herein.

[0016] In some embodiments, the disclosure provides an SMG organoid viable exvivo comprising cells expressing MUC5B, LYZ, and / or LTF in a three dimensional matrix, wherein the SMG organoid comprises branching and / or budding morphogenetic characteristics of in vivo SMG. In some embodiments, the three dimensional matrix is a hydrogel. In some embodiments, the SMG organoid expresses CFTR. In some embodiments, the SMG organoid exhibits hypertrophy, hyperplasia, and / or increased mucus production, optionally wherein the SMG organoid exhibits physical and / or functional characteristics of a hypersecretory airway disorder or disease.

[0017] In some embodiments, the disclosure provides a method of culturing the exvivo SMG organoid in a media effective to induce hypertrophy or hyperplasia of the ex vivo SMG organoid. In some embodiments, the media comprises one or more agonists or ligands of ERBB, optionally wherein the one or more agonists or ligands of ERBB comprise EGF, optionally wherein the ERBB is ERBB3.

[0018] In some embodiments, the culturing results in overexpression of CFTR.

[0019] In some aspects, the disclosure provides an ex vivo SMG organoid producedusing the methods disclosed herein.

[0020] In some aspects, the disclosure provides a method of treating a hypersecretoryairway disorder or disease, or a disorder associated with an increased secretion fromUCH-39925 submucosal glands, in a subject in need thereof, the method comprising administering to the subject an agent that inhibits ERBB.

[0021] In some embodiments, the agent that inhibits ERBB is a gene therapy, a smallmolecule, or a protein. In some embodiments, the agent that inhibits ERBB is an agent that inhibits EGFR, ERBB2, and / or ERBB3, optionally wherein the agent is an agent that inhibits ERBB3. In some embodiments, the agent that inhibits ERBB is an agent that reduces mucus production.

[0022] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands, is cystic fibrosis, asthma, bronchitis (optionally chronic bronchitis), chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), COPD / emphysema, increased airway secretion associated with a microbial infection (optionally viral, bacterial, or fungal infection), an increased airway secretion associated with smoking or second hand smoking, or an increased airway secretion associated with an allergy, or an increased airway secretion associated with an environmental exposure. In some embodiments, the environmental exposure is wildfire smoke, air pollution, burn pit exposures, particulate matter, chemicals, or gases.

[0023] In some aspects, the disclosure provides the use of an agent that inhibitsERBB for treating a subject having a hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands; optionally wherein the agent is TX1-85-1, tucatinib or erlotinib or other ERBB inhibitors including gene therapies, small molecules and antibodies.

[0024] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands, is cystic fibrosis, asthma, bronchitis (optionally chronic bronchitis), chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), COPD / emphysema, increased airway secretion associated with a microbial infection (optionally viral, bacterial, or fungal infection), an increased airway secretion associated with smoking or second hand smoking, or an increased airway secretion associated with an allergy or an increased airway secretion associated with an environmental exposure. In some embodiments, the environmental exposure is wildfire smoke, air pollution, burn pit exposures, particulate matter, chemicals, or gases.UCH-39925 BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1A-1G. show transcriptomic profiling of single cells / nuclei from humanairway submucosa. FIG.1A. SMGs were isolated from the trachea and mainstem bronchi, depicted on the left, of human donor samples for single-cell and single-nuclei RNA sequencing. The SMG epithelium, depicted on the right, is contiguous with the airway surface and comprises a heterogeneous collection of cells organized along a medial-lateral axis. Along this axis, the SMGs can be subdivided into several compartments based on structure and cell composition: ciliated duct (i), collecting duct (ii), tubuloacinar duct (iii), mucous tubules (iv), and serous acini (v). FIG.1B. H&E staining of an intact airway tissue section (i) and airway tissue mechanically depleted of SAE via cytology brush (ii). Airway submucosal cells were isolated from airway samples depleted of SAE in this manner. Scale bars = 100 μm. FIG.1C. Percentage of mitochondrial genes comprising total detected gene expression per transcriptome, visualized by UMAP (uniform manifold approximation and projection). FIG.1D. Number of unique transcripts detected per transcriptome, visualized by UMAP. FIG.1E. UMAP visualization of the complete human airway submucosal transcriptomic dataset, labeled according to sample origin. FIG.1F. Percentage of annotated cell types comprising each donor sample. PS = parasympathetic. FIG.1G. Heatmap display of gene network expression in the annotated submucosal cell types from Fig.1B. Gene networks were based on correlated expression across all of the captured transcriptomes (SA = submucosa all) and categorized by cell type specificity, except in the case of networks with cell cycle signatures or broad enrichment across cell types, which were categorized separately. Each column in the heatmap represents the expression composite of all networks belonging to a particular category merged together (e.g., VA-Basal comprises 13 individual networks enriched specifically in basal cell transcriptomes).

[0026] FIG. 2A-2B shows identification of diverse cell types in the SMG bytranscriptomics. FIG.2A. UMAP of single cell RNA-seq of submucosal glands from human airway, representing 14 donors, numerous cell types, and at least 27,000 cells. It is sorted into four major cell types: endothelial, epithelial, fibroblasts, and immune / blood cells. FIG.2B. UMAP visualization of the complete human airway submucosal transcriptomic dataset, labeled according to cell type annotation. PS = parasympathetic neuron.

[0027] FIG. 3A-K shows representative gene expression networks from thecategories in FIG.1G describing endothelial cells (FIG.3A), macrophages (FIG.3B-C), plasma cells (FIG.3D), B cells (FIG.3E), mast cells (FIG.3F), T cells (FIG.3G),UCH-39925 fibroblasts (FIG.3H-I), smooth muscle (FIG.3J), parasympathetic (PS) and neurons (FIG. 3K). For each network, the following is provided: top gene correlates visualized by UMAP; top gene ontology (GO) term based on the gene set comprising the network.

[0028] FIG. 4 shows that there are several cell types in the SMG tissue

[0029] FIG. 5 shows representative gene expression networks for ionocytes as inFIG.3A-K.

[0030] FIG. 6 demonstrates a heatmap display of gene network expression in theannotated epithelial cell types from FIG.7. Gene networks were based on correlated expression across the epithelial subset of transcriptomes (SE = submucosal epithelia) and categorized by cell type specificity. Each column in the heatmap represents the expression of an individual network belonging to the category specified, ordered sequentially.

[0031] FIG. 7 shows UMAP visualization of the epithelial transcriptomes, labeled bycell type annotations made based on the subsetted dataset.

[0032] FIG. 8 depicts a heatmap display of marker gene expression characterizing theepithelial cell types in FIG.7 normalized by column.

[0033] FIG. 9A-B shows gene expression networks derived from the epithelial subsetof transcriptomes (SE = submucosal epithelia) defining duct basal and MECs.

[0034] FIG. 10A-B shows representative gene expression networks from FIG. 6depicting top gene correlates, UMAP visualization of network enrichment in the epithelial subset of transcriptomes (as shown in FIG.7), and top GO term. The networks represented are enriched in the stem duct basal cells (FIG.10A) and MECs (FIG.10B) gene sets.

[0035] FIG. 11A-B shows gene expression networks derived from the epithelialsubset of transcriptomes (SE = submucosal epithelia) defining suprabasal cells by oxidative phosphorylation (FIG.11A) and cytoplasmic translation (FIG.11B) gene sets.

[0036] FIG. 12A-B shows gene expression networks derived from the epithelialsubset of transcriptomes (SE = submucosal epithelia) defining duct basal cells by wound healing (FIG.12A) and salmonella / E. Coli infection (FIG.12B) gene sets.

[0037] FIG. 13 shows gene expression networks derived from the epithelial subset oftranscriptomes (SE = submucosal epithelia) defining ciliated cells.

[0038] FIG. 14 shows gene expression networks derived from the epithelial subset oftranscriptomes (SE = submucosal epithelia) defining club cells.UCH-39925

[0039] FIG. 15A-B shows gene expression networks derived from the epithelialsubset of transcriptomes (SE = submucosal epithelia) defining duct cells by chemical homeostasis (FIG.15A) and potassium ion transport (FIG.15B) gene sets.

[0040] FIG. 16 shows gene expression networks derived from the epithelial subset oftranscriptomes (SE = submucosal epithelia) defining mucous / serous (secretory) cells.

[0041] FIG. 17A-B shows gene expression networks derived from the epithelialsubset of transcriptomes (SE = submucosal epithelia) defining serous cells by neutrophil degranulation (FIG.17A) and body fluid secretion (FIG.17B) gene sets.

[0042] FIG. 18 shows gene expression networks derived from the epithelial subset oftranscriptomes (SE = submucosal epithelia) defining pre-serous cells.

[0043] FIG. 19 shows IF staining of cells in basal compartment. The cells changemorphologically along the medial-lateral axis. There is a continuum of airway / glandular stem cells. SAE is surface airway epithelium. SMG is submucosal glands. From left to right, DAPI, alpha-SMA, and K17 immunofluorescent staining.

[0044] FIG. 20A-D shows immunohistofluorescence micrographs interrogatingexpression of the specified targets in the SMG duct (i) or tubuloacinar region (ii); scale bars = 50 μm. LGALS7B+ suprabasal cells are found in the SMG ducts but absent from tubuloacini (FIG.20A). SCGB1A1+MUC5B+ club cells are located in the SMG collecting ducts and are distinct from MUC5B+ mucous tubule cells (FIG.20B). KRT23+ TA duct cells are immediately adjacent to the tubuloacinar compartment. The TA ducts lack epithelial pseudostratification observed in the larger diameter ducts located more medially and are populated by cells with cuboidal morphology (FIG.20C). KRT14 expression demarcates basal cells in the SMG ducts and MECs surrounding the tubuloacinar structures (FIG.20D).

[0045] FIG. 21A-B shows gross digest of SMG compared to dissociated tissue. FIG.21A.16-hour gross digest of human airway submucosa, demonstrating isolation of large epithelial structures from the SMG. FIG.21B. Final SMG cell suspension following sequential dissociation in trypsin-EDTA then dispase and DNase I. Scale bar = 100 μm.

[0046] FIG. 22A-B shows immunofluorescence micrographs of cytospins preparedfrom freshly isolated SMG epithelial cells. The SMG isolation protocol produces a final cell suspension containing KRT17+ duct basal, KRT17+αSMA+ MECs, and αSMA+ non- epithelial cells (FIG.22A) in addition to terminally differentiated epithelial cell types, including LTF+ serous, MUC5B+ mucous tubule, and SCGB1A1+ club cells (FIG.22B). Scale bars = 50 μm.UCH-39925

[0047] FIG 23A-C shows a diagram outlining the two-step protocol for human SMG3D cultures. Epithelial aggregates isolated from airway submucosa undergo an initial Expansion Phase in Small Airway Epithelial Cell Growth Media (SAECGM) for 1-2 weeks, followed by the Organoid Phase in SMG Organoid Media for up to 3 weeks. Organoids are subsequently collected for downstream assays, such as wholemount immunostaining and forskolin-induced swelling. FIG 23B. Schematic of culturing organoids from sorted cells or parental populations. FIG 23C. Establishment of the human SMG organoid model from the expansion phase to the differentiation phase.

[0048] FIG. 24 shows SMG isolates one week post-seed in the Expansion Phase.Epithelial cells shown in FIG.22B were seeded in Cultrex 1:1 with small airway epithelial cell media (SAECGM). Scale bar = 1000 μm.

[0049] FIG. 25A-D shows wholemount immunostaining for duct basal / MEC markerKRT14 in SMG cultures collected 16 days post-seeding in the Expansion Phase (i) or 10 days post-seeding in the Organoid Phase (ii). Scale bars = 50 μm.

[0050] FIG. 26 shows the prevalence of CFTR expression in human SMG.

[0051] FIG. 27 shows western blot detection of CFTR after 7 days in the ExpansionPhase or 14 days in the Organoid Phase. Primary intestinal organoids were used as the positive control for CFTR expression; Na / K ATPase was used as the loading control.

[0052] FIG. 28 shows quantification of the forskolin (FSK)-induced swelling assayperformed on SMG organoids 14 days post-seeding in the Organoid Phase; the average change in organoid area over time, determined from confocal imaging, is represented by area under the curve (AUC) for the given treatment conditions. Error bars represent standard deviation. For each condition, n = 6 technical replicates. *p < 0.05, ****p < 0.0001 by Tukey’s test.

[0053] FIG. 29 shows SMG organoids exhibit heterogeneous response to forskolintreatment (i,ii); only a subset of organoids demonstrate forskolin-induced swelling at four hours post-treatment (indicated by arrows). Inhibition of CFTR by CFTRi-172 abrogates swelling, suggesting response is CFTR-dependent (iii,iv).

[0054] FIG. 30A-C shows evaluation of candidate surface markers for fluorescence-activated cell sorting (FACS). Micrographs depict immunofluorescent detection of NGFR (FIG.30A), EGFR (FIG.30B), and ITGA2 (FIG.30C) in SMG duct (i) and tubuloacinar (ii) regions in tissue histology. Image insets show an enlarged view of the region demarcated by the dotted rectangle. Scale bars = 50 μm.UCH-39925

[0055] FIG. 31A-B shows representative FACS plots demonstrating the gatingstrategy used to enrich duct basal and myoepithelial cells isolated from human SMG tissue. Samples are initially gated by detection of EGFR and NGFR (P5 in FIG.31A) then sorted by high (P8 in FIG.31B) and low (P9 in FIG.31B) expression of ITGA2 and NGFR (FIG. 31B). Dotted line in (FIG.31B) indicates threshold for ITGA2 (BV421)-positive signal, based on controls.

[0056] FIG. 32A-B shows cytospins prepared from freshly sorted NGFRHIITGA2HI(ia-iva) and NGFRLOITGA2LOSMG (ib-ivb) cells. NGFRLOITGA2LOsorted cells are primarily comprised of KRT14+KRT17+αSMA- duct basal cells with a small fraction of triple-negative cells (iva), whereas NGFRHIITGA2HIsorted cells are almost entirely enriched for KRT14+KRT17+αSMA+ MECs (ivb). Scale bars = 50 μm.

[0057] FIG. 33 provides an experimental schematic outlining 3D culture of theindicated airway epithelial populations. Each of the four populations, color-coded for reference in the remaining figure panels, was seeded separately as a single-cell suspension to initiate Expansion Phase cultures. After 14 days in the Expansion Phase, cultures were transitioned to the Organoid Phase and continued for another 21 days.

[0058] FIG. 34 shows brightfield micrographs of cultures established from total SMGisolates (i), NGFRHIITGA2HIsorted SMG (ii), NGFRLOITGA2LOsorted SMG (iii), and NGFR+TROP2+ sorted SAE (iv) after 14 days of Expansion Phase or an additional 21 days of Organoid Phase. Scale bars = 500 μm.

[0059] FIG. 35A-B shows average number of 3D epithelial clusters per mm2 andaverage area of distinct clusters determined from brightfield micrographs of Expansion Phase cultures represented in (FIG.35A). Average number of organoids per mm2and average organoid area determined from brightfield micrographs of Organoid Phase cultures represented in (FIG.35B). Graph bars are color-coded to match the four seeded populations in FIG.34. Error bars represent standard error of the mean; n = 8 images (across two biological replicates) for each population. **p < 0.01 by Kruskal-Wallis test.

[0060] FIG. 36 shows UMAP visualization of epithelial organoid single-celltranscriptomes derived from the sorted SMG / SAE and total SMG populations. Cultures established from these four populations were collected at two timepoints: 14 days of Expansion Phase and 21 days of Organoid Phase (35 days total in culture). Clusters are labeled according to cell type annotations and phase of culture; EP = Expansion Phase andUCH-39925 OP = Organoid Phase. Fibroblasts captured at the end of either phase of culture clustered together.

[0061] FIG. 37 shows heatmap display of gene network expression in the annotatedcell types from FIG.36. Gene networks were based on correlated expression across the entire transcriptomic dataset encompassing cultures (CC = cell culture) profiled after 14 days of the Expansion Phase (EP) and 21 days of the Organoid Phase (OP). Networks were categorized based on enrichment in specific subsets of the annotated cell types. Each column in the heatmap represents the expression of an individual network.

[0062] FIG. 38 shows UMAP visualization of basal cell markers KRT5, KRT17,KRT14, and TP63 in the transcriptomes derived from the cultured cell populations represented in FIG.33.

[0063] FIG. 39 shows a heatmap display of marker gene expression for each of theclustered cell types shown in FIG.36 normalized by column.

[0064] FIG. 40A-E shows top enriched gene expression networks defining each ofthe following cell populations: EP Basal 1, OP Basal 2, and OP Secretory 2 (FIG.40A), EP Basal 2 (FIG.40B), EP Basal 3 and OP Basal 3 (FIG.40C), EP Secretory and OP Secretory 1 / 2 (FIG.40D), and Fibroblasts (FIG.40E). Each network is visualized by UMAP (CC = cell culture); top gene ontology (GO) term based on the gene set comprising the network.

[0065] FIG. 41 shows a heatmap display of the percentage of each annotated cell typein FIG.36 comprising cultures established from each of the four seeded populations (i.e., column normalized) profiled at the end of the Expansion Phase.

[0066] FIG. 42A-F shows top enriched gene expression networks, defining OP Basal1 (FIG.42A), OP Basal 4 (FIG.42B), OP Rare / Ionocyte (FIG.42C), OP Secretory 1 (FIG. 42D), OP Secretory 3 (FIG.42E), and OP Ciliated (FIG.42F) cell types.

[0067] FIG. 43 shows a heatmap depicting percent correlation of differentially-expressed genes for each annotated cell population from tissue (“SMG” prefix) or cell culture (“Org” prefix).

[0068] FIG. 44 shows a heatmap display of the percentage of each annotated cell typein FIG. 36 comprising cultures established from each of the four seeded populations profiledat the end of the Organoid Phase.

[0069] FIG. 45A-E shows a heatmap display of predicted receptor-ligand interactionsfor ErbB-family ligands HBEGF (FIG.45A), NRG1 (FIG.45B), AREG (FIG.45C), EREG (FIG.45D), and EGF (FIG.45E), based on transcriptomic data from submucosal tissue.UCH-39925 FIG.45A. Interaction between the HBEGF ligand and the CD44, CD82, CD9, and EGFR receptors in the various cell subpopulations. FIG.45C. Interaction between the AREG ligand and the EGFR and ERBB3 receptors in the various cell subpopulations. Gene expression data is normalized; the scale bar shows relative expression. The top row depicts expression of the ligand across the annotated cell types described in FIG.1A-G; the remaining rows depict expression of the cognate receptors.

[0070] FIG. 46A-D shows immunofluorescent detection of the ErbB-family ligandsfrom FIGs. 45A-D in SMG tissue histology. The SMG tubuloacinar epithelium exhibitsdistinct expression patterns of HBEGF (FIG.46A), NRG1 (FIG.46B), EREG (FIG.46C), and EGF (FIG.46D). Scale bars = 50 μm.

[0071] FIG. 47 shows the experimental timeline for SMG Expansion Phase (EP)treatments with the indicated ErbB-family ligands and SMG Expansion Phase treatments. Wholemount immunofluorescence micrographs show cultures at 10 days post-seeding in SAECGM alone (i) or with supplementation by 100 ng / mL HBEGF (ii), 100 ng / mL NRG1 (iii), or 100 ng / mL (each) HBEGF + NRG1 (iv). Scale bars = 50 μm.

[0072] FIG. 48A-D shows SMG Expansion Phase ErbB-family ligand treatments.FIG.48A. Wholemount immunofluorescence micrographs show cultures at 10 days post- seeding in SAECGM alone (i) or with supplementation by 100 ng / mL HBEGF (ii), 100 ng / mL NRG1 (iii), or 100 ng / mL (each) HBEGF + NRG1 (iv). FIG.48B. Representative images of organoid treatments during expansion phase at 7 days. SMG organoids treated with HBEGF+NRG1 demonstrate increased branching morphogenesis and budding compared to control. FIG.48C. KRT5 and DAPI overlays of SMG organoids treated with HBEGF and NRG1 vs. a no-treatment control. HBEGF+NRG1 treatment during expansion phase patterns stem / progenitor cells. FIG.48D. Relative gene expression quantified by qPCR in SMG organoids. ERBB growth factors guide differentiation features.

