Purified Enriched Population Exosomes Derived From Individuals With A Chronic Progressive Lung Disease For Noninvasive Detection, Staging, And Medical Monitoring Of Disease Progression

Abstract

The present disclosure provides a method for noninvasively diagnosing and staging a progressive chronic lung disease characterized by disease related lung dysfunction by deriving from a biological sample from a subject a purified enriched population of exosomes in the biological sample, wherein dysregulated expression of the two or more microRNAs, compared to a healthy control, comprises a signature of a fibrotic lung disease; and medically managing the diagnosed fibrotic lung disease as early as possible in the course of progression of the disease to reduce or slow its progression. The method may identify interstitial pulmonary fibrosis (IPF) at a stage before standard procedures (e.g., Ashcroft scoring and histology) demonstrate changes consistent with lung fibrosis.

Claims

1. A method for noninvasively diagnosing and staging a progressive chronic lung disease characterized by disease related lung dysfunction comprising: (a) quantifying lung function of a subject with a progressive chronic lung disease characterized by disease-related lung dysfunction; (b) diagnosing and determining a stage of the chronic lung disease by: (i) collecting a biological sample from the subject with the lung disease and from a healthy control, (ii) centrifuging the biological sample at low speed to form a clarified supernatant; (iii) ultracentrifuging the clarified supernatant to pellet the purified enriched population of exosomes; and (iv) characterizing the purified enriched population of exosomes, wherein. (1) the population of exosomes expresses two or more exosome biomarkers selected from the group consisting of CD9, CD63, CD81, or HSP70; (2) size of the exosomes in the population of exosomes ranges from 30 m to 150 μm, inclusive; (3) the population of exosomes comprises a total protein of at least 100-200 μg, inclusive; (4) the population of exosomes comprise a total RNA content of at least 100-200 ng, inclusive; (5) number of exosomes in the population of exosomes comprises at least 10E10 particles; and (6) the population of exosomes comprises a cargo comprising dysregulated expression of two or more microRNAs selected from miR-199, miR Let-7a, miR Let-7b, mir-Let-7d, miR-10a, miR-21, miR-29a, miR-34, miR-101, miR-125, miR-145, miR-146a, miR-181a, miR-181b, miR-181c, miR-199, and miR-142, wherein expression of the two or more microRNAs, compared to a healthy control, comprises a signature of a fibrotic lung disease; and (c) medically managing the diagnosed fibrotic lung disease as early as possible in the course of progression of the disease to reduce or slow its progression by (1) treating the diagnosed fibrotic lung disease; and (2) monitoring over time the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control.

2. The method according to claim 1, wherein the lung function of the subject is quantified by determining forced expiratory volume (FEV1); forced vital capacity (FVC) and FEV/FVC %.

3. The method according to claim 1, wherein the biological sample is a body fluid and the body fluid is amniotic fluid, blood, breast milk, saliva, urine, bile, pancreatic juice, cerebrospinal fluid or a peritoneal fluid.

4. The method according to claim 3, wherein the body fluid is serum or urine

5. The method according to claim 4, wherein the body fluid is serum.

6. The method according to claim 4, wherein the body fluid is urine.

7. The method according to claim 1, wherein the biological sample is obtained from a mammal.

8. The method according to claim 1, wherein the biological sample is obtained from a non-human mammal or a human subject.

9. The method according to claim 1, wherein the healthy control is age and sex matched to the subject.

10. The method according to claim 1, wherein the chronic lung disease if left untreated comprises one or more of a progressive injury, progressive inflammation, progressive fibrosis or a combination thereof.

11. The method according to claim 10, wherein the fibrotic lung disease is interstitial pulmonary fibrosis (IPF).

12. The method according to claim 11, (a) wherein dysregulated miRNA expression of miR-199 and miR-34a in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is fibrotic; or (b) wherein dysregulated miRNA expression of miR-142 in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is antifibrotic; or (c) wherein the dysregulated expression of one or more of miR let-7D, miR-29, or miR-181 in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is decreased, compared to the population of exosomes derived from the healthy control; or (d) wherein dysregulated expression of miR-199 in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is increased and the expression of one or more of miR-let-7d, miR-29, miR-142, miR-181 in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is decreased, or (e) wherein reduced or absent miR Let-7 expression in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control compared to a healthy control is associated with early stage fibrotic lung disease; or (f) wherein micro RNA expression in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control comprises increased miR-Let-7 expression compared to a healthy control and is associated with end-stage fibrotic lung disease.

13. The method according to claim 1, wherein the medical managing modulates one or more of an injury, inflammation, an excess accumulation of extracellular matrix, cell senescence; or a pathway comprising fibrogenic signaling.

14. The method according to claim 13, wherein markers for fibrosis and downstream fibrotic pathways comprise α.sub.v-integrin, collagen type I mRNA expression, c-Jun, AKT expression and MMP-9 activity.

15. The method according to claim 13, wherein the pathway comprising fibrogenic signaling is one or more of a Smad pathway, a mitogen-activated protein kinase pathway, a phosphoinositide 3-kinase pathway; a canonical Wnt-β catenin pathway, and a Notch signaling pathway.

16. The method according to claim 13, wherein the pathway comprising fibrogenic signaling comprises transforming growth factor (TGFβ) signaling.

17. The method according to claim 1, wherein the treating of the fibrotic disease includes administering an active therapeutic agent including an immunomodulator, an analgesic, an anti-inflammatory agent, an anti-fibrotic agent, an anti-viral agent, a proton pump inhibitor, or oxygen therapy.

18. The method according to claim 17, wherein the immunomodulator includes prednisone, azathioprine, mycophenolate, mycophenolate mofetil, colchicine, or interferon-gamma 1b.

19. The method according to claim 17, wherein the analgesic includes capsaisin, codeine, hydrocodone, lidocaine, oxycodone, methadone, resiniferatoxin, hydromorphone, morphine, and fentanyl.

20. The method according to claim 17, wherein the anti-inflammatory agent includes aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sulindac, and tolmetin.

21. The method according to claim 17, wherein the anti-fibrotic agent includes nintedanib or pirfenidone.

22. The method according to claim 17, wherein the anti-viral agent includes acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine/zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, or delavirdine.

23. The method according to claim 17, wherein the proton pump inhibitor includes omeprazole, lansoprazole, dexlansoprazole, esomeprazole, pantoprazole, rabeprazole, or ilaprazole.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0143] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0144] FIG. 1A. Transmission electron microscopy of isolated exosomes. Image magnification Scale bar=50 nm. FIG. 1B shows that isolated exosomes express CD63.

[0145] FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D shows that expression of miR let-7d (A), miR-29a-5p (B), miR 181b-3p (C) and miR-199a-3p (D) in urine-derived exosomes reveals a pattern corresponding to that reported in serum and whole lung of individuals with IPF. PCR was performed on extracted urine-derived exosomes as described in methods. Data are graphed as relative miRNA expression normalized to U6 and percent of control expression. * P<0.05, **P<0.01 compared to control exosomes. Each point represents an individual patient exosome sample. N=5-12 individual samples/group, P values were calculated by Mann-Whitne U test.

[0146] FIG. 2E shows that Urine and serum-derived exosomes isolated from the same individuals with IPF have similar miRNA expression. Exosome isolation, RNA preparation and PCR performed as described in methods. n=5 individual samples of urine and serum-derived exosomes. Paired T test analysis was performed.

[0147] FIG. 3A, FIG. 3B and FIG. 3C illustrate the biodistribution of circulating urine-derived exosomes. Shown are representative in vivo bioluminescence images to study the biodistribution of ExoGlow™ labeled urine-derived exosomes in mice (n=3/group) at the indicated time points. FIG. 3A is a panel showing a mouse injected with labeled U-IPF exo; FIG. 3B is a panel showing a mouse injected with labeled urine-derived exosomes from age and sex-matched control individuals without lung disease. FIG. 3C is a panel showing a mouse injected with PBS. Intensity of luminescence seen in bar from lowest (red) to highest (blue). n=3 individual exosome preparations/group.

[0148] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, and FIG. 4G show representative TEM photos of lung punches. FIG. 4A, FIG. 4B, and FIG. 4C show mouse lung punches injected with gold nanoparticle labeled urine-derived exosomes from age and sex-matched control subjects (without lung disease) or U-IPFexo (FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, FIG. 411). TEM revealed exosomes in alveolar epithelial cells (AEC) type I and type II. Arrows in FIG. 4C, FIG. 4F, and FIG. 4G highlight exosomes containing nanoparticles. n=2 individual exosome preparations/group.

[0149] FIG. 5A FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F show immunofluorescence staining of lung punches injected with exosomes derived from urine (FIG. 5B, FIG. 5C) or myofibroblasts or fibroblasts (FIG. 5E, FIG. 5F). Lung punches were fixed four days post injection with either PBS (FIG. 5A or FIG. 5D) or control urine-derived exosomes (FIG. 5B), U-IPFexo (FIG. 5C), control fibroblast (FIG. 5E) or MF-IPF exosomes (FIG. 5F). Shown are representative merged photographs at 20×, surfactant protein C (SPC, red), αSMC actin (green) and DAPI (blue). n=3 individual exosome preparations/group.

[0150] FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H, FIG. 6I, FIG. 6J, FIG. 6K, and FIG. 6L show that fibrotic pathways [see FIG. 12] are activated in lung punches after injection with urine (U-IPFexo) or myofibroblast-derived (MF-IPFexo) exosomes. Human (FIG. 6A, FIG. 6B, FIG. 6C) and mouse lung (FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H, FIG. 6I, FIG. 6J, FIG. 6K, FIG. 6L, FIG. 6M) punches were injected with PBS alone, U-IPFexo [FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H, FIG. 6I] or MF-IPFexo FIG. 6J, FIG. 6K, FIG. 6L, FIG. 6M or age and sex-matched control urine exosomes or lung fibroblast exosomes from control subjects (without lung disease). Punches were collected 4 days later and processed as described in Methods. Human lung punches were injected with MF-IPFexo or fibroblast cell derived exosomes (FIG. 6A and FIG. 6B) or urine-derived control and U-IPFexo (FIG. 6B and FIG. 6C). n=2 human lung punch isolates, 2 individual exosome preparations/group. Data are graphed as percent PBS control. α.sub.v-integrin (FIG. 6A, FIG. 6D and FIG. 6J) and collagen type 1 (FIG. 6A, FIG. 6E and FIG. 6K) mRNA expression increased in punches injected with U-IPFexo (derived from urine or myofibroblasts). Data are graphed as normalized for 18S content, n=3-7 individual exosome preparations/group. * P<0.05 compared to PBS, **P<0.01 compared to control exosomes. Downstream fibrotic pathways (see FIG. 12); ERα (FIG. 6C, FIG. 6F, and FIG. 6L), activated AKT (FIG. 6H), c-Jun (FIG. 6 G and FIG. 6M), protein expression and MMP-9 activity (FIG. 6 I) were also stimulated by exosomes from individuals with IPF. * P<0.05, **P<0.01 compared to PBS or control exosomes. P values were calculated by Mann Whitney U test.

[0151] FIG. 7A and FIG. 7B show that epithelization in ex vivo wound healing is decreased by urine-derived IPF exosomes (U-IPFexo). Human skin was wounded, injected with U-IPFexo or control (age and sex-matched from individuals without lung disease) exosomes and maintained at the air-liquid interface. Wound healing was assessed at day 4 post-wounding, a time point when exponential epithelialization occurs. FIG. 7A Data are graphed as mean with each data point representing a single wound. Experiments were performed using triplicate technical replicates and 2-3 biological replicates. P<0.005 PBS and control compared to IPF, PBS vs. Control=0.05 P values were calculated by Mann Whitney U test. FIG. 7B, show photos of gross skin showing visual signs of closure and correspond to the histology assessments. Black arrows point to the initial site of wounding, while white arrows point to the wound edge of the migrating epithelial tongue. Scale bars, 500 μm proportional to the image size.

[0152] FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F, FIG. 8G, FIG. 8H, FIG. 8I, FIG. 8J, FIG. 8K, FIG. 8L, FIG. 8M, FIG. 8N, and FIG. 8O illustrate an assessment of fibrosis in Bleomycin (Bleo) treated mice intravenously infused with exosomes derived from the urine of individuals with IPF (U-IPFexo) compared to infusion with urine exosomes derived from age and sex-matched control subjects without lung disease or urine exosomes derived from subjects with non-CF bronchiectasis or asthma (non-fibrotic lung disease). Histological sections of lung tissue were stained with Masson's-Trichrome as described in Materials and Methods. Representative photomicrographs (4×, 10×, and 20×) of lung sections from Bleo+vehicle (FIG. 8A, FIG. 8B, FIG. 8C), Bleo+control exosome injected mice (FIG. 8D, FIG. 8E, FIG. 8F), from Bleo+U-IPFexo injected mice (FIG. 8G, FIG. 8H, FIG. 8I) or from non-fibrotic inducing exosomes (Bronchiectasis, FIG. 8J, FIG. 8K, FIG. 8L). Fibrotic score (FIG. 8M), collagen content (FIG. 8N), avintegrin (FIG. 8O) increased after Bleo+U-IPFexo treatment. FIG. 8M shows Ashcroft scores were used to evaluate the degree of fibrosis. Data are graphed as the mean score of 32 fields/section of lung. FIG. 8N shows collagen content was estimated by hydroxyproline assay as described in Methods. Data are graphed as g/mg of lung tissue. FIG. 8O shows αv-integrin mRNA expression, which was determined by RT-PCR as a marker of fibrosis. Data are graphed normalized for 18S content. Each data point represents an individual mouse, n=3-11/group *p<0.05, ** p<0.001, p<0.0001 compared to control exosome treatment or compared to Bleo+vehicle treatment. Data were analyzed using one-way analysis of variance (ANOVA) and Mann-Whitney U test.

[0153] FIG. 9A and FIG. 9B show lung fluorescence intensity over time of mice injected with urine-derived exosomes from individuals with IPF (U-IPFexo), urine-derived exosomes from individuals without IPF (Control exosomes), and PBS. FIG. 9C shows ex-vivo fluorescence imaging of isolated organs at 48 hours following exosome treatment in mice.

[0154] FIG. 10A, FIG. 10B, FIG. 10C and FIG. 10D show histology and trichrome staining of lung punches from C57BL6 mice. Lung punches from control lungs shown in tissue culture dish (FIG. 10A) have normal histology (FIG. 10B, Trichrome staining 10×mag) and structure by TEM (FIG. 10C, 500 mag). Histology of non-injected lung punch (FIG. 10D).

[0155] FIG. 11 shows that collagen content increases in mice receiving urine derived exosomes from individuals with IPF. Naïve mice were treated with PBS, control or IPF urine-derived exosomes. Mice were sacrificed 21 days later as described in methods. Data are graphed as mean±SEM. Each data point represents an individual mouse (n=2 exosome preps/group). P<0.05 IPF compared to control and PBS, Data were analyzed using Mann Whitney test.

[0156] FIG. 12 shows potential microRNA regulated pathways leading to fibrosis. The genes and biological processes in the network are generated from the IPF vs Control Lung dataset from NCBI GEO (GS21369) of 11 IPF samples and 6 healthy lung samples.

[0157] FIG. 13 shows an overview of the experimental details and design.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0158] As used herein, the term “about” means plus or minus 20% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 40%-60%.

[0159] The term “activation” or “lymphocyte activation” refers to stimulation of lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells resulting in synthesis of RNA, protein and DNA and production of lymphokines; it is followed by proliferation and differentiation of various effector and memory cells. T-cell activation is dependent on the interaction of the TCR/CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule. The molecular events set in motion by receptor engagement are complex. Full responsiveness of a T cell requires, in addition to receptor engagement, an accessory cell-delivered costimulatory activity, e.g., engagement of CD28 on the T cell by CD80 and/or CD86 on the antigen presenting cell (APC).

[0160] The term “activator protein 1 (APi) as used herein refers to a heterodimeric transcription factor composed of members of the Jun and Fos family of basic leucine zipper proteins. These include the Jun proteins c-Jun, JunB and JunD, as well as the Fos proteins c-Fos, Fra2 and Fosb, respectively. C-Jun in combination with protein c-Fos forms the AP-1 early response transcription factor.

[0161] The term “adaptive immunity” as used herein refers to a specific, delayed and longer-lasting response by various types of cells that create long-term immunological memory against a specific antigen. It can be further subdivided into cellular and humoral branches, the former largely mediated by T cells and the latter by B cells. This arm further encompasses cell lineage members of the adaptive arm that have effector functions in the innate arm, thereby bridging the gap between the innate and adaptive immune response.

[0162] The term “adipose stem cell,” “adipose-derived stem cell,” or “ASC” as used herein refers to pluripotent stem cells, mesenchymal stem cells, and more committed adipose progenitors and stroma obtained from adipose tissue.

[0163] “Administering” when used in conjunction with a therapeutic means to give or apply a therapeutic directly into or onto a target organ, tissue or cell, or to administer a therapeutic to a subject, whereby the therapeutic positively impacts the organ, tissue, cell, or subject to which it is targeted. Thus, as used herein, the term “administering”, when used in conjunction with EVs or compositions thereof, can include, but is not limited to, providing EVs into or onto the target organ, tissue or cell; or providing EVs systemically to a patient by, e.g., intravenous injection, whereby the therapeutic reaches the target organ, tissue or cell. “Administering” may be accomplished by parenteral, oral or topical administration, by inhalation, or by such methods in combination with other known techniques.

[0164] The term “alveolus” or “alveoli” as used herein refers to an anatomical structure that has the form of a hollow cavity. Found in the lung, the pulmonary alveoli are spherical outcroppings of the respiratory sites of gas exchange with the blood. The alveoli contain some collagen and elastic fibers. Elastic fibers allow the alveoli to stretch as they fill with air when breathing in. They then spring back during breathing out, in order to expel the carbon dioxide-rich air.

