Bone glycine as lung tissue regenerant

Abstract

The present invention relates to the field of medicine and tissue engineering, and more particularly to means and methods for promoting the repair of epithelial cells, such as damaged lung tissue observed in diseases with defective lung repair, including chronic obstructive pulmonary disease (COPD). In some embodiments, bone glycine (OGN; osteoinducible factor, mimecan) or a functional fragment thereof is provided for use as an agent for promoting regeneration of epithelial cells or epithelial tissues.

Description

Technical Field

[0001] This invention relates to the fields of medicine and tissue engineering. More specifically, this invention relates to apparatus and methods for promoting the repair of soft tissues, such as damaged lung tissue observed in diseases with impaired lung repair, such as chronic obstructive pulmonary disease (COPD). Background Technology

[0002] COPD is one of the most common lung diseases worldwide, characterized by a progressive decline in lung function and airflow limitation that is not entirely reversible. A key issue underlying COPD is abnormal tissue repair. This has two seemingly contradictory components: on one hand, bronchitis and small airway remodeling, accompanied by proliferating connective tissue; and on the other hand, emphysema caused by net destruction of substantial lung tissue. Current treatment options for COPD primarily aim to relieve symptoms and have little effect on the decline in lung function that occurs over time in COPD patients. Standard treatment includes bronchodilators and anti-inflammatory drugs, as well as bronchoscopic / surgical interventions (coil placement, lung volume reduction surgery, transplantation) in more severe cases. None of these treatments support lung tissue regeneration.

[0003] Since current disease treatments at best only alleviate symptoms without altering or positively influencing disease progression, the inventors aim to develop novel therapies for epithelial tissue repair. Specifically, they seek to identify factors that can reactivate endogenous repair mechanisms to stimulate the regeneration of damaged cells in epithelial tissues (e.g., lung parenchyma) and normalize abnormal tissue repair responses.

[0004] Mesenchymal stem cells (MSCs) are known to have therapeutic effects on emphysema in COPD. Both in vitro and in vivo studies have demonstrated beneficial properties of MSCs in COPD, including reducing emphysematous changes, reducing alveolar damage, and promoting the differentiation of type II alveolar-like cells (Cappetta, Stem Cells Int. 2018 Mar 14;2018:9492038). These effects are known to be mediated by soluble factors secreted by the cells and extracellular vesicles (EVs) produced by mesenchymal lung fibroblasts (Van Der Koog et al., ERJ Open Research 2022 8: 2; DOI: 10.1183 / 23120541.LSC-2022.2; Eur. J. Pharm. Vol. 974, 2024, 17661; DOI: 10.1016 / j.ejphar.2024.176612). These secreted soluble factors and extracellular vesicles contain more than 1,000 different proteins. However, the nature of the key factors responsible for regeneration remains unknown.

[0005] The inventors unexpectedly discovered that among the thousands of proteins in secreted soluble factors and EVs, one protein appears to induce an unusually strong effect on the regenerative response of soft tissues. This protein is called osteoglycine (OGN), also known as mimecan. OGN is a leucine-rich endogenous small proteoglycan known to have several regulatory functions in the human body. These functions include regulating fibrosis, tumorigenesis, and inflammation. See, for example, Nulali et al. (Biomolecules. 2022 Nov 11;12(11):1674) and Deckx et al. (FASEB J. 2016 Aug; 30(8):2651-61).

[0006] As shown below, using organoid models (where organoid growth is a marker of soft tissue regeneration), bone glycine was observed to induce approximately 75% more organoid formation than the control, while other proteins tested induced growth of no more than 30%.

[0007] Therefore, this invention relates to bone glycine (OGN) or a functional fragment thereof, which can be used as an agent to promote epithelial tissue regeneration. Specifically, this invention relates to the use of OGN or a functional fragment or derivative thereof in promoting epithelial cell regeneration in lung, intestinal, or liver tissue.

[0008] In other words, the present invention relates to the use of OGN or functional fragments thereof in enhancing epithelial tissue repair and / or regeneration.

[0009] The therapeutic use of OGN in epithelial tissue regeneration is not disclosed or suggested in the prior art. Bone glycine has been reported as a therapeutic agent for heart disease, retinal disease, and osteoporosis. See, for example, Deckx et al. (Matrix Biol. 2018 Mar; 66:110-124); Tasheva et al. (Mol Vis 2002;9) and Chen et al. (BMC Musculoskeletal Disorders 2017 18:423). Shi et al. (Am J Physiol Cell Physiol. 2020 Nov 1;319(5):C895-C905) reported that overexpression of microRNA-140 downregulated bone glycine via the Wnt signaling pathway, thereby inhibiting pulmonary fibrosis in interstitial lung disease. KR2015 / 0059491A discloses the use of a substance that increases bone glycine expression for the treatment or prevention of senile xerosis. It also involves screening methods, kits, and markers for measuring the amount of OGN after treating epidermal cells with a specific substance, and using this to select specific substances as substances for improving age-related xeroderma.

[0010] However, it is worth noting that each of these known applications relates to the (regenerative) effects of fibroblasts and / or osteoblasts, rather than to epithelial cells or epithelial tissue. More specifically, OGN has never been proposed as a therapeutic agent for lung, intestinal, or liver diseases.

[0011] As used herein, the term “OGN or a functional fragment thereof” encompasses any of the three known transcripts of the human OGN gene, as well as N-terminal and / or C-terminal truncated variants and their homologs that exhibit regenerative activity in epithelial cells / epithelial tissues. Regenerative activity can be suitably determined by one or more in vitro assays known in the art, such as using (mouse) organoid model systems, precisely cut lung sections, or mouse models as exemplified below. Exemplary OGN homologs used in this invention include mammalian homologs, such as OGN from cattle, mice, rats, rabbits, or chickens (see Decks et al., FASEB J.. 2016 Aug;30(8):2651-610).

[0012] In a preferred embodiment, the OGN protein or a functional fragment thereof is used. Human OGN polypeptide sequences are available in GenBank under accession number AAH37273.1 (bone glycine [Homo sapiens]), or in the UniProtKB / Swiss-Prot database under accession numbers P20774, Q9UNK5, or in the UniProtKB / TrEMBL database under accession numbers A8K0R3, B4DI63, Q7Z532.

[0013] Human OGN protein sequence:

[0014]

[0015] Residues 1-20 (underlined) form the signal sequence. Residues 21-298 represent the OGN polypeptide sequence. An internal disulfide bond can be formed between Cys residues at positions 255 and 288. OGN possesses a core protein consisting of leucine-rich repeat sequences (LRRs) and an N-terminal cysteine-rich cluster that binds specific glycosaminoglycans (GAGs). The full-length protein is characterized by a long tail and a sequence of seven LRRs (LRR1-LLR7). Positively charged residues within the LRRs, particularly in LRR4, expose regions suitable for interaction with other proteins, suggesting a role in biological activity / signal transduction. LRR4 exhibits significant conservation across species, implying its functional importance.

[0016] This encompasses OGN peptides carrying one or more post-translational modifications, such as N- and / or O-linked glycans. Known glycan modification sites include: an O-link at Thr80 (GalNAc), and an N-link at Asn80, Asn214, and Asn258 (GlcNAc) (keratin sulfate).

[0017] The term "functional fragment" in OGN can mean the portion of an OGN polypeptide that retains its functional activity. It can refer to any polypeptide or oligopeptide containing an OGN fragment capable of exerting a desired regenerative effect on epithelial cells or epithelial tissue, or composed of OGN fragments capable of exerting a desired regenerative effect on epithelial cells or epithelial tissue. The desired regenerative effect of the functional fragment can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the regenerative effect observed in full-length OGN at the same molar concentration. Preferably, the functional OGN fragment retains at least 100% of the functional (regenerative) activity of the OGN.

[0018] Regenerative effects can be readily determined using functional assays and methods known in the art and detailed in the examples herein. Suitable functional assays may involve mammalian organoid systems or precision-cut lung slices (PCLS). In some embodiments, regenerative effects are assessed by determining colony-forming efficiency (CFE) of mouse or human lung organoids, optionally including test settings involving exposure to cigarette smoke. In other embodiments, regenerative effects are evaluated by assessing gene expression of alveolar (AT) type I and / or II cell markers in PCLS treated with elastase. Furthermore, (candidate) functional fragments can also be evaluated in in vivo (mouse) models described below.