[0073] FIG. 49A-B shows SMG Expansion Phase ErbB receptor inhibitor treatments.FIG.49A. Wholemount immunofluorescence micrographs show cultures at 10 days post- seeding in SAECGM. SMG isolates were cultured for 3 days prior to supplementation with 0.1% DMSO (i) (vehicle control), 10 μm EGFR inhibitor Erlotinib (ii), 10 μm ERBB2 inhibitor Tucatinib (iii), or 10 μm ERBB3 inhibitor TX1-85-1 (iv). FIG.49B. KRT5 and DAPI overlays of SMG organoids. ERBB inhibitors (erlotinib and TX1-85-1) curb organoid growth during expansion phase.UCH-39925

[0074] FIG. 50A-C shows effect of treatment of organoids with different ErbB-ligands. FIG.50A. Experimental timeline for SMG Organoid Phase (OP) treatments (top bracket) and combined Expansion Phase and Organoid Phase treatments (bottom bracket). Each of the six total treatment conditions are abbreviated in parentheses at the end of the brackets, with the Expansion Phase and Organoid Phase conditions separated by the slash. C = Control, HN = HBEGF + NRG1, E = EGF, and HEr = HBEGF + EREG. FIG.50B. Representative immunofluorescence micrographs of sectioned SMG organoids following the ligand treatments outlined in (FIG.50A), detecting MUC5B and LYZ expression. Expansion Phase treatments are organized by row: control (i-iii) and HBEGF+NRG1 (iv-vi). Organoid Phase treatments are organized by column: control (i,iv), EGF (ii,v), and HBEGF+EREG (iii,vi). Scale bars = 50 μm. FIG.50C. Relative expression of LTF, LYZ, and MUC5B genes, measured with quantitative PCR in SMG organoid samples. The samples are SMG organoids that have undergone a 7-day expansion phase and a 21-day differentiation phase and half got HBEGF and NRG1 during expansion and half did not. Gene expression is compared against a sample taken at the end of the expansion phase “Control (end of expansion phase) with no additional patterning growth factors”. The addition of EGF in culture increases MUC5B expression, indicating increased SMG mucous cell differentiation and / or enhanced mucus production and this occurred both with and without initial patterning during expansion phase. HBEGF+NRG1 treatment during expansion results in increased organoid patterning but did not affect expression of LTF, LYZ, or MUC5B when administered alone. The combination of HBEGF+NRG1+EGF drives enhanced differentiation towards tubuloacinar cell fates, namely SMG mucous AND serous cells, and increased expression of LTF, LYZ, and MUC5B.

[0075] FIG. 51A-B shows the effect of ErbB ligands on SMG organoid markers.FIG.51A. Percentage of MUC5B+ and LYZ+ cells quantified from the images represented in (L). Error bars represent standard error of the mean; n = 20 images. ***p < 0.001, ****p < 0.0001 by Dunnett’s test. FIG.51B. Averaged quantitative PCR measurements of MUC5B, LYZ, and LTF expression for the six treatment groups outlined in FIG.50A. Error bars represent standard deviation; n = 3 technical replicates from 3 biological replicates. **p < 0.01, ****p < 0.0001 by Dunnett’s test. DETAILED DESCRIPTION

[0076] The present disclosure provides compositions and methods for identifyingagents for treating and / or preventing diseases associated with submucosal gland hyperplasiaUCH-39925 and / or hypertrophy and excessive or abnormal mucus secretions. The present disclosure also identifies specific biological pathways for targeting in the treatment and / or prevention of such diseases, as well as specific agents that can be used for such treatment and / or prevention.

[0077] Submucosal glands (SMGs) represent a fundamental component of properairway function and defense via mucous and serous secretions. Accordingly, discernment of the SMG cellular constituents and their regulatory mechanisms is imperative to advance therapeutic strategies for airway secretory disorders, especially those with pathophysiological manifestations in the SMGs. Disclosed herein is a single-cell / nuclei transcriptomic atlas of human airway SMGs to delineate the composite epithelial populations and to uncover emergent cell-cell interactions that govern expansion and differentiation of these populations. The SMG tissue transcriptomic data indicate two stem cell populations within the SMG that can be readily distinguished. However, histological examination of the airways presents a “basal continuum” along the axis spanning the airway surface to the tubuloacinar structures of the SMG. Morphologically speaking, the stem / progenitor cells comprising this basal layer can be binned into four groups coinciding with distinct anatomical regions, namely airway surface, ciliated (medial) duct, collecting (lateral) duct, and tubuloacini. Along this medial- lateral axis, cells of the basal layer are the stem cells and gradually flatten or stretch prior to acquisition of patent MEC features (e.g., KERATIN 14 / ACTA2 / MYH11 expression.)

[0078] Additionally, regional differences in the luminal epithelium of the SMG ductswere specified. Club cells in the SMG collecting ducts were identified, which likely serve a similar function to typical club cells normally present in the small airways. A subset of SMG duct cells were localized to small-diameter ducts adjacent to the tubuloacinar epithelium, distinguishing them from the preceding collecting ducts. The TA ducts histologically resemble kidney collecting ducts and, based on the gene networks enriched in TA duct cells, may likewise function primarily in osmoregulation and / or acid-base homeostasis, mediated in part by CFTR. If so, this would implicate TA ducts in the modification of mucous secretions arising from the adjacent tubules.

[0079] To interrogate features of the SMG stem / progenitors and their niche, a humanairway SMG organoid model was used to reproduce secretory attributes of the SMG epithelia. Utilizing this culture system, primary SAE basal, SMG duct basal, and MECs were analyzed in their capacity to reconstitute epithelial structures and differentiate into distinct lineages in vitro. The greatest organoid-forming efficiency was found in SAE basal-derived organoids, suggesting that SAE basal cells are quickly mobilized for regeneration and areUCH-39925 likely poised to deal with the higher turnover rate of the surface epithelium. Consequently, SAE basal cells would face a greater likelihood of exhaustion due to a higher frequency of activation. This is consistent with the lower number of cycling surface basal cells seen in CF patients. Based on growth characteristics and transcriptomic profiling of the organoid cultures, MECs had diminished self-renewal capacity and were predisposed to secretory differentiation. The duct basal cells, intermediate between the SAE basal and MECs both in terms of anatomic location and organoid growth dynamics, potentially represent a longer- lived basal population that would be an attractive target for gene correction strategies, like CFTR gene therapy.

[0080] Findings from the predicted receptor-ligand interactions in the SMG tissuetranscriptomics dataset were applied to the SMG organoid model, addressing the prospective function of the ErbB-family ligands present in the SMG tubuloacinar compartment. It was found that HBEGF and NRG1 together drive branching morphogenesis and enhance lumen formation in terminal end buds during SMG organogenesis while EGF, HBEGF, and EREG modulate secretory differentiation and output. Ostensibly, these same factors contribute to SMG hypertrophy and hyperplasia when dysregulated, consistent with studies showing that interplay of ErbB-family ligands drives disease progression of smoking-induced lesions on the airway surface. Similarly, the utility of SMG organoids was for drug screening was demonstrated by application of ErbB inhibitors to stymie expansion of cultured SMG stem / progenitor cells. These inhibitors may be multipurpose in their potential to mitigate SMG hypertrophy and hyperplasia by suppressing stem / progenitor expansion and directing lineage outcomes towards non-secretory fates. Overall, the instant disclosure provides the foundation for a flexible human SMG model to address SMG biology and advance targeted therapeutics. Definitions The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

[0081] A, An, The, Or: As used herein, “a”, “an”, and “the” refer to one or to morethan one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” discloses embodiments of exactly one element and embodiments including more than one element. As used herein, the terms “or” and “and / or”, as conjunctions in a listUCH-39925 of at least two elements, encompass and disclose embodiments in which the listed elements are included in the alternative, together, or in any combination.

[0082] About: As used herein, term “about”, when used in reference to a value,refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0083] Isolated: As used herein, “isolated” refers to a substance and / or entity that hasbeen (a) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (b) designed, produced, prepared, and / or manufactured by human intervention. Isolated substances and / or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated substances and / or entities are at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance and / or entity is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance and / or entity may still be considered “isolated” or “pure” after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance and / or entity is calculated without including such carriers or excipients. For example, duct basal gland cells and / or myoepithelial cells and / or submucosal gland stem and / or progenitor cells are isolated from the proximal airway tissue of a subject.

[0084] Stem, or Progenitor Cells: As used herein, “stem cells” or “progenitor cells”are an undifferentiated cell of a multicellular organism which is capable of giving rise to indefinitely more cells of the same type, and from which certain other cell types arise by differentiation. The stem cells herein are derived from the SMG tissue and express one or more of TROP2, NGFR, EGFR, and / or ITGA2. The stem cells of the SMG tissue are myoepithelial cells and / or duct cells.UCH-39925

[0085] Differentiation: As used herein, “differentiation” refers to a process where aless specialized cell becomes a more specialized cell (e.g., a stem cell becomes a secretory cell). Stem cells in culture may be differentiated by various methods, including by incubation with media that contains one or more factors that promote differentiation. In the instant disclosure, SMG organoid “differentiation media” is provided in Table 2. The “differentiation media” induces cellular differentiation of the organoid from a stem and / or progenitor population to a mature SMG organoid.

[0086] Agent: As used herein, the term “agent” may refer to any chemical entity,including without limitation any of one or more of an atom, molecule, compound, amino acid, polypeptide, nucleotide, nucleic acid, protein, protein complex, liquid, solution, saccharide, polysaccharide, lipid, or combination or complex thereof.

[0087] Expression: As used herein, “expression” refers individually and / orcumulatively to one or more biological process that result in production from a nucleic acid sequence of an encoded agent, such as a polypeptide. Expression specifically includes either or both of transcription and translation. A cell that produces the encoded agent can be said to express the encoded agent.

[0088] The term "Preventing" a disease or condition in a subject, as used herein,refers to administering a therapy (an agent) to the subject prior to the onset of the disease or condition, when administration of the therapy to a statistical sample prior to the onset of the disease or condition reduces the occurrence of the disease or condition in the treated sample relative to an untreated control sample.

[0089] The term “Treating” a disease or condition in a subject, as used herein, refersto administering a therapy (an agent) to the subject having or suspected of having a disease or condition (i.e., after the onset of the disease or condition), such that the therapy partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, and / or features of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result.

[0090] The term, "effective amount" or "effective dose," or similar terms usedherein are intended to mean an amount of an agent that will elicit the desired biological or medical response (e.g., an improvement in one or more symptoms of a hypersecretory disease).UCH-39925

[0091] Agonist: As used herein, the term “agonist” refers to a substance that binds toa receptor and triggers a biological response, in some embodiments mimicking the action of the natural ligand that binds to the receptor. An agonist can be a partial or full agonist.

[0092] Organoid: As used herein, the term “organoid” refers to an in vitro three-dimensional multicellular construct that is developed from submucosal gland cells in a specific 3D organoid culture system. Organoids contain multiple cells types of the in vivo counter parts and organize similarly to the primary tissue. In some embodiments, the organoid culture system includes an organoid culture medium and an extracellular matrix or extracellular matrix substitute.

[0093] ErbB: As used herein, may be interchangeable with “ERBB,” and represents afamily of four receptor tyrosine kinases; ErbB1 (also known as Epidermal Growth Factor Receptor (EGFR)), ErbB2 (also named HER2), ErbB3 (also named HER3), and ErbB4 (also named HER4). Different growth factors are ligands for the ErbB receptors, including Epidermal Growth Factor (EGF) and TGF-^.

[0094] Hydrogel: As used herein, a “hydrogel” is a mixture of porous and permeablesolids and at least 10% of water. The solid phase is a water insoluble three-dimensional network of polymers. Herein, the three-dimensional hydrogel is used to provide a matrix for growth of the organoid into spheroid structures. Potential hydrogels that may be used in the compositions and methods of the disclosure include, but are not limited to, Matrigel or Cultrex.

[0095] Hypertrophy, or Hyperplasia: As used herein, the term “hypertrophy” or“hyperplasia” refers to two distinct ways in which a tissue or organ can grow in size. Hypertrophy involves an increase in the size of individual cells, while hyperplasia refers to an increase in the number of cells.

[0096] Hypersecretory Airway Disease: As used herein, “Hypersecretory AirwayDisease” refers to conditions characterized by excessive mucus production in the airways, leading to airway obstruction and other respiratory problems. Hypersecretory Airway Diseases herein include, but are not limited to, Cystic fibrosis, Asthma, and Chronic Bronchitis.

[0097] Engineered: As used herein, the term “engineered” refers to the aspect ofhaving been manipulated by human intervention. For example, a polynucleotide is considered to be “engineered” when two or more sequences, that are not linked together in that order in nature, are manipulated by human intervention to be linked to one another in theUCH-39925 engineered polynucleotide. Those of skill in the art will appreciate that an “engineered” nucleic acid or amino acid sequence can be a recombinant nucleic acid or amino acid sequence. In some embodiments, an engineered polynucleotide includes a coding sequence and / or a regulatory sequence that is found in nature operably linked with a first sequence but is not found in nature operably linked with a second sequence, which is in the engineered polynucleotide and operably linked in with the second sequence by human intervention. In some embodiments, a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution, deletion, or mating). As is common practice and is understood by those of skill in the art, progeny or copies, perfect or imperfect, of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the direct manipulation was of a prior entity.

[0098] Improve, increase, inhibit, decrease or reduce: As used herein, the terms“improve”, “increase”, “inhibit”, “decrease” and “reduce”, and grammatical equivalents thereof, indicate qualitative or quantitative difference from a reference.

[0099] Level: As used herein with respect to a molecule such as a nucleic acid orpolypeptide, “level” is used to refer to a measure indicative of an amount, concentration, ratio, or activity of the molecule, e.g., in a particular context such as a tissue, sample, organism, or a context representative thereof. An amount can be, for example, a mass or number of molecules. A concentration can be an amount relative to a context value, e.g., per a unit of mass or volume. A ratio can be a relationship between two values, such as an experimental value and a reference control value. Activity can be a measure of a function associated with a molecule, and can in various instances be measured relative to a context value, e.g., per a unit of mass or volume. Those of skill in the art will appreciate that the metric by which a level is expressed can vary depending, e.g., on the assay and purpose. Those of skill in the art will further appreciate that metrics such as amount, concentration, ratio, and activity are often interrelated and / or qualitatively or quantitatively informative of each other.

[0100] Inhibitory Agent or Inhibitor: As used herein, the term “inhibitory agent” or“inhibitor” refers to an entity, condition, or event whose presence, level, or degree correlates with decreased level, expression, or activity of a target). In some embodiments, an inhibitoryUCH-39925 agent may be acting directly (in which case it exerts its influence directly upon its target, for example by binding to the target); in some embodiments, an inhibitory agent may act indirectly (in which case it exerts its influence by interacting with and / or otherwise altering a regulator of the target, so that level and / or activity of the target is reduced). In some embodiments, an inhibitory agent is one whose presence or level correlates with a target level or activity that is reduced relative to a particular reference level or activity (e.g., that observed under appropriate reference conditions, such as presence of a known inhibitory agent, or absence of the inhibitory agent in question, etc.)

[0101] Subject: As used herein, the term “subject” refers to an organism, typically amammal (e.g., a human, rat, or mouse). In some embodiments, a subject is suffering from a disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject is not suffering from a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a subject that has been tested for a disease, disorder, or condition, and / or to whom therapy has been administered. In some instances, a human subject can be interchangeably referred to as a “patient” or “individual.” A subject administered an agent associated with treatment of a disease, disorder, or condition with which the subject is associated can be referred to as a subject in need of the agent, i.e., as a subject in need thereof.

[0102] Unless otherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. Submucosal Glands (SMGs) and SMG Organoids

[0103] SMGs of the proximal airways assume an integral role in host respiratorydefense through production of mucous and serous secretions that augment mucociliary transport on the airway surface. In humans, SMGs in the lower respiratory tract are tortuous structures present throughout the proximal airways (i.e., trachea and bronchi) with uniqueUCH-39925 cellular constituents ordered along a mediolateral axis. The most medial aspect of the SMGs, the ciliated duct, is contiguous with the surface airway epithelium (SAE) and shares a virtually identical cellular makeup from a histological perspective. Moving laterally, the ciliated duct is typically adjoined by a larger collecting duct, ramified extensively along its length by tubuloacinar structures. These structures comprise mucous tubules, which contribute substantially to the polymeric mucin network lining the airway epithelium, as well as serous acini, which produce antimicrobial, anti-inflammatory, and fluid secretions that hydrate the mucus barrier to facilitate clearance. SMG dysfunction is implicated in hypersecretory disorders, chiefly chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), in which SMG hypertrophy and hyperplasia are pathological hallmarks.

[0104] Conjointly with secretory function, the SMGs possess robust regenerativecapacity, constituting a protected niche sheltered from inhaled environmental insults. There are at least two distinct stem / progenitor populations housed within the SMGs; duct basal cells, which line the ciliated and collecting ducts, and myoepithelial cells (MECs), which surround the tubuloacinar network. Both cell types possess a greater breadth of multipotency compared to SAE basal cells, the dedicated stem population of the airway surface epithelium. Indeed, duct basal and MECs are both capable of restoring airway surface and glandular epithelium following severe injury to the SAE. However, isolation and direct comparison of human SMG duct basal and MECs has not been undertaken successfully. As such, the regenerative capacity (i.e., multipotency, self-renewal, and longevity) of these populations has not been established.

[0105] Despite their evident role in respiratory health and disease, there is a lack ofadequate organoid models to study SMGs. Airway SMGs have been primarily studied using in vivo murine models, in which SMG distribution is comparatively restricted, typically spanning the cricoid cartilage to the third or fourth cartilage ring of the trachea. This minimal glandular presence is compounded by a rudimentary architecture that lacks a collecting duct. These species differences can have profound consequences when translating from mouse to human biology. For example, knockout of the cystic fibrosis transmembrane conductance regulator (CFTR) in mice fails to recapitulate CF airway pathology, the major cause of morbidity and mortality in human CF patients. Moreover, culture models that have been applied to SMG-derived cells are primarily restricted to two-dimensional or air-liquid interface systems, which typically produce epithelial layers with airway surface phenotypes. Attempts at producing SMG-derived organoids failed to produce patient cell types of theUCH-39925 SMG, instead generating spheroids that were virtually indistinguishable from those generated by patient-matched SAE basal cells.

[0106] Provided herein are SMG organoids and methods for producing the same exvivo. In some embodiments, an SMG organoid of the disclosure recapitulates an in vivo SMG tissue (e.g., an SMG tissue of a subject such as a human subject). Because the SMG organoid recapitulates in vivo SMG biology, it may be used to identify novel therapeutics.

[0107] An ex vivo SMG organoid may comprise, in some embodiments, cells derivedfrom the proximal airway tissue of a subject, such as a human subject.

[0108] The SMG is comprised of two major stem / progenitor populations:myoepithelial cells (KRT17+KRT14+ACTA2+) and duct basal cells (KRT17+KRT14+). For FACS sorting purposes myopepithelial cells are (NGFRHIITGA2HI), and duct basal cells are (NGFRLOITGA2LO). This is compared to surface airway basal cells which for FACS sorting purposes are (NGFR+ TROP2+), Mature duct cells are comprised of serous, club-like, or ciliated cells. Ionocytes are rare cells in the SMGs and are (CFTR+, FOXI1+). Within the duct basal cells there is variability in the stem cell populations as the compartments change from ciliated to collecting to TA duct.

[0109] The cells of the SMG organoid may express one or more biomarkers. The oneor more biomarkers expressed in the SMG organoid may also be expressed in a native SMG (e.g., an in vivo SMG, such as a native SMG in a subject). For example, in some embodiments, the cells of the SMG organoid express mucin-5B (MUC5B), lysozyme (LYZ), and / or lactoferrin (LTF). In some embodiments, the cells of the SMG organoid express KRT17, ^SMA, CC10, MUC5B, and / or LTF. In some embodiments, the cells of the SMG organoid express HBEGF, NRG1, AREG, EREG, and / or EGF. In some embodiments, the cells of the SMG organoid express CFTR.

[0110] In some embodiments, NGFR and EGFR are predominantly expressed onbasal cells in the duct and myoepithelial cells in the tubuloacinar compartment. In some embodiments, ITGA2 is predominantly expressed on the myoepithelial cells compared to the duct basal cells.

[0111] Expression of biomarkers in the SMG organoid may be determined usingmethods known to those of skill in the art, such as PCR (e.g., qRT-PCR), immunofluorescence staining, single-cell sequencing or single-nuclei sequencing.