[0165] The term “angiotensin II” or “Ang-2” as used herein refers to a vasoactive octapeptide produced by the action of angiotensin-converting enzyme on angiotensin 1; it produces stimulation of vascular smooth muscle, promotes aldosterone production, and stimulates the sympathetic nervous system.

[0166] The terms “animal,” “patient,” and “subject” as used herein include, but are not limited to, humans and non-human vertebrates such as wild, domestic and farm animals. According to some embodiments, the terms “animal,” “patient,” and “subject” may refer to humans. According to some embodiments, the terms “animal,” “patient,” and “subject” may refer to non-human mammals.

[0167] The term “antioxidant” as used herein refers to any substance that can prevent, reduce, or repair the ROS-induced damage of a target biomolecule. In ROS biology and medicine, the target molecules usually include proteins, lipids, and nucleic acids, among others. There are three major modes of action for antioxidants: (i) antioxidants that directly scavenge ROS already formed; (ii) antioxidants that inhibit the formation of ROS from their cellular sources; and (iii) antioxidants that remove or repair the damage or modifications caused by ROS. [Li, R., et al. React. Oxyg. Species (Apex) (2016): 1 (1): 9-21].

[0168] The term “aquaporins” as used herein refers to water-specific membrane channel proteins. Aquaporin 5 (AQP5) is found in airway epithelial cells, type I alveolar epithelial cells and submucosal gland acinar cells in the lungs where it plays a key role in water transport.[Hansel, N N et al. PLoS One (2010) doi.10.1371/journal.pone.0014226, citing Verkman, A S et al. Am. J. Physiol. Lung Cell Mol. Physiol. 278 (5): L867-79] Decreased expression of human AQP5 has been associated with mucus overproduction in the airways of subjects with COPD and lower lung function.[Id., citing Wang, K. et al. Acta Pharmacol. Sin. (2007) 28 (8): 1166-74] Furthermore, smoking has been shown to attenuate the expression of AQP5 in submucosal glands of subjects with COPD.[Id., citing Id, citing Wang, K. et al. Acta Pharmacol. Sin. (2007) 28 (8): 1166-74]

[0169] The term “Argonaute 2” or “AGO2” as used herein refers to an RNA binding protein that can shuttle between the cytoplasm and nucleus in a context-dependent fashion [Sharma, N R et al. J. Biol. Chem. (2016) 291: 2302-9] and is a key effector of RNA-silencing pathways. It is a major component of the RNA-induced silencing complex RISC).

[0170] The term “Ashcroft scale for the evaluation of bleomycin-induced lung fibrosis” refers to the analysis of stained histological samples by visual assessment. A modified Ashcroft scale precisely defines the assignment of grades from 0 to 8 for the increasing extent of fibrosis in lung histological samples. [Hubner, R-H et al. Biotechniques (2008) 44 (4): 507-11].

[0171] The term “biomarkers” (or “biosignatures”) as used herein refers to peptides, proteins, nucleic acids, antibodies, genes, metabolites, or any other substances used as indicators of a biologic state. It is a characteristic that is measured objectively and evaluated as a cellular or molecular indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. The term “indicator” as used herein refers to any substance, number or ratio derived from a series of observed facts that may reveal relative changes as a function of time; or a signal, sign, mark, note or symptom that is visible or evidence of the existence or presence thereof. Once a proposed biomarker has been validated, it may be used to diagnose disease risk, presence of disease in an individual, or to tailor treatments for the disease in an individual (choices of drug treatment or administration regimes). In evaluating potential drug therapies, a biomarker may be used as a surrogate for a natural endpoint, such as survival or irreversible morbidity. If a treatment alters the biomarker, and that alteration has a direct connection to improved health, the biomarker may serve as a surrogate endpoint for evaluating clinical benefit. Clinical endpoints are variables that can be used to measure how patients feel, function or survive. Surrogate endpoints are biomarkers that are intended to substitute for a clinical endpoint; these biomarkers are demonstrated to predict a clinical endpoint with a confidence level acceptable to regulators and the clinical community.

[0172] The term “bleomycin-induced pulmonary fibrosis model” as used herein refers to an animal model of pulmonary fibrosis in rodents. It causes inflammatory and fibrotic reactions within a short period of time, even more so after intratracheal instillation. The initial elevation of pro-inflammatory cytokines (interleukin-1, tumor necrosis factor-α, interleukin-6, interferon-γ) is followed by increased expression of pro-fibrotic markers (transforming growth factor-β1, fibronectin, procollagen-1), with a peak around day 14. [Moeller, A. et al. Int. J. Biochem. Cell Biol. (2008) 40 (3): 362-82]. The “switch” between inflammation and fibrosis appears to occur around day 9 after bleomycin (Id., citing Chaudhary, N I et al. Am. J. Respir. Crit. Care Med. (2006) 173 (7): 769-76). While the bleomycin model has the advantage that it is quite easy to perform, widely accessible and reproducible, and therefore fulfills important criteria expected from a good animal model. the bleomycin model has significant limitations in regard to understanding the progressive nature of human IPF. While bleomycin causes an inflammatory response, triggered by overproduction of free radicals, with induction of pro-inflammatory cytokines and activation of macrophages and neutrophils, thus resembling acute lung injury in some way, the subsequent development of fibrosis, however, is at least partially reversible, independent from any intervention (Izbicki, G et al. Intl J. Exp. Pathol. (2002) 83 (3): 111-19), and the aspect of slow and irreversible progression of IPF in patients is not reproduced in the bleomycin model (Chua, F. et al. Am. J. Respir. Cell Mol. Biol. (2005) 33 (1): 9-13).

[0173] The term “bronchiectasis” as used herein refers to abnormal widening of the bronchi or their branches, causing a risk of infection.

[0174] The term “bronchoalveolar lavage” (BAL) is used herein to refer to a medical procedure in which a bronchoscope is passed through the mouth or nose into the lungs and fluid is squirted into a small part of the lung and then collected for examination. “Bronchoalveolar lavage fluid” (BALF) is used herein to refer to the fluid collected from a BAL procedure. Bronchoalveolar lavage (BAL), performed during fiberoptic bronchoscopy is a useful adjunct to lung biopsy in the diagnosis of nonneoplastic lung diseases. BAL is able to provide cells and solutes from the lower respiratory tract and may provide important information about diagnosis and yield insights into immunologic, inflammatory, and infectious processes taking place at the alveolar level. BAL has been helpful in elucidating the key immune effector cells driving the inflammatory response in IPF (Costabel and Guzman Curr Opin Pulm Med, 7 (2001), pp. 255-261). Increase in polymorphonuclear leukocytes, neutrophil products, eosinophils, eosinophil products, activated alveolar macrophages, alveolar macrophage products, cytokines, chemokines, growth factors for fibroblasts, and immune complexes have been noted in BAL of patients with IPF. [Id.].

[0175] The term “c-jun” as used herein refers to a protein of the activator protein-1 complex, which is involved in numerous cell activities, including proliferation, apoptosis, survival, tumorigenesis and tissue morphogenesis. As a basic leucine zipper (bZIP) transcription factor, it acts as a homo- or heterodimer, binding to DNA and regulating gene transcription. It is able to crosstalk, amplify and integrate different signals for tissue development and disease.[Meng, Q. and Xia, Y. Protein Cell (2011) 2 (11): 889-98].

[0176] The term “cargo” as used herein refers to a load or that which is conveyed. With respect to exosomes and or extracellular vesicles, the term cargo refers to a substance encapsulated in the exosome and or extracellular vesicle. The compound or substance can be, e.g., a nucleic acid (e.g., nucleotides, DNA, RNA), a polypeptide, a lipid, a protein, or a metabolite, or any other substance that can be encapsulated in an exosome and or extracellular vesicle.

[0177] The term “cargo profile” as used herein refers to measurements of cargo components that characterize a population of extracellular vesicles

[0178] The term “caveolins (Cavs)” as used herein refers to integrated plasma membrane proteins that are complex signaling regulators with numerous partners and whose activity is highly dependent on cellular context (Boscher, C, Nabi, I R. Adv. Exp. Med. Biol. (2012) 729: 29-50). Cavs are both positive and negative regulators of cell signaling in and/or out of caveolae, invaginated lipid raft domains whose formation is caveolin expression dependent. Caveolins and rafts have been implicated in membrane compartmentalization; proteins and lipids accumulate in these membrane microdomains where they transmit fast, amplified and specific signaling cascades. The term “caveolin 1 (CAV1)”, refers to a scaffolding protein that links integrin subunits to the tyrosine kinase FYN, an initiating step in coupling integrins to the Ras-ERK pathway and promoting cell cycle progression.

[0179] “Cluster of Differentiation” or “cluster of designation” (CD) molecules are utilized in cell sorting using various methods, including flow cytometry. Cell populations usually are defined using a “+” or a “−” symbol to indicate whether a certain cell fraction expresses or lacks a particular CD molecule.

[0180] The term “CD9” as used herein refers to a member of the tetraspanin protein family whose crystal structure shows a reversed cone-like molecular shape, which generates membrane curvature in the crystalline lipid layers. (Umeda, R. et al. Nature Communic. (2020) 11: article 1606).

[0181] The term “CD29” as used herein refers to integrin R31.

[0182] The term “CD37” as used herein refers to a member of the tetraspanin protein family exclusively expressed on immune cells. (Zuidscherwoude, M. et al. Scientific Reports (2015) 5: 12201).

[0183] The term “CD44” as used herein refers to a cell adhesion molecule (HCAM) found on monocytes, neutrophils, fibroblasts and memory T cells, which is involved in lymphocyte homing.

[0184] The term “CD63” as used herein refers to a member of the tetraspanin protein family, the C-terminal domain of which interacts with several subunits of adaptor protein (AP) complexes, linking the traffic of this tetraspanin to clathrin-dependent pathways (Andreu, Z. & Yanez-Mo, M., citing Rous, B A et al. Mol. Biol. Cell (2002) 13 (3): 1071-82). Among intracellular interacting proteins, CD63 was shown to directly bind to syntenin-1, a double PDZ domain-containing protein (Id., citing Latysheva, N. et al. Mol. Cell Biol. (2006) 26 (20): 7707-18). A major role in exosome biogenesis has been reported for Syntenin-1 (Id., citing Baietti, M F et al. Nat. Cell Biol. (2012) 14 (7): 677-85).

[0185] The term “CD81” as used herein refers to a member of the tetraspanin protein family whose crystal structure shows a reversed teepee-like arrangement of the four transmembrane I helices, which create a central pocket in the intramembranous region that appears to bind cholesterol in the central cavity. (Zimmerman, B. et al. Cell (2016) 167: 1041-51). During development, CD81 regulates the trafficking of CD19, an essential co-stimulatory molecule of lymphoid B cells and a well-characterized CD81 partner, along the secretory pathway. (Shoham, T. et al. J. Imunol. (2003) 171: 4062-72). CD9 and CD81 have been shown to regulate several cell-cell fusion processes. (Charrin, S. et al. J. Cell Science (2014) 127: 3641-48).

[0186] The term “CD82” as used herein refers to a member of the tetraspanin protein family that has been implicated in the regulation of protein sorting into EVs and in antigen presentation by antigen presenting cells. (Andreu, Z. and Yanez-Mo, M. Front. Immunol. (2014) doi.org/10.3389/fimmu.2014.00442).

[0187] The term “CD105” refers to endoglin, a cell membrane glycoprotein and part of the transforming growth factor-β receptor complex, which plays a role in angiogenesis.

[0188] The term “cell reprogramming” as used herein refers to a process by which transcription factors inducing cells to revert to an earlier stage of development. It includes reverting mature differentiated cells into immature stem or progenitor cells and then differentiating those stem or progenitor cells; a process of converting somatic cells into other cell types without the need for an intermediate pluripotent state (direct cell reprogramming); or direct conversion of one differentiated cell type into another, also known as transdifferentiation. [Wilmut, I. et al. Philos. Trans. R. Soc. London B. Biol. Sci. (2011) 366 (1575): 2183-97].

[0189] The term “cellular senescence” as used herein refers to a process that results from a variety of stresses and leads to a state of irreversible growth arrest. A variety of cell-intrinsic and -extrinsic stresses can activate the cellular senescence program. These stressors engage various cellular signaling cascades but ultimately activate p53, p16Ink4a, or both. Cells exposed to mild damage that can be successfully repaired may resume normal cell-cycle progression. On the other hand, cells exposed to moderate stress that is chronic in nature or that leaves permanent damage may resume proliferation through reliance on stress support pathways (green arrows). This phenomenon (termed assisted cycling) is enabled by p53-mediated activation of p21. Thus, the p53-p21 pathway can either antagonize or synergize with p16Ink4a in senescence depending on the type and level of stress. Cells undergoing senescence induce an inflammatory transcriptome regardless of the senescence inducing stress. [van Deursen, J M. Nature (2014) 509 (7501): 439-46]. Senescent cells accumulate in tissues and organs with age [Id., citing Lawless, C. Exp. Gerontol. (2010) 45: 772-78; Krishnamurthy, J. et al Nature (2006) 443: 453-57; Jeyapalan, J C et al. Mech Ageing Dev. (2007) 128: 36-44] as well as at sites of tissue injury and remodeling [Id., citing Rajagopalan, S and Long, E O. Proc. Natl. Acad. Sci. USA (2012) 109: 20596-601; Munoz-Espin, D. et al. Cell (2013) 155: 1104-18; Storer, M. et al. Cell (2013) 155: 1119-30; Jun, J I and Lau, L F. Nature Cell Biol. (2010) 12: 676-85; Krizhanovsky, V. et al. Cell (2008) 134: 657-67].

[0190] The term “Chronic Obstructive Pulmonary Disease” as used herein refers to a lung disease that causes obstructed airflow in the lungs and results in breathing problems. It is used to describe such lung diseases as emphysema, chronic bronchitis, and severe asthma. COPD affects 9-10% of the adult population in the United States; it is the third leading cause of death and the 12.sup.th leading cause of morbidity. By 2030, it is expected to be the 4.sup.th leading cause of death worldwide, representing over 4.5 million deaths annually.

[0191] The term “contact” and its various grammatical forms as used herein refers to a state or condition of touching or of immediate or local proximity.

[0192] The term “cytokine” as used herein refers to small soluble protein substances secreted by cells which have a variety of effects on other cells. Cytokines mediate many important physiological functions including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in target cells. Generally, cytokines act locally. They include type I cytokines, which encompass many of the interleukins, as well as several hematopoietic growth factors; type II cytokines, including the interferons and interleukin-10; tumor necrosis factor (“TNF”)-related molecules, including TNFα and lymphotoxin; immunoglobulin super-family members, including interleukin 1 (“IL-1”); and the chemokines, a family of molecules that play a critical role in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on a cell depending on the state of the cell. Cytokines often regulate the expression of, and trigger cascades of, other cytokines.

[0193] The terms “D value” or “mass division diameter” as used herein, refer to the diameter which, when all particles in a sample are arranged in order of ascending mass, divides the sample's mass into specified percentages. The percentage mass below the diameter of interest is the number expressed after the “D”. For example, the D10 diameter is the diameter at which 10% of a sample's mass is comprised of smaller particles, and the D50 is the diameter at which 50% of a sample's mass is comprised of smaller particles. The D50 is also known as the “mass median diameter” as it divides the sample equally by mass. The D90 diameter is the diameter at which 90% of a sample's mass is comprised of smaller particles. While D-values are based on a division of the mass of a sample by diameter, the actual mass of the particles or the sample does not need to be known. A relative mass is sufficient as D-values are concerned only with a ratio of masses. This allows optical measurement systems to be used without any need for sample weighing.

[0194] From the diameter values obtained for each particle a relative mass can be assigned according to the following relationship:

Mass of a sphere=π/6d.sup.3ρ

[0195] Assuming that ρ is constant for all particles and cancelling all constants from the equation:

Relative mass=d.sup.3

[0196] i.e., each particle's diameter is therefore cubed to give its relative mass. These values can be summed to calculate the total relative mass of the sample measured. The values may then be arranged in ascending order and added iteratively until the total reaches 10%, 50% or 90% of the total relative mass of the sample. The corresponding D value for each of these is the diameter of the last particle added to reach the required mass percentage.

[0197] As used herein, the term “derived from” is meant to encompass any method for receiving, obtaining, or modifying something from a source of origin.

[0198] As used herein, the terms “detecting”, “determining”, and their other grammatical forms, are used to refer to methods performed for the identification or quantification of a biomarker, such as, for example, the presence or level of miRNA, or for the presence or absence of a condition in a biological sample. The amount of biomarker expression or activity detected in the sample can be none or below the level of detection of the assay or method.

[0199] The terms “disease” or “disorder” as used herein refer to an impairment of health or a condition of abnormal functioning. The term “fibrotic disease” as used herein refers to a condition marked by an increase of interstitial fibrous tissue. The terms “lung tissue disease” or “lung disease” as used herein refers to a disease that affects the structure of the lung tissue, for example, without limitation, pulmonary interstitium. Scarring or inflammation of lung tissue makes the lungs unable to expand fully (“restrictive lung disease”). It also makes the lungs less capable of taking up oxygen (oxygenation) and releasing carbon dioxide. Examples of lung tissue diseases include, but are not limited to, idiopathic pulmonary fibrosis (IPF), acute lung injury (ALI), radiation-induced fibrosis in the lung, a fibrotic condition associated with lung transplantation, and sarcoidosis, a disease in which swelling (inflammation) occurs in the lymph nodes, lungs, liver, eyes, skin, or other tissues. According to some embodiments, pulmonary fibrosis is due to acute lung injury caused by viral infection, including, without limitation, influenza, SARS-CoV, MERS, COVID-19, and other emerging respiratory viruses.