[0019] Preferred functional fragments contain up to 200, 180, 170, or 160 amino acids. In one embodiment, the OGN functional fragment is an N-terminal truncated variant, lacking at least residues 1-10, preferably at least residues 1-20. However, a larger portion of the N-terminus can be deleted without impairing the therapeutic efficacy of OGN. For example, this can be intended to limit the effect of OGN on collagen crosslinking while preserving the regenerative / signal transduction properties of OGN. For example, the functional fragment may lack N-terminal residues 1-50, 1-100, 1-150, 1-160, 1-170, or 1-179. Good results have been obtained using fragments of approximately 15 kDa consisting of residues 180-298. In some embodiments, the fragment contains or is composed of one or more leucine-rich repeat (LRR) motifs found in OGN. Preferably, the fragment comprises or consists of one or more LRR4 (residues 180-199), LRR5 (residues 200-225), LRR6 (residues 226-246), and LRR7 (residues 247-277). For example, at least motifs LRR4, LRR5, LRR6, or LRR7 are present. A functional fragment may comprise one or more copies of a given LRR, such as LRR4, LRR5, LRR6, and / or LRR7. In one aspect, the fragment comprises or consists of LRR4-LRR7 (residues 180-277). In a preferred embodiment, the functional OGN fragment comprises or consists of the amino acid sequence of motif LRR4 (LKLPVLPPKLTLFNAKYNKI). Variant OGNs (fragments) containing one or more (conserved) amino acid substitutions compared to the wild-type OGN sequence are also covered. For example, one or more Lys residues may be replaced by Arg or His.

[0020] In one specific aspect, the functional fragment contains at least residues Leu180-Phe298. In another embodiment, the OGN functional fragment is a C-terminal truncated variant lacking at least residue Phe298, or residues Tyr297 and Phe298, or residues Ser296-Tyr297-Phe298. In one specific aspect, the functional fragment contains at least residues Leu180-Gly295.

[0021] According to the present invention, OGN or a functional fragment thereof is used as a regenerative agent in epithelial cells or epithelial tissues.

[0022] Epithelial cells can be squamous, cuboidal, or columnar in shape and can be arranged in a single or multiple layer. Simple cuboidal epithelium is found in glandular tissue and renal tubules. Simple columnar epithelium covers the stomach and intestines. Pseudostratified columnar epithelium covers parts of the respiratory tract and some ducts of the male reproductive tract. Most epithelial cells self-renew through a process called tissue homeostasis, in which the number of cell divisions within the tissue compensates for the number of cells lost. Tissue homeostasis is maintained by the presence of stem cells (SCs) located in a special microenvironment called a niche. After injury, epithelial tissue undergoes different stages of hemostasis, inflammation, proliferation, and remodeling, typically leading to fibrosis and scar formation.

[0023] The epithelial cells of the lungs are located at the interface between the environment and the body and perform many important functions, including barrier protection, fluid balance, gas exchange, particulate matter clearance, initiation of immune response, production of mucus and surfactant, and repair after injury.

[0024] Liver epithelial cells clump together to form functional units called liver lobules. The main functions of hepatocytes are to synthesize and secrete bile, form and store proteins, remove toxins, and store and release carbohydrates.

[0025] Intestinal epithelial cells (IECs) form a selective permeability barrier that separates the contents of the intestinal lumen from the underlying tissue. Intestinal epithelial injury leads to a wound healing process. Intestinal wound healing depends on a balance of three cellular events: the recovery, proliferation, and differentiation of epithelial cells near the site of injury. The occurrence of tissue damage and incomplete healing of the intestinal epithelium are prerequisites for immune activation and are a cause of the relapsing, chronically progressive phenotype of inflammatory bowel disease (IBD).

[0026] In a preferred embodiment, the present invention provides the use of OGN or its functional fragments or derivatives for promoting the regeneration of epithelial cells in lung, (gastrointestinal) or liver tissue.

[0027] As illustrated in the following examples, this invention provides the use of OGN for enhancing the expansion of (distal) lung stem cells into airway and / or alveolar epithelial cells.

[0028] In a preferred aspect, the present invention provides an OGN or a functional fragment thereof for use in a method of treating diseases involving defective lung tissue repair, defective intestinal tissue repair, and / or defective liver tissue repair. A method for treating a (human) subject with a designed defective lung tissue repair, defective intestinal tissue repair, and / or defective liver tissue repair is also provided, comprising administering a therapeutically effective amount of an OGN or a functional fragment thereof to the subject. Preferably, the method comprises administering the OGN or a functional fragment thereof via pulmonary delivery.

[0029] Preferably, OGN or a functional fragment thereof is used to treat diseases or conditions involving impaired lung tissue repair and / or decreased lung function. For example, diseases may include COPD, pulmonary fibrosis, cystic fibrosis, post-pneumonia lung repair, acute lung injury / ARDS, chronic COVID-19, and sarcoidosis.

[0030] According to the present invention, OGN or a functional fragment thereof may be used as a protein (administered) or as DNA or mRNA encoding the protein.

[0031] In one embodiment, OGN or a functional fragment thereof is administered as a protein. The OGN protein may be recombinantly produced, for example, using eukaryotic organisms such as yeast or mammalian host cells. In some embodiments, OGN or a functional OGN fragment is synthesized. The synthesized OGN product may contain protein-derived and / or non-protein-derived amino acids. OGN may be used as a regenerant in glycosylated or non-glycosylated forms. In one embodiment, OGN is glycosylated, preferably N- and O-glycosylated.

[0032] Further methods involve pharmaceutical compositions comprising an OGN polypeptide or a functional fragment thereof, or OGN mRNA or a functional fragment thereof, and a pharmaceutically acceptable carrier, medium, or diluent, which preferably stabilizes the protein or mRNA or enhances its therapeutic activity. In further embodiments, the OGN polypeptide or a functional fragment thereof, or OGN mRNA or a functional fragment thereof, is formulated into a suitable dosage form for administration via a desired route of administration, for example, as a liquid formulation for injection or a liquid suitable for nasal administration. In a preferred embodiment, the composition is formulated for pulmonary delivery. Pulmonary drug delivery is achieved by inhaling the pharmaceutical formulation through the mouth, for example, in a scenario where a patient uses an inhaler to inhale their drug. The inhaled drug is further deposited in the lower respiratory tract, where absorption of the drug into the bloodstream via the lung mucosa is the primary objective of this route of administration. In some embodiments, the OGN polypeptide or a functional fragment thereof is a powder for inhalation or a liquid for nebulization.

[0033] OGN peptides or their functional fragments may be formulated as solutions containing functional excipients, such as, but not limited to, buffers such as phosphates or acetates, stabilizers such as sugars, polyols or amino acids, surfactants such as polysorbate 80, and antioxidants such as ascorbic acid.

[0034] In dry form, OGN peptides or their functional fragments can be stabilized by incorporating the protein into a matrix containing sugars (such as trehalose), and / or polysaccharides (such as inulin or pullulan), and / or polyols (such as mannitol), and / or buffers and / or amino acids.

[0035] Dry powder medications can be pure formulations consisting solely of OGN as the active pharmaceutical ingredient (API), or formulations may contain other substances for different purposes, such as excipients to improve product stability, increase API bioavailability, and / or bioactivity. APIs may also be incorporated into an excipient matrix before being formulated into the final dosage form, in which one or more excipients may be used again. Pharmacologically inert excipients may be included to dilute potent APIs, act as API carriers, or improve the flowability of the formulation to enhance powder metering and filling properties.

[0036] Powders with a suitable particle size for inhalation, i.e., particles with an aerodynamic diameter (AD) in the range of 0.5-5 μm, tend to aggregate, in other words, to form smaller or larger aggregates. These aggregates must be deagglomerated before the particles enter the user's respiratory tract. Deagglomeration refers to the dispersion of aggregated powder by introducing energy (e.g., electrical, mechanical, pneumatic, or aerodynamic energy). The aerodynamic diameter of particles of any shape is defined as the particle size at a density of 1 g / cm³. ³ The diameter of a spherical particle that has the same inertial properties in air as the particle of interest.