[0112] In some embodiments, the SMG organoid may comprise a three-dimensionalmatrix. The three-dimensional matrix provides structural support for cell growth. Thus, inUCH-39925 some embodiments the SMG organoid may comprise cells in a three-dimensional matrix. In some embodiments, the cells may be adhered to the three-dimensional matrix. In some embodiments, the cells may be dispersed within the three-dimensional matrix. In some embodiments, the three-dimensional matrix is a hydrogel. Potential hydrogels include, but are not limited to, Matrigel or Cultrex.

[0113] In some embodiments, the SMG organoid comprises branching and / orbudding morphogenetic characteristics of in vivo SMG. In some embodiments, the SMG organoid comprises highly branched structures (e.g., ducts) at a similar level or to a similar extent as in an in vivo SMG. In some embodiments, the SMG organoid comprises tubuloacinar structures (budding) at a similar level or to a similar extent as in an in vivo SMG. Branching and / or budding morphogenetic characteristics may be determined using techniques known to those of skill in the art, such as microscopy. In some embodiments, the SMG organoid produces mucus secretions at a level that is comparable with an in vivo SMG tissue. In some embodiments, the SMG organoid produces at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% as much mucus as an in vivo SMG tissue. In some embodiments, the SMG organoid produces mucus that has substantially the same composition as the mucus produced by an in vivo SMG tissue. For example, in some embodiments, the mucus produced by the SMG organoid comprises one or more proteins also found in the mucus produced by an in vivo SMG tissue, such as MUC55, lysozyme, or lactoferrin. In some embodiments, the mucus produced by the SMG organoid comprises the same level or substantially the same level of one or more proteins found in the mucus produced by an in vivo SMG tissue.

[0114] In some embodiments, immunofluorescent staining is conducted to quantifythe number of cells expressing MUC5B, LYZ, and / or LTF in the SMG organoid. In some embodiments, immunofluorescent staining is conducted to identify MUC5B, LYZ, and / or LTF in the SMG organoid. In some embodiments, RT-qPCR is conducted to quantify the number of cells expressing MUC5B, LYZ, and / or LTF in the SMG organoid. In some embodiments, RT-qPCR is conducted to quantify the expression levels of MUC5B, LYZ, and / or LTF in the SMG organoid. In some embodiments, expression of MUC5B, LYZ, and / or LTF in the ex vivo SMG organoid is associated with mucus production.

[0115] In some embodiments, the ex vivo SMG organoid exhibits hypertrophy,hyperplasia, and / or increased mucus production. The mucus production may be increased relative to an in vivo SMG of a healthy subject. In some embodiments, the ex vivo SMGUCH-39925 organoid exhibits physical and / or functional characteristics of a hypersecretory airway disorder or disease.

[0116] In some embodiments, CFTR is expressed in the TA duct and pre-serous cellpopulations of SMG tissue. In some embodiments, CFTR expression in SMG tissue is analyzed by single cell sequencing and / or single-nuclei sequencing. In some embodiments, CFTR specific function is present in the SMG organoid. CTFR specific function may be tested, for example, using a forskolin swelling assay with and without a CFTR inhibitor. In some embodiments, SMG organoids express KRT14 and lack mature cell markers during the Expansion Phase. In some embodiments, SMG organoids express KRT14, MUC5B and LYZ during the Organoid Phase.

[0117] In some embodiments, the ex vivo SMG organoid is viable ex vivo (e.g., can bemaintained ex vivo for a prolonged period of time, e.g., at least, or more than, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, or 6 months). In some embodiments, the SMG organoid is grown and exists in a three-dimensional matrix ex vivo as described herein. Engineering Submucosal Gland (SMG) Organoids

[0118] Provided herein are methods for engineering a SMG organoid ex vivo. Alsoprovided herein are SMG organoids produced using the methods of the disclosure.

[0119] In some embodiments of these methods, SMG tissue is used to develop anorganoid. In some embodiments, the SMG tissue is obtained from a subject. In some embodiments, the subject is a human donor. In some embodiments, the SMG organoid is developed from human proximal airway tissue. In some embodiments, the SMG organoid is developed from murine proximal airway tissue. In some embodiments, the SMG organoid is developed from primate proximal airway tissue. In some embodiments, the proximal airway tissue is from a human donor undergoing a lung transplant.

[0120] In one aspect, the present disclosure provides ex vivo methods of engineering asubmucosal gland (SMG) organoid, comprising (i) obtaining a population of cells from the airway tissue of a subject (e.g., a human); (ii) isolating from the obtained population of cells (a) submucosal gland basal cells and / or myoepithelial cells, and / or (b) submucosal gland stem and / or progenitor cells; (iii) culturing the isolated cells in an expansion media comprising one or more growth agents promoting branching and / or budding morphogenesis; and (iv) culturing the isolated cells in a differentiation media comprising one or more agonists or ligands of ERBB, thereby engineering an SMG organoid ex vivo.UCH-39925 Step (i)- obtaining cells from an airway tissue sample

[0121] In some embodiments, an ex vivo method for engineering a SMG organoidcomprises a step (i): obtaining a population of cells from the airway tissue of a subject.

[0122] In some embodiments, the airway tissue from which the cells are obtained instep (i) is the proximal airway tissue. In some embodiments, the airway tissue is derived from the intercartilaginous zone tracheae or bronchi of a subject. In some embodiments, the airway tissue is derived from or comprises cells of cartilaginous airway basal epithelia. In some embodiments, the airway tissue comprises submucosal gland duct basal cells and / or myoepithelial cells. Step (ii)- isolating human SMG cells from the airway tissue sample

[0123] In some embodiments, an ex vivo method for engineering a SMG organoidcomprises a step (ii): isolating from the obtained population of cells (a) submucosal gland basal cells and / or myoepithelial cells, and / or (b) submucosal gland stem and / or progenitor cells.

[0124] In some embodiments, the isolating in step (ii) comprises isolating submucosalgland duct basal cells and / or myoepithelial cells. In some embodiments, the method comprises isolating submucosal gland stem and / or progenitor cells. In some embodiments, submucosal gland basal cells are isolated from the population of cells. In some embodiments, myoepithelial cells are isolated from the population of cells. In some embodiments, submucosal gland stem and / or progenitor cells are isolated from the population of cells.

[0125] In some embodiments, the isolating in step (ii) comprises isolating stem and / orprogenitor cells that express on the cell surface one, two, three or all four of: TROP2, NGFR, EGFR and / or ITGA2 (TROP2+, NGFR+, EGFR+, and / or ITGA2+). In some embodiments, the isolating in step (ii) comprises isolating stem and / or progenitor cells that express TROP2. In some embodiments, the isolating in step (ii) comprises isolating stem and / or progenitor cells that express NGFR. In some embodiments, the isolating in step (ii) comprises isolating stem and / or progenitor cells that express EGFR. In some embodiments, the isolating in step (ii) comprises isolating stem and / or progenitor cells that express ITGA2.

[0126] In some embodiments, the isolating submucosal gland stem and / or progenitorcells in step (ii) comprises mechanically manipulating the proximal airway tissue to deplete surface epithelium, exposing the depleted tissue to one or more enzymes to digest extracellular matrix, and staining the exposed tissue with one or more markers needed toUCH-39925 isolate the submucosal gland stem and / or progenitor cells. In some embodiments, the submucosal gland stem and / or progenitor cells are TROP2+, NGFR+, EGFR+, and ITGA2+.

[0127] In some embodiments, the isolation of SMG cells captures a diversity ofepithelial cell types, including duct basal [KRT17+αSMA-], MECs [KRT17+αSMA+], club [CC10+], mucous [MUC5B+], and serous cells [LTF+].

[0128] The cells can be isolated by means of cell surface marker identifications (suchas using marker(s) described herein and, e.g., FACS). Step (iii) and (iv)- culturing the SMG cells to develop an organoid

[0129] In some embodiments, an ex vivo method for engineering a SMG organoidcomprises a step (iii): culturing the isolated cells in an expansion media comprising one or more growth agents promoting branching and / or budding morphogenesis. In some embodiments, an ex vivo method for engineering a SMG organoid comprises a step (iv): culturing the isolated cells in a differentiation media comprising one or more agonists or ligands of ERBB, thereby engineering an SMG organoid ex vivo.

[0130] In some embodiments, the one or more growth agents promoting branchingand / or budding morphogenesis in step (iii) comprise HBEGF. In some embodiments, the one or more growth agents promoting branching and / or budding morphogenesis comprise Heregulinβ-1 (NRG1). In some embodiments, the expansion media comprises HBEGF at a concentration of about 10, 50, 100, 250, 500, 750, 1000 ng / mL (or any value in between these values), or about 10-1000 ng / mL, or about 100 ng / mL. In some embodiments, the expansion media comprises HBEGF at a concentration of 100 ng / mL. In some embodiments, the expansion media comprises NRG1 at a concentration of about 10, 50, 100, 250, 500, 750, 1000 ng / mL (or any value in between these values), or about 10-1000 ng / mL, or about 100 ng / mL. In some embodiments, the expansion media comprises NRG1 at a concentration of 100 ng / ml. In some embodiments, the expansion media comprises HBEGF at a concentration of 100 ng / ml and NRG1 at a concentration of 100 ng / ml.

[0131] In some embodiments, the organoid is treated with HBEGF and / or NRG1. Insome embodiments, treatment of the organoid with HBEGF promotes branching and / or budding morphogenesis. In some embodiments, treatment of the organoid with NRG1 promotes branching and / or budding morphogenesis. In some embodiments, treatment of the organoid with HBEGF and NRG1 promotes significantly enhanced branching and / or budding morphogenesis compared to treatment with HBEGF alone. In some embodiments, treatment of the organoid with HBEGF and NRG1 promotes significantly enhanced branching and / orUCH-39925 budding morphogenesis compared to treatment with NRG1 alone. In some embodiments, treatment of the organoid with HBEGF stimulates branching. In some embodiments, treatment of the organoid with NRG1 augments growth and lumen formation in budded clusters. In some embodiments, treatment with HBEGF and / or NRG1 maintains KRT5 expression in the organoid.

[0132] In some embodiments, the SMG organoid is first cultured in the ExpansionPhase (step iii) and then cultured in the Organoid Phase (step iv). In some embodiments, the Expansion Phase promotes dome formation and ensures gelation of the SMG organoid culture. In some embodiments, the Organoid Phase promotes differentiation of the SMG organoid culture. In some embodiments, the isolated cells are cultured for 14 days in the Expansion Phase and 21 days in the Organoid Phase. In some embodiments, the isolated cells are cultured for 7 days in the Expansion Phase and 14 days in the Organoid Phase. In some embodiments, the isolated cells are cultured for 10 days in the Expansion Phase and 10 days in the Organoid Phase. In some embodiments, the isolated cells are cultured for 14 days in the Expansion Phase and 10 days in the Organoid Phase. In some embodiments, the isolated cells are cultured for 7 days in the Expansion Phase and 21 days in the Organoid Phase. In some embodiments, the isolated cells are cultured for 5-14 days in the Expansion Phase and 5-28 days in the Organoid Phase.

[0133] In some embodiments, the Expansion Phase in step (iii) comprises culturingthe organoid in expansion media. In some embodiments, the expansion media in step (iii) is Small Airway Epithelial Cell Growth Medium (SAECGM) supplemented with 1% FBS and 10uM ROCK inhibitor.

[0134] In some embodiments, the expansion media comprises amphiregulin (AREG)and / or epiregulin (EREG). In some embodiments, the expansion media comprises HBEGF and / or NRG1, and further comprises AREG and / or EREG. In some embodiments, the expansion media does not comprise AREG. In some embodiments, the expansion media does not comprise EREG. In some embodiments, the culturing comprises use of media comprising AREG and / or EREG (whether in the expansion or differentiation media).

[0135] In some embodiments, the culturing in the expansion media in step (iii) is forat least (or more than) 5 or 7 days. In some embodiments, the culturing in the expansion media in step (iii) is for at least (or more than) 5, 7, 10 or 14 days. In some embodiments, the culturing in the expansion media in step (iii) is for 5 to 14 days. In some embodiments, the culturing in the expansion media in step (iii) is for at least 14 days. In some embodiments, theUCH-39925 culturing in the expansion media in step (iii) is for 7 days. In some embodiments, the culturing in the expansion media in step (iii) is for 10 days. In some embodiments, the culturing in the expansion media in step (iii) is for 14 days. In some embodiments, the culturing is for 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days.

[0136] In some embodiments, the culturing in the expansion media and / or in thedifferentiation media is conducted without cell passaging (e.g., for the time periods indicated herein. In some embodiments, the expansion media and / or in the differentiation media is changed every 3 days. In some embodiments, the expansion media and / or in the differentiation media is changed every 1 day. In some embodiments, the expansion media and / or in the differentiation media is changed every 2 days. In some embodiments, the expansion media and / or in the differentiation media is changed every 3 days. In some embodiments, the expansion media and / or in the differentiation media is changed every 10 days.

[0137] In some embodiments, the cells are first cultured in the expansion media, andthen in the differentiation media. In some embodiments, the cells are cultured in the differentiation media, and then cultured in the expansion media. In some embodiments, the media is used in which the expansion media and differentiation media components are present simultaneously.

[0138] In some embodiments, the differentiation media comprises R-Spondin 1(250ng / mL), FCF 7 (5ng / mL), FGF 10 (20ng / mL), Noggin (100ng / mL), A83-01 (500nM), Y- 27632 (10mM), SB202190 (1mM), B27 Supplement (1X), N-Acetylcysteine (1.25mM), Nicotinamide (10mM), GlutaMax 100x (1X), Hepes (10mM), Penicillin / Streptomycin (100 mg / ml or 100 U / mL), Primocin (50 mg / ml), and Advanced DMEM / F12 (1X). In some embodiments, the differentiation media comprises 100-300 ng / mL R-Spondin 1. In some embodiments, the differentiation media comprises 1-10 ng / mL FCF 7. In some embodiments, the differentiation media comprises 10-30ng / mL FCF 10. In some embodiments, the differentiation media comprises 50-150ng / mL Noggin. In some embodiments, the differentiation media comprises 250-1000nM A83-01. In some embodiments, the differentiation media comprises 5-20mM Y-27632. In some embodiments, the differentiation media comprises 0.5-5mM SB202190. In some embodiments, the differentiation media comprises 1-5mM N-Acetylcysteine. In some embodiments, the differentiation media comprises 5-20mM Nicotinamide. In some embodiments, the differentiation media comprisesUCH-39925 5-20mM Hepes. In some embodiments, the differentiation media comprises 50-250mg / mL Penicillin / Streptomycin.

[0139] In some embodiments, the differentiation media of step (iv) comprises one ormore agonists or ligands of ERBB. In some embodiments, the one or more agonists or ligands of ERBB comprise (or is) EGF. In some embodiments, the differentiation media comprises one or more agonists or ligands of ERBB (e.g., EGF) at a concentration of about 10-1000 ng / mL, about 10-100 ng / mL, about 10-500 ng / mL, about 100-500 ng / mL, about 250-750 ng / mL, or about 500-1000 ng / mL. In some embodiments, the differentiation media comprises one or more agonists or ligands of ERBB (e.g., EGF) at a concentration of about 10, about 50, about 100, about 250, about 500, about 750, about 1000 ng / mL (or any value in between these values).In some embodiments, ERBB is EGFR, ERBB2, and / or ERBB3. In some embodiments, ERBB is ERBB3.

[0140] In some embodiments, the culturing in the differentiation media in step (iv) isfor at least (or more than) about 5 or 7 days. In some embodiments, the culturing in the differentiation media in step (iv) is for about 5 to 14 days. In some embodiments, the culturing in the differentiation media in step (iv) is for about 5 to 21 days. In some embodiments, the culturing in the differentiation media in step (iv) is for about 7 to 21 days. In some embodiments, the culturing in the differentiation media in step (iv) is for about 7 to 28 days. In some embodiments, the culturing in the differentiation media in step (iv) is for at least 14 days. In some embodiments, the culturing is for 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0141] In some embodiments, the culturing step (iii) and / or (iv) can be in a three-dimensional matrix such as but not limited to hydrogel (e.g., Matrigel or Culturex). In some embodiments, the ex vivo SMG organoids (engineered using cells derived from a human donor) are grown in a three-dimensional matrix.

[0142] In some embodiments, the cells are isolated based on marker expression, e.g.,based by expression of cell surface markers identified by fluorescence-activated cell sorting (FACS).

[0143] In some embodiments, the transcriptome of an SMG organoid is analyzed bysingle-cell sequencing. In some embodiments, the transcriptome of an SMG organoid is analyzed by single-nuclei sequencing. In some embodiments, the gene expression of the SMG organoid is analyzed by PCR. In some embodiments, the SMG organoid is analyzed for protein expression. In some embodiments, the protein expression of the SMG organoid isUCH-39925 analyzed by immunofluorescence. In some embodiments, the protein expression of the SMG organoid is analyzed by Western Blot.

[0144] In some embodiments, NGFR+EGFR+ cells are first isolated and then sortedbased on high (HI) versus low (LO) expression of both ITGA2 and NGFR (i.e., NGFRHIITGA2HIvs. NGFRLOITGA2LO). In some embodiments, the NGFRHIITGA2HIpopulation is highly enriched for MECs. In some embodiments, the NGFRLOITGA2LOpopulation is primarily enriched for duct basal cells. In some embodiments, NGFR+ TROP2+ cells are surface airway epithelium cells.

[0145] In some embodiments, MEC-enriched organoids are generated by seedingsingle NGFRHIITGA2HISMG cells. In some embodiments, duct basal-enriched organoids are generated by seeding NGFRLOITGA2LOSMG cells. In some embodiments, SAE basal- enriched organoids are generated by seeding NGFR+TROP2+SAE cells. In some embodiments, NGFRHIITGA2HIcells produced fewer 3D clusters in the Expansion Phase than NGFRLOITGA2LOcells. In some embodiments, NGFRHIITGA2HIcells produced fewer organoids in the Organoid Phase than NGFRLOITGA2LOcells. In some embodiments, the organoids express KRT5, a marker of undifferentiated stem / progenitors. In some embodiments, using the methods described herein an ex vivo SMG organoid is obtained and / or maintained. In some embodiments, the cells of the SMG organoid express mucin-5B (MUC5B), lysozyme (LYZ), and / or lactoferrin (LTF), e.g., as determined by PCR (such as qRT-PCR). In some embodiments, the cells of the SMG organoid express mucin-5B (MUC5B), lysozyme (LYZ), and / or lactoferrin (LTF), e.g., as determined by immunofluorescence staining. In some embodiments, the cells of the SMG organoid express mucin-5B (MUC5B), lysozyme (LYZ), and / or lactoferrin (LTF), e.g., as determined by single- cell sequencing or single-nuclei sequencing.

[0146] In some embodiments, the cells of the SMG organoid express KRT17, ^SMA,CC10, MUC5B, and / or LTF as determined by qRT-PCR, immunofluorescence staining, and / or single-cell sequencing. In some embodiments, the cells of the SMG organoid express HBEGF, NRG1, AREG, EREG, and / or EGF, as determined by single-cell sequencing or single-nuclei sequencing. In some embodiments, the cells of the SMG organoid express HBEGF, NRG1, AREG, EREG, and / or EGF, as determined by immunofluorescence staining.

[0147] In some embodiments, the SMG organoid is treated with EGF. In someembodiments, the SMG organoid is treated with HBEGF and EREG. In some embodiments, treatment of the organoid with EGF during the Organoid Phase significantly increasesUCH-39925 MUC5B and LYZ gene expression and the proportion of MUC5B+and LYZ+cells compared to untreated control cultures. In some embodiments, treatment with HBEGF and EREG results in increased LYZ+cells and LYZ expression with fewer MUC5B+cells and less MUC5B expression. In some embodiments, treatment with HBEGF and NRG1 during the Expansion Phase followed by treatment with EGF during the Organoid Phase upregulates LTF gene expression in the SMG organoid. In some embodiments, treatment with HBEGF and NRG1 during the Expansion Phase followed by treatment with HBEGF and EREG during the Organoid Phase upregulates LTF gene expression in the SMG organoid. Culturing SMG Organoids

[0148] In another aspect, the present disclosure provides methods of culturing the exvivo SMG organoids described herein in a media effective to induce hypertrophy or hyperplasia of the ex vivo SMG organoid. In some embodiments, the media comprises an agonist or ligand of ERBB, e.g., ERBB3, such as EGF. Such SMG organoids can be used for screening and identifying agents that can be effective in the treatment or prevention of hypersecretory diseases or disorder and / or that can reduce airway mucus production.