[0200] The term “dispersion”, as used herein, refers to a two-phase system, in which one phase is distributed as droplets in the second, or continuous phase. In these systems, the dispersed phase frequently is referred to as the discontinuous or internal phase, and the continuous phase is called the external phase and comprises a continuous process medium. For example, in course dispersions, the particle size is 0.5 μm. In colloidal dispersions, size of the dispersed particle is in the range of approximately 1 nm to 0.5 μm. A molecular dispersion is a dispersion in which the dispersed phase consists of individual molecules; if the molecules are less than colloidal size, the result is a true solution.

[0201] The term “dry powder inhaler” or “DPI” as used herein refers to a device similar to a metered-dose inhaler, but where the drug is in powder form. The patient exhales out a full breath, places the lips around the mouthpiece, and then quickly breathes in the powder. Dry powder inhalers do not require the timing and coordination that are necessary with MDIs.

[0202] The term “Drosha” as used herein refers to a nuclear RNase III that cleaves primary miRNAs to release hairpin-shaped pre-miRNAs that are subsequently cut by the cytoplasmic RNase III Dicer to generate mature miRNAs.

[0203] The term “Endosomal Sorting Complexes required for transport” (ESCRTs) refers to components involved in multivesicular body (MVB) and intraluminal vesicle (ILV) biogenesis. ESCRTs consist of approximately twenty proteins that assemble into four complexes (ESCRT-0, -I, -II and -III) with associated proteins (VPS4, VTA1, ALIX), which are conserved from yeast to mammals (Colombo, M. et al. J. Cell Science (2013) 126: 5553-65, citing Henne, W. M., et al. (2011). Dev. Cell 21, 77-91; Henne et al. (2011) Roxrud, I. et al. (2010). ESCRT & Co. Biol. Cell 102, 293-318). The ESCRT-0 complex recognizes and sequesters ubiquitylated proteins in the endosomal membrane, whereas the ESCRT-I and -II complexes appear to be responsible for membrane deformation into buds with sequestered cargo, and ESCRT-III components subsequently drive vesicle scission (Id., citing Hurley, J. H. and Hanson, P. I. (2010). Nat. Rev. Mol. Cell Biol. 11, 556-566; Wollert, T. et al. Nature (2009) 458: 172-77). ESCRT-0 comprises HRS protein that recognizes the mono-ubiquitylated cargo proteins and associates in a complex with STAM, Eps15 and clathrin. HRS recruits TSG101 of the ESCRT-I complex, and ESCRT-I is then involved in the recruitment of ESCRT-III, through ESCRT-II or ALIX, an ESCRT-accessory protein. Finally, the dissociation and recycling of the ESCRT machinery requires interaction with the ATPase associated with various cellular activities (AAA-ATPase) Vps4; Vps4 releases ESCRT-III from the MVB membrane for additional sorting events. It is unclear whether ESCRT-II has a direct role in ILV biogenesis or whether its function is limited to particular cargo (Id., citing Bowers, K. et al. (2006) J. Biol. Chem. 281, 5094-5105; Malerod, L. et al. Traffic 8, 1617-1629).

[0204] Concomitant depletion of ESCRT subunits belonging to the four ESCRT complexes does not totally impair the formation of MVBs, indicating that other mechanisms may operate in the formation of ILVs and thereby of exosome and or extracellular vesicles (Id., citing Stuffers, S. et al., (2009) Traffic 10, 925-937). One of these pathways requires a type II sphingomyelinase that hydrolyses sphingomyelin to ceramide (Id., citing Trajkovic, K. et al. (2008) Science 319, 1244-1247). Although the depletion of different ESCRT components does not lead to a clear reduction in the formation of MVBs and in the secretion of proteolipid protein (PLP) associated to exosomes and or extracellular vesicles, silencing of neutral sphingomyelinase expression with siRNA or its activity with the drug GW4869 decreases exosome and or extracellular vesicle formation and release. However, whether such dependence on ceramides is generalizable to other cell types producing exosomes and or extracellular vesicles and additional cargos has yet to be determined. The depletion of type II sphingomyelinase in melanoma cells does not impair MVB biogenesis (Id., citing van Niel, G. et al., (2011) Dev. Cell 21, 708-721) or exosome and or extracellular vesicle secretion, but in these cells the tetraspanin CD63 is required for an ESCRT-independent sorting of the luminal domain of the melanosomal protein PMEL (van Niel et al., (2011) Dev. Cell 21, 708-721). Moreover, tetraspanin-enriched domains have been proposed to function as sorting machineries allowing exosome and or extracellular vesicle formation (Perez-Hernandez, D. et al. J. Biol. Chem. (2013) 288, 11649-11661).

[0205] Despite evidence for ESCRT-independent mechanisms of exosome and or extracellular vesicle formation, proteomic analyses of purified exosomes and or extracellular vesicles from various cell types have identified ESCRT components (TSG101, ALIX) and ubiquitylated proteins (Id., citing Buschow, S. et al., (2005) Blood Cells Mol. Dis. 35, 398-403; Thery, C. et al., (2006). Curr. Protoc. Cell Biol. Chapter 3, Unit 3.22). It has also been reported that the ESCRT-0 component HRS could be required for exosome and or extracellular vesicle formation and/or secretion by dendritic cells (DCs), and thereby impact on their antigen-presenting capacity (Id., citing Tamai, K. et al., (2010) Biochem. Biophys. Res. Commun. 399, 384-390). The transferrin receptor (TfR) in reticulocytes that is generally fated for exosome and or extracellular vesicle secretion, although not ubiquitylated, interacts with ALIX for MVB sorting (Id., citing Geminard, C. et al., (2004). Traffic 5, 181-193). It was also shown that ALIX is involved in exosome and or extracellular vesicle biogenesis and exosomal sorting of syndecans through its interaction with syntenin (Id., citing Baietti, M F et al., (2012). Nat. Cell Biol. 14, 677-685). Silencing of genes for two components of ESCRT-0 (HRS, STAM1) and one of ESCRT-I (TSG101), as well as a late acting component (VPS4B) induced consistent alterations in exosome and or extracellular vesicle secretion. [Colombo, M. et al. J. Cell Sci. (2013) 126: 5553-65].

[0206] As used herein, the term “enrich” is meant to refer to increasing the proportion of a desired substance, for example, to increase the relative frequency of a subtype of cell or cell component compared to its natural frequency in a cell population. Positive selection, negative selection, or both are generally considered necessary to any enrichment scheme. Selection methods include, without limitation, magnetic separation and fluorescence-activated cell sorting (FACS).

[0207] Estrogen receptors. Estrogen receptors alpha (ERα) and beta (ERβ) are nuclear transcription factors that are involved in the regulation of many complex physiological processes in humans. They act in the cell nucleus, regulating transcription of specific target genes by binding to associated DNA regulatory sequences. In humans, both receptor subtypes are expressed in many cells and tissues, and they control key physiological functions in various organ systems, such as reproductive, skeletal, cardiovascular and central nervous systems, as well as in specific tissues (such as breast and sub-compartments of prostate and ovary). ERα is present mainly in mammary gland, uterus, ovary (thecal cells), bone, male reproductive organs (testes and epididymis), prostate (stroma), liver, and adipose tissue. ERβ is found mainly in the prostate (epithelium), bladder, ovary (granulosa cells), colon, adipose tissue, and immune system. Both subtypes are markedly expressed in the cardiovascular and central nervous systems. There are some common physiological roles for the two ERs, such as in the development and function of the ovaries, and in the protection of the cardiovascular system. The alpha subtype has a more prominent role on the mammary gland and uterus, as well as on the preservation of skeletal homeostasis and the regulation of metabolism. The beta subtype seems to have a more profound effect on the central nervous and immune systems, and it generally counteracts the ERα-promoted cell hyperproliferation in tissues such as breast and uterus. [Paterni, I. et al. Steroids (2014) 0: 13-29. Doi: 10.1016/j.steroids.2014.06.012].

[0208] As used herein, the term “expression” and its various grammatical forms refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may also refer to the post-translational modification of a polypeptide or protein.

[0209] The term “extracellular vesicles” or “EVs” as used herein includes exosomes and microvesicles that carry bioactive molecules, such as proteins, RNAs and microRNAs, that may be released into and influence the extracellular environment. Microvesicles are small membrane-enclosed sacs thought to be generated by the outward budding and fission of membrane vesicles from the cell surface. Exosomes originate predominantly from preformed multivesicular bodies that are released upon fusion with the plasma membrane.

[0210] The term “exosomes” as used herein refers to extracellular bilayered membrane-bound vesicles of endosomal origin in a size range of ˜40 to 160 nm in diameter (˜100 nm on average) generated by all cells that are actively secreted.

[0211] When used to describe the expression of a gene or polynucleotide sequence, the terms “down-regulation”, “disruption”, “inhibition”, “inactivation”, and “silencing” are used interchangeably herein to refer to instances when the transcription of the polynucleotide sequence is reduced or eliminated. This results in the reduction or elimination of RNA transcripts from the polynucleotide sequence, which results in a reduction or elimination of protein expression derived from the polynucleotide sequence (if the gene comprised an ORF). Alternatively, down-regulation can refer to instances where protein translation from transcripts produced by the polynucleotide sequence is reduced or eliminated. Alternatively still, down-regulation can refer to instances where a protein expressed by the polynucleotide sequence has reduced activity. The reduction in any of the above processes (transcription, translation, protein activity) in a cell can be by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to the transcription, translation, or protein activity of a suitable control cell. Down-regulation can be the result of a targeting event as disclosed herein (e.g., indel, knock-out), for example.

[0212] The term “clinical efficacy” as used herein refers to the therapeutic effectiveness of a drug or therapeutic in humans using appropriate outcome measures.

[0213] The term “extracellular matrix” as used herein refers to a scaffold in a cell's external environment with which the cell interacts via specific cell surface receptors. The extracellular matrix serves many functions, including, but not limited to, providing support and anchorage for cells, segregating one tissue from another tissue, and regulating intracellular communication. The extracellular matrix is composed of an interlocking mesh of fibrous proteins and glycosaminoglycans (GAGs). Examples of fibrous proteins found in the extracellular matrix include collagen, elastin, fibronectin, and laminin. Examples of GAGs found in the extracellular matrix include proteoglycans (e.g., heparin sulfate), chondroitin sulfate, keratin sulfate, and non-proteoglycan polysaccharide (e.g., hyaluronic acid). The term “proteoglycan” refers to a group of glycoproteins that contain a core protein to which is attached one or more glycosaminoglycans.

[0214] The term “extracellular vesicles (EVs)” as used herein refers to nanosized, membrane-bound vesicles released from cells that can transport cargo-including DNA, RNA, and proteins-between cells as a form of intercellular communication. Different EV types, including microvesicles (MVs), exosomes, oncosomes, and apoptotic bodies, have been characterized on the basis of their biogenesis or release pathways. Microvesicles bud directly from the plasma membrane, are 100 nanometers (nm) to 1 micrometer (μm) in size, and contain cytoplasmic cargo (Zaborowski, M P et al. BioScience (2015) 65 (8): 783-97, citing Heijnen, H F et al. Blood (1999) 94: 3791-99). Another EV subtype, exosomes, is formed by the fusion between multivesicular bodies and the plasma membrane, by which multivesicular bodies release smaller vesicles (exosomes) whose diameters range from 40 to 120 nm (Id., citing El Andaloussi, S. et al. Nature Reviews Drug Discovery (2013) 12: 347-57; Cocucci, E. and Meldolesi J. Trends in Cell Biology (2015) 25: 364-72). Dying cells, release vesicular apoptotic bodies (50 nm-2 m) that can be more abundant than exosomes and or extracellular vesicles or MVs under specific conditions and can vary in content between biofluids (Id., citing Thery, C. et al. J. Immunology (2001) 1666: 7309-18; El Andaloussi, S. et al. Nature Reviews Drug Discovery (2013) 12: 347-57). Membrane protrusions can also give rise to large EVs, termed oncosomes (1-10 m), which are produced primarily by malignant cells in contrast to their nontransformed counterparts (Id., citing Di Vizio, D. et al. Am. J. Pathol. (2012) 181: 1573-84; Morello, M. et al. Cell Cycle (2013) 12: 3526-36).

[0215] The term “forced expiratory volume” or “FEV1” as used herein refers to the volume of air that an individual can exhale during a forced breath in 1 second. The normal value (95% confidence interval) for FEV1 is 80% to 120%.

[0216] The term “forced vital capacity” or “FVC” as used herein refers to the maximal volume of gas that can be exhaled from full inhalation by exhaling as forcefully and rapidly as possible. The normal value (95% confidence interval) for FVC is 80% to 120%.

[0217] The FEV1/FVC ratio or Tiffeneau Index is the amount of air exhaled in the first second divided by all of the air exhaled during a maximal exhalation. The ratio FEV1/FVC is between 70% and 80% in normal adults; a value less than 70% indicates airflow limitation.

[0218] The term “free radical” is defined as any chemical species capable of independent existence that contains one or more unpaired electrons. An unpaired electron refers to the one that occupies an atomic or molecular orbital by itself. Examples of oxygen radicals include superoxide, hydroxyl, peroxyl, and alkoxyl radicals.

[0219] The term “growth factor” as used herein refers to extracellular polypeptide molecules that bind to a cell-surface receptor triggering an intracellular signaling pathway, leading to proliferation, differentiation, or other cellular response that stimulate the accumulation of proteins and other macromolecules, e.g., by increasing their rate of synthesis, decreasing their rate of degradation, or both. Exemplary growth factors include fibroblast growth factor (FGF), insulin-like growth factor (IGF-1), transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF)

[0220] Fibroblast Growth Factor (FGF). The fibroblast growth factor (FGF) family currently has over a dozen structurally related members. FGF1 is also known as acidic FGF; FGF2 is sometimes called basic FGF (bFGF); and FGF7 sometimes goes by the name keratinocyte growth factor. Over a dozen distinct FGF genes are known in vertebrates; they can generate hundreds of protein isoforms by varying their RNA splicing or initiation codons in different tissues. FGFs can activate a set of receptor tyrosine kinases called the fibroblast growth factor receptors (FGFRs). Receptor tyrosine kinases are proteins that extend through the cell membrane. The portion of the protein that binds the paracrine factor is on the extracellular side, while a dormant tyrosine kinase (i.e., a protein that can phosphorylate another protein by splitting ATP) is on the intracellular side. When the FGF receptor binds an FGF (and only when it binds an FGF), the dormant kinase is activated, and phosphorylates certain proteins within the responding cell, activating those proteins.

[0221] FGFs are associated with several developmental functions, including angiogenesis (blood vessel formation), mesoderm formation, and axon extension. While FGFs often can substitute for one another, their expression patterns give them separate functions. For example, FGF2 is especially important in angiogenesis, whereas FGF8 is involved in the development of the midbrain and limbs.

[0222] The term “hepatocyte growth factor” (or HGF) as used herein refers to a pleiotrophic growth factor, which induces cellular motility, survival, proliferation, and morphogenesis, depending upon the cell type. In the adult, HGF has been demonstrated to play a critical role in tissue repair, including in the lung. Administration of HGF protein or ectopic expression of HGF has been demonstrated in animal models of pulmonary fibrosis to induce normal tissue repair and to prevent fibrotic remodeling. HGF-induced inhibition of fibrotic remodeling may occur via multiple direct and indirect mechanisms including the induction of cell survival and proliferation of pulmonary epithelial and endothelial cells, and the reduction of myofibroblast accumulation. [Panganiban, R A M and Day, R M, Acta Pharmacol. Sin. (2011) 32 (1): 12-20].

[0223] Insulin-Like Growth Factor (IGF-1). IGF-1, a hormone similar in molecular structure to insulin, has growth-promoting effects on almost every cell in the body, especially skeletal muscle, cartilage, bone, liver, kidney, nerves, skin, hematopoietic cell, and lungs. It plays an important role in childhood growth and continues to have anabolic effects in adults. IGF-1 is produced primarily by the liver as an endocrine hormone as well as in target tissues in a paracrine/autocrine fashion. Production is stimulated by growth hormone (GH) and can be retarded by undernutrition, growth hormone insensitivity, lack of growth hormone receptors, or failures of the downstream signaling molecules, including tyrosine-protein phosphatase non-receptor type 11 (also known as SHP2, which is encoded by the PTPN11 gene in humans) and signal transducer and activator of transcription 5B (STAT5B), a member of the STAT family of transcription factors. Its primary action is mediated by binding to its specific receptor, the Insulin-like growth factor 1 receptor (IGF1R), present on many cell types in many tissues. Binding to the IGF1R, a receptor tyrosine kinase, initiates intracellular signaling; IGF-1 is one of the most potent natural activators of the AKT signaling pathway, a stimulator of cell growth and proliferation, and a potent inhibitor of programmed cell death. IGF-1 is a primary mediator of the effects of growth hormone (GH). Growth hormone is made in the pituitary gland, released into the blood stream, and then stimulates the liver to produce IGF-1. IGF-1 then stimulates systemic body growth. In addition to its insulin-like effects, IGF-1 also can regulate cell growth and development, especially in nerve cells, as well as cellular DNA synthesis.

[0224] IGF-1 was shown to increase the expression levels of the chemokine receptor CXCR4 (receptor for stromal cell-derived factor-1, SDF-1) and to markedly increase the migratory response of MSCs to SDF-1 (Li, Y, et al. 2007 Biochem. Biophys. Res. Communic. 356(3): 780-784). The IGF-1-induced increase in MSC migration in response to SDF-1 was attenuated by PI3 kinase inhibitor (LY294002 and wortmannin) but not by mitogen-activated protein/ERK kinase inhibitor PD98059. Without being limited by any particular theory, the data indicate that IGF-1 increases MSC migratory responses via CXCR4 chemokine receptor signaling which is PI3/Akt dependent.