[0037] Compositions for pulmonary delivery may further comprise one or more additional therapeutic agents selected from bronchodilators and anti-inflammatory compounds. Further exemplary agents include Saba (e.g., salbutamol), Laba (e.g., formoterol), Lama (tiotropium bromide), and corticosteroids (e.g., budesonide).

[0038] In another embodiment, the drug formulation is in the form of a formulation that can deliver the OGN (protein) intact to the site of intestinal injury. This can be achieved via an oral route using site-specific drug delivery or a prodrug pathway; or via a parenteral route using specific drug targeting techniques.

[0039] In another embodiment, for example when OGN is used for liver repair, the composition utilizes a technique capable of site-specific delivery of therapeutic proteins to the liver. See, for example, those described by Böttger et al., Advanced Drug Delivery Reviews 154–155 (2020) 79–101 or Poelstra et al., Journal of Controlled Release 161 (2012) 188–197.

[0040] OGN polypeptides may be present in the composition in a dissolved or dried state. Preferably, they are stabilized by one or more excipients, such as those selected from polar solutes, surfactants, sugars, sugar alcohols, polysaccharides, and buffers; or stabilized by incorporation into an amorphous sugar glass containing monosaccharides, disaccharides or polysaccharides, buffers, amino acids, or inert peptides or proteins.

[0041] Other exemplary pharmaceutical compositions according to the invention comprise OGN mRNA. For example, non-viral delivery systems known in the art for RNA therapy can be used. See, for example, Paunovska et al. (Nature Reviews Genetics Volume 23, pg. 265–280 (2022)). These involve synthetic materials encapsulating RNA, such as polymers, lipids, and lipid nanoparticles (LNPs). Typically, LNP formulations consist of cationic lipids, neutral lipids, and / or cholesterol, as well as PEG-lipids. The present invention is particularly interested in LNPs that are preferentially taken up by alveolar epithelial cells, such as LNPs composed of DOTAP, DPPC, cholesterol, DLin-MC3-DMA, and DSPE-PEG. See also Lokugamage et al. (Nature Biomedical Eng. Volume 5, pg. 1059–1068 (2021)) who reported the design of LNPs for the efficient delivery of therapeutic RNA to the lungs via nebulization. Liquid formulations of nanoparticles may also contain sugars such as sucrose.

[0042] In another embodiment, OGN mRNA is administered using an LNP that is preferentially taken up by the liver, such as an LNP incorporating a lipid containing tri(2-aminoethyl)amine (TREN) and three linoleic acid chains (referred to as TRENL3), which optionally further contains unsaturated fatty acids (Yu et al., Biomaterials. 2012 Sep; 33(25): 5924–5934).

[0043] Medical devices comprising pharmaceutical compositions according to the invention are also provided. For example, the device is a self-injector containing a stabilized OGN solution (aqueous solution). As another example, the medical device is a nebulizer, metered-dose inhaler (MDI), or dry powder inhaler (DPI) containing OGN formulated for pulmonary delivery.

[0044] DPI devices have been accepted by healthcare professionals because they deliver an effective dose in a single inhalation; they are reliable, generally quite small, and easy for the user to operate. Two types are common: multi-dose dry powder inhalers and single-dose dry powder inhalers. The advantage of multi-dose devices is that the inhaler stores enough powder for multiple doses and dispenses a dose from the reservoir shortly before the inhalation is needed. Single-dose inhalers use a pre-measured dose, and these inhalers contain a limited number of individually packaged doses of the expected amount, with each dose package or container opened shortly before the inhalation of the contained dose occurs.

[0045] Other aspects involve individually packaged (sealed) expected doses of OGN used in conjunction with inhalation devices, wherein each dose package or container is opened shortly before the inhalation of the dose contained therein occurs. Attached Figure Description

[0046] Figure 1 Representative bright-field images of mouse organ cultures on day 14 after treatment with different recombinant growth factors. Scale bar = 500 µm

[0047] Figure 2: (A) Colony formation efficiency of rodent organs on day 14 of drug screening (mean ± SEM, n = 3–5, paired Friedman test). (B) Logarithm of rodent organ diameter on day 14 of drug screening (showing median, N = 3–5, Kolmogorov-Smirnov test (Bonferroni corrected: α = 0.00417)). *p < 0.05 and **p < 0.01 indicate statistical significance compared to the control.

[0048] Figure 3: (A) Representative bright-field images of mouse lung organoids. (B) Colony formation efficiency (mean ± SEM, N=8, paired Friedman test) and log diameter (showing median, N=8, Kolmogorov-Smirnov test (Bonferroni corrected: α=0.0017)) of mouse organoids treated with increased concentrations of OGN or fragments on day 14.

[0049] Figure 4: (A) Representative bright-field images of CSE-exposed mouse lung organoids. (B) Colony formation efficiency (mean ± SEM, N = 8–11, paired Friedman test) and logarithmic diameter (showing median, N = 8–11, Kolmogorov-Smirnov test (Bonferroni corrected: α = 0.0013)) of CSE-exposed mouse organoids treated with increased concentrations of OGN or fragments on day 14. (C) Representative immunofluorescence images of organoids stained for alveolar type (surfactant protein C, green channel) and nucleus (DAPI, blue channel). (D) Quantitative analysis of immunohistochemical staining for prosurfactant protein C in CSE-exposed mouse organoids on day 14 for identification of alveolar type organoids (mean ± SEM, N = 7–8, paired Friedman test).

[0050] Figure 5: (A) Representative bright-field images of TGF-β-exposed mouse lung organoids. (B) Colony formation efficiency (mean ± SEM, N=8, paired Friedman test) and log diameter (showing median, N=8, Kolmogorov-Smirnov test (Bonferroni corrected: α=0.0013)) of TGF-β-exposed mouse organoids treated with increased concentrations of OGN or fragments on day 14.

[0051] Figure 6: (A) Representative bright-field images of mouse lung organoids treated with degenerated OGN. (B) Normalized organoid counts (mean ± SEM, N=5, paired T-test) and sizes (showing median, N=8–11, Kolmogorov-Smirnov test) of mouse organoids treated with degenerated OGN.

[0052] Figure 7 Colony formation efficiency (mean ± SEM, N=5, paired Friedman test) and log diameter (showing median, N=5, Kolmogorov-Smirnov test (with Bonferroni correction: α=0.025) of human COPD type IV lung organoids treated with OGN (300 nm) or fragments (300 nm) on day 14.

[0053] Figure 8: (A) Gene expression levels of alveolar epithelial cell markers (Aqp5, Rage, Con43, Sftpc) in precisely cut lung sections treated with elastase (16 h) and / or OGN (40 h) (N=2). (B) Gene expression levels of Ogn in precisely cut lung sections treated with elastase (16 h) (mean ± SEM, N=5, paired t-test on log-transformed data).

[0054] Figure 9: (A) Example images of PCLS stained with F-actin filaments (green) and DAPI (blue) after treatment with the mediator control, elastase, or elastase + OGN or fragments (scale bar = 100 µm). (B) Mean linear intercept measurements after treatment, expressed in µm (mean ± SEM, N = 6, one-way ANOVA followed by Sidak multiple comparisons). **p < 0.01 and ****p < 0.0001 indicate statistical significance compared to the control, and ##p < 0.01 and ###p < 0.001 indicate statistical significance compared to elastase.

[0055] Figure 10: (A) Schematic diagram of the in vivo mouse model of elastase-induced lung injury. (B to D) Lung function parameters: 0.2-second forced expiratory flow (FEF0.2), forced vital capacity (FVC), and 50% forced vital capacity (FEF_50%FVC) measured with FlexiVent (median ± minimum and maximum data points, N=7–8, one-way ANOVA followed by Sidak multiple comparisons). (E) Pressure-volume loop used to assess lung expandability (N=7–8, two-way ANOVA, Dunnett multiple comparisons). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 represent statistically significant comparisons.