[0149] In some embodiments, the ex vivo SMG organoid described herein expressesCFTR. In some embodiments, culturing of the ex vivo SMG organoid results in overexpression of CFTR. In some embodiments, the ex vivo SMG organoid described herein overexpresses CFTR. In some embodiments, the ex vivo SMG organoid described herein expresses or overexpresses CFTR during the differentiation stage. In some embodiments, CFTR expression is assessed in the SMG organoid during the Expansion Phase and Organoid Phase by Western Blot. In some embodiments, the SMG organoid does not express CFTR protein during the Expansion Phase.

[0150] In some embodiments, CFTR function in the SMG organoid is assessed by theForsoklin-induced swelling (FIS) assay. In some embodiments, the FIS assay is used to assess functional capacity of the CTFR in an SMG organoid. In some embodiments, the SMG organoid has a functional CFTR, as assessed by the FIS assay. Use of SMG Organoids for Therapeutic Development

[0151] In another aspect, the present disclosure provides methods of identifying anagent that reduces airway secretions and mucus production in general. In some embodiments, the methods may comprise (i) culturing an ex vivo SMG organoid; (ii) administering one orUCH-39925 more agents to the ex vivo SMG organoid; and (iii) assessing whether the one or more agents reduce the production of mucus in the ex vivo SMG organoid. In some embodiments, if any agent of the one or more agents reduce the production of mucus the agent is identified as an agent that reduces mucosal mucus production. In some embodiments, the agent or any one or more agents tested may be used for the treatment of a subject with a hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands.

[0152] In some embodiments, the one or more agents in step (iii) are known to or areexpected to inhibit ERBB. In some embodiments, the organoid is treated with an ErbB inhibitor. In some embodiments, the organoid is treated with an EGFR inhibitor. In some embodiments, the organoid is treated with an ERBB2 inhibitor. In some embodiments, the organoid is treated with an ERBB3 inhibitor. In some embodiments, the organoid is treated with Erlotinib. In some embodiments, the organoid is treated with Tucatinib. In some embodiments, the organoid is treated with TX1-85-1. In some embodiments, treatment with any of the ErbB inhibitors prevent organoid expansion, budding, branching, and secretions.

[0153] In some embodiments, the organoid is treated with an ErbB inhibitor duringthe Expansion Phase. In some embodiments, the organoid is treated with an ErbB inhibitor during the Organoid Phase. In some embodiments, the organoid is treated with 1-20uM Erlotinib. In some embodiments, the organoid is treated with 10uM Erlotinib. In some embodiments, the organoid is treated with 1-20uM Tucatinib. In some embodiments, the organoid is treated with 10uM Tucatinib. In some embodiments, the organoid is treated with 1-20uM TX1-85-1. In some embodiments, the organoid is treated with 10uM TX1-85-1. In some embodiments, the organoid is treated with one or more of Erlotinib, Tucatinib, TX1-85- 1, or any combination. Methods of Treatment and Prevention

[0154] In another aspect, the present disclosure provides methods of treating ahypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands, in a subject (e.g., a mammal such as a human), comprising administering to the subject an agent that inhibits ERBB. In some embodiments, the agent that inhibits ERBB is an agent that reduces mucosal mucus production.

[0155] The agent can be any agent that inhibits ERBB, e.g., a gene therapy, a smallmolecule or a protein. In some embodiments, the agent is TX1-85-1, tucatinib or erlotinib.UCH-39925 In some embodiments, the agent is any agent of a class to which any one of TX1-85-1, tucatinib and erlotinib belongs, which is capable of inhibiting ERBB. In some embodiments, the agent is TX1-85-1 or a derivative thereof that inhibits ERBB. In some embodiments, the agent is tucatinib or a derivative thereof that inhibits ERBB. In some embodiments, the agent is erlotinib or a derivative thereof that inhibits ERBB. In some embodiments, any agent identified by a methodology known in the art or described herein as an inhibitor of ERBB can be used in the methods described herein. In some embodiments, the subject is in need of treatment (e.g., suffers from or has been diagnosed with the disease or disorder). The agent may, in some embodiments, be comprised in a pharmaceutical composition.

[0156] In another aspect, the present disclosure provides methods of preventing ahypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands, in a subject (e.g., a mammal such as a human), comprising administering to the subject an agent that inhibits ERBB. In some embodiments, the agent that inhibits ERBB is an agent that reduces mucus production. The agent can be any agent that inhibits ERBB, e.g., a gene therapy, a small molecule or a protein. In some embodiments, the agent is TX1-85-1, tucatinib or erlotinib. In some embodiments, the agent is any agent of a class to which any one of TX1-85-1, tucatinib and erlotinib belongs, which is capable of inhibiting ERBB. In some embodiments, the agent is TX1-85-1 or a derivative thereof that inhibits ERBB. In some embodiments, the agent is tucatinib or a derivative thereof that inhibits ERBB. In some embodiments, the agent is erlotinib or a derivative thereof that inhibits ERBB. In some embodiments, any agent identified by a methodology known in the art or described herein as an inhibitor of ERBB can be used in the methods described herein. In some embodiments, the subject is at risk of the disease or disorder. The agent may, in some embodiments, be comprised in a pharmaceutical composition.

[0157] In another aspect, the present disclosure provides compositions comprising anagent that inhibits ERBB for treating a subject (e.g., a mammal such as a human) having a hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands. In some embodiments, the agent is TX1-85-1, tucatinib or erlotinib.

[0158] In another aspect, the present disclosure provides compositions comprising anagent that inhibits ERBB for preventing a hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands, in a subject (e.g., aUCH-39925 mammal such as a human). In some embodiments, the agent is TX1-85-1, tucatinib or erlotinib.

[0159] In another aspect, the present disclosure provides uses of an agent that inhibitsERBB for treating a subject (e.g., a mammal such as a human) having a hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands. In some embodiments, the agent is TX1-85-1, tucatinib or erlotinib.

[0160] In another aspect, the present disclosure provides uses of an agent that inhibitsERBB for preventing a hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands, in a subject (e.g., a mammal such as a human). In some embodiments, the agent is TX1-85-1, tucatinib or erlotinib.

[0161] In some embodiments, the agent that inhibits ERBB is an agent that inhibitsEGFR, ERBB2, and / or ERBB3. In some embodiments, the agent that inhibits ERBB is an agent that inhibits ERBB3.

[0162] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands, is cystic fibrosis.

[0163] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands, is asthma.

[0164] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands, is bronchitis (optionally chronic bronchitis).

[0165] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands, is chronic obstructive pulmonary disease (COPD).

[0166] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands, is primary ciliary dyskinesia (PCD).

[0167] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands, is emphysema.

[0168] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands, is an increased airway secretion associated with a microbial infection (e.g., viral, bacterial, or fungal infection).UCH-39925

[0169] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands, is an increased airway secretion associated with smoking or second-hand smoking.

[0170] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands, is an increased airway secretion associated with an allergy.

[0171] In some embodiments, the hypersecretory airway disorder or disease, or adisorder associated with an increased secretion from submucosal glands is an increased airway secretion associated with an environmental exposure. In some embodiments, the hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands is an increased airway secretion associated with exposure to wildfire smoke. In some embodiments, the hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands is an increased airway secretion associated with exposure to air pollution. In some embodiments, the hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands is an increased airway secretion associated with exposure to burn pits. In some embodiments, the hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands is an increased airway secretion associated with exposure to particulate matter. In some embodiments, the hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands is an increased airway secretion associated with exposure to chemicals. In some embodiments, the hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands is an increased airway secretion associated with exposure to gases. Pharmaceutical Compositions

[0172] The compositions and methods of the present disclosure may be utilized totreat a subject or an individual in need thereof.

[0173] In some embodiments, a composition may comprise an agent that inhibitsERBB. In some embodiments, the agent may be TX1-85-1. In some embodiments, the agent may be tucatinib. In some embodiments, the agent may be erlotinib. In some embodiments, the composition may be for treating a subject having a hypersecretory airway disorder or disease. In some embodiments, the composition may be for treating a subject having aUCH-39925 disorder associated with an increased secretion from submucosal glands. The hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands, may be, for example, cystic fibrosis, asthma, bronchitis (optionally chronic bronchitis), chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), COPD / emphysema, increased airway secretion associated with a microbial infection (optionally viral, bacterial, or fungal infection), an increased airway secretion associated with smoking or second hand smoking, an increased airway secretion associated with an allergy, or an increased airway secretion associated with an environmental exposure. In some embodiments, the environmental exposure causing increased airway secretion is wildfire smoke. In some embodiments, the environmental exposure causing increased airway secretion is air pollution. In some embodiments, the environmental exposure causing increased airway secretion is burn pit exposures. In some embodiments, the environmental exposure causing increased airway secretion is particulate matter. In some embodiments, the environmental exposure causing increased airway secretion is chemicals. In some embodiments, the environmental exposure causing increased airway secretion is gases.

[0174] The subject receiving this treatment may be any animal in need. For example,in some embodiments the subject may be a primate or another mammal, such as equines, cattle, swine, sheep, cats, dogs, or poultry. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal.

[0175] When administered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In some embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free.

[0176] A pharmaceutically acceptable carrier can contain physiologically acceptableagents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextran, antioxidants, such as ascorbic acid or glutathione, chelatingUCH-39925 agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition.

[0177] The phrase "pharmaceutically acceptable" is employed herein to refer to thosecompounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0178] A pharmaceutical composition (preparation) can be administered to a subjectby any of a number of routes of administration including, for example, orally, parenterally, or topically.

[0179] Formulations suitable for oral administration may be in the form of capsules(including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid.

[0180] The phrases "parenteral administration" and "administered parenterally" asused herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0181] These compositions may also contain adjuvants such as preservatives, wettingagents, emulsifying agents, and dispersing agents.

[0182] Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.UCH-39925

[0183] The selected dosage level will depend upon a variety of factors including theactivity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0184] A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” it is meant that the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound disclosed herein. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

[0185] In general, a suitable daily dose of the compositions and methods of thedisclosure will be that amount of the composition that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0186] In certain embodiments, compositions of the disclosure may be used alone orconjointly administered with another type of therapeutic agent. Kits

[0187] In some embodiments, this disclosure provides a kit for producing an SMGorganoid. In some embodiments, the kit comprises reagents for (i) obtaining a population of cells from the proximal airway tissue of a subject;UCH-39925 (ii) isolating from the obtained population of cells (a) submucosal gland basal cellsand / or myoepithelial cells, and / or (b) submucosal gland stem and / or progenitor cells; (iii) culturing the isolated cells in an expansion media comprising one or more growth agents promoting branching and / or budding morphogenesis; and (iv) culturing the isolated cells in a differentiation media comprising one or more agonists or ligands of ERBB.

[0188] In some embodiments, the kit comprises reagents for isolating human SMGtissue, comprising mechanical and enzymatic digestion. In some embodiments, the kit comprises reagents for culturing an SMG organoid. In some embodiments, the kit comprises reagents for treating the SMG organoid with an ErbB-family ligand.

[0189] For use in the therapeutic applications described herein, kits and articles ofmanufacture are also provided. In some embodiments, such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) including one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic.

[0190] The kits provided herein contain packaging materials. Packaging materials foruse in packaging pharmaceutical products include, e.g., U.S. Pat. Nos.5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. A wide array of formulations of the compounds and compositions provided herein are contemplated as are a variety of treatments for any disorder that benefit by an agent that inhibits ERBB.

[0191] In some embodiments, a container(s) of a kit will include one or morecompounds described herein, optionally in a composition or in combination with another agent as disclosed herein. The container(s) optionally have a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprising a compound with an identifying description or label or instructions relating to its use in the methods described herein.UCH-39925

[0192] A kit will typically include one or more additional containers, each with one ormore of various materials (such as reagents, optionally in concentrated form, and / or devices) desirable from a commercial and user standpoint for use of a compound described herein. Non-limiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0193] In some embodiments, a label is on or associated with the container. A labelcan be on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. A label can be used to indicate that the contents are to be used for a specific therapeutic application. The label can also indicate directions for use of the contents, such as in the methods described herein.

[0194] In some embodiments, the compositions of the disclosure can be presented ina pack or dispenser device which can contain one or more unit dosage forms containing a compound provided herein. The pack can, for example, contain metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration or use. The pack or dispenser can also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or research materials. Such notice, for example, can be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. EXAMPLES Example 1- Transcriptomic profiling of human airway submucosal glands (SMGs)

[0195] To better elucidate the cellular constituents comprising the human airwaysubmucosa, primary cells from the intercartilaginous region of multiple donor tracheae and bronchi were isolated for single-cell and single-nuclei sequencing (FIG.1A). The surface airway epithelium was stripped to ensure transcriptomic profiling was restricted to the airway submucosa, then the tissue was dissociated to yield cells for scRNA-seq or homogenized toUCH-39925 obtain nuclei for snRNA-seq (FIG.1B). Because of the dense irregular connective tissue encapsulating cells in the submucosa, several approaches were utilized to isolate single cells, including microdissection, EDTA chelation, and acetic acid / methanol maceration, with variation. Methods Human Tissue Procurement

[0196] Cartilaginous airway tissues were acquired from de-identified, human donorsafter lung transplantation at Ronald Reagan UCLA Medical Center (RRUMC) and from the International Institute for the Advancement of Medicine (IIAM). Deidentified tissues were procured under Institutional Review Board-approved protocols at the David Geffen School of Medicine at UCLA, IRB exemption# 21-000390. Human airway SMG isolation

[0197] Donor proximal airway tissue was handled according to the initial steps for theACME tissue dissociation to generate minced tissue depleted of surface epithelium. Minced tissue was transferred to a 500 mL Erlenmeyer flasks with gross digestion buffer (Collagenase / Hyaluronidase (STEMCELL Technologies 07912) diluted 1:10 in EpiCult-C media, supplemented with Primocin, Amphotericin B (1 μg / mL), Levofloxacin (10 μg / mL), and 10 μM ROCK inhibitor Y-27632), adding 20 mL of buffer for every ~5 mL volume of tissue. The flasks were sealed with parafilm and incubated in an orbital shaker at 37℃ for 14 hours, shaking at 100 rpm. Following digestion, samples were passed through a 300 μm cell strainer into a 50 mL tube to remove large debris and cartilage fragments, and the flask was washed with an additional volume of EpiCult-C media, which was also passed through the cell strainer and collected with the sample filtrate. The samples were then centrifuged at 200xg for 3 minutes to enrich for epithelial cells, comprising larger ductal and acinar fragments as well as smaller multiplets and single cells. At this point, samples were either cryopreserved in EpiCult-C media with 50% FBS and 6% DMSO or processed further for organoid culture.

[0198] To generate small epithelial aggregates for organoid culture, samples wereresuspended in 5 mL of 0.25% trypsin-EDTA pre-warmed in a 37℃ water bath and triturated for 3 minutes with a 1 mL pipette. After the addition of 10 mL of ice-cold HBSS with 2% FBS (HF), samples were centrifuged at 350xg for 5 minutes at 4℃. Next, the samples wereUCH-39925 resuspended in 2 mL of pre-warmed dispase (STEMCELL Technologies 07913, 5 U / mL) with 200 μL of DNase I (STEMCELL Technologies 07900, 1 mg / mL) and triturated for 1 minute with a 1 mL pipette. The resultant cell suspension was diluted with an additional 10 mL of HF and filtered through a 70 μm cell strainer. The cells then underwent a final centrifugation at 350xg for 5 minutes at 4℃, and the pellet was resuspended in cold expansion media (small airway epithelial cell growth media (SAECGM, PromoCell C-21170) supplemented with 1% FBS and 10 μM ROCK inhibitor) and kept on ice. Human surface airway epithelium (SAE) dissociation

[0199] Following resection of the ICZ for SMG isolation, the remaining proximalairway tissue was trimmed into smaller pieces (roughly 2 cm x 2 cm surface area) for ease of handling. Trimmed tissue was transferred to a 50 mL tube containing pre-warmed dispase (Corning 354235, 50 u / mL) with DNase I (Sigma DN25, 1 mg / mL) and incubated for 1 hour in a 37℃ water bath. Following enzymatic digestion, the 50 mL tube containing tissue was placed directly on ice; individual tissue pieces were transferred to a 10 cm dish on ice with cold DPBSabx, stripping the surface epithelium via cell scraper. Stripped epithelium was collected into a fresh tube and centrifuged at 300xg for 5 minutes at 4℃. The pellet was resuspended in pre-warmed 0.25% trypsin-EDTA and incubated for 15 minutes in a 37℃ water bath. After neutralization with 10% serum, the cell suspension was triturated several times with a 1 mL pipette and passed through a 70 μm cell strainer. Cells were centrifuged at 300xg for 5 minutes at 4℃ and resuspended in BamBanker (GC Lymphotec CS-02-001) for cryopreservation. Single Cell Isolation and Sequencing

[0200] Human tracheae and bronchi were processed by several methods to generatesingle-cell suspensions for transcriptomic sequencing, outlined in the subheadings below. For each method, the sample ID(s) pertaining to that isolation method are listed in the subheading and Table 1.UCH-39925 Table 1. Tissue Sample MetadataUCH-39925UCH-39925 Method A: SMG Microdissection (Samples: SMG1, SMG2, SMG3)

[0201] Bronchial tissue was dissected and cleaned of excess blood, adipose, andconnective tissues in ice-cold DPBS supplemented with Primocin. Cleaned tissue was treated with dispase (50 U / mL in HBSS, Corning 354235) for 45 minutes at room temperature, followed by DNase I (10 mg / mL in PBS, Roche 11284932001) for an additional 45 minutes. The surface airway epithelium was then stripped from the tissue using a cell scraper. The remaining tissue after stripping was incubated overnight at 4℃ in Liberase TM (1:40 dilution in PBS from 2.5 mg / mL stock solution, Roche 5401119001), and SMGs were recovered the following day via microdissection. The isolated SMGs were subsequently dissociated in 0.25% trypsin-EDTA (Gibco 25200072) for 30 minutes at 37℃, after which an equal volume of media containing 10% FBS was added to neutralize the trypsin. After neutralization, samples were filtered through a 40 µm cell strainer to generate a single-cell suspension. The sample filtrate was then spun at 600xg for 5 minutes at 4℃ and resuspended in 200 µL of DPBS. To this cell suspension, 800 µL of chilled methanol was added dropwise while stirring with the pipette tip to prevent cell clumping. Samples were stored in 80% methanol at -80℃ until ready for preparation of sequencing libraries. Immediately prior to library prep, samples were equilibrated on ice for 5 minutes, centrifuged at 1000xg for 5 minutes at 4℃, and resuspended in Wash-Resuspension Buffer (0.04% BSA, 1mM DTT, 0.2 U / µL RNase inhibitor SUPERase-In in 3X SSC Buffer prepared in nuclease-free water). The samples were then loaded onto the 10X Genomics Chromium X instrument, targeting 10,000 cells for recovery. Sequencing libraries were prepared according to 10X Genomics Single Cell 3’ v3.1 User Guide and sequenced via NovaSeq 6000. Method B: Cold Chelation with Collagenase / Dispase Dissociation (Sample: SMG4)

[0202] The intercartilaginous zone (ICZ) was resected from the bronchi of donorproximal airways in ice-cold DPBS supplemented with Primocin, Amphotericin B (1 μg / mL), and Levofloxacin (10 μg / mL) (DPBSabx). Resected ICZ tissue was mechanically depleted of its surface epithelium by scraping with a cytology brush, washed in cold DPBS, and finely minced via razor blade. The minced tissue was then incubated on a rocking shaker overnight at 4℃ in chelation buffer (distilled water with 10 mM EDTA, 5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCl, 1.6 mM KCl, 43.4 mM sucrose, 54.9 mM d-sorbitol, and 0.5 mM DL-dithiothreitol) supplemented with DNase I (1 mg / mL) and ROCK inhibitor Y-27632 (10 µM). The following day, samples were centrifuged at 300xg for 5 minutes at 4℃,UCH-39925 resuspended in collagenase / dispase (2 mg / mL in PBS, Roche 10269638001) supplemented with DNase I (1 mg / mL) and ROCK inhibitor (10 µM), and incubated for 1 hour at 37℃ while rotating. After, samples were filtered through a 40 µm cell strainer to generate a single- cell suspension. At this point, the sample filtrate was handled as described above in Method A for methanol fixation, storage, and sequencing. Method C: Cold Chelation without Enzymatic Dissociation (Samples: SMG5a, SMG6a, SMG7a)