[0225] The term “platelet derived growth factor” or “PDGF” as used herein refers to a major mitogen for connective tissue cells and certain other cell types. It is a dimeric molecule consisting of disulfide-bonded, structurally similar A- and B-polypeptide chains, which combine to homo- and heterodimers. The PDGF isoforms exert their cellular effects by binding to and activating two structurally related protein tyrosine kinase receptors, denoted the alpha-receptor and the beta-receptor. Activation of PDGF receptors leads to stimulation of cell growth, but also to changes in cell shape and motility; PDGF induces reorganization of the actin filament system and stimulates chemotaxis, i.e., a directed cell movement toward a gradient of PDGF. In vivo, PDGF has important roles during the embryonic development as well as during wound healing. Moreover, overactivity of PDGF has been implicated in several pathological conditions. Helden, C H and Westermark, B. Physiol. Rev. (1999) 79 (4): 1283-316].

[0226] Transforming Growth Factor Beta (TGF-β). There are over 30 structurally related members of the TGF-β superfamily, and they regulate some of the most important interactions in development. The proteins encoded by TGF-β superfamily genes are processed such that the carboxy-terminal region contains the mature peptide. These peptides are dimerized into homodimers (with themselves) or heterodimers (with other TGF-β peptides) and are secreted from the cell. The TGF-β superfamily includes the TGF-β family, the activin family, the bone morphogenetic proteins (BMPs), the Vg-1 family, and other proteins, including glial-derived neurotrophic factor (GDNF, necessary for kidney and enteric neuron differentiation) and Müllerian inhibitory factor, which is involved in mammalian sex determination. TGF-β family members TGF-β1, 2, 3, and 5 are important in regulating the formation of the extracellular matrix between cells and for regulating cell division (both positively and negatively). TGF-β1 increases the amount of extracellular matrix epithelial cells make both by stimulating collagen and fibronectin synthesis and by inhibiting matrix degradation. TGF-βs may be critical in controlling where and when epithelia can branch to form the ducts of kidneys, lungs, and salivary glands.

[0227] The term “tumor necrosisfactor-alpha” (“TNF-α”) as used herein refers to a potent pro-inflammatory cytokine exerting pleiotropic effects on various cell types and plays a critical role in the pathogenesis of chronic inflammatory diseases, Transmembrane TNF-α, a precursor of the soluble form of TNF-α, is expressed on activated macrophages and lymphocytes as well as other cell types. After processing by TNF-α-converting enzyme (TACE), the soluble form of TNF-α is cleaved from transmembrane TNF-α and mediates its biological activities through binding to Types 1 and 2 TNF receptors (TNF-R1 and -R2) of remote tissues. Accumulating evidence suggests that not only soluble TNF-α, but also transmembrane TNF-α is involved in the inflammatory response. [Horiuchi, T. et al. Rheumatology (Oxford)](2010) 49 (7): 1215-28].

[0228] Vascular Endothelial Growth Factor (VEGF). VEGFs are growth factors that mediate numerous functions of endothelial cells including proliferation, migration, invasion, survival, and permeability. The VEGFs and their corresponding receptors are key regulators in a cascade of molecular and cellular events that ultimately lead to the development of the vascular system, either by vasculogenesis, angiogenesis, or in the formation of the lymphatic vascular system. VEGF is a critical regulator in physiological angiogenesis and also plays a significant role in skeletal growth and repair.

[0229] VEGF's normal function creates new blood vessels during embryonic development, after injury, and to bypass blocked vessels. In the mature established vasculature, the endothelium plays an important role in the maintenance of homeostasis of the surrounding tissue by providing the communicative network to neighboring tissues to respond to requirements as needed. Furthermore, the vasculature provides growth factors, hormones, cytokines, chemokines and metabolites, and the like, needed by the surrounding tissue and acts as a barrier to limit the movement of molecules and cells.

[0230] The term “healthy control” as used herein refers to a subject in a state of physical well-being without signs or symptoms of a fibrotic lung disease or process.

[0231] Hydroxyproline assay. Collagen content is assessed by quantifying hydroxyproline, an amino acid present in appreciable quantities in collagen.

[0232] The term “high throughput screening” or “HTS” as used herein refers to the use of automated equipment to rapidly test thousands to millions of samples for biological activity at the model organism, cellular, pathway, or molecular level.

[0233] The term “immune system” as used herein refers to a complex arrangement of cells and molecules that maintain immune homeostasis to preserve the integrity of the organism by elimination of all elements judged to be dangerous. Responses in the immune system may generally be divided into two arms, referred to as “innate immunity” and “adaptive immunity.” The two arms of immunity do not operate independently of each other, but rather work together to elicit effective immune responses.

[0234] The term “inflammation” as used herein refers to the physiologic process by which vascularized tissues respond to injury. See, e.g., FUNDAMENTAL IMMUNOLOGY, 4th Ed., William E. Paul, ed. Lippincott-Raven Publishers, Philadelphia (1999) at 1051-1053, incorporated herein by reference. During the inflammatory process, cells involved in detoxification and repair are mobilized to the compromised site by inflammatory mediators. Inflammation is often characterized by a strong infiltration of leukocytes at the site of inflammation, particularly neutrophils (polymorphonuclear cells). These cells promote tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue. Traditionally, inflammation has been divided into acute and chronic responses.

[0235] The term “acute inflammation” as used herein refers to the rapid, short-lived (minutes to days), relatively uniform response to acute injury characterized by accumulations of fluid, plasma proteins, and neutrophilic leukocytes. Examples of injurious agents that cause acute inflammation include, but are not limited to, pathogens (e.g., bacteria, viruses, parasites), foreign bodies from exogenous (e.g. asbestos) or endogenous (e.g., urate crystals, immune complexes), sources, and physical (e.g., burns) or chemical (e.g., caustics) agents.

[0236] The term “chronic inflammation” as used herein refers to inflammation that is of longer duration and which has a vague and indefinite termination. Chronic inflammation takes over when acute inflammation persists, either through incomplete clearance of the initial inflammatory agent or as a result of multiple acute events occurring in the same location. Chronic inflammation, which includes the influx of lymphocytes and macrophages and fibroblast growth, may result in tissue scarring at sites of prolonged or repeated inflammatory activity. The term “innate immunity” as used herein refers to a nonspecific fast response to pathogens that is predominantly responsible for an initial inflammatory response before adaptive immunity is induced. These include such mechanisms as anatomical barriers, antimicrobial peptides, the complement system and the chemokine/cytokine system; macrophages and neutrophils carrying nonspecific pathogen-recognition receptors, and a number of specialized cell types, including innate lymphoid cells (TLCs, including natural killer (NK) cells) mast cells and dendritic cells (DCs). Innate immunity is present in all individuals at all times, does not increase with repeated exposure to a given pathogen, and discriminates between groups of similar pathogens, rather than responding to a specific pathogen.

[0237] The term “infuse” and its other grammatical forms as used herein refers to introduction of a fluid other than blood into a vein.

[0238] The term “inhalation delivery device” as used herein refers to a machine/apparatus or component that produces small droplets or an aerosol from a liquid or dry powder aerosol formulation and is used for administration through the mouth in order to achieve pulmonary administration of a drug, e.g., in solution, powder, and the like. Examples of inhalation delivery device include, but are not limited to, a nebulizer, a metered-dose inhaler, and a dry powder inhaler (DPI).

[0239] The terms “inhibiting”, “inhibit” or “inhibition” are used herein to refer to reducing the amount or rate of a process, to stopping the process entirely, or to decreasing, limiting, or blocking the action or function thereof. Inhibition may include a reduction or decrease of the amount, rate, action function, or process of a substance by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%.

[0240] The term “inhibitor” as used herein refers to a molecule that reduces the amount or rate of a process, stops the process entirely, or that decreases, limits, or blocks the action or function thereof. Enzyme inhibitors are molecules that bind to enzymes thereby decreasing enzyme activity. Inhibitors may be evaluated by their specificity and potency.

[0241] The term “insufflation” as used herein refers to the act of delivering air, a gas, or a powder under pressure to a cavity or chamber of the body. For example, nasal insufflation relates to the act of delivering air, a gas, or a powder under pressure through the nose.

[0242] The term “interleukin” as used herein refers to a cytokine secreted by white blood cells as a means of communication with other white blood cells. For example, interleukin-8 (or “IL-8”) is produced by phagocytes and mesenchymal cells exposed to inflammatory stimuli (e.g., interleukin-1 or tumor necrosis factor) and activates neutrophils inducing chemotaxis, exocytosis and the respiratory burst. In vivo, IL-8 elicits a massive neutrophil accumulation at the site of injection. Interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 form a family of cytokines based on their sharing the common cytokine receptor γ chain (γc). IL-2 and IL-15 both share IL-2Rβ and γc and activate the same Janus kinase (JAK)1/JAK3/signal transducers and activators of transcription (STAT)5 pathway. Although many different cell types can express IL-15 messenger RNA (mRNA), IL-15 protein is mainly produced by dendritic cells (DCs) and monocytes in response to Toll-like receptor (TLR) activation and binds to receptors on these cells. IL-15 plays critical roles in the development and/or maintenance of memory CD8+ T cells and preferentially can expand central memory phenotype T cells in vivo, with Il 15−/− or Il15ra−/− mice having profound loss of memory phenotype CD8+ T cells, intestinal intraepithelial lymphocytes, NKT cells, and NK cells. IL-15 receptors are composed of IL-15Rα, IL-2Rβ, and γc. IL-15Rα is expressed on a wide range of cells, including immune cells (T cells, B cells, macrophages, and stromal-cell lines) and nonimmune cells (keratinocytes and skeletal muscle cells). IL-15 binds to IL-15Rα with high affinity (K.sub.d≈25 pm), much higher than the affinity of IL-2 for IL-2Rα, whereas IL-15 binds IL-2Rβ and γc with a K.sub.d≈1 nm, similar to the affinity of IL-2 to IL-2Rβ and γc. Like IL-2Rα, IL-15Rα does not transduce IL-15 signals, but IL-15Rα-expressing APCs, such as DCs and monocytes, can bind IL-15 and trans-present the cytokine to lymphocytes that express IL-2Rβ and 7c; this trans signaling is the dominant mode for IL-15 action. [Lin, J-X and Leonard, WJ. Cold Spring Harb. Persp. Biol. 2018] 10 (9): a028449]

[0243] The term “interleukin-1 receptor associated kinase” or IRAK-1″ IRAK-4, refer to protein kinases that are part of the intracellular signaling pathways leading from TLRs.

[0244] The term “invasive” and its other grammatical forms as used herein means requiring medical instruments to enter or penetrate the body or to disturb body tissue. The term “noninvasive” and its various grammatical forms accordingly means not requiring medical instruments to enter or penetrate the body or to disturb body tissue.

[0245] The term “isolated” is used herein to refer to material, such as, but not limited to, a nucleic acid, peptide, polypeptide, or protein, which is: (1) substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment. The terms “substantially free” or “essentially free” are used herein to refer to considerably or significantly free of, or more than about 95%, 96%, 97%, 98%, 99% or 100% free. The isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human intervention to a composition and/or placed at a location in the cell (e.g., genome or subcellular organelle) not native to a material found in that environment. The alteration to yield the synthetic material may be performed on the material within, or removed, from its natural state.

[0246] The term “JAG1” refers to the gene that encodes protein jagged-1. Jagged-1 is the ligand for multiple Notch receptors and is involved in the mediation of Notch signaling.

[0247] The term “long noncoding RNA” (“lncRNAs”) as used herein refers to a class of transcribed RNA molecules that are longer than 200 nucleotides and yet do not encode proteins. LncRNAs can fold into complex structures and interact with proteins, DNA and other RNAs, modulating the activity, DNA targets or partners of multiprotein complexes. Crosstalk of lncRNAs with miRNAs creates an intricate network that exerts post-transcriptional regulation of gene expression. For example, lncRNAs can harbor miRNA binding sites and act as molecular decoys or sponges that sequester miRNAs away from other transcripts. Competition between lncRNAs and miRNAs for binding to target mRNAs has been reported and leads to de-repression of gene expression (Zampetaki, A. et al. Front. Physiol. (2018) doi.org/10.3389/fphys.2018.01201, citing Yoon, J H et al. Semin. Cell Dev. Bio. (2014) 34: 9-14; Ballantyne, M D et al. Clin. Pharmacol. Ther. (2016) 99: 494-501). Finally, lncRNAs may contain embedded miRNA sequences and serve as a source of miRNAs (Id., citing Piccoli, M T et al. Cir. Res. (2017) 121: 575-83).

[0248] The terms “lung function” or “pulmonary function” are used interchangeably to refer to the process of gas exchange called respiration (or breathing). In respiration, oxygen from incoming air enters the blood, and carbon dioxide, a waste gas from the metabolism, leaves the blood. A reduced lung function means that the ability of lungs to exchange gases is reduced.

[0249] The terms “lung interstitium” or “pulmonary interstitium” are used interchangeably herein to refer to an area located between the airspace epithelium and pleural mesothelium in the lung. Fibers of the matrix proteins, collagen and elastin, are the major components of the pulmonary interstitium. The primary function of these fibers is to form a mechanical scaffold that maintains structural integrity during ventilation.

[0250] The abbreviation “MAPK” as used herein refers to Mitogen-Activated Protein Kinase (MAPK) signaling which activates a three-tiered cascade with MAPK kinase kinases (MAP3K) activating MAPAK kinases (MAP2K) and finally MAPK. MAPKs are protein Ser/Thr kinases that convert extracellular stimuli into a wide range of cellular responses. (Cargnello, M. and Roux, PP, Microbiol. Mol. Biol. Rev. (2011) 75(1): 50-83). The major MAPK pathways involved in inflammatory diseases are extracellular regulating kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (INK). All three MAPK pathways may be activated by TGF-β, and signaling through these cascades can further regulate the expression of Smad proteins and mediate Smad-independent TGF-β responses. These three MAPK pathways are all involved in TGF-β-induced fibrosis. [He, W. and Dai, C. Curr. Pathobiol. Rep. (2015) 3: 183-92, citing Tsou, P S et al. Am. J. Physiol. Cell Physiol. (22014) 307: C2-13; Kamato, D. et al. Cell Signal (2013) 25: 2017-24; Pannu, J. et al. J. Biol. Chem. (2007) 282: 10405-13; Yu, L. et al. J. Biol. Chem. (2002) EMBO J. 21: 3749-59].

[0251] Upstream kinases include TGFβ-activated kinase-1 (TAK1) and apoptosis signal-regulating kinase-1 (ASK1). Downstream of p38 MAPK is MAPK activated protein kinase 2 (MAPKAPK2 or MK2). TGF-β can signal in a noncanonical manner via the MAPK family.

[0252] The term “matrix metalloproteinases” as used herein refers to a collection of zinc-dependent proteases involved in the breakdown and the remodeling of extracellular matrix components (Guiot, J. et al. Lung (2017) 195(3): 273-280, citing Oikonomidi et al. Curr Med Chem. 2009; 16(10): 1214-1228). MMP-1 and MMP-7 seem to be primarily overexpressed in plasma of IPF patients compared to hypersensitivity pneumonitis, sarcoidosis and COPD with a possible usefulness in differential diagnosis (Id., citing Rosas I O, et al. PLoS Med. 2008; 5(4): e93). They are also involved in inflammation and seem to take part to the pathophysiological process of pulmonary fibrosis (Id., citing Vij R, Noth I. Transl Res. 2012; 159(4): 218-27; Dancer RCA, et al. Eur Respir J. 2011; 38(6): 1461-67). The most studied is MMP-7, which is known as being significantly increased in epithelial cells both at the gene and protein levels and is considered to be active in hyperplastic epithelial cells and alveolar macrophages in IPF (Id., citing Fujishima S, et al. Arch Pathol Lab Med. 2010; 134(8): 1136-42). There is also a significant correlation between higher MMP-7 concentrations and disease severity assessed by forced vital capacity (FVC) and diffusing capacity of the lungs for carbon monoxide (DLCO) (Id., citing Rosas I O, et al. PLoS Med. 2008; 5(4): e93). Higher levels associated to disease progression and worse survival (>4.3 ng/ml for MMP-7) (Id.). The MMP2 gene provides instructions for making matrix metallopeptidase 2. This enzyme is produced in cells throughout the body and becomes part of the extracellular matrix, which is an intricate lattice of proteins and other molecules that forms in the spaces between cells. One of the major known functions of MMP-2 is to cleave type IV collagen, which is a major structural component of basement membranes, the thin, sheet-like structures that separate and support cells as part of the extracellular matrix.

[0253] MMPs play a critical role in neuroinflammation through the cleavage of ECM proteins, cytokines and chemokines. (Ji. R-R et al, US Neurology, Touch Briefings (2008) 71-74). MMP-2 is constitutively expressed and normally present in brain and spinal cord tissues. In contrast, MMP-9 is normally expressed at low levels, but upregulated in many injury and disease states such as spinal cord injury and brain trauma (Id., citing Rosenberg, G A. Glia (2002) 39: 279-91); it is also induced in the crushed sciatic nerve and causes demyelination, a condition associated with neuropathic pain, by the cleavage of myelin basic protein. (Id., citing Chattopadhyay, S. et al. Brain Behav. Immun. (20007) 21: 561-8). Besides targeting matrix, because MMPs can process a variety of growth factors and other extracellular cytokines and signals, they may contribute to the neurovascular remodeling that accompanies chronic CNS injury. (Id., citing Zhao, B Q, et al. Nat. Med. (2006) 12: 441-45).