[0056] Figure 11: (AC) Example images of OGN staining in whole lung tissue from non-smokers, current smokers, and quitters (scale bar = 4000 µm). (DE) Percentage of positive OGN staining area in whole lung tissue and parenchyma from non-smokers, current smokers, and quitters (%). (FG) Staining intensity of OGN in whole lung tissue and parenchyma from non-smokers, current smokers, and quitters. (H) Example image of OGN staining in whole lung tissue from moderate to severe COPD (COPD II / III) (scale bar = 4000 µm). (I) Example image of OGN staining in whole lung tissue from severe early-onset (SEO) COPD (scale bar = 4000 µm). (JK) Percentage of positive OGN staining area in whole lung tissue and parenchyma from COPD II / III patients (%). (LM) Staining intensity of OGN in whole lung tissue and parenchyma from COPD II / III patients. (NO) Percentage of positive OGN staining area in whole lung tissue and parenchyma from SEO-COPD patients (%). (PQ) Staining intensity of OGN in whole lung tissue and parenchyma of SEO-COPD patients. *p<0.05 and **p<0.01 indicate that the comparison is statistically significant.

[0057] Figure 12: (AD) Measured gene expression of α-smooth muscle actin, collagen 1a1, fibronectin, and TGF-β in human lung fibroblasts (MRC5) by RT-qPCR. MRC5 fibroblasts were treated with TGF-β, OGN (10 µg / mL), and / or fragments (4.5 µg / mL). Values ​​are presented as 2-ΔΔCt values ​​relative to the mean of the mediator controls (mean ± standard error, N = 5–6, one-way ANOVA followed by Sidak multiple comparisons). *p < 0.05 and **p < 0.01 indicate statistical significance.

[0058] Figure 13 Cellular characteristics of fibroblasts and myofibroblasts based on transcriptomics of mouse fibroblasts, which were treated with mediators or re-sorted from organoids using OGN and subjected to bulk RNA sequencing (mean ± minimum and maximum, N=4, one-way ANOVA followed by Dunnett multiple comparisons).

[0059] Figure 14 Normalized organoid counts (mean ± SEM, N=6, paired Friedman test) and organoid sizes (median shown, N=6, Kolmogorov-Smirnov test (Bonferroni corrected: α=0.017)) after 14 days of treatment with (fresh) OGN fragments or spray-dried fragments (using inulin or mannitol as excipients). Spray-dried fragments were added to organoid cultures immediately after spray-drying to test whether they retained their organoid support potential after drying.

[0060] Figure 15 Normalized organoid counts (mean ± SEM, N=6, paired Friedman test) and organoid sizes (showing median, N=6, Kolmogorov-Smirnov test (Bonferroni corrected: α=0.017)) after 14 days of treatment with (fresh) OGN fragments or spray-dried fragments (using inulin or mannitol as excipients). Spray-dried fragments were stored for one week under two different conditions: 60°C, 0% relative humidity; and 30°C, 43% relative humidity. This experiment aimed to evaluate the organoid support potential of spray-dried fragments under extreme storage conditions.

[0061] Figure 16 Proteomics-guided drug target strategies.

[0062] Experimental Section

[0063] Materials and Methods

[0064] animal

[0065] Mouse experiments for organoid research and PCLS were conducted at the Central Animal Facility (CDP) of the University Medical Center Groningen (UMCG), in accordance with national guidelines, and the experimental procedures were approved by the CDP and the Institutional Animal Care and Use Committee of the University of Groningen (IACUC). Animals were routinely housed with a 12-hour light / dark cycle and had free access to food and water.

[0066] Human body material

[0067] Human lung tissue was obtained from lung transplant donors and strictly adhered to UMCG research guidelines (as described at https: / / umcgresearch.org / w / research-code-umcg and the national ethics and professional guidelines, Code of Conduct for Health Research (https: / / www.coreon.org / wp-content / uploads / 2023 / 06 / Code-of-Conduct-for-Health-Research-2022.pdf)). As confirmed by the UMCG Medical Ethics Committee, the use of residual lung tissue in this study was not subject to the Dutch Act on Human Subjects in Medical Research and was therefore exempt from licensing under national law (Dutch Law: Article 458 of the Medical Treatment Protocols Act (WGBO) / Article 9 of the GDPR / Article 24 of the UAVG). Human lung tissue was obtained from residual tissue following lung surgery (such as lung resection and lung transplantation) in quantities exceeding those required for clinical therapeutic purposes. All samples and clinical information were coded pre-experimentally, and any identifiable information was not visible to researchers.

[0068] Cell culture

[0069] Mouse fibroblast CCL206 cells (ATCC, Mlg2908) were cultured in DMEM / F12 medium (Gibco) supplemented with 10% (v / v) fetal bovine serum (FBS) (Sigma Aldrich, 12103C), 100 U / ml penicillin / streptomycin (Gibco, 15070-063), 2 mM L-glutamine (Gibco, 25030-024), and 1% amphotericin B (Gibco, 15290026) at 37°C in a humid environment of 5% CO2 / 95% air. Human fetal mesenchymal lung fibroblast MRC-5 cell line (SigmaAldrich, 05081101) was cultured in Ham's F12 medium (Thermo Fisher, 11320033) supplemented with 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin / streptomycin and 2 mM L-glutamine at 37°C in a humid environment of 5% CO2 / 95% air.

[0070] For organoid experiments, two types of fibroblasts (CCL206 and MRC5) were incubated with mitomycin C (10 µg / mL) in growth medium (Sigma Aldrich, M4287-5X2MG) for 2 hours before co-culturing with primary epithelial cells to inactivate fibroblast proliferation. After incubation with mitomycin C, the fibroblasts were washed with warm PBS and then recovered in medium without mitomycin C for 1 hour.

[0071] organoid culture

[0072] Primary murine alveolar epithelial cells (in short, Epcam) + Cells, CD31 - / CD45 - / CD326 + The isolation of the cells was based on a previously published protocol (Wu et al., Front Pharmacol. 2021 Jan 20;11:609509). Briefly, mouse lungs were flushed with PBS via the heart, perfused with a dispersing enzyme (Corning, 354235), and incubated at room temperature for 45 minutes. Lung tissue was then digested with DNase I (VWR, A3778.0500) to obtain a single-cell suspension. QuadroMACS was used. ™ A mixture of a separator (Miltenyi Biotec, 130-091-051) and antibody-bound magnetic microbeads was used to perform negative selection of cell suspensions against CD31 (Miltenyi Biotec, 130-097-418) and CD45 (Miltenyi Biotec, 130-052-301). Subsequently, to obtain Epcam... + Cells were treated with anti-mouse CD326 microbeads (Miltenyi Biotec, 130-105-958) to inhibit CD31. - / CD45 - Cells undergo positive selection.

[0073] For human organs, adult donor tissue was isolated from histologically normal areas of lung tissue specimens obtained from 5 patients with Gold stage IV COPD at UMCG. Human lung tissue was stored in MACS® tissue storage buffer (Miltenyi Biotech, 130-100-008) until further processing. The lung tissue was cut into small pieces (approximately 1 mm). ³The cells were transferred to a dissociation mixture containing 1% penicillin / streptomycin in PBS, 1 mg / mL collagenase / dispersin (Roche, 11097113001), and 1.8 µg / mL DNase I. The tissue was further dissociated for 20 min at 37°C using a gentleMACS™ Octo dissociator with a heater (130-096-427, Miltenyi Biotec). The resulting single-cell suspension was washed and lysed for 10 min at 4°C using a lysis buffer (ammonium chloride (155 mM), potassium bicarbonate (1 mM), Titriplex III (0.001 mM), and 10 µg / mL DNase I in ultrapure water). EpCAM + The selection of cells is similar to that described above for mouse lung tissue.