[0203] Minced tracheal / bronchial tissue depleted of surface epithelium was generatedas described in Method B. The minced tissue was transferred to a 50 mL tube and quickly washed with 2 mL of 7.5% N-acetyl cysteine in PBS before directly adding 38 mL of chelation buffer. The tissue was then incubated for 2-4 hours at 4℃ on a nutating mixer. Following incubation, the tubes were placed on ice, allowing large tissue pieces to settle by gravity (~1 minute). The chelation buffer was removed via 10 mL pipette and passed through a 100 µm cell strainer into a fresh 50 mL tube. Ice-cold DPBS was added to the remaining tissue pieces and the tube was inverted 5-10 times to dislodge loose cells. After allowing the tissue to settle, the DPBS was collected into the chelation buffer through the same 100 µm strainer, and the resultant filtrate was additionally passed through a 40 µm cell strainer. At this point, the filtered cell suspension was handled as described above in Method A. Method D: Acetic Acid / Methanol (ACME) Maceration without Mechanical Dissociation (Samples: SMG5b, SMG5c, SMG6b)

[0204] Tissue samples were handled exactly as described for Method C, with thefollowing exception: instead of chelation buffer, 38 mL of ACME buffer (13:3:2:2 ratio of DNase / RNase-free water, methanol, glacial acetic acid, and glycerol) was added to minced tissue resuspended in 7.5% N-acetyl cysteine, and samples were incubated for 2-4 hours at room temperature on a see-saw rocking shaker. Method E: ACME Maceration with Cold Chelation (Sample: SMG7b)

[0205] The tissue sample was handled as described for Method D, with the followingalteration: after addition of the ACME buffer, the sample was instead incubated for 1 hour, centrifuged at 300xg for 5 minutes, resuspended in chelation buffer, and incubated for 2 hours at 4℃ on a nutating mixer.UCH-39925 Method F: ACME Maceration with gentleMACS Dissociation (Samples: SMG9, SMG10, SMG11)

[0206] Tissue samples were handled as described for Method D, up to the addition ofACME buffer. After adding ACME buffer, the tissue suspension was transferred into gentleMACS C-tubes, incubated for 10 minutes at room temperature on a see-saw rocking shaker, and loaded onto the GentleMACS Octo dissociator to run program “B.” Subsequently, the following process was repeated three times: the C-tubes were returned to the rocking shaker for 10 minutes, then loaded onto the GentleMACS Octo dissociator to run program “Multi_A_1.” The macerated tissue was then transferred to a 50 mL tube and centrifuged at 1000xg for 5 minutes to remove ACME buffer. The pellet was resuspended in 30 mL of cold wash buffer (DPBS + 1% BSA) and passed through a cell strainer stack (100, 70, and 40 μm PluriSelect strainers) on ice. The cell strainer stack was washed with an additional 15 mL of wash buffer, and the filtrate was centrifuged at 1000xg for 5 minutes at 4℃. The pellet was then resuspended in 900 μL of wash buffer, followed by 100 μL DMSO; the samples were stored directly at -80℃ prior to sorting via FACS. For FACS, ACME-fixed samples were thawed on ice, centrifuged at 1000xg for 5 minutes at 4℃, washed once with 1 mL of cold washing buffer, centrifuged again, and resuspended in wash buffer supplemented with RNase inhibitor (40 U / mL), nuclear dye DRAQ5 (1 μL / mL; 5mM stock), and cytoplasmic dye Concanavalin-A AlexaFluor 488 conjugate (2 μL / mL; 1 mg / mL stock.) The samples were stained on ice in the dark for 45 minutes, then sorted via BD FACS Aria (SMG9, SMG10) or On-chip Sort (SMG11), collecting DRAQ5-positive, Concanavalin-A singlets into wash buffer with RNase inhibitor. Post-sort, samples were centrifuged at 1000xg for 5 minutes at 4℃, resuspended in wash buffer with RNase inhibitor, and taken immediately for preparation of sequencing libraries. Library preparation and sequencing were performed as described in Method A. Method G: Methanol Pre-Fixation with Trypsin-EDTA and gentleMACS Dissociation (Sample: SMG8)

[0207] Minced bronchial tissue depleted of surface epithelium was generated asdescribed in Method B. The minced tissue was incubated in 80% methanol (in DPBS) for 1 hour at 4℃ on a nutating mixer. After, the tissue was centrifuged at 1000xg for 5 minutes at 4℃, washed once with DPBS, and centrifuged again. The tissue was then transferred toUCH-39925 gentleMACS C-tubes, resuspended in 0.25% trypsin-EDTA, and loaded onto the gentleMACS Octo dissociator to run the “37C_Multi_B” program. After, the dissociated tissue was passed sequentially through 100 µm and 40 µm cell strainers to yield a single-cell suspension. At this point, the sample filtrate was handled as described in Method A. Single-nuclei isolation and sequencing

[0208] Donor proximal airway tissue was mechanically depleted of its surfaceepithelium by cytology brush, minced via razor blade, and cryopreserved in EpiCult-C media with 50% FBS and 6% DMSO. For nuclei isolation, samples were thawed quickly in a 37℃ water bath, washed with cold DPBS, and centrifuged at 300xg for 5 minutes at 4℃. The tissue was then transferred to a pre-cooled 15 mL glass dounce (Corning Pyrex 772215), on ice, containing 5 mL of nuclei isolation buffer (0.25 M sucrose, 5 mM MgCl2, 25 mM KCl, 10 mM Tris (pH 8), 1 mM DTT, 0.1% Triton X-100, cOmplete EDTA-free protease inhibitor cocktail (Roche 04693132001), and RNase inhibitors RNasin (Promega N2615, 0.4 U / μL) and SUPERase-In (Invitrogen AM2694, 0.2 U / μL)). Nuclei were isolated via tissue homogenization, using 15 strokes each of pestle A then pestle B. Subsequently, the resultant tissue homogenate was passed through a 40 μm cell strainer and nuclei were collected by centrifugation at 1000xg at 4℃ for 10 minutes. Nuclei were resuspended in 500 mL nuclei storage buffer (DPBS with 4% BSA and RNasin (0.2 U / μL)), followed by the addition of 1 drop of NucBlue Live ReadyProbes (Invitrogen R37605) and a 30 minute incubation at 4℃. Hoechst (NucBlue)-stained singlets were separated from debris via BD FACS Aria and collected in storage buffer. Post-sort, samples were centifuged at 1000xg for 10 minutes at 4℃, resuspended in storage buffer, counted, and loaded onto 10X Genomics Chromium X instrument, targeting 10,000 cells for recovery. Sequencing libraries were prepared according to 10X Genomics Single Cell 3’ v3.1 User Guide and sequenced via NovaSeq 6000. Single-Cell Processing and Analysis

[0209] Single cell libraries were mapped to the human reference genome GRCh38using CellRanger 7.1.0 and processed and analyzed in R using the library Seurat for normalization, dimensionality reduction, scaling, and clustering. Cells were filtered for at least 500 reads and genes were filtered for expression in at least 10 cells per library and 5% of cells were excluded as putative doublets using the DoubletFinder library. Cells with more than 12.5% expression from mitochondrial genes were removed. Louvain clusters andUCH-39925 differential expression from the Seurat FindClusters and FindAllMarkers function were used to characterize cell types. Cell subtypes were further informed using gene-gene clustering; Genes were clustered using correlation to form groups. The script for this correlation grouping can be found on the World Wide Web at the website identified by placing https: / / before github.com / Teneth / GEND_Script. Ligand-receptor pairs enriched in the dataset were found using the iTalk package. Data was displayed using the pheatmap and ggplot libraries. Gene groups were displayed by calculating the average expression value for the set of genes in each cell. Results

[0210] Single cell RNA-seq analysis was conducted to identify gene expressionpatterns across the submucosal glands of donors. After removal of low-quality transcriptomes (FIG.1C-D), over 23,000 cells from 12 donors were analyzed, which varied in number and type of cells captured depending on the isolation method (Table 1 FIG.1E-F). The resultant transcriptomes were clustered and annotated based on enrichment of correlated geneexpression networks to identify cell types (FIG. 1G). UMAP clustering sorted the cells intofour major cell types: endothelial, epithelial, fibroblasts, and immune / blood cells (FIG.2A). Further annotation of the captured transcriptomes revealed a diversity of cell types which exhibited unique gene network signatures (FIG.2B), including endothelial cells (FIG.3A). Among the immune cell types, macrophages (FIG.3B-C), plasma (FIG.3D), B (FIG.3E), mast (FIG.3F), and T cells (FIG.3G) were identified, while the mesenchymal cell typesincluded fibroblasts (FIG. 3H-I) and smooth muscle cells (FIG 3J). Additionally, a clusterdefined by expression of neuronal genes likely representing parasympathetic ganglia was identified, consistent with their presence near the adventitial surface of the trachealis (FIG. 3K). Visualization of expression (VOX) was used to enable analysis of expression in different cell subtypes. This enabled the identification of specific transcriptional profiles for duct basal stem cells, transitioning duct cells, pre-serous and serous cells, myoepithelial cells, mucus cells, and intermediate secretory cells (FIG.4).

[0211] Within the epithelial compartment, cell types were categorized based onmarker genes and gene expression networks across the entire dataset (i.e., including non- epithelial cell types, from which basal, ciliated, secretory, and rare cell types were identified. Of the rare cell types documented in the airways, ionocytes [ASCL3+CFTR+] were readily identified (FIG.5) but no pulmonary neuroendocrine or tuft cells were found. GeneUCH-39925 expression was then re-analyzed exclusively in the epithelial subset of the transcriptomic data (FIG.6), excluding the ionocyte population based on their distinctive clustering separate from the remaining epithelial cells, in order to further resolve cell types in this compartment (FIG.7). Both of the recognized stem / progenitor populations in this subset, namely MECs [TP63+ACTA2+MYH11+] and stem duct basal cells [KRT17+TP63+BCAM+] were identified (FIG.8). These two populations share gene expression networks defined by TCF / LEF transcription factors and developmental ontology related to tube morphogenesis (FIG.9A-B), consistent with the role of canonical Wnt signaling in SMG organogenesis. Conversely, these two populations also retain unique patterns of gene expression, including networks with ontologies such as plasma membrane projections for stem duct basal and muscle contraction for myoepithelial cells (FIG.10A-B).

[0212] Within the basal population, two additional subtypes were identified:suprabasal and duct basal. The suprabasal population [LGALS7B+S100A2+PLP2+] retained expression of basal markers but at decreased levels compared to the stem duct basal cells (FIG.8) and exhibited overlapping gene network enrichments with club and ciliated cells (FIG.6), suggesting these are differentiating basal cells. Moreover, the top network enrichments within this population featured prominent expression of genes associated with oxidative phosphorylation (FIG.11A) and translation (FIG.11B). The other basal subtype, duct basal, demonstrated further downregulation of canonical basal markers and was instead dominated by expression of genes indicative of squamous metaplasia, including DSG3, EMP1, and SFN (FIG.8). Enriched ontology terms for this cluster further indicate gene expression related to immunogenic and injury responses (FIG.12A-B), suggesting this is a population of duct basal cells reacting to environmental stressors. Together, the data indicate a range of basal cell states but do not seem to reflect a distinct basal cell type, i.e., region- specific basal stem cell subtypes are not resolved in the SMG duct.

[0213] In contrast, differentiated cell types of the duct epithelium that do have region-specific distribution were identified along the duct. The ciliated cells [TMEM190+DNAH12+FOXJ1+] (FIG.8), which are restricted to the medial aspect of the duct that connects the SMG to the SAE, express genes related to cilium organization and motility (FIG.13). Beyond the ciliated ducts, a club cell [SCGB1A1+WFDC2+CEACAM6+] secretory population was identified (FIG.8), which localizes to the collecting duct. Consistently, this population is characterized by expression of gene networks related to humoral immune response and secretion of antimicrobial peptides (FIG.14). Additionally, aUCH-39925population of cells expressing SMG duct markers WNT5B and KRT23 was found that also hasappreciable CFTR expression (FIG.8), which is termed tubuloacinar (TA) duct cells. Gene network enrichments for this population are described by ontologies related to chemical homeostasis and transport of small molecules (FIG 15A-B). The associated genes comprising these networks encode membrane transporters of Na+, Cl-, K+, and HCO3- ions as well as the transcription factor TFCP2L1 and the mineralocorticoid receptor (NR3C2) (FIG.15A-B). TFCP2L1 coordinates developmental patterning of kidney collecting duct principal and intercalated cells, which regulate water-electrolyte and acid-base homeostasis, respectively. Moreover, the mineralocorticoid receptor is a ligand-dependent transcription factor, functioning as an effector of aldosterone activity to increase expression of protein channels involved in ion and water transport. Taken together, the TA ducts likely regulate the composition of luminal contents by adjusting extracellular fluid and / or pH levels.

[0214] Regarding differentiated cell types of the tubuloacinar compartment, bothmucous and serous cells were identified in the transcriptomic dataset. The top gene network enrichments defining the mucous tubule population were networks associated with either the club (FIG.14) or serous (FIG.16) cell populations, suggesting overlapping gene expression patterns across the secretory populations. Thus, the mucous tubule cells were primarily distinguished by differential expression of canonical marker genes such as MUC5B, TFF3, and NKX3-1 (FIG.8). The serous cells [LYZ+LTF+PIP+] (FIG.8) were defined by enrichment of gene expression networks with ontology related to neutrophil degranulation (FIG.17A) and body fluid secretion (FIG.17B), conforming with their antimicrobial function and contribution to airway surface liquid. Additionally, a “pre-serous” cell population that shares overlapping expression of serous-associated transcripts (FIG.8) and networks (FIG.17A) but does not express the full complement of characteristic serous markers, such as LYZ or PIP was identified. Moreover, these pre-serous cells exhibit enrichment for a gene expression network with ontology corresponding to glandular development (FIG.18) and CFTR expression comparable to the TA duct population (FIG. 8), consistent with reports that CFTR demonstrates predominant expression across serous cells within the SMGs.

[0215] Overall, these data further elucidate the diverse complement of epitheliacomprising the glandular compartment which impart the unique secretory features requisite for respiratory homeostasis and defense.UCH-39925 Example 2- Confirmation of SAE and SMG Cell Types by Immunofluorescence

[0216] Expression of cell surface markers across diverse cell types were thenconfirmed using immunofluorescence. Basal cells from surface airway epithelium (SAE) and submucosal gland (SMG) ducts and myoepithelial cells were compared. Methods Cytocentrifugation and Immunocytofluorescence

[0217] Freshly isolated total SMG cell suspensions or sorted SMG populations wereimmediately centrifuged onto microscope slides at 1000 RPM for 5 minutes using a Cytospin 4 (Thermo Scientific). Cytospun cells were allowed to dry at room temperature overnight, fixed in methanol for 10 minutes, dried for an additional 2 hours, then rehydrated in TBS for 10 minutes. Next, cells were permeabilized and blocked for 1 hour at room temperature in antibody signal enhancer blocking buffer prepared in Dako serum-free block (5% donkey serum, 50 mM glycine, 0.05% Tween 20, 0.1% Triton X-100, 0.1% BSA in Dako blocking solution (Agilent X090930-2.) After, cells were incubated overnight at 4℃ with primary antibodies diluted in antibody incubation solution prepared in Dako serum-free block (10 mM glycine, 0.05% Tween 20, 0.1% Triton X-100, 0.1% H2O2 in Dako blocking solution); the following primary antibodies were used: KRT17 Alexa Fluor 488 conjugate (Abcam ab185032, 1:100), αSMA (Thermo Fisher 14-9760-82, 1 µg / mL), LTF (Bioss BS-5810R, 1:200), MUC5B (Santa Cruz sc393952, 1:500), SCGB1A1 (R&D Systems MAB4218, 15 µg / mL), and KRT14 (BioLegend 906004, 1 µg / mL). Cells were then washed three times for 5 minutes in TBS with 0.025% Triton X-100, followed by a 1 hour incubation at room temperature with appropriate secondary antibodies (Donkey anti-Mouse IgG Alexa Fluor 488, Invitrogen A21202; Donkey anti-Rabbit IgG Alexa Fluor 594, Invitrogen A21207; Donkey anti-Mouse IgG Alexa Fluor 594, Invitrogen A21203; Donkey anti-Mouse IgG Alexa Fluor 647, Invitrogen A31571; Donkey anti-Rat IgG Alexa Fluor 647, Invitrogen A21247; Donkey anti-Chicken IgY Alexa Fluor 647, Invitrogen A78952) diluted 1:1000 in Dako with 0.1% Tween 20. Subsequently, sections were washed for 5 minutes twice in TBS with 0.025% Triton X-100, washed for 10 minutes once in TBS with DAPI (2.5 µg / mL), incubated for 3 minutes in TrueView autofluorescence quenching reagent (Vector SP-8400- 15), washed again for 5 minutes in TBS, and mounted in VectaShield Vibrance Antifade Mounting Medium (Vector H-1700-10.) Mounted cells were imaged using a Zeiss AxioUCH-39925 Imager. D1 epi-fluorescent microscope equipped with a 20X objective lens and AxioCam MR3 camera. Tissue Immunohistofluorescence

[0218] Immunohistofluorescence was performed on formaldehyde-fixed paraffin-embedded (FFPE) human trachea and bronchi sections prepared by UCLA Translational Pathology Core Laboratory (TPCL). FFPE sections were baked on a heat block at 65℃ for 1 hour prior to deparaffinization via HistoClear II (Electron Microscopy Sciences 641110) and rehydration via ethanol, decreasing from 100% to 0% in water in a stepwise series. Tissue sections then underwent heat-induced epitope retrieval in a sodium citrate buffer (pH 6.0) for 1 hour in a steamer (IHC World), after which, slide containers were placed on ice for 20 minutes. Next, tissue sections were permeabilized and blocked for 1 hour at room temperature, using antibody signal enhancer blocking buffer (described above). After, sections were incubated overnight at 4℃ with primary antibodies diluted in antibody incubation solution; the following primary antibodies were used: NGFR (Abcam ab52987, 1:100), EGFR (Cell Signaling Technology 4267, 1:50), ITGA2 (Abcam ab133557, 1:200), KRT17-AlexaFluor488 conjugate (Abcam ab185032, 1:100), HBEGF (Invitrogen PA5- 121922, 1:100), MUC5B (Santa Cruz sc393952, 1:500), NRG1 (EMD Millipore ABT179, 1:50), EREG (Invitrogen PA5-46969, 15 µg / mL), EGF (Abcam ab9695, 1:100), αSMA (Thermo Fisher 14-9760-82, 1 µg / mL), LTF (Bioss BS-5810R, 1:200). After primary antibody incubation, sections were handled as described above for immunocytofluorescence, with the following exceptions: Donkey anti-Goat IgG Alexa Fluor 647 (Invitrogen A21447) was used when appropriate and TruView incubation was extended to 5 minutes. Results

[0219] Epithelial cell types were validated using immunofluorescent staining in SAEand SMG tissues. The basal layer of cells show a continuum of airway / glandular stem cells. The cells in the basal compartment change morphologically along the medial-lateral axis. IF staining for KRT17 and alpha-SMA was performed (FIG.19)

[0220] Additional targets in the SMG duct or tubuloacinar region were analyzedillustrating distinct cell types within each compartment. In airway SMG tissue, a subset of LGALS7B+basal cells in the SMG ducts were detected via immunostaining (FIG.20A), including cells that are displaced from the basal lamina, further indicating these are aUCH-39925 transitioning suprabasal population. The club cell [SCGB1A1+WFDC2+CEACAM6+] secretory population (FIG.8) was found to localize to the collecting duct based on SCGB1A1 immunostaining (FIG.20B). KRT23 immunostaining reveals that tubuloacinar (TA) duct cells consist of luminal cells with cuboidal / squamous morphology; moreover, these cells comprise small-diameter ducts adjoining the tubuloacinar epithelium but are absent from the larger diameter collecting ducts (FIG.20C) In addition, LGALS7B+ suprabasal cells are found in the SMG but absent from tubuloacini, SCGB1A1+MUC5B+ club cells are located in the SMG collecting ducts and are distinct from MUC5B+ mucous tubule cells, and KRT23+ TA duct cells are immediately adjacent to the tubuloacinar compartment (FIG.20). Lastly, KRT14 expression demarcates basal cells in the SMG ducts and MECs surrounding the tubuloacinar structures (FIG.20D). Example 3- Methods for culturing ex-vivo SMG organoids Methods ACME tissue dissociation, sorting, and single-cell sequencing