[0254] The term “mesenchymal stem cells” (MSCs) (also known as bone marrow stromal stem cells or skeletal stem cells) are non-blood adult stem cells found in a variety of tissues. They are characterized by their spindle-shape morphologically; by the expression of specific markers on their cell surface; and by their ability, under appropriate conditions, to differentiates along a minimum of three lineages (osteogenic, chondrogenic, and adipogenic) [Najar M. et al., “Mesenchymal stromal cells and immunomodulation: A gathering of regulatory immune cells”, Cytotherapy, Vol. 18(2): 160-171, (2016)]. No single marker that definitely delineates MSCs in vivo has been identified due to the lack of consensus regarding the MSC phenotype, but it generally is considered that MSCs are positive for cell surface markers CD105, CD166, CD90, and CD44 and that MSCs are negative for typical hematopoietic antigens, such as CD45, CD34, and CD14. As for the differentiation potential of MSCs, studies have reported that populations of bone marrow-derived MSCs have the capacity to develop into terminally differentiated mesenchymal phenotypes both in vitro and in vivo, including bone, cartilage, tendon, muscle, adipose tissue, and hematopoietic supporting stroma. Studies using transgenic and knockout mice and human musculoskeletal disorders have reported that MSC differentiate into multiple lineages during embryonic development and adult homeostasis [Najar M. et al., “Mesenchymal stromal cells and immunomodulation: A gathering of regulatory immune cells”, Cytotherapy, Vol. 18(2): 160-171, (2016)].

[0255] The term “metered-dose inhaler”, “MDI”, or “puffer” as used herein refers to a pressurized, hand-held device that uses propellants to deliver a specific amount of medicine (“metered dose”) to the lungs of a patient. The term “propellant” as used herein refers to a material that is used to expel a substance usually by gas pressure through a convergent, divergent nozzle. The pressure may be from a compressed gas, or a gas produced by a chemical reaction. The exhaust material may be a gas, liquid, plasma, or, before the chemical reaction, a solid, liquid or gel. Propellants used in pressurized metered dose inhalers are liquefied gases, traditionally chlorofluorocarbons (CFCs) and increasingly hydrofluoroalkanes (HFAs). Suitable propellants include, for example, a chlorofluorocarbon (CFC), such as trichlorofluoromethane (also referred to as propellant 11), dichlorodifluoromethane (also referred to as propellant 12), and 1,2-dichloro-1,1,2,2-tetrafluoroethane (also referred to as propellant 114), a hydrochlorofluorocarbon, a hydrofluorocarbon (HFC), such as 1,1,1,2-tetrafluoroethane (also referred to as propellant 134a, HFC-134a, or HFA-134a) and 1,1,1,2,3,3,3-heptafluoropropane (also referred to as propellant 227, HFC-227, or HFA-227), carbon dioxide, dimethyl ether, butane, propane, or mixtures thereof. In other embodiments, the propellant includes a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or mixtures thereof. In other embodiments, a hydrofluorocarbon is used as the propellant. In other embodiments, HFC-227 and/or HFC-134a are used as the propellant.

[0256] The term “microRNA” (or “miRNA” or “miR”) as used herein refers to a class of small, 18- to 28-nucleotide-long, noncoding RNA molecules.

[0257] The term “modulate” as used herein means to regulate, alter, adapt, or adjust to a certain measure or proportion.

[0258] The terms “monitor” or “clinically monitor” are used interchangeably herein to refer to a process of watching so as to provide a warning of the occurrence of events, operations or circumstances.

[0259] As used herein the term “natural killer (NK) cells” refers to lymphocytes in the same family as T and B cells, classified as group I innate lymphocytes. They have an ability to kill tumor cells without any priming or prior activation, in contrast to cytotoxic T cells, which need priming by antigen presenting cells. NK cells secrete cytokines such as IFNγ and TNFα, which act on other immune cells, like macrophages and dendritic cells, to enhance the immune response. Activating receptors on the NK cell surface recognize molecules expressed on the surface of cancer cells and infected cells and switch on the NK cell. Inhibitory receptors act as a check on NK cell killing. Most normal healthy cells express MHCI receptors, which mark them as “self.” Inhibitory receptors on the surface of the NK cell recognize cognate MHCI, which switches off the NK cell, preventing it from killing. Once the decision is made to kill, the NK cell releases cytotoxic granules containing perforin and granzymes, which leads to lysis of the target cell. Natural killer reactivity, including cytokine secretion and cytotoxicity, is controlled by a balance of several germ-line encoded inhibitory and activating receptors such as killer immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs). The presence of the MHC Class I molecule on target cells serves as one such inhibitory ligand for MHC Class I-specific receptors, the Killer cell Immunoglobulin-like Receptor (KIR), on NK cells. Engagement of KIR receptors blocks NK activation and, paradoxically, preserves their ability to respond to successive encounters by triggering inactivating signals. Therefore, if a KIR is able to sufficiently bind to MHC Class I, this engagement may override the signal for killing and allows the target cell to live. In contrast, if the NK cell is unable to sufficiently bind to MHC Class I on the target cell, killing of the target cell may proceed. Consequently, those tumors which express low MHC Class I and which are thought to be capable of evading a T-cell-mediated attack may be susceptible to an NK cell-mediated immune response instead.

[0260] The abbreviation “NFκB” as used herein refers to which is a proinflammatory transcription factor. It switches on multiple inflammatory genes, including cytokines, chemokines, proteases, and inhibitors of apoptosis, resulting in amplification of the inflammatory response [Barnes, PJ, (2016) Pharmacol. Rev. 68: 788-815]. The molecular pathways involved in NF-1B activation include several kinases. The classic (canonical) pathway for inflammatory stimuli and infections to activate NF-κB signaling involve the IKK (inhibitor of KB kinase) complex, which is composed of two catalytic subunits, IKK-α and IKK-β, and a regulatory subunit IKK-γ (or NFκB essential modulator [Id., citing Hayden, M S and Ghosh, S (2012) Genes Dev. 26: 203-234]. The IKK complex phosphorylates Nf-κB-bound IκBs, targeting them for degradation by the proteasome and thereby releasing NF-κB dimers that are composed of p65 and p50 subunits, which translocate to the nucleus where they bind to KB recognition sites in the promoter regions of inflammatory and immune genes, resulting in their transcriptional activation. This response depends mainly on the catalytic subunit IKK-β (also known as IKK2), which carries out IκB phosphorylation. The noncanonical (alternative) pathway involves the upstream kinase NF-κB-inducing kinase (NTK) that phosphorylates IKK-α homodimers and releases RelB and processes p100 to p52 in response to certain members of the TNF family, such as lymphotoxin-β [Id., citing Sun, S C. (2012) Immunol. Rev. 246: 125-140]. This pathway switches on different gene sets and may mediate different immune functions from the canonical pathway. Dominant-negative IKK-β inhibits most of the proinflammatory functions of NF-κB, whereas inhibiting IKK-α has a role only in response to limited stimuli and in certain cells such as B-lymphocytes. The noncanonical pathway is involved in development of the immune system and in adaptive immune responses. The coactivator molecule CD40, which is expressed on antigen-presenting cells, such as dendritic cells and macrophages, activates the noncanonical pathway when it interacts with CD40L expressed on lymphocytes [Id., citing Lombardi, V et al. (2010) Int. Arch. Allergy Immunol. 151: 179-89].

[0261] The term “Notch” refers to a signaling pathway that has been implicated in abnormal differentiation of respiratory epithelial cells in progressive IPF or secondary pulmonary fibrosis. [He, W. and Dai, C. Curr. Pathobiol. Resp. (2015) 3: 183-92, citing Plantier, L. et al. Thorax (2011) 66: 651-57]. Notch proteins are single-pass transmembrane receptors with conserved expression among animal species during evolution. Their principal function is the regulation of many developmental processes, including proliferation, differentiation, and apoptosis. Mammals possess four different Notch receptors, referred to as Notch 1-4. The Notch receptor consists of an extracellular domain, which is involved in ligand binding, and an intracellular domain that works in signal transduction. Notch ligands also are single-pass transmembrane proteins named Jagged (Jag1 and 2) and Delta (Dll1, 3, and 4) [Id., citing Sharma, S. et al. Curr. Opin. Nephrol Hypertens. (2011) 20: 56-61; Bray, SJ. Nat. Rev. Mol. Cell Biol. (2006) 7: 678-89]. Activation of this signaling pathway requires cell-cell contact. Interaction of ligands with the Notch receptors triggers a series of proteolytic cleavages, by a metalloprotease of the ADAM family (TACE; tumor necrosis factor-α-converting enzyme) and finally by the γ-secretase complex. The final cleavage leads to the release of Notch intracellular domain (NICD), which travels to the nucleus and binds to other transcriptional regulators (mainly of the CBF1/RBP-Jκ, SU(H), Lagl family) to trigger the transcription of the target genes, classically belonging to the Hes and Hey family. This core signal transduction pathway is used in most Notch-dependent processes and is known as the canonical Notch pathway [Id., citing Sharma, S. et al. Curr. Opin. Nephrol. Hypertens. (2011) 20: 56-61; Kavian N. et al. Open Rheumatol. J (2012) 6: 96-102; Fortini, M E. Dev. Cell (2009) 16: 633-47]. During the past few years, activation of Notch signaling has shown fibrogenic effects in a wide spectrum of diseases, including systemic sclerosis (SSc) [Id., citing Dees, C. et al. Ann. Rheum. Dis. (2011) 70: 1304-10; Kavian, N. et al. Arthritis Rheum. (2010) 62: 3477-87], scleroderma, idiopathic pulmonary fibrosis (IPF) [Id., citing Plantier, L. et al. Thorax (2011) 66: 651-7], kidney fibrosis [Id., citing Sharma, S. et al. Curr. Opin. Nephrol. Hpertens. (2011) 20: 56-61; Niranjan, T. et al. Nat. Med. (2008) 14: 290-98; Murea, M. et al. Kidney Int. (2010) 78: 514-22], and cardiac fibrosis [Id., citing Kavian, N. et al. Open Rheumatol J. (2012) 6: 96-102].

[0262] The term “organ” as used herein refers to a differentiated structure consisting of cells and tissues and performing some specific function in an organism.

[0263] The term “oxidative stress” as used herein refers to a condition where the levels of ROS significantly overwhelm the capacity of antioxidant defenses, leading to potential damage in a biological system. Oxidative stress condition can be caused by either increased ROS formation or decreased activity of antioxidants or both. not any increases in ROS levels in a biological system are associated with injury. Under certain circumstances, small transient increases in ROS levels can be employed as a signaling mechanism, leading to physiological cellular responses. [Li, R. et al. React. Oxyg. Species (Apex) (2016) 1 (1): 9-21].

[0264] As used herein, the term “paracrine signaling” refers to short range cell-cell communication via secreted signal molecules that act on adjacent cells.

[0265] The term “parenteral” as used herein refers to introduction into the body by way of an injection (i.e., administration by injection), including, for example, subcutaneously (i.e., an injection beneath the skin), intramuscularly (i.e., an injection into a muscle), intravenously (i.e., an injection into a vein), intrathecally (i.e., an injection into the space around the spinal cord or under the arachnoid membrane of the brain), intrasternal injection or infusion techniques.

[0266] The term “particles” as used herein refers to refers to an extremely small constituent (e.g., nanoparticles, microparticles, or in some instances larger) in or on which is contained the composition as described herein.

[0267] The term “particulate” as used herein refers to fine particles of solid or liquid matter suspended in a gas or liquid.

[0268] The term “pathogen” as used herein refers to a causative agent of disease. It includes, without limitation, viruses, bacteria, fungi and parasites.

[0269] The term “pathogenesis” as used herein refers to the pathologic, physiologic or biochemical mechanism resulting in the development of a disease.

[0270] The term “pharmaceutical composition” is used herein to refer to a composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition or disease. The terms “formulation” and “composition” are used interchangeably herein to refer to a product of the described invention that comprises all active and inert ingredients.

[0271] The term “pharmaceutically acceptable,” is used to refer to the carrier, diluent or excipient being compatible with the other ingredients of the formulation or composition and not deleterious to the recipient thereof. The carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the subject being treated. The carrier further should maintain the stability and bioavailability of an active agent. For example, the term “pharmaceutically acceptable” can mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0272] “Phosphopinositide 3-Kinase Pathway, AKT/mTOR and PAK2/c-Abl”. The phosphoatidylinositol 3-kinase (PI3K) pathway is a non-Smad pathway contributing to TGF-β induced fibrosis. It induces two profibrotic pathways: Akt-mammalian target of rapamycin (mTOR) and p21-activated kinase 2 (PAK2)/Abelson kinase (c-Abl). [He, W and Dai, C. Curr. Pathobiol. Rep. (2015) 3: 183-92, citing Biernacka, A, et al. Growth Factors (2011) 29: 196-202; Tsou, P S et a. Am. J. Physiol. Cell Physiol. (2014) 307: C2-C13; Kamato, D. et al. Cell Signal (2013) 25: 2017-24; Wilkes, M C et al. Cancer Res. (2005) 65: 10431-40]. The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTor) signaling pathways are crucial to many aspects of cell growth and survival. [Porta, C. et al., “Targeting PI2K/Akt/mTor signaling in cancer. Frontiers in Oncology (2014) doi.10.3389/fpmc.2014.00064). They are so interconnected that they could be regarded as a single pathway that, in turn, heavily interacts with many other pathways, including that of hypoxia inducible factors (HIFs).

[0273] PI3Ks constitute a lipid kinase family characterized by the capability to phosphorylate inositol ring 3′—OH group in inositol phospholipids. (Id., citing Fruman, D A et al., Phosphoinositide kinases. Annu. Rev. Biochem. (1998) 67: 481-507). Class I PI3Ks are heterodimers composed of a catalytic (CAT) subunit (i.e., p110) and an adaptor/regulatory subunit (i.e., p85). This classis further divided into two subclasses: subclass IA (PI3Kα, β, and δ), which is activated by receptors with protein tyrosine kinase activity, and subclass IB (PI3Kγ), which is activated by receptors coupled with G proteins (Id., citing Fruman, D A et al., Phosphoinositide kinases. Annu. Rev. Biochem. (1998) 67: 481-507).

[0274] Activation of growth factor receptor protein tyrosine kinases results in autophosphorylation on tyrosine residues. PI3K is then recruited to the membrane by directly binding to phosphotyrosine consensus residues of growth factor receptors or adaptors through one of the two SH2 domains In the adaptor subunit. This leads to allosteric activation of the CAT subunit. PI3K activation leads to the production of the second messenger phosphatidylinositol-4,4-bisphosphate (PI3,4,5-P3) from the substrate phosphatidylinositol-4,4-bisphosphate (PI-4,5-P2). PI3,4,5-P3 then recruits a subset of signaling proteins with pleckstrin homology (PH) domains to the membrane, including protein serine/threonine kinase-3′-phosphoinositide-dependent kinase I (PDK1) and Akt/protein kinase B (PKB) (Id., citing Fruman, D A et al., Phosphoinositide kinases. Annu. Rev. Biochem. (1998) 67: 481-507, Fresno-Vara, J A, et al., PI3K/Akt signaling pathway and cancer. Cancer Treat. Rev. (2004) 30: 193-204). Akt/PKB, on its own, regulates several cell processes involved in cell survival and cell cycle progression.

[0275] Akt. Akt (also known as protein kinase B) is a 60 kDa serine/threonine kinase. It is activated in response to stimulation of tyrosine kinase receptors such as platelet-derived growth factor (PDGF), insulin-like growth factor, and nerve growth factor (Shimamura, H, et al., J. Am. Soc. Nephrol. 14: 1427-1434, 2003; Datta K, Franke T F, Chan T O, Makris A, Yang S I, Kaplan D R, Morrison D K, Golemis E A, Tsichlis P N, Mol Cell Biol 15: 2304-2310, 1995; Kulik G, Klippel A, Weber M J, Mol Cell Biol 17: 1595-1606, 1997; Yao R, Cooper G M, Science 267: 2003-2006, 1995). Stimulation of Akt has been shown to be dependent on phosphatidylinositol 3-kinase (PI3-kinase) activity (Fruman D A, Meyers R E, Cantley L C, Annu Rev Biochem 67: 481-507, 1998; Choudhury G G, Karamitsos C, Hernandez J, Gentilini A, Bardgette J, Abboud H E, Am J Physiol 273: F931-938, 1997, Franke T F, Yang S I, Chan T O, Datta K, Kazlauskas A, Morrison D K, Kaplan D R, Tsichlis P N, Cell 81: 727-736, 1995; Franke T F, Kaplan D R, Cantley L C, Cell 88: 435-437, 1997).

[0276] Akt has been shown to act as a mediator of survival signals that protect cells from apoptosis in multiple cell lines (Brunet A, Bonni A, Zigmond M J, Lin M Z, Juo P, Hu L S, Anderson M J, Arden K C, Blenis J, Greenberg M E, Cell 96: 857-868, 1999; Downward J, Curr Opin Cell Biol 10: 262-267, 1998). For example, phosphorylation of the pro-apoptotic Bad protein by Akt was found to decrease apoptosis by preventing Bad from binding to the anti-apoptotic protein Bcl-XL (Dudek H, Datta S R, Franke T F, Birnbaum M J, Yao R, Cooper G M, Segal R A, Kaplan D R, Greenberg M E, Science 275: 661-665, 1997; Datta S R, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg M E, Cell 91: 231-241, 1997). Akt was also shown to promote cell survival by activating nuclear factor-kB (NF-kB) (Cardone M H, Roy N, Stennicke H R, Salvesen G S, Franke T F, Stanbridge E, Frisch S, Reed J C, Science 282: 1318-1321, 1998; Khwaja A, Nature 401: 33-34, 1999) and inhibiting the activity of the cell death protease caspase-9 (Kennedy S G, Kandel E S, Cross T K, Hay N, Mol Cell Biol 19: 5800-5810, 1999).