[0074] Epithelial organoid culture

[0075] For rodent organs, freshly isolated Epcam+ cells were conjugated with CCL206 mouse lung fibroblasts at a 1:1 ratio (10,000 cells each) in DMEM / F12 containing 10% (v / v) FBS. The cell suspension was diluted 1:1 (v / v) with Corning® Matrigel® membrane matrix (Corning, 356234) and then seeded into transwell inserts (Greiner, 662641) in 24-well plates (100 µl / insert). Similarly, human organs were generated by co-culturing freshly isolated EpCAM+ cells with proliferation-inactivated MRC-5 lung fibroblasts. Matrigel was allowed to... ™Cure at 37°C for 30 minutes. After curing, add 410 µl of organoid culture medium (DMEM / Ham's F12, supplemented with 5% FBS, 1% penicillin / streptomycin, 1% L-glutamine, 1% amphotericin B, 0.025‰ epidermal growth factor (EGF) (Sigma Aldrich, SRP3196-500UG), 1% insulin-transferrin-selenium (Gibco, 51300044), and 1.75‰ bovine pituitary extract (Thermo Fisher, 11568866)) to the bottom of the chamber. On the day of inoculation, add 10 µM Y-27632 dihydrochloride (Axon, 1683) to selectively inhibit Rho kinase. Organoid culture is performed at 37°C and 5% CO2. Change the medium every 2-3 days. Add the treatment agent to the organoid culture medium at the bottom of the culture chamber. Full-length recombinant human bone glycine (OGN) produced in yeast (Orbyt, ORB383003) or a labeled functional fragment of His (aa 180-298) produced in E. coli (LSBio, LS-G15022) were used at the indicated concentrations. Fourteen days after inoculation, the total number of organoids in each well was manually counted using an optical microscope at 20x magnification. Organoid diameter was measured on the same day using NIS-Elements software. The number of organoids represents the ability of alveolar epithelial progenitor cells to be activated and form organoids, while organoid size measures the swelling or proliferation of organoids.

[0076] Cigarette smoke extract

[0077] To generate 100% cigarette smoke extract (CSE), smoke from two unfiltered 3R4F research cigarettes (University of Kentucky Tobacco Research Institute, Lexington, KY) was introduced into 25 mL of warm fibroblast culture medium. The smoke was delivered into the medium at 45 rpm using a peristaltic pump (Watson Marlow 323 E / D, Rotterdam, The Netherlands). CSE was freshly prepared before each experiment. For organoid experiments, 5% CSE was used in the organoid growth medium.

[0078] Organoids were re-sorted to re-obtain fibroblasts and epithelial cells.

[0079] In the organoid re-sorting, 300,000 Epcams were selected. +A mixture of cells and 300,000 CCL206 fibroblasts was seeded in 1 mL of Matrigel solution (Matrigel diluted 1:1 (v / v) with DMEM / F12 supplemented with 10% FBS) and added to one well of a 6-well plate. After Matrigel solidification for 1 hour, 2 mL of organoid culture medium containing OGN (10 µg / mL) was added above the Matrigel. Three days later, dispersant enzyme (Corning, 354235) was added to each well for 30 minutes at 37°C to dissociate the Matrigel. MACS buffer (MACS wash buffer (Milteny Biotec, 130-091-222) premixed with BSA (Milteny Biotec, 130-091-376)) was added to terminate the dispersant enzyme activity. Organoids were collected and centrifuged at 300 g for 5 minutes. The cell pellet was resuspended at 37°C in 5 mL of diluted trypsin (1:5 dilution in PBS, v / v) (Sigma-Aldrich, T7409) for 5 minutes, followed by the addition of 9 mL of DMEM / F-12 supplemented with 10% FBS to neutralize the trypsin. The cell pellet was incubated with CD326 beads for 20 minutes and then resuspended in MACS buffer. The cell suspension was then introduced into QuadroMACS. ™ Separator system to obtain CD326 derived from organoids - Fibroblasts and CD326+ (Epcam) + Epithelial cells were then used for further experiments.

[0080] Batch RNA sequencing analysis

[0081] Total RNA was extracted from re-sorted cells from organoids using the NucleoSpin RNA Isolation Kit (Bioké, 740955.50) according to the manufacturer's instructions. The re-sorted Epcam RNA from organoids was sequenced using an Illumina NovaSeq 6000 sequencer via GenomeScan (www.genomescan.nl). +Batch RNA sequencing (RNAseq) was performed on cells and fibroblasts. The analysis procedure included several steps, including data quality control, adapter trimming, short read alignment, and feature counting. To ensure the integrity of library preparation, calculations were performed to assess ribosome (and globin) content. Additionally, checks were performed to identify potential sample and barcode contamination. A set of standardized quality metrics was established for the raw dataset using quality control tools such as FastQC v0.34 and FastQA. Before alignment, adapter sequences in reads were removed using Trimmomatic v0.30. Reads for each sample were aligned against the global mouse reference sequence GRCm38 (patch 6). Principal component analysis was performed in R using the DESeq2 package to visualize the overall effect and batch effect of experimental covariates. Differentially expressed genes (DEGs) between control and treated samples were identified using the same R package and standard normalization procedures. Gene set enrichment analysis (GSEA) was performed on the top 50 differentially regulated genes using ShinyGO 0.77 (www.bioinformatics.sdstate.edu / go / ). The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used as a reference database, and pathway enrichment was reported as FDR q values ​​<0.05.

[0082] Immunohistochemical staining of human lung tissue sections

[0083] Lung tissues from control and COPD patients were obtained from surplus materials at the Mayo Clinic St. Mary's Hospital and UMCG in Rochester, Minnesota. This staining was part of the HOLLAND (Pathology of Tissues of Aging and COPD in the Lungs) cohort. Lung tissues were paraffin-embedded and sectioned into 6 µm thick sections. These sections were dewaxed and rehydrated, followed by antigen retrieval with 10 mM citrate buffer (pH 6). Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide (H2O2) followed by overnight incubation at 4°C with OGN primary antibody (1:400) (Abcam, ab168348) in 1% BSA-PBS. Sections were then washed and incubated with a secondary antibody conjugated to horseradish peroxidase (HRP) diluted 1:100 in 1% BSA-PBS containing 2% normal human serum. Staining was visualized by incubation with VectorNovaRED substrate (Vector Laboratories, SK-4800) for 5 min. The sections were then counterstained with hematoxylin, fixed, and scanned at 40x magnification using a Hamamatsu NanoZoomer 2.0HT digital slide scanner.

[0084] OGN expression analysis in human lung sections

[0085] In summary, images were extracted from the scans using Aperio ImageScope software V.12.4.3 (Leica Biosystems). For whole lung tissue analysis, artifact-free scan images were used. Following this step, specific regions, including airway walls, bronchial epithelium, and blood vessels, were extracted using Adobe Photoshop software (Adobe Inc. CA) to analyze OGN expression in the parenchyma. The intensity and area of ​​OGN-positive staining in the whole tissue and lung parenchyma were quantified using Fiji / ImageJ software. Analysis of stained human lung sections was performed according to Ngassie et al. (Am J Physiol Lung Cell Mol Physiol. 2023 June 1;324(6):L799-L814). The formula used to calculate the percentage of area positively stained for the protein is as follows:

[0086]

[0087] Precisely cut lung slices

[0088] PCLS was obtained from young C57BL / 6J mice (8 to 14 weeks old). A total of 12 mice (female to male ratio 1:1) were used for PCLS experiments. The mouse lungs were inflated with 1.5 mL of 1.5% (w / v) low-melting-point agarose solution (Gerbu Biotechnik). After inflating, the agarose was cured at 4°C for 15 minutes, and then the lung tissue was harvested. Lung sections with a thickness of 250 µm were cut using a tissue slicer (Leica VT 1000 S Vibratome series). Lung tissue sections were thoroughly washed and then cultured in 12-well plates in DMEM medium (Gibco, 42430-025) supplemented with sodium pyruvate (1 mM), a MEM non-essential amino acid mixture (1:100, Gibco, 11140-050), gentamicin (45 µg / mL, Merck, G1397), penicillin / streptomycin (100 U / mL), and amphotericin B (1.5 µg / mL, Gibco, 15290-026), using 3 sections per well. Sections from the same mouse were matched according to lung region (middle of the left lobe or middle of the right upper lobe). To induce emphysema changes, matched sections were treated with 2.5 µg / mL elastase for 16 hours. Sections were then treated with 10 µg / mL OGN or 4.48 µg / mL OGN active fragment for 40 hours, overlapping with the 16-hour elastase treatment.