[0221] The intercartilaginous zone (ICZ) was resected from the trachea and mainstembronchi of donor proximal airways in ice-cold DPBS supplemented with Primocin, Amphotericin B (1 μg / mL), and Levofloxacin (10 μg / mL) (DPBSabx). Resected ICZ tissue was mechanically depleted of its surface epithelium by scraping with a cytology brush, washed in cold DPBS, and finely minced via razor blade. Minced tissue was then fixed and dissociated via acetic acid / methanol (ACME) maceration as previously described (García- Castro et al.) Briefly, minced tissue was transferred to a 50 mL tube and quickly washed with 2 mL 7.5% N-acetyl cysteine in PBS before adding 38 mL of ACME buffer (13:3:2:2 ratio of DNase / RNase-free water, methanol, glacial acetic acid, and glycerol.) The tissue in ACME buffer was then divided into GentleMACS C-tubes, 5 mL to each tube, incubated for 10 minutes at room temperature on a see-saw rocking shaker, and loaded onto the GentleMACS Octo dissociator to run program “B.” Subsequently, the following process was repeated three times: the C-tubes were returned to the rocking shaker for 10 minutes, then loaded onto the GentleMACS Octo dissociator to run program “Multi_A_1.” The macerated tissue was then transferred to a 50 mL tube and centrifuged at 1000xg for 5 minutes to remove ACME buffer. The pellet was resuspended in 30 mL of cold wash buffer (DPBS + 1% BSA) and passed through a cell strainer stack (100, 70, and 40 μm PluriSelect strainers) on ice. The cellUCH-39925 strainer stack was washed with an additional 15 mL of wash buffer, and the filtrate was centrifuged at 1000xg for 5 minutes at 4℃. The pellet was then resuspended in 900 μL of wash buffer, followed by 100 μL DMSO; the samples were stored directly at -80℃ prior to sorting. For FACS, ACME-fixed samples were thawed on ice, centrifuged at 1000xg for 5 minutes at 4℃, washed once with 1 mL of cold washing buffer, centrifuged again, and resuspended in wash buffer supplemented with RNase inhibitor (40 U / mL), nuclear dye DRAQ5 (1 μL / mL; 5mM stock), and cytoplasmic dye Concanavalin-A AlexaFluor 488 conjugate (2 μL / mL; 1 mg / mL stock.) The samples were stained on ice in the dark for 45 minutes, then sorted via BD FACS Aria, collecting DRAQ5-positive, Concanavalin-A singlets into wash buffer with RNase inhibitor. Post-sort, samples were centrifuged at 1000xg for 5 minutes at 4℃, resuspended in wash buffer with RNase inhibitor, counted, and loaded onto 10X Genomics Chromium X instrument, targeting 10,000 cells for recovery. Sequencing libraries were prepared according to 10X Genomics Single Cell 3’ v3.1 User Guide and sequenced via NovaSeq 6000. Human airway SMG organoid culture

[0222] Isolated airway SMG epithelial aggregates resuspended in expansion media(SAECGM) with 1% FBS and 10uM ROCK inhibitor were mixed 1:1 with Cultrex UltiMatrix RGF basement membrane extract (R&D systems BME001-10) on ice. SMG aggregates were plated as 8 x 30 μL droplets in 6-well plates pre-warmed at 37℃. The plates were then quickly flipped upside down and placed in a tissue culture incubator (37℃, 20% O2, 5% CO2) for ~2 hours to promote dome formation and ensure gelation, respectively, after which, 2 mL of expansion media was added to each well. Organoids were routinely passaged every ~10 days (depending on growth kinetics), unless otherwise stated.

[0223] To initiate SMG organoid differentiation, SAECGM expansion media wasaspirated and 2 mL TrypLE was added to a single well; Cultrex domes were washed off the bottom of the well and triturated 10 times with a 1 mL pipette pre-coated with anti-adherence rinse solution (STEMCELL Technologies 07010) before transferring to the adjacent well, repeating the process until 3 wells were pooled together. Pooled organoids were then transferred to a 15 mL tube (pre-coated with anti-adherence rinse solution) on ice containing 10 mL of advanced DMEM / F12 supplemented with 1x GlutaMax and 10 mM HEPES (adDMEM / F12++). Each well was subsequently washed with 1 mL of cold adDMEM / F12++UCH-39925 to collect any remaining organoids, and the tubes were centrifuged at 300xg for 5 minutes at 4℃. Pellets were resuspended in cold SAECGM supplemented with 1% FBS and 10 μMROCK inhibitor and thereafter mixed 1:1 with Cultrex, plating droplets as described above.Following gelation, 2 mL of differentiation media (see table below for formulation) was added to each well. SMG Organoid Media consisted of R-Spondin 1 (PeproTech 120-38, 250 ng / mL), FGF7 (PeproTech 100-19, 5 ng / mL), FGF10 (PeproTech 100-26, 20 ng / mL), Noggin (PeproTech 120-10C, 100 ng / mL), A83-01 (Tocris 2939, 500 nM), ROCK inhibitor Y-27632 (10 μM), SB202190 (Sigma S7067, 1 mM), 1x B27 supplement (Gibco 17504-44), N-Acetyl cysteine (Sigma A9165, 1.25 mM), Nicotinamide (Sigma N0636, 10 mM), 1x GlutaMax, HEPES (10 mM), 1x Penicillin / Streptomycin (Invitrogen 15140-122), andPrimocin (100 µg / mL) in Advanced DMEM / F12 (as in Table 2). Organoids were cultured for7 to 21 days, changing media every 3 days. Table 2. SMG Organoid differentiation mediaFluorescence-activated cell sorting for organoid culture

[0224] SMG tissue isolate cryopreserved after gross digestion was thawed, washed inadDMEM / F12++, and centrifuged at 300xg for 5 minutes at 4℃ to remove DMSO. Thawed samples then underwent sequential enzymatic dissociation detailed above for SMG isolation with the following modifications: trypsin-EDTA incubation was extended to 5 minutes and dispase / DNase incubation was extended to 3 minutes. Diluted cell suspensions were filtered through a 40 μm cell strainer, centrifuged at 300xg for 5 minutes at 4℃, resuspended in aUCH-39925 minimal volume of FACS buffer (DPBS with 2% HI FBS, 25 mM HEPES, 1 mM EDTA, Primocin, DNase I (50 μg / mL), and 10 μM ROCK inhibitor), and set aside on ice. Meanwhile, donor-matched SAE samples were thawed and washed to remove DMSO. SAE pellets were resuspended in dispase / DNase solution and triturated for 1 minute. After diluting with HF, cell suspensions were filtered, centrifuged, and resuspended in FACS buffer. Human BD Fc Block (BD Pharmingen 564219) was added directly to SMG and SAE cell suspensions (1 μL per 100 μL reaction), which were then incubated at room temperature for 10 minutes. The following primary-conjugated antibodies were added at the specified volume per 100 μL sample reaction: 5 μL ITGA2-BV421 (BD OptiBuild 742644), 5 μL TROP2- AF488 (R&D Systems FAB650G), 20 μL CD271-PE (BD Pharmingen 557196), and 5 μL EGFR-APC (BioLegend 352906). Cells were incubated with primary antibodies at 4℃ for 30 minutes on a nutating mixer. After, samples were washed twice with FACS buffer, then resuspended in a minimal volume of FACS buffer with DAPI (1 μg / mL final concentration). Cells were then sorted using a BD FACSAria cell sorter with BD FACSDiva software into collection tubes containing SAECGM supplemented with 10% FBS and 10 μM ROCK inhibitor. Sorted cells or matched total SMG single-cell suspensions (parent population from which SMG cells were sorted) were centrifuged at 300xg for 5 minutes at 4℃, resuspended in SAECGM supplemented with 1% FBS and 10 μM ROCK inhibitor, mixed with an equivalent volume of Cultrex, and immediately plated in 30 μL droplets, seeded at a density of 1,000 cells per droplet. Seeded cells were then handled as described above for SMG cultures. Established cultures underwent a 14 day Expansion Phase followed by a 21 day Organoid Phase.

[0225] The following reference provides background on ACME dissociation methodsand is hereby incorporated by reference in its entirety: García-Castro H, Kenny NJ, Iglesias M, Álvarez-Campos P, Mason V, Elek A, Schönauer A, Sleight VA, Neiro J, Aboobaker A, Permanyer J, Irimia M, Sebé-Pedrós A, Solana J. ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics. Genome Biol.2021 Apr 8;22(1). Organoid Immunohistofluorescence, Image Acquisition and Quantification

[0226] SMG organoids subjected to combined Expansion + Organoid Phase ErbB-family ligand treatments were recovered from Cultrex via trituration in TrypLE as described above for SMG cultures. After pooling organoids in cold adDMEM / F12++ and centrifugation, organoids were resuspended in 4% PFA in PBS (Santa Cruz sc-281692) andUCH-39925 incubated for 24 hours at 4℃ with agitation. Next, fixed organoids were washed once in IF buffer (0.1% BSA, 0.2% Triton X-100, 0.05% Tween 20 in PBS) and once in PBS. Samples were stored at 4℃ in PBS until ready for embedding. Fixed organoids were resuspended in melted HistoGel (Epredia HG4000012) and dispensed into Cryomolds (Tissue-Tek 4557) on ice to cool. Once set, HistoGel blocks were transferred to tissue cassettes (Fisher Scientific 22-272417) and incubated in formalin (Sigma-Aldrich HT501128) for 14 hours at room temperature. Cassettes were subsequently transferred to 70% ethanol and delivered to UCLA TPCL for preparation of FFPE sections.

[0227] Immunohistofluorescence was performed on sectioned organoids as describedabove, using primary antibodies targeting MUC5B (Santa Cruz sc-393952, 1:500) and LYZ (Abcam ab108508, 1:250), and ERBB2 (R&D Systems AF1129, 15 μg / mL). Mounted sections were imaged using a Zeiss Axio Imager.D1 epi-fluorescent microscope equipped with a 20X objective lens and AxioCam MR3 camera. For figure images, the dynamic range was scaled identically across images for each channel in ZEN 3.2 (blue edition). Raw images were converted into 8-bit TIFF files in ZEN 3.2 and quantified in FIJI (ImageJ). For each ligand treatment condition, 20 images were quantified by counting the number of nuclei and MUC5B / LYZ-expressing cells, using ERBB2 surface expression to demarcate cell boundaries (data not shown). The percentage of MUC5B+and LYZ+cells was calculated for each image by dividing the sum of MUC5B / LYZ-expressing cells by the total number of nuclei counted. These values were then averaged for each condition and the mean with SEM was plotted in GraphPad Prism. Two-way ANOVA followed by Dunnett’s multiple comparisons test was used to compare treatment groups to CTRL / CTRL (Expansion + Organoid Phase untreated control) organoids. Recovered Expansion / Organoid Phase Wholemount Immunostaining

[0228] Epithelial clusters (Expansion Phase) or organoids (Organoid Phase) wererecovered from Cultrex as described then resuspended in 4% PFA in PBS (Santa Cruz sc- 281692) and incubated for 1 hour at room temperature on a see-saw rocking shaker. After fixation, clusters / organoids were assayed following the protocol described by Stem Cell Technologies for immunocytochemical staining of epithelial organoids (See the website identified by placing https: / / before www.stemcell.com / performing-icc-staining-epithelial- organoids.html). Briefly, fixed clusters / organoids were washed once in IF buffer (0.1% BSA, 0.2% Triton X-100, 0.05% Tween 20 in PBS) and once in PBS, allowing organoids to settleUCH-39925 by gravity in between washes. Clusters / organoids were then resuspended in citrate buffer (pH 6.0) and incubated at 98℃ in a heat block for 20 minutes, after which, the heat block was switched off and the samples were allowed to cool for an additional 20 minutes. Next, clusters / organoids were washed with 0.3M glycine for 30 minutes prior to incubation in Permeabilization / Blocking solution (5% donkey serum, 1% Triton X-100 in PBS) for 5 hours at room temperature on a see-saw rocking shaker. The clusters / organoids were subsequently washed 3 times in IF buffer then incubated with primary antibody prepared in IF buffer for 24 hours at room temperature on a see-saw rocking shaker; the following primary antibodies were used: mouse anti-MUC5B (1:200, Santa Cruz sc-393952) and rabbit anti-LYZ (1:250, Abcam ab108508.) Clusters / organoids were then washed again 3 times in IF buffer before returning to the shaker to incubate with secondary antibodies (Donkey anti-Mouse IgG Alexa Fluor 488, Invitrogen A21202; Donkey anti-Rabbit IgG Alexa Fluor 594, Invitrogen A21207) prepared in IF buffer with 10% donkey serum for 16 hours at room temperature. To counterstain, DAPI was added directly to the clusters / organoids with secondary antibodies to a final concentration of 2.5 µg / mL and incubated for 20 minutes before washing once with water and once with PBS. Stained clusters / organoids were optically cleared by incubation in 50% methanol in PBS for 1 hour followed by incubation in 100% methanol for 1 hour, rocking samples for both incubations. After clearing, clusters / organoids were resuspended in 50 µL VectaShield Vibrance and transferred to a 96-well glass-bottom plate (Cellvis P96- 1.5H-N) for visualization. In Situ Expansion Phase Wholemount Immunostaining

[0229] To preserve epithelial structures and visualize branching morphogenesis, SMGepithelial aggregates were seeded in 50 µL Cultrex mixed 1:1 with SAECGM supplemented with 1% FBS and 10 µM ROCK inhibitor into 96-well glass-bottom plates, which were then placed in a tissue culture incubator for ~2 hours to allow gelation. For ErbB-family ligand treatments, 150 µL of SAECGM (with 1% FBS and ROCK inhibitor) alone (untreated control) or with HBEGF (100 ng / mL), NRG1 (100 ng / mL), or HBEGF and NRG1 (100 ng / mL each) was added to each well after gelation of the Cultrex domes. For ErbB inhibitor treatments, 150 µL of SAECGM (with 1% FBS and ROCK inhibitor) was added to each well after Cultrex gelation; after three days, media was additionally supplemented with DMSO (vehicle control, 0.1% v / v), Erlotinib (MedChemExpress HY-50896, 10 µM), Tucatinib (MedChemExpress HY-16069, 10 µM), or TX1-85-1 (MedChemExpress HY-100848, 10UCH-39925 µM). In either case, seeded SMG aggregates were cultured for 10 days total, changing media every 3 days. In Situ staining of 3D cultures was performed. Briefly, media was aspirated from each well and 150 µL 4% PFA was added directly onto the Cultrex gel containing epithelial clusters. The cultures were fixed for 15 minutes at room temperature on an orbital shaker then washed thrice in PBS for 10 minutes each. The fixed cultures were then incubated in a permeabilization / blocking buffer (10% donkey serum, 0.5% Triton X-100 in PBS) for 4 hours at room temperature on an orbital shaker, after which, they were incubated 48 hours at 4℃ on an orbital shaker with rabbit anti-KRT5 primary antibody (1:100, Abcam ab52635) diluted in PBS with 10% donkey serum and 1% BSA. The cultures were then washed 3 times in PBS for 15 minutes each on an orbital shaker. Next, cultures were incubated 10 hours on an orbital shaker at room temperature with Donkey anti-Rabbit Alexa Fluor 594 F(ab’)2 secondary antibody (Jackson ImmunoResearch 711-586-152) diluted 1:800 in the same diluent used for the primary antibody. DAPI was subsequently diluted directly into the secondary antibody solution in each well to a final concentration of 2.5 μg / mL, and the cultures were incubated for an additional 2 hours. After, the cultures were washed three times with PBS for 15 minutes each, then mounted in VectaShield Vibrance applied directly to each well. Confocal Microscopy

[0230] Tissue immunohistofluorescence and Expansion / Organoid Phase wholemountimmunostaining images were acquired using a Zeiss LSM880 confocal microscope with a 20X or 10X objective lens, respectively. Maximum intensity projections were created for each image in ZEN (black edition). For figure images, the dynamic range was scaled identically for image sets for each channel in ZEN 3.2 (blue edition), and scale bars were superimposed onto images using ZEN’s Graphics functions. Brightfield Microscopy

[0231] Brightfield micrographs were captured using a Zeiss Axiovert 40 CFLequipped with a 5X objective lens and an AxioCam MRm digital camera. For figure images, the dynamic range was automatically adjusted using “Min / Max” in ZEN 3.2 (blue edition), and scale bars were superimposed onto images using ZEN’s Graphics functions. Brightfield Image QuantificationUCH-39925

[0232] For cultures established from the sorted SAE / SMG populations and theunsorted total SMG isolates, brightfield micrographs were acquired after 14 days in the Expansion Phase or after an additional 21 days in the Organoid Phase. Captured images were converted into 8-bit TIFF files in ZEN 3.2 and the number and area of distinct epithelial clusters (Expansion Phase) or organoids (Organoid Phase) were quantified in FIJI (ImageJ). Four independent fields of view with equivalent dimensions (3.58 mm x 2.68 mm) were quantified for each population at the end of the 14 day Expansion Phase for two separate human donors; five fields of view were quantified for each population at the end of the complete 35 day culture period for the same two donors. In FIJI, individual clusters / organoids were outlined using the polygon selection tool for each field of view; the number of objects and area for each selection was then calculated from the ROI manager. For each population, the mean and standard error of the mean (SEM) of the area across all measured clusters / organoids were plotted in GraphPad Prism; the four populations were then compared against each other using the Kruskal-Wallis test. As a proxy for organoid-forming efficiency, the number of clusters / organoids per mm2was determined by dividing the number of objects by the image area for each field of view. For each population, the mean and SEM of this value across all fields of view were plotted in GraphPad Prism, and the four populations were compared against each other using the Kruskal-Wallis test. Results

[0233] To enable functional evaluation of human SMG epithelium, a tractableorganoid model from total SMG isolates derived from human tracheal and bronchial donor samples was developed. Immunohistochemistry (IHC) for surface markers to identify and sort by specific cell populations was used for FACS. These markers can help distinguish duct basal stem cells, myoepithelial cells, and other populations. The sorted or unsorted cells can then be used to seed organoid culture. To isolate gross SMG epithelial structures, overnight collagenase / hyaluronidase digestion of airway tissue samples that were mechanically depleted of SAE was completed (FIG.21A). Sequential trypsinization and dispase treatments further dissociated these large epithelial fragments into smaller multicellular aggregates (FIG.21B). Evaluation of the resultant SMG epithelial aggregates immediately after this sequential dissociation step revealed that the SMG isolation captured a diversity of epithelial cell types, including duct basal [KRT17+αSMA-], MECs [KRT17+αSMA+], club [CC10+], mucous [MUC5B+], and serous cells [LTF+] (FIG.22A-B). These SMG epithelial aggregatesUCH-39925 were seeded in three-dimensional (3D) droplets of Cultrex basement membrane extract and proceeded through a two-step culture scheme comprising a 3D Expansion Phase and an Organoid Phase (FIG.23A-C). In the Expansion Phase, the cells were cultured in Small Airway Epithelial Cell Growth Medium (SAECGM) to promote replication and maintenance of the isolated SMG stem populations. Subsequently, the cultures were transitioned into SMG Organoid Media to initiate differentiation. During the Expansion Phase, the SMG epithelial cells grew as budded clusters (FIG.24) composed entirely of undifferentiated cells, indicated by KRT14 expression, which demarcates duct basal and MECs within SMG tissue (FIG.20), and lack of mature cell type markers (FIG.25A, FIG.25C). However, αSMA was not detected in these cells, suggesting that any MECs present in the SMG isolate likely acquired a basal-like phenotype in culture, a phenomenon shown in lineage-traced murine MECs grown in vitro. In contrast, cultures transitioned into SMG Organoid Media demonstrated structured reordering of KRT14+cells along the organoid surface with expression of mucous and serous cell markers MUC5B and LYZ in cells internal to this outer KRT14+stem / progenitor layer (FIG.25B, FIG.25D). Overall, human SMG isolates are amenable to expansion of epithelial stem / progenitors with basal-like phenotypes, which may be subsequently differentiated into cells expressing SMG tubuloacinar mucous and serous secretory proteins. Example 4- SMG-Derived Organoids as a Model of Cystic Fibrosis Methods Forsolin swelling assay

[0234] Forskolin induced swelling (FIS) assays were performed by removing SMGorganoids from Matrigel GFR droplets after 14 days of the Organoid Phase, then plated into 96-well plates using 3.5 microliters of undiluted Matrigel GFR per well. Assays were run 24 hours after replating of organoids. All conditions use 6 wells as technical replicates. Organoids were treated with 30 uM Calcein AM (InvitrogenTM) for 45 minutes before placement into an LSM-780 confocal microscope (Zeiss) with live chamber installations. Each individual well of the 96-well plate was then manually set an X / Y / Z plane position before serial dilutions of forskolin were applied. Plates were then scanned for Calcein AM (Thermo Scientific) fluorescence every 20 minutes over 4 hours to detect increases in surface area post forskolin addition.UCH-39925

[0235] Fluorescent images were processed in ZEN Blue to establish binarythresholding based on Calcein AM signal versus devoid areas using ZEN blue's customizable macros. Calcein AM signal was converted to pixel values for each image per 96-well position and scan time interval. All pixel values were then exported into comma separated value files and analyzed using R-shiny, producing averaged by condition kinetic curves of organoid area change over scan time, including area under the curve (AUC) values. Mean AUC values + / - SD were plotted in GraphPad Prism. Each dose of forskolin was compared to either the vehicle control or its respective CFTRi-172 treatment via two-way ANOVA with Tukey’s test. Results

[0236] The SMG tissue transcriptomic data demonstrated CFTR expression in the TAduct and pre-serous cell populations (FIG.8, FIG.26). Accordingly, CFTR expression and function was assessed in the SMG culture model. CFTR expression in both Expansion Phase and SMG Organoid Phase cultures was analyzed via Western Blot (FIG.27). Cultures evaluated after 7 days of Expansion Phase lacked detectable CFTR protein expression, consistent with the absence of CFTR in SMG stem / progenitor populations. In contrast, cultures interrogated after 14 days of SMG Organoid Phase had robust CFTR expression, suggesting that the CFTR+differentiated SMG cell types captured in the tissue transcriptomic dataset are recapitulated in the SMG organoids.