[0277] mTOR signaling pathway: Mechanistic target of rapamycin (mTOR) is an atypical serine/threonine kinase that is present in two distinct complexes. The first, mTOR complex 1 (mTORC1), is composed of mTOR, Raptor, GOL, and DEPTOR and is inhibited by rapamycin. It is a master growth regulator that senses and integrates diverse nutritional and environmental cues, including growth factors, energy levels, cellular stress, and amino acids. It couples these signals to the promotion of cellular growth by phosphorylating substrates that potentiate anabolic processes such as mRNA translation and lipid synthesis, or limit catabolic processes such as autophagy. The small GTPase Rheb, in its GTP-bound state, is a necessary and potent stimulator of mTORC1 kinase activity, which is negatively regulated by its GTPase-activating protein (GAP), the tuberous sclerosis heterodimer TSC1/2. TSC1 and TSC2 are the tumour-suppressor genes mutated in the tumour syndrome TSC (tuberous sclerosis complex). Their gene products form a complex (the TSC1-TSC2 (hamartin-tuberin) complex), which, through its GAP activity towards the small G-protein Rheb (Ras homologue enriched in brain), is a critical negative regulator of mTORC1 (mammalian target of rapamycin complex 1). (Huang, J. Manning B D, Biochem J. (2008) 412(2): 179-90). Most upstream inputs are funneled through Akt and TSC1/2 to regulate the nucleotide-loading state of Rheb. In contrast, amino acids signal to mTORC1 independently of the PI3K/Akt axis to promote the translocation of mTORC1 to the lysosomal surface where it can become activated upon contact with Rheb. This process is mediated by the coordinated actions of multiple complexes, including the v-ATPase, Ragulator, the Rag GTPases, and GATOR1/2. The second complex, mTOR complex 2 (mTORC2), is composed of mTOR, Rictor, GOL, Sin1, PRR5/Protor-1, and DEPTOR. mTORC2 promotes cellular survival by activating Akt, regulates cytoskeletal dynamics by activating PKCa, and controls ion transport and growth via SGK1 phosphorylation. Aberrant mTOR signaling is involved in many disease states

[0278] PI3K also acts as a branch point in response to TGF-β, leading to activation of PAK2/c-Abl, which stimulates collagen gene expression in normal fibroblasts, and induces fibroblast proliferation, thereby increasing the number of myofibroblast precursors. [[He, W and Dai, C. Curr. Pathobiol. Rep. (2015) 3: 183-92 citing Wilkes, M C and Leof, EB. J. Biol. Chem. (2006) 281: 27846-54]. PAK2/c-Abl promotes fibrosis through its downstream mediators, including PKCδ/Fli-1 and early growth response (Egr)-1, -2, and -3 {Id., citing Tsou, P S et al. A. J. Physiol. Cell Physiol. (2014) 307: C2-C13; Bhattacharyya, S. et al. J. Pathol. (2013) 229: 286-97; Fang, F. et al. Am. J. Pathol. (2013) 183: 1197-1208}.

[0279] The term “Plasma-Lyte” or Plasma-Lyte 148” as used herein refers to an isotonic, buffered intravenous crystalloid solution with a physiochemical composition that closely reflects human plasma.

[0280] The term “potency” and its various grammatical forms as used herein, refers to power or strength of a formulation.

[0281] The term “pulmonary compliance” as used herein refers to the change in lung volume per unit change in pressure. Dynamic compliance is the volume change divided by the peak inspiratory transthoracic pressure. Static compliance is the volume change divided by the plateau inspiratory pressure. Pulmonary compliance measurements reflect the elastic properties of the lungs and thorax and are influenced by factors such as degree of muscular tension, degree of interstitial lung water, degree of pulmonary fibrosis, degree of lung inflation, and alveolar surface tension (Doyle D J, O'Grady K F. Physics and Modeling of the Airway, D, in Benumof and Hagberg's Airway Management, 2013). Total respiratory system compliance is given by the following calculation:

C=ΔV/ΔP

where ΔV=change in lung volume, and ΔP=change in airway pressure This total compliance may be related to lung compliance and thoracic (chest wall) compliance by the following relation:

[00001]$\frac{1}{C_T}=\frac{1}{C_L}+\frac{1}{C_{\mathrm{Th}}}$ [0282] where CT=total compliance (e.g., 100 mL/cm H.sub.2O) [0283] CL=lung compliance (e.g., 200 mL/cm H.sub.2O) [0284] CTh=thoracic compliance (e.g., 200 mL/cm H.sub.2O)
The values shown in parentheses are some typical normal adult values that can be used for modeling purposes (Id.).

[0285] It has been reported that soon after onset of respiratory distress from COVID, patients initially retain relatively good compliance despite very poor oxygenation. [Marini, J J and Gattinoni, L., JAMA Insights (2020) doi: 10.1001/jama.2020.6825, citing Grasselli, G. et al., JAMA (2020) doi: 10.1001/jama.2020.5394; Arentz, M. et al. JAMA (2020) doi: 10.1001/jama.2020.4326]. Minute ventilation is characteristically high. Infiltrates are often limited in extent and, initially, are usually characterized by a ground-glass pattern on CT that signifies interstitial rather than alveolar edema. Many patients do not appear overtly dyspneic. These patients can be assigned, in a simplified model, to “type L,” characterized by low lung elastance (high compliance), lower lung weight as estimated by CT scan, and low response to PEEP. {Id., citing Gattinoni, L. et al. Intensive Care Med. (2020) doi: 10.1007/s00134-020-06033-2}. For many patients, the disease may stabilize at this stage without deterioration while others, either because of disease severity and host response or suboptimal management, may transition to a clinical picture more characteristic of typical ARDS. These can be defined as “type H,” with extensive CT consolidations, high elastance (low compliance), higher lung weight, and high PEEP response. Types L and H are the conceptual extremes of a spectrum that includes intermediate stages, in which their characteristics may overlap.

[0286] The term “potency” as used herein and its various grammatical forms is an expression of the activity of a drug in terms of the concentration or amount of the drug required to produce a defined effect.

[0287] The term “precision medicine” as used herein refers to an approach for disease treatment and prevention that takes into account individual variability in genes, environment and lifestyle. A precision medicine approach allows for a more accurate prediction of which treatment and prevention strategies for a particular disease will work in which groups of patients. This is in contrast to a one-size-fits-all approach, in which disease treatment and prevention strategies are developed for the average person with less consideration for differences between individuals.

[0288] The term “progression” as used herein refers in medicine to the course of a disease as it becomes worse or spreads in the body.

[0289] The term “purification” and its various grammatical forms as used herein refers to the process of isolating or freeing from foreign, extraneous, or objectionable elements.

[0290] The term “reactive oxygen species” as used herein refers to oxygen-containing reactive species. It is a collective term to include superoxide (O2.—), hydrogen peroxide (H2O2), hydroxyl radical (OH.), singlet oxygen (1O2), peroxyl radical (LOO.), alkoxyl radical (LO.), lipid hydroperoxide (LOOH), peroxynitrite (ONOO—), hypochlorous acid (HOCl), and ozone (O3), among others. [Li, R. et al. React. Oxyg. Species (Apex) (2016) 1 (1): 9-21]

[0291] The term “recombinant” as used herein refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

[0292] The term “rejuvenate” and its various grammatical forms as used herein refer to making young or youthful again. The term “resuscitate” as used herein refers to being restored to life or being revived.

[0293] The term “RISC” or RNA-induced silencing complex, as used herein, refers to a multiprotein complex that incorporates one strand of a small interfering RNA (siRNA) or micro RNA (miRNA). RISC uses the siRNA or miRNA as a template for recognizing complementary mRNA. When it finds a complementary strand, it activates RNase and cleaves the RNA. This process is important both in gene regulation by microRNAs and in defense against viral infections, which often use double-stranded RNA as an infectious vector.

[0294] Redox signaling refers to a physiological process, where ROS act as second messengers to mediate responses that are required for proper function and survival of the cell. On the other hand, redox modulation (or redox regulation) refers to a process wherein ROS alter the activity or function of the redox-sensitive molecular targets, including signaling proteins and metabolic enzymes, leading to either physiological or pathophysiological responses. When pathophysiological responses occur it is also known as oxidative stress. [Li, R. et al. React. Oxyg. Species (Apex) (2016) 1 (1): 9-21].

[0295] The term “repair” as used herein as a noun refers to any correction, reinforcement, reconditioning, remedy, making up for, making sound, renewal, mending, patching, or the like that restores function. When used as a verb, it means to correct, to reinforce, to recondition, to remedy, to make up for, to make sound, to renew, to mend, to patch or to otherwise restore function.

[0296] The term “reverse” as used herein refers to turning backward or in an opposite direction.

[0297] The terms “staging” or “clinical staging” are used interchangeably herein to refer to a process of determining details about the extent of a disease that guide decisions about treatment.

[0298] The term “signal transducers and activators of transcription” or “STATS” refers to a family of seven transcription factors activated by many cytokine and growth factor receptors. There are seven STATs (1-4, 5a, 5b, and 6), which reside in the cytoplasm in an inactive form until activated by cytokine receptors. Before activation, most STATS form homodimers, due to a specific homotypic interaction between domains present at the amino termini of the individual STAT proteins. The receptor specificity of each STAT is determined by the recognition of the distinctive phosphotyrosine sequence on each activated receptor by the different SH2 domains within the various STAT proteins. Recruitment of a STAT to the activated receptor brings the STAT close to an activated Janus kinase (JAK), which can then phosphorylate a conserved tyrosine residue in the carboxy terminus of the particular STAT. This leads to a rearrangement, in which the phosphotyrosine of each STAT protein binds to the SH2 domain of the other STAT, forming a configuration that can bind DNA with high affinity. Activated STATS predominantly form homodimers, with cytokine typically activating one type of STAT. For example, IFN-gamma activates STAT1 and generates STAT 1 homodimers, while IL-4 activates STAT6, generating STAT1 homodimers. Other cytokine receptors can activate several STATS, and some STAT heterodimers can be formed. The phosphorylated STAT dimer enters the nucleus, where it acts as a transcription factor to initiate the expression of selected genes that can regulate growth and differentiation of particular subsets of lymphocytes. [Janeway's Immunobiology, 9.sup.th Ed., Murphy K. & Weaver, C. Eds. Garland Science, New York (2017) at 110-111].

[0299] The term “signature” as used herein refers to a specific and complex combination of biomarkers that reflect a biological state.

[0300] The term “Sirt1” as used herein refers to a member of the sirtuin family. Sirtuins are evolutionarily conserved proteins that use nicotinamide adenine dinucleotide (NAD+) as a co-substrate in their enzymatic reactions. There are seven proteins (SIRT1-7) in the human sirtuin family, among which SIRT1 is the most conserved and characterized. Sirt1 is a nicotinamide adenosine dinucleotide (NAD)-dependent deacetylase that removes acetyl groups from several transcription factors and regulatory proteins that are involved in inflammation, antioxidant expression, DNA repair, mitochondrial function, proteostasis, including autophagy. It inhibits cellular senescence and PI3K-mTOR signaling and restores defective autophagy. More specifically, Sirt1 activates FOXo3a, which regulates antioxidants (superoxide dismutases and catalase), activates PGC-1α, a transcription factor that maintains mitochondrial function, inhibits p53 induced senescence, and inhibits NF-κB thereby suppressing the senescence-associated secretory phenotype (SASP). The SASP response is activated by p2.sup.CIP1, which results in activation of p38 mitogen activated protein (MAP) kinase and Janus-activated kinases (JAK), which results in the activation of NF-κB and secretion of proinflammatory cytokines (e.g., IL-1β, IL-6, TNFα), growth factors (e.g., VEGF, TGF-β), chemokines (e.g., CXCL1, CXCL8, CCL2) and matrix metalloproteinases (e.g., MMP-2, MMP-9), which are all increased in age-related diseases, including COPD. [Barnes, P J et al. Am L. Respir. Crit. Care Med. (2019) 200 (5): 556-64]. Plasminogen activator inhibitor-1 (PAI-1), another characteristic SASP protein, is increased in the sputum, sputum macrophages, and alveoli of patients with COPD [Id., citing To, M. et al. Chest (2013) 144: 515-21] and in IPF (Id., citing Schlliga, M. et al. Int. J. Biochem. Cell Biol. (2018) 97: 108-117).

[0301] Sirt1 has been implicated in a broad range of physiological functions, including control of gene expression, metabolism and aging [Rahman, S. and Islam, R. Cell Communication & Signaling (2011) article 11, citing Michan, S. & Sinclair, D. Biochem. J (2007) 404: 1-13; Haigis, M C and Guarente, LP. Genes Dev. (2006) 20: 2913-21; Yamamoto, H. et al. Mol. Endocrinol. (2007) 21: 1745-55]. Whereas an increase in the expression of the SIRT1 protein has been observed in cancer [Elibol, B. and Kilic, U. Front. Endocrinol. (Laussane) (2018) 9: 614, citing Chen, W Y et al. Cell (2005) 123: 437-48; Wang, C. et al. Nat. Cell Biol. (2006) 8: 1025-31], reductions in the SIRT1 level are more common in other diseases such as Alzheimer's Diseases (AD), Parkinson Disease (PD), obesity, diabetes, and cardiovascular diseases [Id., citing Lutz, M I et al. Neuromol. Med. (2014) 16: 405-14; Costa dos Santos, C. et al. Obes. Surg. (2010) 20: 633-9; Singh, P. et al. BMC Neurosci. (2017) 18: 46; Chan, S H et al. Redox Biol. (2017) 13: 301-9; Aditya, R. et al. Curr. Phar. Des. (2017) 23: 2299-307]. Recent developments elucidated the relation between downregulation of SIRT1 levels and disease progression as an increase in oxidative stress and inflammation [Id., citing Singh, P. et al. BMC Neurosci. (2017) 18: 46; Chan, S H et al. Redox Biol. (2017) 13: 301-9]. The list of Sirt1 substrates is continuously growing and includes several transcription factors: the tumor suppressor protein p53, members of the FoxO family (forkhead box factors regulated by insulin/Akt), HES1 (hairy and enhancer of split 1), HEY2 (hairy/enhancer-of-split related with YRPW motif 2), PPARγ (peroxisome proliferator-activated receptor gamma), CTIP2 [chicken ovalbumin upstream promoter transcription factor (COUPTF)-interacting protein 2], p300, PGC-1α (PPARγ coactivator), and NF-κB (nuclear factor kappa B) [Rahman, S. and Islam, R. Cell Communication & Signaling (2011) article 11, citing Michan S. and Sinclair D. Biochem. J. (2007) 404: 1-13; Haigis, M C and Guarente, LP. Genes Dev. (2006) 20: 2913-21; Yamamoto, H. et al. Mol. Endocrinol. (2007) 21: 1745-55].

[0302] The term “skeletal muscle satellite cells” as used herein refers to myogenic stem cells residing between the myofiber plasmalemma and basal lamina that can self-renew and produce differentiated progeny. Skeletal muscle satellite cells may be identified by the specific expression of the paired box transcription factor Pax-7. [Yablonka-Reuveni, Z. J. Histochem. Cytochem. (2011) 59 (12): 1041-59].

[0303] The term “slow” as used herein refers to holding back progress or development.

[0304] The terms “soluble” and “solubility” refer to the property of being susceptible to being dissolved in a specified fluid (solvent). The term “insoluble” refers to the property of a material that has minimal or limited solubility in a specified solvent. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.

[0305] A “solution” generally is considered as a homogeneous mixture of two or more substances. It is frequently, though not necessarily, a liquid. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent.

[0306] The term “solvate” as used herein refers to a complex formed by the attachment of solvent molecules to that of a solute.

[0307] The term “solvent” as used herein refers to a substance capable of dissolving another substance (termed a “solute”) to form a uniformly dispersed mixture (solution).

[0308] The term “stem cells” refers to undifferentiated cells having high proliferative potential with the ability to self-renew that can generate daughter cells that can undergo terminal differentiation into more than one distinct cell phenotype. The term “renewal” or “self renewal” as used herein, refers to the process by which a stem cell divides to generate one (asymmetric division) or two (symmetric division) daughter cells having development potential indistinguishable from the mother cell. Self-renewal involves both proliferation and the maintenance of an undifferentiated state.

[0309] The term “adult (somatic) stem cells” as used herein refers to undifferentiated cells found among differentiated cells in a tissue or organ. Their primary role in vivo is to maintain and repair the tissue in which they are found. Adult stem cells, which have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscles, skin, teeth, gastrointestinal tract, liver, ovarian epithelium, and testis, are thought to reside in a specific area of each tissue, known as a stem cell niche, where they may remain quiescent (non-dividing) for long periods of time until they are activated by a normal need for more cells to maintain tissue, or by disease or tissue injury. Mesenchymal stem cells are an example of adult stem cells.

[0310] As used herein, the phrase “subject in need” of treatment for a particular condition is a subject having that condition, diagnosed as having that condition, or at risk of developing that condition. According to some embodiments, the phrase “subject in need” of such treatment also is used to refer to a patient who (i) will be administered a composition of the described invention; (ii) is receiving a composition of the described invention; or (iii) has received at least one a composition of the described invention, unless the context and usage of the phrase indicates otherwise.

[0311] The terms “surfactant protein A (SP-A)” and “surfactant protein D (SP-D)” refer to hydrophobic, collagen-containing calcium-dependent lectins, with a range of nonspecific immune functions at pulmonary and cardiopulmonary sites. SP-A and SP-D play crucial roles in the pulmonary immune response, and are secreted by type II pneumocytes, nonciliated bronchiolar cells, submucosal glands, and epithelial cells of other respiratory tissues, including the trachea and bronchi. SP-D is important in maintaining pulmonary surface tension, and is involved in the organization, stability, and metabolism of lung parenchyma (Wang K, et al. Medicine (2017) 96 (23): e7083). An increase of 49 ng/mL (1 SD) in baseline SP-A level was associated with a 3.3-fold increased risk of mortality in the first year after presentation. SP-A and SP-D are predictors of worse survival in a one year mortality regression model (Guiot, J. et al. Lung (2017) 195(3): 273-280).