[0089] Immunofluorescence staining of PCLS

[0090] PCLS were fixed with 4% paraformaldehyde (Sigma Aldrich, P6148) at 4°C for 15 minutes, and then washed with PBS. To visualize the solid tissue of PCLS, actin filaments were spliced ​​using Alexa Fluor. ™ 488 phalloidin (ThermoFisher, A12379) was stained for 15 minutes at room temperature. After incubation, sections were washed with PBS and transferred to slides with two drops of DAPI-containing mounting medium (Abcam, 104139). Fluorescence imaging was performed using a confocal laser scanning microscope equipped with a true confocal scanner (SP8 Leica) at a 20x objective. All images were acquired within the linear range at a resolution of 1024 × 1024 pixels, with a pinhole size of 1 Airy unit to avoid local saturation. The presented images represent a single z-axis scan.

[0091] mRNA isolation and real-time PCR

[0092] Total RNA was isolated from PCLS using a Maxwell 16 instrument and the corresponding Maxwell 16 LEV simply RNA tissue kit, which is designed for automated purification according to the manufacturer's instructions. Total RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer. Equal volumes of total mRNA were then reverse transcribed (Promega, Madison, USA) and cDNA was subjected to real-time qPCR. Real-time PCR was performed on a QuantStudio 7 Flex real-time PCR system (Applied Biosystems) using SYBR Green as the DNA-binding dye (Roche Applied Science, Mannheim, Germany), with 45 cycles of denaturation at 94°C for 30 seconds, annealing at 59°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a final extension at 72°C for 10 minutes. Real-time qPCR data were analyzed using the Ct method. The amount of the target gene was normalized to the endogenous reference genes B2M and 18S. The nucleotide sequences of the forward and reverse primers used are listed in Table 1.

[0093] Table 1: Primer sequences target gene forward primer reverse primer B2M 5'-ATGGGAAGCCGAACATACTG-3' 5'-CAGTCTCAGTGGGGGTGAAT-3' 18S 5'-AAACGGCTACCACATCCAAG-3' 5'-CCTCCAATGGATCCTCGTTA-3 Aqp5 5'-CTCACTGGGTCTTCTGGGTAG-3' 5'-TGCCGGTCAGTGTGCC-3 Rage 5'-CACAGGCTCTGTGGGTGAG-3' 5'-TTCAGCTCTGCACGTTCCTC-3' Con43 5'-TCCTTTTCGTTTGACTTCAGCCTC-3' 5'TCTGAAAATGAAGAGCACCGACA-3' Sftpc 5'-GGAGCACCGGAAACTCAGAA-3' 5'-GGAGCCGCTGGTAGTCATAC-3'

[0094] In vivo elastase mouse model

[0095] This study included male and female C57BL / 6J mice (1:1 ratio) and randomly assigned them to six experimental groups. Emphysema was induced on day 0 by intratracheal infusion of 40 µL porcine pancreatic elastase (40 U / kg body weight) in sterile PBS. Animals were treated every other day (6.75 µg or 20.25 µg) with OGN fragments (for a total of 5 treatments) from day 0 to day 9. Ten days later, mice were euthanized by exsanguination under anesthesia, and the therapeutic effect was then assessed. To ensure objective analysis, in vivo samples were blinded before analysis.

[0096] Lung function measurement

[0097] Respiratory function was measured using FlexiVent System Module 2 (Scireq). Mice were anesthetized with dexmedetomidine (Dexdomitor®) and ketamine (Ketamine®) and administered the muscle relaxant rocuronium bromide (FreseniusKabi, 10 mg / mL). Mice were ventilated at a tidal volume of 10 mL / kg and a frequency of 150 breaths / min to approximate mean lung volume to spontaneous breathing. Lung function parameters were assessed using Flexiware V8.3.0 software with a pre-specified protocol employing SnapShot, Primewave perturbation, and forced expiratory volume strategies. Recordings were taken three times per animal.

[0098] Fibrosis in human fibroblasts by OGN and its active fragments

[0099] Human fetal lung mesenchymal fibroblast MRC-5 cell line (Sigma Aldrich, 05081101) was cultured at 37°C in a humidified atmosphere of 5% CO2 / 95% air in Ham's F12 medium (Thermo Fisher, 11320033) supplemented with 10% (v / v) FBS, 100 U / mL penicillin / streptomycin (P / S), and 2 mM L-glutamine. Once the cells reached confluence, 300,000 fibroblasts were seeded into 6-well plates (Greiner Bio-one, 657160). After 72 hours, the MRC-5 cells were starved for 24 hours in DMEM / F12 containing 0.5% HI-FBS, 2% P / S, and 1% L-glutamine. The following day, the cells were treated for 48 hours as follows:

[0100] RT-qPCR in MRC5 cells

[0101] Add TRIzol (Invitrogen, Waltham, MA, USA, 15596018) to each well to induce cell lysis. mRNA was extracted into TRIzol, pipetted into Eppendorf cuvettes, and centrifuged at 4°C, 12,000 rcf for 10 min using a 5427R centrifuge (Eppendorf SE, Hamburg, Germany). The supernatant was poured into new cuvettes, and 1-bromo-3-chloropropane (Sigma-Aldrich, B9673) was added to each cuvette. After vortexing, the cuvettes were incubated at room temperature for 10 min, then centrifuged at 4°C, 12,000 rcf for 15 min. Next, the aqueous phase was transferred to new cuvettes, and ice-cold isopropanol (Biosolve, Valkenswaard, The Netherlands, 162606) was added. The cuvettes were vortexed again, placed on ice for 10 min, and then centrifuged at 4°C, 12,000 rcf for 8 min. Carefully aspirate the isopropanol, leaving a clear precipitate. To wash the mRNA precipitate, first add 80% ice-cold RNase-free ethanol (VWR international BV, Leuven, Belgium, 8025.2500), then centrifuge at 4°C, 7,500 rcf for 5 minutes, and finally discard the ethanol. Repeat these washing steps once or more. After the ethanol evaporates, resuspend the mRNA precipitate in 30 µL of RNase-free water (Macherey-Nagel, Dueren, Germany, 740378.1000).

[0102] An equal volume of total mRNA (1 µg) was reverse transcribed, and the resulting cDNA underwent real-time (RT) qPCR. RT-qPCR was performed using a QuantStudio 7 Flex real-time PCR system (Applied Biosystems) with RealQ Plux 2xMaster Mix Green as the DNA-binding dye (Ampliqon, 5000840-1250). The cycle consisted of 45 cycles: denaturation at 94°C for 30 seconds, annealing at 64°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 10 minutes. RT qPCR data were analyzed using the Ct assay and normalized relative to endogenous reference genes B2M and 18S. The specific forward and reverse primers (1 µM) used are listed in Table 2 below.

[0103] Table 2: PCR primer sequences

[0104] Statistical analysis

[0105] All data are expressed as mean ± SEM (SEM). All data were assessed for statistical significance using one-way ANOVA and corrected for Geisser-Greenhouse variance. Statistical analysis was performed using GraphPad Prism 10 software.

[0106] Example 1: Ligand screening in rodent organs

[0107] We used proteomics analysis to identify a variety of different growth factors and cytokines present in the secretome of alveolar fibroblasts. Based on a proteomics-guided ligand targeting strategy and literature search, we identified potential therapeutic targets for further investigation in our epithelial organoid model. Figure 16 This represents a ligand-targeting strategy used to identify potential therapeutic targets. Since the corresponding receptor for bone glycine is currently unknown, we included this protein in our drug screening.

[0108] Figure 1 Bright-field images of organoid cultures on day 14 are shown. A control panel shows organoid cultures that received no treatment. Each panel shows organoid cultures treated with several identified recombinant growth factors at each change in culture medium. Colony formation efficiency was quantified on day 14, and the size of the resulting organoids was determined (Figure 2).

[0109] Surprisingly, among all the proteins tested in the lung fibroblast secretome, one protein appeared to have an unusually strong effect on the regenerative response in our organoid cultures. This protein is called osteoglycine (OGN).