[0237] CFTR-dependent secretory function in SMG organoids was then assessed viathe forskolin-induced swelling (FIS) assay. Briefly, forskolin induces cAMP-dependent phosphorylation of CFTR, opening the CFTR channel to allow chloride ion transport into the organoid lumen. The resultant osmotic gradient drives the transepithelial flow of water into the organoid, causing swelling. Forskolin treatment induced SMG organoid swelling in a dose-dependent manner compared to untreated controls (FIG.28), indicating functional CFTR protein expression in the organoids. Notably, examination of individual organoids imaged over the duration of the assay revealed that only a subset of SMG organoids exhibited swelling in response to forskolin (FIG.29), suggesting heterogeneity in cellular composition or differentiation. Further, the FIS response was diminished by the CFTR inhibitor, CFTRi- 172, congruent with CFTR-dependent function (FIG.28, FIG.29). Taken together, the human SMG epithelial organoid model has tissue-specific secretory features that can be utilized to further our understanding of the SMG proper. In addition, the SMG organoids hadUCH-39925 functional expression of CFTRs indicating that they would be an effective model to test the impact of various treatments on CFTR activity, which is relevant for cystic fibrosis. Example 5- Enhanced Regenerative Potential of SMG Duct Basal Cells Compared to MECs in Organoid Culture

[0238] Following the generation of SMG organoids from tissue aggregates, organoidswere established from each of the stem / progenitor populations spanning the airway surface and SMGs, namely, SAE basal, duct basal, and MECs, to compare their regenerative potential. Specifically, each population was evaluated for its capacity to generate organoids from single-cell suspensions, self-renew and propagate, and differentiate into distinct lineages. To isolate SAE basal cells, the surface epithelium of patient-matched airway tissue was enzymatically stripped, dissociated and sorted using established SAE basal markers NGFR and TROP2.

[0239] To separate the two SMG stem / progenitor populations,immunohistofluorescence candidate surface markers were identified that could be leveraged for fluorescence-activated cell sorting (FACS) (FIG.30A-C). NGFR (FIG.30A) and EGFR (Fig.3B) were predominantly expressed on basal cells in the duct and MECs in the tubuloacinar compartment, whereas ITGA2 (FIG.30C) had much stronger expression in the MECs compared to duct basal cells.

[0240] Thus, NGFR and EGFR could together be utilized to isolate both duct basaland MECs via FACS, and ITGA2 could be used to further separate these two populations. Accordingly, this marker panel was implemented to perform FACS on SMG isolates derived from patient-matched tissue from which the SAE basal cells were isolated, first gating the NGFR+EGFR+double-positive cells (FIG.31A). Interestingly, within the ITGA2+cells in this gated population, ITGA2 expression was positively correlated with NGFR expression (FIG.31B, right side of dotted line). Therefore, the NGFR+EGFR+gated population were sorted (FIG.31A) based on high (HI) versus low (LO) expression of both ITGA2 and NGFR (i.e., NGFRHIITGA2HIvs. NGFRLOITGA2LO) (FIG.31B). Immediately after sorting, cellular identities were interrogated via cytocentrifugation with immunofluorescent staining and the NGFRHIITGA2HIpopulation was found to be highly enriched for MECs [KRT17+KRT14+ACTA2+] (FIG.32A). Conversely, the NGFRLOITGA2LOpopulation was primarily enriched for duct basal cells [KRT17+KRT14+ACTA2-] but contained a fraction ofUCH-39925 lineage-negative cells and rare MECs (FIG.32B), indicating that the sorting strategy successfully isolated and enriched the two SMG stem / progenitor populations.

[0241] After sorting, the following populations were seeded as single cells atequivalent densities to initiate Expansion Phase cultures: unsorted total SMG (parent population for sorted SMG cells), NGFRHIITGA2HISMG (MEC-enriched), NGFRLOITGA2LOSMG (duct basal-enriched), and NGFR+TROP2+SAE basal cells (FIG. 33). Established cultures proceeded through 14 days of Expansion Phase followed by 21 days of Organoid Phase. At the end of each phase, cultures were examined by brightfield microscopy to compare growth characteristics across the seeded populations (FIG.34). Notably, NGFRHIITGA2HIcells produced fewer 3D clusters in Expansion Phase (FIG.35A) and fewer Organoids in Organoid Phase (FIG.35B) compared to the other sorted populations, suggesting a limited competency for self-renewal and propagation. Conversely, NGFR+TROP2+SAE basal cells had the highest cluster / organoid-forming efficiencies of the sorted stem / progenitor populations. During the Expansion Phase, all four populations were morphologically indistinguishable, presenting as budded clusters (FIG.34) comparable in size across each population (FIG.35A). In contrast, NGFRHIITGA2HIcells in the Organoid Phase generated organoids that were significantly larger than those derived from the other seeded populations (FIG.35B). Moreover, organoids derived from NGFRLOITGA2LOcells more closely resembled those established from NGFR+TROP2+SAE basal cells (FIG.34) both in appearance and size (FIG.35B). Interestingly, cultures derived from total SMG isolates infrequently contained large, distinct organoids (FIG.34) that resembled those generated by the NGFRHIITGA2HIcells, suggesting these organoids were produced by MECs present in the SMG isolate. However, the majority of the organoids derived from total SMG isolates were also similar in appearance and size to those generated by the NGFRLOITGA2LOcells (FIG.35B). This discrepancy suggests that duct basal cells either outnumbered MECs in the initial seeding or outcompeted MECs during culture, consistent with the observed difference in cluster / organoid-forming efficiency between NGFRHIITGA2HIand NGFRLOITGA2LOcells. Overall, these data suggest that SAE basal cells have a greater capacity for reestablishment of epithelial structures, consistent with a higher turnover rate of the surface epithelium in tissue. Moreover, in this capacity, duct basal cells represent an intermediate between SAE basal and MECs.UCH-39925 Example 6- SMG Organoids Derived from Myoepithelial Cells are Biased Towards Differentiation of Secretory Lineages Methods Single Cell Sequencing of SAE and SMG Expansion / Organoid Cultures

[0242] Cultures derived from sorted SAE, sorted SMG, and unsorted total SMGisolates were recovered from Cultrex after either 14 days in the Expansion Phase or after a subsequent 21 days in the Organoid Phase and dissociated to single cell suspensions via TrypLE with intermittent trituration. For samples collected after the Expansion Phase, Cultrex domes were pooled together and triturated 10 times in TrypLE with a 1 mL pipette, followed by a 25 minute incubation at room temperature with trituration every 5 minutes. Afterward, samples were diluted in adDMEM / F12++ and centrifuged at 300xg for 5 minutes at 4℃. The resultant single cell suspensions were then fixed in 4% PFA and stored at -80℃ prior to preparation of sequencing libraries using the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit according to manufacturer instructions. The instructions can be found on the World Wide Web at the website identified by placing https: / / before 10xgenomics.com / support / single-cell-gene-expression-flex / documentation / steps / sample- prep / fixation-of-cells-and-nuclei-for-chromium-single-cell-gene-expression-flex. Samples collected after the Organoid Phase were handled identically with the following exceptions: samples were incubated in a 37℃ water bath for the TrypLE dissociation and filtered through a 40 µm cell strainer after dilution in adDMEM / F12++. Fixed frozen samples were delivered to UCLA Technology Center for Genomics and Bioinformatics (TCGB) for sequencing library construction following the Chromium Fixed RNA Profiling Reagent Kits user guide for multiplexed samples. The user guide can be found on the World Wide Web at the website identified by placing https: / / before 10xgenomics.com / support / single-cell-gene-expression- flex / documentation / steps / library-prep / chromium-single-cell-gene-expression-flex-reagent- kits-for-multiplexed-samples). Libraries were sequenced as 50 base-pair paired-end reads on a NovaSeq X Plus instrument (Illumina). Single-Cell Sequencing Processing and Analysis

[0243] Single cell libraries were mapped to the human reference genome GRCh38using CellRanger 7.1.0 and processed and analyzed in R using the library Seurat for normalization, dimensionality reduction, scaling, and clustering. Cells were filtered for atUCH-39925 least 500 reads and genes were filtered for expression in at least 10 cells per library and 5% of cells were excluded as putative doublets using the DoubletFinder library. Cells with more than 12.5% expression from mitochondrial genes were removed. Louvain clusters and differential expression from the Seurat FindClusters and FindAllMarkers function were used to characterize cell types. Cell subtypes were further informed using gene-gene clustering; Genes were clustered using correlation to form groups.

[0244] The script for this correlation grouping can be found on the World Wide Webat the website identified by placing https: / / before github.com / Teneth / GEND_Script. Ligand- receptor pairs enriched in the dataset were found using the iTalk package. Data was displayed using the pheatmap and ggplot libraries. Gene groups were displayed by calculating the average expression value for the set of genes in each cell. Results

[0245] Next, single-cell RNA sequencing was performed on cultures derived fromeach of the seeded populations represented in FIG.33 to assess maintenance of cells with stem / progenitor characteristics and evaluate differentiation potential. Cultures were collected at two timepoints for profiling: half of each sample was collected after 14 days at the end of the Expansion Phase and the remaining half was transitioned into Organoid Phase and collected after 21 days. To identify unique subsets of cells (FIG.36), marker genes were used to determined networks of correlated gene expression and their enrichment was evaluated across the dataset (FIG.37). In the Expansion Phase, two major epithelial cell types were distinguished: basal and secretory. The Expansion Phase basal cells (EP Basal) were defined by expression of basal markers KRT5, KRT14, KRT17, and TP63 (FIG.38). For the basal cell types, three subsets were resolved: EP Basal 1, EP Basal 2, and EP Basal 3. The EP Basal 1 subset is characterized by networks with cell cycle ontology and associated transcripts, such as TOP2A, MKI67, and CDK1 (FIG.39, FIG.40A). The EP Basal 2 subset is dominated by networks related to extracellular matrix organization and adhesion, expressing transcripts encoding various laminin, collagen, and integrin subunits (FIG.39, FIG.40B). The EP Basal 3 subset enriches for the network defined by keratinization ontology and gene correlates KRT16, DSG3, and SFN, suggesting a squamous or hillock cell-like phenotype (FIG.39, FIG.40C). The Expansion Phase secretory cells (EP Secretory) comprise a single subset with enrichment for networks with features related to club and serous cell secretion, including expression of PIP, DEFB1, and CEACAM6 and ontology related to neutrophil degranulation,UCH-39925 xenobiotic metabolism, and innate immunity (FIG.39, FIG.40D). Additionally, a population of fibroblasts was identified that persisted across both phases of culture (i.e., fibroblast transcriptomes were captured at both collection timepoints), which express PDGFRB and DCN and enrich for networks related to secretion of extracellular matrix components (FIG.39, FIG.40E).

[0246] To assess the composition of cultures derived from each of the seededpopulations in FIG.33, the proportion of single cell transcriptomes derived from each population was analyzed at the end of the Expansion Phase comprising each of the annotated subsets in FIG.36, which is reported as the percent distribution across each annotated subset on a per sample basis (FIG.41). Notably, the presence of the EP Secretory subset is negligible in all samples except cultures established from the unsorted total SMG, likely deriving from secretory cells present in the initial isolate that were maintained in culture. Similarly, the fibroblast subset is unique to the total SMG sample, in agreement with observations made by brightfield microscopy during culture (FIG.34). Across all samples, however, the majority of cells profiled at the end of the Expansion Phase are basal-like stem / progenitors, consistent with outgrowth of undifferentiated epithelial clusters during this phase. Together, these data demonstrate that the seeded populations converge to a similar basal-like phenotype in culture, with the most overt difference being the proportion of transcriptomes with detectable cell cycle gene signatures (i.e., EP Basal 1 vs. EP Basal 2).

[0247] Utilizing the same approach to identify cell subsets, four major epithelial celltypes were identified in the Organoid Phase: basal, secretory, ciliated, and rare / ionocyte-like. Similar to the EP Basal cells, the Organoid Phase basal (OP Basal) subsets were unified by expression of KRT5 and TP63 (FIG.38). Based on enrichment of gene networks, we further classified the OP Basal cells into four subsets. The OP Basal 1 subset is defined by networks correlated to BCAM and NGFR expression and corresponding ontology related to epithelial development (FIG.38, FIG.42A). The OP Basal 2 subset exhibits enrichment for genes / networks related to DNA replication and cell cycle (FIG.38, FIG.40A). The OP Basal 3 subset, like EP Basal 3, is characterized by enrichment of genes / networks related to keratinization and epithelial stratification (FIG.38, FIG.40C). The OP Basal 4 subset is enriched for networks with ontology related to regulation of secretion and the Wnt signaling pathway (FIG.42A-B). Additionally, this basal subset is characterized by expression of CXCL14 and LGR6 transcripts (FIG.38); together with the Wnt signaling ontology, these features are consistent with a subset of basal cells in airway organoids which are transitioningUCH-39925 to rare cell types (i.e., tuft, ionocyte, and pulmonary neuroendocrine cells). Accordingly, the rare / ionocyte-like cells (OP Rare / Ionocyte) comprise a single subset enriched for networks with correlated expression of FOXI1, POU2F3, CFTR, and HEPACAM2, indicating these cells are differentiating ionocytes (FIG.38, FIG.42C). Moreover, this subset exhibits the highest correlation to the ionocyte population identified in the SMG tissue transcriptomic dataset, based on comparison of differential gene expression profiles (FIG.43). The OP secretory cells were further resolved into three subsets. OP Secretory 1 exhibits expression of mixed secretory cell type markers, such as PIGR, CEACAM6, WFDC2, BPIFB1, and MUC16 (FIG.38), and enrichment for networks with ontologies corresponding to humoral immune response, ion transport, neutrophil degranulation, and secretion, consistent with club cell identity (FIG.42D). Further, this subset shows comparable correlation to both the club and mucous populations from the SMG tissue (FIG.43). The OP Secretory 2 subset shows overlapping enrichment for the same secretory networks as OP Secretory 1 and networks related to cell division, suggesting these are dividing secretory cells (FIG.38, FIG.40A, FIG.42D). The OP Secretory 3 subset is characterized by the network expressing TFF3 and MUC5B and represents the secretory subset with the most distinct mucous identity (FIG.38, FIG.42E). The ciliated cells (OP Ciliated) also constitute a single subset, defined by FOXJ1 expression and ontology related to cilium assembly and movement (FIG.38, FIG.42F).

[0248] After establishing cell subsets within the transcriptomic data, the proportion ofcell transcriptomes belonging to each of the seeded populations in FIG.33 present in the identified subsets (FIG.44) was assessed. Notably, organoids derived from theNGFRHIITGA2HI MEC-enriched cells are predominantly skewed towards secretory fates,particularly OP Secretory 1, at the expense of basal cell maintenance. In contrast, the other seeded populations exhibit similar proportions across the identified subsets, with the majority of profiled cells distributed amongst the OP Basal subsets. Moreover, all three sorted populations are competent to produce ciliated and rare / ionocyte-like cell types, albeit infrequently. Taken together, the sorted cells populations demonstrate a comparable breadthof multipotency in organoid culture; however, the NGFRHIITGA2HI MEC-enriched cells havea diminished capacity for self-renewal, instead preferentially generating secretory progeny. Example 7- ERBB-Family Receptors are Present in the SMG

[0249] In the proximal, cartilaginous airways, glandular structures are embeddedwithin the underlying submucosa and are contiguous with the surface airway epitheliumUCH-39925 (SAE) via gland ducts. Submucosal glands (SMGs) produce mucous and antimicrobial secretions that contribute to clearance of pathogens and particulate matter from the airways. SMGs are implicated in hypersecretory disorders, which are typified by glandular hypertrophy and hyperplasia.

[0250] The SMG niche consists of immune, mesenchymal, and endothelial cell typesthat likely interact with the SMG epithelium to regulate tissue maintenance and differentiation. Maintenance of the airway epithelium relies on regional populations of stem and progenitor cells within defined anatomical compartments. There are multiple stem / progenitor cell populations within the mouse SMGs, namely myoepithelial cells (MECs) and duct basal cells.

[0251] To understand these cues from the SMG niche, the transcriptomic datasetderived from the primary airway tissue was leveraged to interrogate cognate receptor-ligand pairs. Single-cell mRNA sequencing (scRNA-seq) was used to profile gene expression of human airway submucosa derived from the intercartilaginous zone of donor or research-grade tracheae and bronchi. Single-cell trajectories and receptor-ligand interaction were analyzed via the STREAM pipeline and iTALK package, respectively. Because robust expression of EGFR was observed in the SMG epithelia (FIG.30B), signaling mediated by ErbB receptors, the family of receptor tyrosine kinases which includes EGFR, was interrogated. Predicted receptor-ligand interactions indicated signaling via multiple ligands of ErbB receptors, including HBEGF, NRG1, AREG, EREG, and EGF (FIG.45A-E). Protein expression of these ErbB-family ligands was next evaluated in the SMG tissue to determine if they had a specific spatial distribution in the SMG niche. Within the tubuloacinar structures of the SMGs, HBEGF is highly expressed in KRT17+αSMA+MECs (FIG.46A), whereas NRG1 is present within the luminal compartment, in both MUC5B+mucous tubule cells and surrounding serous cells (FIG.46B). Moreover, EREG was highly enriched in LTF+serous cells (FIG.46C), while EGF exhibited broader distribution across the epithelia, though expression appeared restricted to the basolateral surfaces in MUC5B+mucous tubule cells (FIG.46D). These data demonstrated a differential localization of these ligands within the tubuloacinar epithelium. Example 8- ERBB-Family Ligands Promote Branching Morphogenesis and Secretory Differentiation in SMG Organoids MethodsUCH-39925 Treatment with ERBB-Ligands

[0252] Cultures were generated according to the methods in Example 3. For ERBB-family ligand treatments, expansion media was supplemented with HBEGF (PeproTech 100- 47, 100 ng / mL) and NRG1 (PeproTech 100-03, 100 ng / mL), and differentiation media was supplemented with EGF (PeproTech AF-100-15, 100 ng / mL). Organoid cultures were maintained in expansion media for 7 days before directly transitioning to differentiation media; i.e., media was exchanged on established cultures without passaging at the onset of differentiation in order to preserve branched structures formed during expansion. Organoids were differentiated for 7 days, at which point wells were collected for analysis or passaged into Cultrex mixed 1:1 with differentiation media either with or without EGF to continue differentiation for an additional 14 days.