[0312] The term “suspension” as used herein refers to a dispersion (mixture) in which a finely-divided species is combined with another species, with the former being so finely divided and mixed that it doesn't rapidly settle out. In everyday life, the most common suspensions are those of solids in liquid.

[0313] The term “symptom” as used herein refers to a sign or an indication of disorder or disease, especially when experienced by an individual as a change from normal function, sensation, or appearance.

[0314] As used herein, the term “therapeutic agent” or “active agent” refers to refers to the ingredient, component or constituent of the compositions of the described invention responsible for the intended therapeutic effect.

[0315] The term “therapeutic component” as used herein refers to a therapeutically effective dosage (i.e., dose and frequency of administration) that eliminates, reduces, or prevents the progression of a particular disease manifestation in a percentage of a population. An example of a commonly used therapeutic component is the ED50, which describes the dose in a particular dosage that is therapeutically effective for a particular disease manifestation in 50% of a population.

[0316] The term “therapeutic effect” as used herein refers to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect may include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect may also include, directly or indirectly, the arrest, reduction, or elimination of the progression of a disease manifestation.

[0317] The term “therapeutic signature” as used herein refers to a specific and complex combination of biomarkers that reflect a biological state that leads to a specific therapeutic effect.

[0318] As used herein, the term “tissue” refers to a collection of similar cells and the intercellular substances surrounding them. For example, adipose tissue is a connective tissue consisting chiefly of fat cells surrounded by reticular fibers and arranged in lobular groups or along the course of smaller blood vessels. Connective tissue is the supporting or framework tissue of the body formed of fibrous and ground substance with numerous cells of various kinds. It is derived from the mesenchyme, and this in turn from the mesoderm. The varieties of connective tissue include, without limitation, areolar or loose; adipose; sense, regular or irregular, white fibrous; elastic; mucous; lymphoid tissue; cartilage and bone.

[0319] The term “tissue inhibitors of metalloproteases” or “TIMPs” as used herein refers to key regulators of the metalloproteinases that degrade the extracellular matrix and shed cell surface molecules. [Brew, K. and Nagase, H. Biochim. Biophys. Acta (2010) 1803 (1): 55-71]. TIMPs can undergo changes in molecular dynamics induced by their interactions with proteases. TIMPs also have biological activities that are independent of metalloproteinases; these include effects on cell growth and differentiation, cell migration, anti-angiogenesis, anti- and pro-apoptosis, and synaptic plasticity. The human genome has 4 paralogous genes encoding TIMPs (TIMPs-1 to -4). All four TIMPs inhibit MMPs, but with affinities that vary for different inhibitor-protease pairs. The four human TIMPs are, in general terms, broad-spectrum inhibitors of the 23 MMPs found in humans, but there are some differences in specificity among them. TIMP-1 is more restricted in its inhibitory range than the other three TIMPs, having a relatively low affinity for the membrane-type MMPs, MMP-14, MMP-16, and MMP-24 as well as for MMP-19. There are some relatively subtle differences between the affinities of different TIMPs for other MMPs. For example, TIMPs-2 and -3 are weaker inhibitors than TIMP-1 for MMP-3 and MMP-7, contrasting with their affinities for other MMPs [Id., citing Hamze, Abe et al. Protein Sci. (2007) 16: 1905-13]]. TIMP-3 is unique among the mammalian TIMPs in inhibiting a broader array of metalloproteinases including several members of the aggrecanase ADAM and ADAMTS families [Id., citing Amour, A. et al. FEBS Lett. (1998) 435: 39-44; Kashiwagi, M. et al. J. Biol. Chem. (2001) 276: 12501-4; Amour, A. et al. FEBS Lett. (2000) 473: 275-9; Hashimoto, G. et al. FEBS Lett. (2001) 494: 192-5; Wang, W M et al. Biochem. J. (2006) 398: 515-19; Jacobssen, J. et al. Biochemistry (2008) 47: 537-47]. Other TIMPs have limited inhibitory activities for ADAMs: TIP-1 and TIMP-2 inhibit ADAM10 [Id., citing Amour, A. et al. FEBS Lett. (2000) 473: 275-9] and ADAM12 [Id., citing Jacobsen, J. et al. Biochemistry (2008) 47: 537-47], respectively. TIP-3 and N-TIMP-4, but not full-length TIMP-4, inhibit ADAM17 [Id., citing Lee, M H et al. J. Biol. Chem. (2005) 280: 15967-75]. TIMP-4 was also reported to inhibit ADAM28 [Mochizuki, S. et al. Biochem. Biophys. Res. Commun. (2004) 315: 79-84]. ADAM metalloproteinases differ from the MMPs in domain structures and are highly divergent in catalytic domain sequences: ADAMs are membrane-bound enzymes containing disintegrin, cysteine-rich, EGF-like and transmembrane domains C-terminal to their catalytic domains [Id., citing Edwards, D R et al. Mol. Aspects Med. (2008) 29: 258-89]; and ADAMTS (disintegrin-metalloproteinases with thrombospondin motifs) are secreted proteins with a disintegrin domain and variable numbers of thrombospondin type 1 motifs and other domains in their C-terminal regions [Id., citing Porter, S. et al. Biochem. J. (2005) 386: 15-27]. The term “toll-like receptor” or “TLRs” as used herein refers to innate receptors on macrophages, dendritic cells and some other cells that recognize pathogens and their products, such as bacterial lipopolysaccharide (LPS). Recognition stimulates the receptor-bearing cells to produce cytokines that help initiate immune responses. For example, TLR-1 is a cell surface toll-like receptor that acts in a heterodimer with TLR-2 to recognize lipoteichoic acid and bacterial lipoproteins. TLR-2 is a cell surface toll-like receptor that acts in a heterodimer with either TLR-1 or TLR-6 to recognize lipoteichoic acid and bacterial lipoproteins. TLR-4 is a cell surface toll-like receptor that, in conjunction with accessory proteins MD-2 and CD14, recognizes bacterial lipopolysaccharide and lipoteichoic acid. TLR5 is a cell surface toll-like receptor that recognizes the flagellin protein of bacterial flagella. TLR 6 is a cell surface toll-like receptor that acts in a heterodimer with TLR2 to recognize lipoteichoic acid and bacterial lipoproteins. TLR3 is an endosomal toll-like receptor that recognizes double-stranded viral RNA. TLR-7 is an endosomal toll-like receptor that recognizes single-stranded viral RNA. TLR-8 is an endosomal toll-like receptor that recognizes single-stranded viral RNA. TLR-9 is an endosomal toll-like receptor that recognizes DNA containing unmethylated CpG.

[0320] Smad-dependent pathway for TGF-β signaling. The classical Smad-dependent pathway for transforming growth factor-β (TGFβ) signaling occurs when TGF-β receptor type 2, which is constitutively active, transphosphorylates and forms a complex with the TGF-β-bound TGF-β receptor type 1. This complex then phosphorylates serine residues of cytoplasmic receptor-activated Smad (R-Smad), a complex of Smad2 and Smad3. These two heterodimerize and bind to the common mediator Smad (Co-Smad) Smad4, and the whole complex translocates across the nuclear membrane to interact with specific cis-acting elements in the regulatory regions of its target genes [(He, W. and Dai, C. Curr. Pathobiol. Rep. (2015) 3(2): 183-92), citing Tsou, P S et al. Am. J. Physiol. Cell Physiol. (2014) 307: C2-13], recruiting coactivators such as p300 and CBP; corepressors such as c-Ski, SnoN, transforming growth-inhibiting factor, and Smad nuclear-interacting protein 1; or transcription factors such as AP-1 and Sp1 to modulate gene expression [Id., citing Biernacka, A. et al. Growth Factors (2011) 29: 196-202]. Inhibitory Smad (I-Smad) Smad6 or Smad7, acting as negative regulators, not only antagonizes the TGF-β/Smad pathway by binding to TGF-β1 or competing with activated R-Smad for binding to Co-Smad, but also recruits the E3 ubiquitin-protein ligases Smurf1 and Smurf2, which target Smad proteins for proteasomal degradation, thereby blocking Smad2/3 activation, facilitating receptor degradation, and eventually terminating Smad-mediated signaling.

[0321] The term “transcriptome” as used herein refers to the full range of messenger RNA (or mRNA) molecules expressed by an organism. The term “transcriptome” also refers to the array of mRNA transcripts produced in a particular cell or tissue type.

[0322] The terms “treat,” “treated,” or “treating” as used herein refers to both therapeutic treatment and/or prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0323] The term “tumor necrosis factor receptor-associated factor 6” or TRAF6” as used herein refers to an E3 ligase that produces a K63 polyubiquitin signaling scaffold in TLR-4 signaling to activate the NFκB pathway.

[0324] The term “tumor susceptibility gene 101 or Tsg101” as used herein refers to a housekeeping gene highly conserved between mouse and human for which significant variations in high or low protein expression levels in normal tissues or cancer cells are likely a consequence of post-transcriptional or post-translational mechanisms. It has been suggested to function as a negative regulator of ubiquitin-mediated protein degradation [Ferraiuolo, R-M, et al., Cancers (Basel) (2020) 12 (20: 450, citing Koonin, E V and Abagyan, RA. Nat. Genet. (1997) 75: 467-69) as well as a mediator for the intracellular movement of ubiquinated proteins [Id., citing Katzmann, D J et al. Cell (2001) 106: 145-55].

[0325] The terms “usual interstitial pneumonia” or “UIP” pattern are used interchangeably herein to refer to a morphologic entity defined by a combination of 1) patchy interstitial fibrosis with alternating areas of normal lung, (2) temporal heterogeneity of fibrosis characterized by scattered fibroblastic foci in the background of dense acellular collagen, and (3) architectural alteration due to chronic scarring or honeycomb change [Am. Thoracic Society (ATS)?European Respiratory Society (ERS), Am. J. Resp. Crit. Care Med. (2002) 165(2): 277-304. UIP is not entirely synonymous with IPF, and diagnosis of IPF requires an exclusion of possible underlying clinical conditions, including collagen vascular disease, drug toxicity, chronic hypersensitivity pneumonitis, asbestosis, familial IPF, and Hermansky-Pudlak syndrome. [Id.]

[0326] The term “WNT1 (Wnt Family Member 1)” as used herein refers to a protein coding gene and a member of the WNT gene family. Protein Wnt-1 is encoded by the WNT1 gene and is very conserved in evolution. It acts in the canonical Wnt signaling pathway by promoting beta-catenin-dependent transcriptional activation. Activation of Wnt/P catenin signaling has been reported in skin, kidney, liver, lung and cardiac fibrosis. [He, W and Dai, C. Curr. Pathobiol. Rep (2015) 3: 183-92, citing Wynn, T A and Ramalingam, TR. Nat. Med. (2012) 18: 1028-40; Lam, A P and Gottardi, CJ. Curr. Opin. Rheumatol. (2011) 23: 562-7]. Wnt proteins deliver their signal across the plasma membrane by interacting with Fizzled receptors and coreceptors. Once Wnts bind to their receptors/coreceptors, they initiate a chain of downstream signaling events leading to dephosphorylation of β-catenin [Id., citing Liu, Y. J. Am. Soc. Nephrol. (2010) 21: 212-22]. Escaping from degradation mediated by the ubiquitin/proteasome system stabilized β catenin accumulates in the cytoplasm and translocates into the nucleus, where it interacts with its DNA-binding partner known as T cell factor (TCF)/lymohpcyte enhancer-binding factor 1 (LEF1) to stimulate the transcription of Wnt target genes. [Id., citing Lam, A P and Gottardi, CJ. Curr. Opin. Rheumatol. (2011) 23: 562-67; Liu, Y. J. Am. Soc. Nephrol. (2010) 21: 212-22; Huang, H. & He, X. Curr. Opin. Cell Biol. (2008) 20: 119-25}.

[0327] The term “wound healing” refers to the process by which the body repairs trauma to any of its tissues, especially those caused by physical means and with interruption of continuity.

[0328] A wound-healing response often is described as having three distinct phases-injury, inflammation and repair. Generally speaking, the body responds to injury with an inflammatory response, which is crucial to maintaining the health and integrity of an organism. If, however, it goes awry, it can result in tissue destruction.

[0329] Although these three phases are often presented sequentially, during chronic or repeated injury, these processes function in parallel, placing significant demands on regulatory mechanisms. (Wilson and Wynn, Mucosal Immunol., 2009, 3(2): 103-121).

Phase I: Injury

[0330] Injury caused by factors including, but not limited to, autoimmune or allergic reactions, environmental particulates, or infection or mechanical damage, often results in the disruption of normal tissue architecture, initiating a healing response. Damaged epithelial and endothelial cells must be replaced to maintain barrier function and integrity and prevent blood loss, respectively. Acute damage to endothelial cells leads to the release of inflammatory mediators and initiation of an anti-fibrinolytic coagulation cascade, temporarily plugging the damaged vessel with a platelet and fibrin-rich clot. For example, lung homogenates, epithelial cells or bronchoalveolar lavage fluid from idiopathic pulmonary fibrosis (IPF) patients contain greater levels of the platelet-differentiating factor, X-box-binding protein-1, compared with chronic obstructive pulmonary disease (COPD) and control patients, suggesting that clot-forming responses are continuously activated. In addition, thrombin (a serine protease required to convert fibrinogen into fibrin) is also readily detected within the lung and intra-alveolar spaces of several pulmonary fibrotic conditions, further confirming the activation of the clotting pathway. Thrombin also can directly activate fibroblasts, increasing proliferation and promoting fibroblast differentiation into collagen-producing myofibroblasts. Damage to the airway epithelium, specifically alveolar pneumocytes, can evoke a similar anti-fibrinolytic cascade and lead to interstitial edema, areas of acute inflammation, and separation of the epithelium from the basement membrane.

[0331] Platelet recruitment, degranulation and clot formation rapidly progress into a phase of vasoconstriction with increased permeability, allowing the extravasation (movement of white blood cells from the capillaries to the tissues surrounding them) and direct recruitment of leukocytes to the injured site. The basement membrane, which forms the extracellular matrix underlying the epithelium and endothelium of parenchymal tissue, precludes direct access to the damaged tissue. To disrupt this physical barrier, zinc-dependent endopeptidases, also called matrix metalloproteinases (MMPs), cleave one or more extracellular matrix constituents allowing extravasation of cells into, and out of, damaged sites.

Phase H: Inflammation

[0332] Once access to the site of tissue damage has been achieved, chemokine gradients recruit inflammatory cells. Neutrophils, eosinophils, lymphocytes, and macrophages are observed at sites of acute injury with cell debris and areas of necrosis cleared by phagocytes.

[0333] The early recruitment of eosinophils, neutrophils, lymphocytes, and macrophages providing inflammatory cytokines and chemokines can contribute to local TGF-β and IL-13 accumulation. Following the initial insult and wave of inflammatory cells, a late-stage recruitment of inflammatory cells may assist in phagocytosis, in clearing cell debris, and in controlling excessive cellular proliferation, which together may contribute to normal healing. Late-stage inflammation may serve an anti-fibrotic role and may be required for successful resolution of wound-healing responses. For example, a late-phase inflammatory profile rich in phagocytic macrophages, assisting in fibroblast clearance, in addition to IL-10-secreting regulatory T cells, suppressing local chemokine production and TGF-β, may prevent excessive fibroblast activation.

[0334] The nature of the insult or causative agent often dictates the character of the ensuing inflammatory response. For example, exogenous stimuli like pathogen-associated molecular patterns (PAMPs) are recognized by pathogen recognition receptors, such as toll-like receptors and NOD-like receptors (cytoplasmic proteins that have a variety of functions in regulation of inflammatory and apoptotic responses), and influence the response of innate cells to invading pathogens. Endogenous danger signals also can influence local innate cells and orchestrate the inflammatory cascade.

[0335] The nature of the inflammatory response dramatically influences resident tissue cells and the ensuing inflammatory cells. Inflammatory cells themselves also propagate further inflammation through the secretion of chemokines, cytokines, and growth factors. Many cytokines are involved throughout a wound-healing and fibrotic response, with specific groups of genes activated in various conditions. Fibrotic lung disease (such as idiopathic pulmonary fibrosis) patients more frequently present pro-inflammatory cytokine profiles (including, but not limited to, interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), and platelet-derived growth factors (PDGFs)). Each of these cytokines has been shown to exhibit significant pro-fibrotic activity, acting through the recruitment, activation and proliferation of fibroblasts, macrophages, and myofibroblasts.

Phase III: Tissue Repair and Contraction

[0336] The closing phase of wound healing consists of an orchestrated cellular reorganization guided by a fibrin (a fibrous protein that is polymerized to form a “mesh” that forms a clot over a wound site)-rich scaffold formation, wound contraction, closure and re-epithelialization. The vast majority of studies elucidating the processes involved in this phase of wound repair have come from dermal wound studies and in vitro systems.

[0337] Myofibroblast-derived collagens and smooth muscle actin (α-SMA) form the provisional extracellular matrix, with macrophage, platelet, and fibroblast-derived fibronectin forming a fibrin scaffold. Collectively, these structures are commonly referred to as granulation tissues. Primary fibroblasts or alveolar macrophages isolated from IPF patients produce significantly more fibronectin and α-SMA than control fibroblasts, indicative of a state of heightened fibroblast activation. It has been reported that IPF patients undergoing steroid treatment had similar elevated levels of macrophage-derived fibronectin as IPF patients without treatment. Thus, similar to steroid resistant IL-13-mediated myofibroblast differentiation, macrophage-derived fibronectin release also appears to be resistant to steroid treatment, providing another reason why steroid treatment may be ineffective. From animal models, fibronectin appears to be required for the development of pulmonary fibrosis, as mice with a specific deletion of an extra type III domain of fibronectin (EDA) developed significantly less fibrosis following bleomycin administration compared with their wild-type counterparts.