[0110] These data confirmed the significant effect of OGN (10.0 µg / mL) treatment, which significantly increased the number of organoids by 73.00 ± 12.75% (p = 0.018). Compared with control organoids, the size of OGN-treated organoids was significantly reduced (189.80 ± 9.63 µm vs. 157.50 ± 6.20 µm, p = 0.0021).

[0111] Example 2: Dose-response curves of OGN and its fragments in rodent organs.

[0112] This example demonstrates that OGN and its active fragment induce their supportive effect in a concentration-dependent manner. The C-terminal 15.1 kDa fragment of OGN (containing residues 180-298, including LRR4-7) is commercially available from LS Bio, Shirley, USA. Three different equimolar concentrations of OGN and the fragment were used: 3 nM, 30 nM, and 300 nM.

[0113] An increase in the number of organoids formed was observed with increasing doses of OGN or OGN fragments (see Figures 3A and 3B). Treatment with the highest concentrations of OGN or its fragments significantly improved the CFE of the organoids (Figure 3A).

[0114] Example 3: Dose-response curves of OGN and fragments in mouse organs in the presence of cigarette smoke extract and TGF-β.

[0115] This example demonstrates that both the full-length OGN protein and its C-terminal 15.1 kDa fragment (residues 180-298) have regenerative effects in a mouse organoid model, an effect maintained in the presence of CSE (Figure 4). Three different equimolar concentrations of OGN and its fragment were used: 3, 30, and 300 nM. As shown in Figure 4, increasing the concentration of OGN protein or the OGN fragment resulted in a significant increase in CFE in organoids generated in the presence of CSE. Furthermore, staining for the alveolar marker—prosuractive protein C—revealed that OGN and its active fragment increased the proportion of organoids expressing this marker. This indicates an increase in the number of alveolar organoids within the treated organoids.

[0116] In addition to CSE, organoid cultures were also exposed to the COPD-associated cytokine transforming growth factor β (TGF-β), which led to a significant reduction in organoid number (Figure 5). Increasing the concentration of OGN offset the adverse effects of TGF-β on CSE without affecting organoid diameter (Figure 6). In the presence of TGF-β, OGN treatment increased SPC. + The proportion of organoids (Figure 6).

[0117] Example 4: Degenerated OGN did not improve organoid formation

[0118] As a negative control, OGN (300 nM) was denatured by heating at 95 °C for 10 minutes. Treatment of organoids with denatured OGN did not result in significant changes in the number and size of organoids (Figure 6).

[0119] Example 5: The regenerative effect of OGN and its fragments on human organs.

[0120] Next, the supporting role of OGN and its fragments in organoid formation derived from lung tissue of COPD stage IV patients was investigated. Figure 7 As shown, OGN and its active fragments supported the formation of human alveolar epithelial organoids from five donors, a highly unusual effect in this human organ model. It appears to have no effect on organoid size in the human organ model.

[0121] Example 6: Effects of OGN and its fragments on gene expression in precision-cut lung slices (PCLS)

[0122] While organoids are valuable and easily controlled models, their complexity and translational value remain somewhat limited compared to tissue explants or animal models. Therefore, we also used precisely cut mouse lung slices (PCLS) to validate the regenerative potential of OGNs. The advantage of PCLS is that all lung cell types are present in their native structure, and cell-cell contacts and cell-matrix interactions are preserved. Our previous experience with PCLS has shown that elastase induces emphysema and alters gene expression of alveolar epithelial markers (Van Dijk et al., Front Physiol. 2017 Jan 4;7:657).

[0123] Lung sections were treated with elastase for 16 hours to induce emphysema. The total treatment time was 40 hours from the start of OGN (300 nM). Figure 8A shows the gene expression levels of type I alveolar (ATI) cell markers (Aqp5, Rage, and Con43) and ATII cell markers (Stfpc). Elastase treatment decreased the gene expression of ATI and ATII cell markers, while the presence of OGN during enzyme treatment prevented this effect. Since these genes are markers of alveolar epithelial cells, the decreased expression of these markers induced by elastase treatment indicates parenchymal damage. OGN treatment increased the expression of alveolar epithelial cell markers, indicating a protective effect against parenchymal damage.

[0124] Furthermore, we determined the expression of OGN in elastase-treated versus control lung sections (Figure 8B). Interestingly, elastase-treated sections of emphysematous lungs showed decreased bone glycine expression.

[0125] Example 7: Effects of OGN and its active fragments on elastase-induced damage in PCLS

[0126] After 16 hours of elastase incubation, lung tissue damage indicative of emphysema was induced, as evidenced by a significant increase in LMI (Figure 9). Interestingly, treatment with OGN or its active fragment for 40 hours (with the first 16 hours accompanied by elastase exposure) effectively prevented the induction of lung tissue damage (Figure 9).

[0127] Example 8: Effect of OGN fragment on elastase-induced damage in mice

[0128] We evaluated whether the OGN fragment could improve lung function parameters in a mouse model of elastase-induced lung injury (Eur. J. Pharm. Vol. 974, 2024, 17661). Mice were given low-dose (4.5 µg) or high-dose (20.5 µg) functional OGN fragments after elastase-induced lung injury (Fig. 10A). We evaluated several lung function parameters. Elastase treatment significantly increased forced expiratory volume in 0.2 seconds (FEF0.2), while forced vital capacity (FVC) and forced expiratory flow at 50% of forced vital capacity (FEF_50%FVC) significantly decreased (Fig. 10B-D). High-dose OGN fragment treatment significantly reversed the negative effects of elastase on lung function (Fig. 10B-D). Furthermore, elastase treatment led to a decrease in elastic recoil force, showing a typical significant upward shift as illustrated in the pressure-volume ring of emphysematous mice (Fig. 10E), indicating that elastase reduces lung extensibility. The upward shift of the pressure-volume loop observed in mice with emphysema was absent in mice treated with high doses of the OGN fragment. In summary, the active OGN fragment improves lung function in mice with elastase-induced lung injury.

[0129] Example 9: OGN expression in human lung tissue sections.

[0130] To investigate the effect of smoking on OGN expression, OGN expression in lung tissues from never-smokers, current smokers, and quitters was determined (Figures 11A-C). Image analysis of whole lung tissue and parenchymal sections from never-smokers, current smokers, and quitters revealed that the percentage of OGN-positive regions in the tissues of current smokers was proportionally reduced compared to never-smokers (Figures 11D-E).

[0131] Interestingly, mean OGN expression in the lung tissue of smokers who had quit smoking was lower than that in never-smokers (not statistically significant), suggesting a persistent effect of cigarette smoke on OGN expression. Quantitative analysis of mean OGN staining intensity showed no overall difference between lung tissues of never-smokers, current smokers, and smokers who had quit smoking, either in whole lung tissue or in the parenchyma (Fig. 11F-G). Next, we assessed whether OGN expression changed in patients with moderate-to-severe COPD or severe early-onset (SEO-) COPD compared to lung tissues of smokers who had quit smoking (Fig. 11H-I). No significant differences in OGN expression were observed in whole lung or parenchyma in patients with moderate-to-severe COPD or SEO-COPD (Fig. 11J-M and 11N-Q). However, mean expression in the lung parenchyma of SEO-COPD patients tended to be lower (p=0.09) (Fig. 11O). Interestingly, OGN intensity tended to increase in whole lung tissue (p=0.09), while mean staining intensity in the parenchyma was significantly enhanced (Fig. 11P-Q).

[0132] In summary, this suggests that the lungs of emphysematous patients may lack OGN, which could reduce the regenerative capacity of the lungs in patients with COPD.

[0133] Example 10: OGN lacks fibrosis.

[0134] Although OGN has not been previously associated with COPD, it has been suggested in the art that it may be related to pulmonary fibrosis. More specifically, Shi et al. (Am J Physiol Cell Physiol. 2020 Nov 1;319(5):C895-C905) reported that microRNA-140 inhibits lung fibroblast proliferation and promotes lung fibroblast apoptosis by directly inhibiting OGN expression. Therefore, the inventors set out to investigate the potential negative impact of OGN protein on induced pulmonary fibrosis. To this end, the gene expression of fibrosis markers after treatment with OGN or its active fragment was evaluated using human lung fibroblasts (MRC5). As expected, treatment with the known fibrotic TGF-β (2 ng / mL) increased the gene expression of α-smooth muscle actin and fibronectin, and showed a trend toward increased collagen 1a1 expression (Fig. 12). TGF-β gene expression remained unchanged. Notably, treatment with OGN or its active fragment did not alter the gene expression of fibrosis markers (Fig. 12). Interestingly, combined treatment with TGF-β and OGN fragments restored the increased expression of fibrosis marker genes to normal.