[0253] Table 3. Growth Factors / Inhibitors and concentrations used in SMG organoidculturesIn Situ Expansion Phase Wholemount Immunostaining

[0254] To preserve epithelial structures and visualize branching morphogenesis, SMGepithelial aggregates were seeded in 50 µL Cultrex mixed 1:1 with SAECGM supplemented with 1% FBS and 10 µM ROCK inhibitor into 96-well glass-bottom plates, which were then placed in a tissue culture incubator for ~2 hours to allow gelation. For ErbB-family ligand treatments, 150 µL of SAECGM (with 1% FBS and ROCK inhibitor) alone (untreated control) or with HBEGF (100 ng / mL), NRG1 (100 ng / mL), or HBEGF and NRG1 (100 ng / mL each) was added to each well after gelation of the Cultrex domes. For ErbB inhibitor treatments, 150 µL of SAECGM (with 1% FBS and ROCK inhibitor) was added to each well after Cultrex gelation; after three days, media was additionally supplemented with DMSO (vehicle control, 0.1% v / v), Erlotinib (MedChemExpress HY-50896, 10 µM), TucatinibUCH-39925 (MedChemExpress HY-16069, 10 µM), or TX1-85-1 (MedChemExpress HY-100848, 10 µM). In either case, seeded SMG aggregates were cultured for 10 days total, changing media every 3 days. In Situ staining of 3D cultures was performed. Briefly, media was aspirated from each well and 150 µL 4% PFA was added directly onto the Cultrex gel containing epithelial clusters. The cultures were fixed for 15 minutes at room temperature on an orbital shaker then washed thrice in PBS for 10 minutes each. The fixed cultures were then incubated in a permeabilization / blocking buffer (10% donkey serum, 0.5% Triton X-100 in PBS) for 4 hours at room temperature on an orbital shaker, after which, they were incubated 48 hours at 4℃ on an orbital shaker with rabbit anti-KRT5 primary antibody (1:100, Abcam ab52635) diluted in PBS with 10% donkey serum and 1% BSA. The cultures were then washed 3 times in PBS for 15 minutes each on an orbital shaker. Next, cultures were incubated 10 hours on an orbital shaker at room temperature with Donkey anti-Rabbit Alexa Fluor 594 F(ab’)2 secondary antibody (Jackson ImmunoResearch 711-586-152) diluted 1:800 in the same diluent used for the primary antibody. DAPI was subsequently diluted directly into the secondary antibody solution in each well to a final concentration of 2.5 μg / mL, and the cultures were incubated for an additional 2 hours. After, the cultures were washed three times with PBS for 15 minutes each, then mounted in VectaShield Vibrance applied directly to each well. RNA Extraction and cDNA Synthesis

[0255] Total RNA was extracted from SMG Organoids using Norgen Total RNAPurification Kit (Norgen Biotek 37500) following manufacturer’s instructions. Following RNA extraction, samples were subjected to DNAse I treatment in-solution (Norgen Biotek 25710) prior to column-based clean-up and concentration (Norgen Biotek 23600.) cDNA was prepared using Transcriptor High Fidelity cDNA Synthesis Kit (Roche 5081955001) according to manufacturer’s directions. qPCR

[0256] Gene expression was assessed by quantitative RT-PCR using TaqMan FastAdvanced Master Mix (Applied Biosystems 4444557) and predesigned TaqMan Gene Expression Assays targeting human genes (Applied Biosystems 4331182 (FAM, target genes) and 4448485 (VIC_PL, endogenous control)): MUC5B (Hs00861595_m1), LYZ (Hs00426232_m1), LTF (Hs00914334_m1), and RNA18S5 (Hs03928985_g1). qPCR wasUCH-39925 performed using the QuantStudio 5 instrument (Applied Biosystems) with default settings for the Relative Quantification Analysis Module using TaqMan probes and Fast Run Mode. Relative gene expression was evaluated for ErbB-family ligand-treated SMG cultures derived from three independent donors, with three technical replicates for each TaqMan Assay. Relative quantification (RQ) fold change was averaged over the three donor samples and plotted as mean with standard deviation (SD) in GraphPad Prism. Two-way ANOVA followed by Dunnett’s multiple comparisons test was used to compare treatment groups to CTRL / CTRL (Expansion + Organoid Phase untreated control) organoids. Results

[0257] The impact of identified ligand interactions was assessed using the novelhuman SMG epithelial organoid model. This scRNA-seq dataset captures a diverse collection of epithelial, mesenchymal, immune, and endothelial lineages comprising a multitude of cell types and states, including multiple distinct fibroblast and secretory populations and divergent transitioning epithelial precursors. Interrogation of the human airway submucosa reactome via receptor-ligand analysis revealed that activation and differentiation of SMG stem / progenitor cells is mediated by ERBB family members, namely EGFR and ERBB3.

[0258] After sorting, the cells were then grown in organoid culture and scRNA-seqwas performed on the organoids in both expansion and differentiation phases. Expression around two receptor ligands in the ERBB family (HBEGF and AREG) was therefore further analyzed. To explore the effect of these ErbB-family ligands on SMG epithelium, the SMG culture system was used to screen ligands individually and in combination. Treatment with ERBB-family ligands during the Expansion Phase

[0259] Initially, cultures were treated during the Expansion Phase (FIG. 47) toascertain if these ligands could influence growth patterns or drive precocious differentiation. Accordingly, 3D structures and cell identity were assessed via whole mount immunostaining in treated cultures. Whereas control cultures grew as typical budded clusters of KRT5+stem / progenitor cells, unique growth behavior was observed in cultures supplemented with either HBEGF or NRG1 (FIG.47). Specifically, HBEGF stimulated branching while NRG1 augmented growth and lumen formation in budded clusters (FIG.48A). Interestingly, HBEGF and NRG1 together produced a combinatorial effect, promoting branching morphogenesis concomitant with enhanced lumen formation and radial growth of terminalUCH-39925 buds (FIG.47, FIG.48A-D). Moreover, these expansion phase treatments maintained undifferentiated stem / progenitors, indicated by robust KRT5 expression (FIG.47).

[0260] To address the dispensability of the receptors mediating these ligandinteractions, cultures were treated with ErbB inhibitors, namely, the EGFR inhibitor Erlotinib, ERBB2 (the preferred heterodimerization partner of all other ErbB receptors) inhibitor Tucatinib, and ERBB3 inhibitor TX1-85-1. Conversely, treating the cultures with ERBB inhibitors (e.g., erlotinib or TX1-85-1) prevented the organoids from developing thebudding and branching features (FIG. 49A-B). This indicates that activation of ERBB, eitherdirectly or via the addition of its ligands HBEGF and NRG1, is important for creating SMG organoids with budding and branching features. These data helped illuminate that treating the organoids during expansion with the ligands HBEGF and NRG1 would increase budding and branching features associated with SMG hyperplasia and hypertrophy. The SMG organoids undergo branching morphogenesis, mimicking development in the body. This is a unique form of patterning that occurs in several organs during organogenesis and is regulated by developmental signals. The budding is the appearance of the acinar / grape-like structures that is the hallmark of the SMG glandular structure. During expansion of human SMG epithelial organoids, HBEGF and NRG1 promote branching morphogenesis of undifferentiated stem / progenitor cells. Conversely, treatment of SMG organoids with EGFR or ERBB3 inhibitors results in attenuated growth. Treatment with ERBB-family ligands during the Organoid Phase

[0261] The ErbB-family ligands from FIG. 45A-E were screened during theOrganoid Phase (FIG.50A, top bracket) to evaluate their effect on differentiation outcomes. Specifically, the ability of ligand treatments were assessed for their ability to modulate expression of mucous marker MUC5B and serous markers LYZ and LTF in organoids, through which two treatment conditions were identified: 1) EGF and 2) HBEGF+EREG.

[0262] This morphological change (increase budding and branching features) wascorrelated with changes in expression of key genes, as measured by RT-qPCR. Treatment with EGF throughout the Organoid Phase significantly increased MUC5B and LYZ gene expression and the proportion of MUC5B+and LYZ+cells compared to untreated control cultures, whereas treatment with HBEGF+EREG resulted in a similar increase in LYZ+cells and LYZ expression with fewer MUC5B+cells and less MUC5B expression (FIG.50B, FIG. 51A-C). Hence, EGF promotes secretory differentiation with a marked increase in mucinUCH-39925 output, whereas the combination of HBEGF and EREG preferentially increases the number of LYZ-expressing cells, suggesting a bias towards serous differentiation. However, neither treatment was sufficient to stimulate LTF expression, indicating incomplete recapitulation of serous cell characteristics. The expression and secretion of lactoferrin (LTF) and lysozyme (lysozyme) are unique to SMGs, as these products are not expressed on the surface airway epithelium. Therefore, this expression confirmed that the organoids are truly SMGs, and not surface airway epithelium cultures. Expression changes were confirmed with IF staining.

[0263] Based on ligand expression in SMG tissue and the morphological effectsobserved during the Expansion Phase treatment, it was hypothesized that HBEGF and NRG1 may pattern tubuloacinar fate specification. Accordingly, the Expansion Phase treatment with HBEGF+NRG1 was followed by the Organoid Phase treatment conditions, EGF or HBEGF+EREG (FIG.50A, bottom bracket). Notably, the HBEGF+NRG1 Expansion Phase treatment followed by either EGF or HBEGF+EREG treatment during the Organoid Phase upregulated LTF expression, with the latter condition (HBEGF+EREG) producing the greatest increase in LTF (FIG.51B). Moreover, the ratio of LYZ+to MUC5B+cells was increased in these two conditions relative to matched Expansion Phase controls (FIG.51A). Taken together, these results suggest that HBEGF+NRG1 treatment during the Expansion Phase followed by sustained ErbB signaling during the Organoid Phase through EGF or HBEGF+EREG further actualizes serous cell differentiation. Overall, ErbB-family ligands are capable of regulating growth and differentiation of the SMG epithelium. The cartilaginous airway basal epithelial compartment comprises a continuum of stem / progenitor cells. Basal cells of the SMG duct are uniquely positioned between the airway surface and SMG terminal structures; these cells seemingly constitute a stem / progenitor population with broad multipotency and capacity to regenerate both surface and SMG epithelia. Mobilization and differentiation of these cells is regulated by ERBB signaling.

[0264] The present disclosure identifies several advances in understanding andengineering of submucosal glands. Single cell RNA seq of human submucosal glands and their surrounding cells was performed, identifying stem and progenitor cells and their receptor-ligand interactions. Pathways that regulate submucosal gland (SMG) hypertrophy and hyperplasia were identified, including some compounds that regulate gland development and mucus production. A method for FACS was developed to separate out the stem and progenitor cells and culture them in the dish. It has previously not been possible to effectively isolate SMG stem cells and grow SMGs organoids in the dish.UCH-39925

[0265] Small molecules are used to activate and inhibit the signaling pathwaysidentified from single cell RNA sequencing. These compounds promoted extreme branching and budding of the SMG organoids with excess mucus production. Other compounds prevented branching and budding and reduced mucus production. These compounds all have potential clinical applications to modulate mucus production in the airway in patients. The compounds all target EGFR signaling via ERBB family members and regulate SMG branching. budding and mucus production.

[0266] It was found that ERBB-family ligands drive SMG stem / progenitormorphogenesis and differentiation. These ERBB factors can therefore cause SMG hyperplasia and hypertrophy (i.e., can make larger and more complex SMG organoids). Because SMG hyperplasia and hypertrophy is classically seen in many mucus diseases like COPD and CF, this discovery indicates that manipulating the SMG organoids via ERBB- family ligands can enable the creation of organoids relevant to disease models. SMG hyperplasia and hypertrophy also occurs after a viral infection or smoke exposure and can regress after the infection / exposure has been cured. OTHER EMBODIMENTS

[0267] It will be appreciated that the scope of the present disclosure is to be definedby that which may be understood from the disclosure and claims rather than by the specific embodiments that have been presented by way of example. Elements described with respect to one aspect or embodiment of the present disclosure are also contemplated with respect to other aspects or embodiments of the present disclosure. Moreover, recitation of claim elements in connection with a particular independent claim support recitation of such elements in connection with other independent claims. Throughout the disclosure and claims, where compositions or methods are described as having, including, or comprising specific elements, compositions that consist essentially of, consist of, or do not comprise the recited elements are likewise hereby disclosed. INCORPORATION BY REFERENCE

[0268] All publications, patents, and patent applications mentioned herein are herebyincorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

Claims

UCH-39925 CLAIMS WHAT IS CLAIMED IS:

1. A method of engineering a submucosal gland (SMG) organoid ex vivo, the method comprising: (i) obtaining a population of cells from the proximal airway tissue of a subject; (ii) isolating from the obtained population of cells (a) submucosal gland basal cells and / or myoepithelial cells, and / or (b) submucosal gland stem and / or progenitor cells; (iii) culturing the isolated cells in an expansion media comprising one or more growth agents promoting branching and / or budding morphogenesis; and (iv) culturing the isolated cells in a differentiation media comprising one or more agonists or ligands of ERBB; thereby engineering an SMG organoid ex vivo.

2. The method of claim 1, wherein the isolating in step (ii) comprises isolating stem and / or progenitor cells that express on the cell surface TROP2, NGFR, EGFR and / or ITGA2.

3. The method of any one of claims 1-2, wherein the one or more growth agents promoting branching and / or budding morphogenesis comprise HBEGF and / or Heregulinβ-1 (NRG1); optionally wherein the expansion media comprises (a) HBEGF at a concentration of 10-1000 ng / mL or about 100 ng / mL and (b) NRG1 at a concentration of 10-1000 ng / mL or about 100 ng / mL.

4. The method of any one of claims 1-3, wherein the one or more agonists or ligands of ERBB comprise EGF; optionally wherein the differentiation media comprises EGF at a concentration of 10-1000 ng / mL or about 100 ng / mL.

5. The method of any one of claims 1-4, wherein the ERBB is ERBB3.

6. The method of any one of claims 1-5, wherein the culturing in the differentiation media in step (iv) is for at least 5 or 7 days, is for between 5 and 14 days, or is for between 5 and 21 days.UCH-39925 7. The method of any one of claims 1-6, wherein the expansion media further comprises amphiregulin (AREG) and / or epiregulin (EREG).

8. The method of any one of claims 1-7, wherein the culturing in the expansion media in step (iii) is for at least 5 or 7 days, or is between 5 and 14 days, optionally without cell passaging.

9. The method of any one of claims 1-8, wherein the culturing in step (iii) and / or (iv) is in a three-dimensional matrix.

10. The method of claim 9, wherein the three-dimensional matrix is a hydrogel (e.g., Matrigel or Culturex).

11. The method of any one of claims 1-10, wherein the proximal airway tissue is derived from the intercartilaginous zone tracheae or bronchi.

12. The method of any one of claims 1-10, wherein the proximal airway tissue comprises cells of cartilaginous airway basal epithelia.

13. The method of any one of claims 1-10, wherein the proximal airway tissue comprises submucosal gland duct basal cells and / or myoepithelial cells.

14. The method of any one of claims 1-13, wherein the isolating submucosal gland stem and / or progenitor cells in step (ii) comprises mechanically manipulating the proximal airway tissue to deplete surface epithelium, exposing the depleted tissue to one or more enzymes to digest extracellular matrix, and straining the exposed tissue with one or more markers needed to isolate the submucosal gland stem and / or progenitor cells.

15. The method of any one of claims 1-14, wherein the isolating submucosal gland stem and / or progenitor cells in step (ii) comprises fluorescence-activated cell sorting (FACS).

16. The method of any one of claims 1-15, wherein the submucosal gland stem and / or progenitor cells are TROP2+, NGFR+, EGFR+, and ITGA2+.UCH-39925 17. The method of any one of claims 1-16, wherein cells of the SMG organoid express mucin-5B (MUC5B), lysozyme (LYZ), and / or lactoferrin (LTF), and / or wherein the SMG organoid comprises branching and / or budding morphogenetic characteristics of in vivo SMG.

18. The method of any one of claims 1-17, wherein the subject is a human.

19. The method of any one of claims 1-18, wherein the SMG organoid exhibits hypertrophy, hyperplasia, and / or increased mucus production, optionally wherein the SMG organoid exhibits physical and / or functional characteristics of a hypersecretory airway disorder or disease.

20. An SMG organoid engineered using the method of any one of claims 1-19.

21. An SMG organoid viable ex vivo comprising cells expressing MUC5B, LYZ, and / or LTF in a three-dimensional matrix, wherein the SMG organoid comprises branching and / or budding morphogenetic characteristics of in vivo SMG.

22. The SMG organoid of claim 21, wherein the three-dimensional matrix is a hydrogel.

23. The SMG organoid of claim 21 or 22, which expresses CFTR.

24. The SMG organoid of any one of claims 21-23, which exhibits hypertrophy, hyperplasia, and / or increased mucus production, optionally wherein the SMG organoid exhibits physical and / or functional characteristics of a hypersecretory airway disorder or disease.

25. A method of culturing the ex vivo SMG organoid of any one of claims 21-23 in a media effective to induce hypertrophy or hyperplasia of the ex vivo SMG organoid.

26. The method of claim 25, wherein the media comprises one or more agonists or ligands of ERBB, optionally wherein the one or more agonists or ligands of ERBB comprise EGF, optionally wherein the ERBB is ERBB3.UCH-39925 27. The method of claim 25 or 26, wherein the culturing results in overexpression of CFTR.

28. An ex vivo SMG organoid produced using the method of any one of claims 25-27.

29. A method of identifying an agent that reduces airway mucus production, the method comprising: (i) culturing the ex vivo SMG organoid according to any one of claims 25-27 or using the ex vivo SMG organoid of any one of claims 19, 24 or 28; (ii) administering one or more agents to the ex vivo SMG organoid; and (iii) assessing whether the one or more agents reduce the production of mucus in the ex vivo SMG organoid; if any agent of the one or more agents reduce the production of mucus, the agent is identified as an agent that reduces mucus production.

30. The method of claim 29, wherein the one or more agents are agents known or expected to inhibit ERBB.

31. A method of treating a hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands, in a subject in need thereof, the method comprising administering to the subject an agent that inhibits ERBB.

32. The method of claim 30 or 31, wherein the agent that inhibits ERBB is a gene therapy, a small molecule, or a protein.

33. The method of any one of claims 30-32, wherein the agent that inhibits ERBB is an agent that inhibits EGFR, ERBB2, and / or ERBB3, optionally wherein the agent is an agent that inhibits ERBB3.

34. The method of claim 33, wherein the agent that inhibits ERBB is TX1-85-1, tucatinib or erlotinib.

35. The method of claim 34, wherein the agent that inhibits ERBB is an agent that reduces mucus production in accordance with claim 29 or 30.UCH-39925 36. The method of any one of claims 31-35, wherein the hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands, is cystic fibrosis, asthma, bronchitis (optionally chronic bronchitis), chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), COPD / emphysema, increased airway secretion associated with a microbial infection (optionally viral, bacterial, or fungal infection), an increased airway secretion associated with smoking or second hand smoking, an increased airway secretion associated with an allergy or an increased airway secretion associated with an environmental exposure.

37. The method of claim 36, wherein the environmental exposure is wildfire smoke, air pollution, burn pit exposures, particulate matter, chemicals, or gases.

38. The method of any one of claims 31-37, wherein the subject is a human.

39. A composition comprising an agent that inhibits ERBB for treating a subject having a hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands; optionally wherein the agent is TX1-85-1, tucatinib or erlotinib.

40. A use of an agent that inhibits ERBB for treating a subject having a hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands; optionally wherein the agent is TX1-85-1, tucatinib or erlotinib.

41. The composition of claim 39 or the use of claim 40, wherein the hypersecretory airway disorder or disease, or a disorder associated with an increased secretion from submucosal glands, is cystic fibrosis, asthma, bronchitis (optionally chronic bronchitis), chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), COPD / emphysema, increased airway secretion associated with a microbial infection (optionally viral, bacterial, or fungal infection), an increased airway secretion associated with smoking or second hand smoking, an increased airway secretion associated with an allergy, or an increased airway secretion associated with an environmental exposure.UCH-39925 42. The composition of claim 41, wherein the environmental exposure is wildfire smoke, air pollution, burn pit exposures, particulate matter, chemicals, or gases.

Images