[0135] Furthermore, transcriptomic analysis of fibroblasts re-sorted from OGN-treated organoids predicted the ratio of fibroblasts to myofibroblasts. To this end, a gene signature based on the top ten genes associated with either fibroblasts or myofibroblasts was constructed. This analysis showed that the proportion of fibroblasts with myofibroblast gene signatures was lower compared to the mediators. Figure 13 In summary, these data indicate that, at least at the transcriptomic level, OGN and its active fragments do not alter the fibrotic state of lung fibroblasts.

[0136] Example 11: Spray drying of active fragments

[0137] Spray drying of proteins using excipients provides stable formulations suitable for inhalation administration. For spray drying of the active fragment of OGN, the fragment was dissolved in 20 mM HEPES at pH 7.4, which contained either 4 kDa inulin or 2.5 mM mannitol and leucine as stabilizers. Spray drying was performed using a Büchi B-290 mini spray dryer in a closed-loop configuration, equipped with a high-performance cyclone separator, a B-295 inert circulation loop, and a B-296 dehumidifier.

[0138] To investigate whether the therapeutic potential of the active fragment was preserved during spray drying, we treated mouse lung organoids and assessed organoid counts. The fragments were spray-dried with inulin or mannitol and immediately added to organoid culture medium. Treatment with spray-dried OGN fragments using mannitol or inulin as excipients increased organoid formation. The number of organoids formed was comparable to that of unspray-dried "fresh" fragments. Figure 14 ).

[0139] To test the stability of the obtained spray-dried formulation, the spray-dried fragments were stored for one week under two different conditions: 60°C, 0% relative humidity and 30°C, 43% relative humidity. After one week of storage under extreme conditions, the spray-dried OGN fragments still induced organoid formation, indicating that the formulation was stable. Figure 15 ).

[0140] Example 12: Pharmaceutical composition containing OGN.

[0141] A. Dry powder composition for OGN peptide inhalation

[0142] An inulin solution was prepared by dissolving 1.75 g of inulin in 28.4 mL of microporous water at 80 °C. The solution was cooled and 47 mg of sodium chloride was added. Subsequently, 700 μL of 0.1 M HEPES buffer solution (pH 7.5) and 700 μL of 0.01% (v / v) Tween 80 solution were added. Finally, 10 mL of aqueous OGN solution (1.77 mg / mL) was added to the solution to obtain a formulation containing OGN:inulin at a ratio of 1:99 (w / w).

[0143] The solution was sprayed into a stainless steel container containing liquid nitrogen using a 0.5 mm dual-fluid nozzle approximately 5 cm above the surface of the liquid nitrogen. The resulting suspension was transferred to a freeze dryer (Christ Alpha 2–4, Salm & Kipp, Breukelen, The Netherlands) pre-cooled at a shelf temperature of -80°C. After the liquid nitrogen evaporated, primary drying was performed for 32 hours at 0.220 mbar pressure, gradually increasing the temperature from -40°C to 5°C. Secondary drying was then performed for 12 hours at 0.055 mbar pressure, gradually increasing the temperature from 5°C to 20°C. The resulting powder is suitable for inhalation using a Twincer® dry powder inhaler, a cyclone separator-based dry powder inhaler.

[0144] B. The prepared liquid composition for delivering OGN mRNA

[0145] Lipid nanoparticles (LNPs) containing mRNA encoding OGN with an N / P ratio of 11.8 were prepared. Lipids 1,2-distearyl-sn-glycerol-3-phosphocholine, (6Z,9Z,28Z,31Z)-heptadecano-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate, cholesterol, and 1,2-distearyl-rac-glycerol-3-methoxy polyethylene glycol-2000 were dissolved in ethanol at a molar ratio of 8:52:39:1. The OGN-encoding mRNA was dissolved at 7.5 μg / mL in 20 mM citrate buffer (pH 3.2) as the aqueous phase. The two phases were rapidly mixed at a volume ratio of 3:1 (mRNA:lipid) using a Nanoassemblr® Benchtop (Precision NanoSystems, Inc.) at a total flow rate of 4 mL / min. After overnight dialysis in phosphate-buffered saline (PBS, pH 7.4), the solution was finally concentrated by ultrafiltration and resuspended in PBS.

Claims

1. Osteoglycine (OGN; bone-inducing factor; mimecan) or its functional fragments, which are used as agents to promote lung tissue regeneration.

2. The OGN or its functional fragment according to claim 1, used to enhance the expansion of lung stem cells into airway and / or alveolar epithelial cells.

3. OGN or a functional fragment thereof, for the treatment of diseases involving defective lung tissue repair.

4. The OGN or a functional fragment thereof according to claim 3, wherein the disease involves defective lung tissue repair and / or decreased lung function.

5. The OGN or a functional fragment thereof according to claim 4, wherein the disease is selected from COPD, pulmonary fibrosis, cystic fibrosis, post-pneumonia lung repair, acute lung injury / ARDS, chronic COVID-19, and sarcoidosis.

6. The OGN used according to any of the preceding claims, wherein the OGN or a functional fragment thereof comprises at least the residues Leu180-Phe298 of the sequence available in GenBank under the serial number AAH37273.1 (bone glycine [Homo sapiens]).

7. The OGN used according to any one of the preceding claims includes administering the OGN or a functional fragment thereof as a protein or as DNA or mRNA encoding the OGN protein or a functional fragment thereof.

8. The OGN used according to claim 7, wherein the OGN or a functional fragment thereof is administered as a protein.

9. A pharmaceutical composition comprising an OGN polypeptide or a functional fragment thereof and a pharmaceutically acceptable carrier, medium or diluent, wherein the composition is formulated for pulmonary delivery.

10. The pharmaceutical composition according to claim 9, wherein the composition is formulated as a powder for inhalation or as a liquid for nebulization.

11. The pharmaceutical composition according to claim 9 or 10, comprising an OGN polypeptide or a functional fragment thereof in a dissolved or dried state, said OGN polypeptide or functional fragment thereof being stabilized by one or more excipients preferably selected from polar solutes, surfactants, sugars, sugar alcohols or buffers, or by incorporation into an amorphous sugar glass containing monosaccharides, disaccharides or polysaccharides, buffers, amino acids or inert peptides or proteins.

12. The pharmaceutical composition of claim 11, comprising an OGN polypeptide or a functional fragment thereof in a dry state, said OGN polypeptide or functional fragment thereof being stabilized by incorporation into a matrix comprising sugars such as trehalose, and / or polysaccharides such as inulin or pullulan, and / or polyols such as mannitol, and / or buffers and / or amino acids.

13. The pharmaceutical composition according to any one of claims 9-12, further comprising one or more additional therapeutic agents, preferably selected from bronchodilators and anti-inflammatory compounds.

14. A medical device comprising a pharmaceutical composition according to any one of claims 9-13.

15. The medical device according to claim 14, wherein it is a nebulizer, a metered-dose inhaler (MDI), or a dry powder inhaler (DPI).

16. A packaged quantity of OGN composition, the dosage of which is used in combination with the medical device according to claim 15.

17. A method for treating a disease involving defective lung tissue repair, the method comprising administering a therapeutically effective amount of OGN or a functional fragment thereof to a subject in need.

18. The method of claim 17, comprising administering OGN or a functional fragment thereof via pulmonary delivery.

19. The method according to claim 17 or 18, comprising administering the pharmaceutical composition according to any one of claims 9-13.

20. The method according to any one of claims 17-19, wherein the disease is selected from COPD, pulmonary fibrosis, cystic fibrosis, post-pneumonia lung repair, acute lung injury / ARDS, chronic COVID-19, and sarcoidosis, preferably wherein the disease is COPD.

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