[0338] In addition to fibronectin, the provisional extracellular matrix consists of glycoproteins (such as PDGF), glycosaminoglycans (such as hyaluronic acid), proteoglycans and elastin. Growth factor and TGF-β-activated fibroblasts migrate along the extracellular matrix network and repair the wound. Within skin wounds, TGF-β also induces a contractile response, regulating the orientation of collagen fibers. Fibroblast to myofibroblast differentiation, as discussed above, also creates stress fibers and the neo-expression of α-SMA, both of which confer the high contractile activity within myofibroblasts. The attachment of myofibroblasts to the extracellular matrix at specialized sites called the “fibronexus” or “super mature focal adhesions” pull the wound together, reducing the size of the lesion during the contraction phase. The extent of extracellular matrix laid down and the quantity of activated myofibroblasts determines the amount of collagen deposition. To this end, the balance of matrix metalloproteinases (MMPs) to tissue inhibitor of metalloproteinases (TIMPs) and collagens to collagenases vary throughout the response, shifting from pro-synthesis and increased collagen deposition towards a controlled balance, with no net increase in collagen. For successful wound healing, this balance often occurs when fibroblasts undergo apoptosis, inflammation begins to subside, and granulation tissue recedes, leaving a collagen-rich lesion. The removal of inflammatory cells, and especially α-SMA-positive myofibroblasts, is essential to terminate collagen deposition. Interestingly, in IPF patients, the removal of fibroblasts can be delayed, with cells resistant to apoptotic signals, despite the observation of elevated levels of pro-apoptotic and FAS-signaling molecules.

[0339] Several studies also have observed increased rates of collagen-secreting fibroblast and epithelial cell apoptosis in IPF, suggesting that yet another balance requires monitoring of fibroblast apoptosis and fibroblast proliferation. From skin studies, re-epithelialization of the wound site re-establishes the barrier function and allows encapsulated cellular re-organization. Several in vitro and in vivo models, using human or rat epithelial cells grown over a collagen matrix, or tracheal wounds in vivo, have been used to identify significant stages of cell migration, proliferation, and cell spreading. Rapid and dynamic motility and proliferation, with epithelial restitution from the edges of the denuded area, occur within hours of the initial wound. In addition, sliding sheets of epithelial cells can migrate over the injured area assisting wound coverage. Several factors have been shown to regulate re-epithelialization, including serum-derived transforming growth factor alpha (TGF-α), and matrix metalloproteinase-7 (MMP-7) (which itself is regulated by TIMP-1).

[0340] Collectively, the degree of inflammation, angiogenesis, and amount of extracellular matrix deposition all contribute to ultimate development of a fibrotic lesion.

EMBODIMENTS

Method of Preparing a Purified, Enriched Population of Exosomes

[0341] According to one aspect, the present disclosure provides a method of preparing a purified, enriched population of exosomes derived from a biological sample, wherein the biological sample is derived from a subject with a chronic lung disease, the method comprising: [0342] (a) collecting a biological sample from the subject with the lung disease and from a healthy control; [0343] (b) centrifuging the biological sample at low speed to form a clarified supernatant; [0344] (c) ultracentrifuging the clarified supernatant to pellet the purified enriched population of exosomes; and [0345] (d) characterizing the purified enriched population of exosomes, wherein. [0346] (i) the population of exosomes expresses two or more exosome biomarker selected from the group consisting of CD9, CD63, CD81, or HSP70; [0347] (ii) size of the exosomes in the population of exosomes ranges from 30 μm to 150 μm, inclusive; [0348] (iii) the population of exosomes comprises a total protein of at least about 100-200 μg, inclusive; [0349] (iv) the population of exosomes comprise a total RNA content of at least about 100-200 ng, inclusive; and [0350] (v) the population of exosomes comprises a cargo comprising dysregulated expression of two or more microRNAs selected frommiR-199, miR Let-7a, miR Let-7b, mir-Let-7d, miR-10a, miR-21, miR-29a, miR-34, miR-101, miR-125, miR-145, miR-146a, miR-181a, miR-181b, miR-181c, miR-199, and miR-142.

[0351] According to some embodiments, the biological sample is a body fluid. According to some embodiments, the body fluid is blood, breast milk, saliva, urine, bile, pancreatic juice, cerebrospinal fluid, amniotic fluid, or a peritoneal fluid. According to some embodiments, the body fluid is serum or urine. According to some embodiments, the body fluid is serum. According to some embodiments, the body fluid is urine. According to some embodiments, the number of exosomes in the population of exosomes comprises at least 10E10 particles.

[0352] According to some embodiments, the biological sample is obtained from a mammal. According to some embodiments, the biological sample is obtained from a non-human mammal or a human subject. According to some embodiments, the human subject is over 50 years of age. According to some embodiments, the healthy control is age and sex matched to the subject.

[0353] An exemplary protocol for isolation of a population of exosomes from a biological sample is as follows. A 4° C. biological sample is centrifuged at 3,000×g for 20 min at room temperature in a swinging bucket rotor to remove large cells and debris. The clarified supernatant is collected and ultracentrifuged at 100,000×g for 2 hours, in a fixed angle rotor at 4° C., to pellet exosomes. The exosome pellet is resuspended in a minimum volume of DPBS (approximately 120 uL/ultracentrifugation tube). Exosomes are characterized using a Thermo NanoDrop spectrophotometer for protein determination and approximate RNA concentration by direct absorbance; exosomes were not lysed, stained, or RNA extracted prior to taking these measurements. Particle diameter and concentration is assessed by tunable resistive pulse sensing (TRPS; (qNano, Izon Science Ltd) using a NP150 nanopore membrane at a 47 mm stretch. The concentration of particles is standardized using multi-pressure calibration with carboxylated polystyrene beads of a defined size (nm diameter) and at a defined concentration (particles/mL).

[0354] According to some embodiments, size of the population of exosomes ranges from about 30 μm to about 150 μm, inclusive, in diameter, i.e., about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm, about 100 μm, about 105 μm, about 110 μm, about 120 μm, about 125 μm, about 130 μm, about 135 μm, about 140 μm, about 145 μm, or about 150 am, in diameter.

[0355] According to some embodiments, the population of exosomes comprises a total protein content of at least about 100-200 μg, inclusive, i.e., at least about 100 μg, at least about 105 μg, at least about 110 μg, at least about 115 μg, at least about 120 μg, at least about 125 μg, at least about 130 μg, at least about 135 μg, at least about 140 μg, at least about 145 μg, at least about 150 μg, at least about 155 μg, at least about 160 μg, at least about 165 μg, at least about 170 μg, at least about 175 μg, at least about 180 μg, at least about 185 μg, about 190 μg, at least about 195 μg, or at least about 200 μg.

[0356] For RNA isolation a Norgen Preserved Blood RNA Purification Kit I can be used according to manufacturer's directions. Briefly, for every 100 μl of exosome preparation, 300 μL of lysis solution is added. After a short vortex, 400 μL of 95-100% ethanol is added. 600 μl of the lysate containing ethanol is loaded onto the column and centrifuged for 1 minute at >3,500×g (˜6,000 RPM). The column is washed three times with wash solution and centrifuged each time for 1 minute (14,000×g). The column is spun for an additional two minutes in order to thoroughly dry the resin and the column contents eluded with 50 μl of Elution Solution by centrifuging for 2 minutes at 200×g (˜2,000 RPM), followed by 1 minute at 14,000×g. According to some embodiments, the population of exosomes comprises a total RNA content of from at least about 100 ng to at least about 200 ng, inclusive, i.e., at least about 100 ng, at least about 105 ng, at least about 110 ng, at least about 115 ng, at least about 120 ng, at least about 125 ng, at least about 130 ng, at least about 135 ng, at least about 140 ng, at least about 145 ng, at least about 150 ng, at least about 155 ng, at least about 160 ng, at least about 165 ng, at least about 170 ng, at least about 175 ng, at least about 180 ng, at least about 185 ng, at least about 190 ng, at least about 195 ng, or at least about 200 ng.

[0357] According to some embodiments, the miRNA expression is a signature of a fibrotic lung disease. According to some embodiments, dysregulated miRNA expression of miR-199 and miR-34a in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is fibrotic. According to some embodiments, dysregulated miRNA expression of miR-142 in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is antifibrotic. According to some embodiments, dysregulated expression of miR-199 in the population of exosomes derived from the biological sample of a subject diagnosed with IPF compared to a population of exosomes derived from a biological sample derived from a healthy control is increased compared to a population of exosomes derived from a healthy control. According to some embodiments, the dysregulated expression of one or more of let-7D, miR-29, or miR-181 in the population of exosomes derived from the biological sample of the subject diagnosed with IPF compared to a population of exosomes derived from a biological sample derived from a healthy control is decreased, compared to the population of exosomes derived from the healthy control. According to some embodiments, dysregulated expression of miR-199 in the population of exosomes derived from the biological sample of the subject diagnosed with IPF compared to a population of exosomes derived from a biological sample derived from a healthy control and the dysregulated expression of one or more of let-7D, miR-29, miR-142, miR-181 derived from the population of exosomes derived from a biological sample from the subject diagnosed with IPF is decreased, compared to the population of exosomes derived from the healthy control. According to some embodiments, reduced or absent microRNA Let-7 expression in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is associated with early stage fibrotic lung disease. According to some embodiments, microRNA Let-7 expression in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is associated with end-stage fibrotic lung disease.

[0358] Examples of methods for exosome product characterization analysis include, without limitation, nanoparticle size and concentration analysis; flow cytometry analysis of exosome specific protein markers (CD63, HSP70), and pro-angiogenic and pro-migratory potency. Other exemplary analytics include, without limitation:cytokine testing using a 9-panel human angiogenesis multiplex ELISA comprising: Ang-2, FGF, HGF, IL-8, PDGF, TIMP-1, TIMP-2, and VEGF at standard dilutions of 1:2, 1:20, 1:200 and 1:800; Mycoplasma testing by multiplex detection by polymerase chain reaction; sterility testing for media by membrane filtration; and metabolite and pH analysis on the NOVA Flex 2 for Gln, Glu, Gluc, Lac, NH.sub.4+, Na+, K+, Ca++, pH, pCO.sub.2, pO.sub.2 and osmolality.

[0359] According to some embodiments, the purified enriched population of exosomes provides enhanced therapeutic targeting/precision; improved cell selectivity/tropism, and directed pharmacology at desired cellular or tissue sites. According to some embodiments, the purified enriched population of exosomes provides enhanced therapeutic targeting/precision. According to some embodiments, the purified enriched population of exosomes provides improved cell selectivity/tropism. According to some embodiments, the purified enriched population of exosomes provides directed pharmacology at desired cellular or tissue sites.

[0360] According to some embodiments, markers for fibrosis (ay-integrin, collagen type 1 mRNA, TGF-β mRNA expression, and other downstream fibrotic pathways (e.g., c-Jun, AKT expression and MMP-9 activity) can be stimulated in an ex vivo assay by the population of exosomes derived from the biological sample (e.g., urine or serum) from a subject diagnosed with the fibrotic lung disease compared to a healthy control. According to some embodiments, the ex vivo assay is a human lung punch assay. According to some embodiments, decreased wound closure by the population of exosomes derived from the biological sample (e.g., urine or serum) from a subject diagnosed with the fibrotic lung disease compared to a healthy control can be measured in an ex vivo wound healing assay

Methods for Diagnosis, Prognosis, and Treatment

[0361] According to another aspect, the present disclosure provides a method for noninvasively diagnosing and staging a progressive chronic lung disease characterized by disease related lung dysfunction comprising: [0362] (a) quantifying lung function of a subject with a progressive chronic lung disease characterized by disease-related lung dysfunction; [0363] (b) determining a stage of the chronic lung disease by: [0364] (i) collecting a biological sample from the subject with the lung disease and from a healthy control; [0365] (ii) centrifuging the biological sample at low speed to form a clarified supernatant; [0366] (iii) ultracentrifuging the clarified supernatant to pellet the purified enriched population of exosomes; and [0367] (iv) characterizing the purified enriched population of exosomes, wherein. [0368] (1) the population of exosomes expresses two or more exosome biomarker selected from the group consisting of CD9, CD63, CD81, or HSP70; [0369] (2) size of the exosomes in the population of exosomes ranges from 30 μm to 150 μm, inclusive; [0370] (3) the population of exosomes comprises a total protein of at least about 100 μg-200 μg, inclusive; [0371] (4) the population of exosomes comprise a total RNA content of at least about 100 ng-200 ng, inclusive; and [0372] (5) the population of exosomes comprises a cargo comprising dysregulated expression of two or more microRNAs selected from miR-199, miR Let-7a, miR Let-7b, mir-Let-7d, miR-10a, miR-21, miR-29a, miR-34, miR-101, miR-125, miR-145, miR-146a, miR-181a, miR-181b, miR-181c, miR-199, and miR-142, and

[0373] wherein expression of the two or more microRNAs, compared to a healthy control, comprises a signature of a fibrotic lung disease; and [0374] (c) medically managing the diagnosed fibrotic lung disease as early as possible in the course of progression of the disease to reduce or slow its progression by [0375] (1) treating the diagnosed fibrotic lung disease; and [0376] (2) monitoring over time the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control.

[0377] According to some embodiments, the lung function of the subject is quantified by determining forced expiratory volume (FEV1); forced vital capacity (FVC) and FEV/FVC %.

[0378] According to some embodiments, the biological sample is a body fluid. According to some embodiments, the body fluid is blood, breast milk, saliva, urine, bile, pancreatic juice, cerebrospinal fluid, amniotic fluid or a peritoneal fluid. According to some embodiments, the body fluid is serum or urine. According to some embodiments, the body fluid is serum. According to some embodiments, the body fluid is urine. According to some embodiments, the number of exosomes in the population of exosomes comprises at least 10E10 particles. According to some embodiments, the biological sample is obtained from a mammal. According to some embodiments, the biological sample is obtained from a non-human mammal or a human subject. According to some embodiments, the human subject is over 50 years of age. According to some embodiments, the healthy control is age and sex matched to the subject.

[0379] According to some embodiments, the population of exosomes comprises a total protein content of at least about 100-200 μg, inclusive, i.e., at least about 100 μg, at least about 105 μg, at least about 110 μg, at least about 115 μg, at least about 120 μg, at least about 125 μg, at least about 130 μg, at least about 135 μg, at least about 140 μg, at least about 145 μg, at least about 150 μg, at least about 155 μg, at least about 160 μg, at least about 165 μg, at least about 170 μg, at least about 175 μg, at least about 180 μg, at least about 185 μg, about 190 μg, at least about 195 μg, or at least about 200 μg.

[0380] According to some embodiments, the population of exosomes comprises a total RNA content of from at least about 100 ng to at least about 200 ng, inclusive, i.e., at least about 100 ng, at least about 105 ng, at least about 110 ng, at least about 115 ng, at least about 120 ng, at least about 125 ng, at least about 130 ng, at least about 135 ng, at least about 140 ng, at least about 145 ng, at least about 150 ng, at least about 155 ng, at least about 160 ng, at least about 165 ng, at least about 170 ng, at least about 175 ng, at least about 180 ng, at least about 185 ng, at least about 190 ng, at least about 195 ng, or at least about 200 ng.

[0381] According to some embodiments, selected miRNAs are expressed in the population of exosomes derived from subjects diagnosed with IPF when compared to healthy controls. According to some embodiments this selective miRNA expression can identify IPF at a stage before standard procedures (e.g., Ashcroft scoring and histology) demonstrate changes consistent with lung fibrosis.

[0382] According to some embodiments, the miRNA expression is a signature of a fibrotic lung disease. According to some embodiments:dysregulated miRNA expression of miR-199 and miR-34a in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is fibrotic. According to some embodiments:dysregulated miRNA expression of miR-142 in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is antifibrotic. According to some embodiments, dysregulated expression of miR-199 in the population of exosomes derived from the biological sample of a subject diagnosed with IPF compared to a population of exosomes derived from a biological sample derived from a healthy control is increased compared to a population of exosomes derived from a healthy control. According to some embodiments, the dysregulated expression of one or more of let-7D, miR-29, or miR-181 in the population of exosomes derived from the biological sample of the subject diagnosed with IPF compared to a population of exosomes derived from a biological sample derived from a healthy control is decreased, compared to the population of exosomes derived from the healthy control. According to some embodiments, dysregulated expression of miR-199 in the population of exosomes derived from the biological sample of the subject diagnosed with IPF compared to a population of exosomes derived from a biological sample derived from a healthy control and the dysregulated expression of one or more of let-7D, miR-29, miR-142, miR-181 derived from the population of exosomes derived from a biological sample from the subject diagnosed with IPF is decreased, compared to the population of exosomes derived from the healthy control. According to some embodiments, reduced or absent microRNA Let-7 expression in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is associated with early stage fibrotic lung disease. According to some embodiments, microRNA Let-7 expression in the population of exosomes derived from the biological sample of the subject compared to a population of exosomes derived from a biological sample derived from a healthy control is associated with end-stage fibrotic lung disease.

[0383] According to some embodiments, the purified enriched population of exosomes provides enhanced therapeutic targeting/precision; improved cell selectivity/tropism, and directed pharmacology at desired cellular or tissue sites. According to some embodiments, the purified enriched population of exosomes provides enhanced therapeutic targeting/precision. According to some embodiments, the purified enriched population of exosomes provides improved cell selectivity/tropism. According to some embodiments, the purified enriched population of exosomes provides directed pharmacology at desired cellular or tissue sites.

[0384] According to some embodiments, the progressive chronic lung disease if left untreated comprises one or more of a progressive injury, progressive inflammation, progressive fibrosis or a combination thereof.

[0385] According to some embodiments, the fibrotic lung disease is pulmonary fibrosis. According to some embodiments, the fibrotic lung disease is idiopathic pulmonary fibrosis. According to some embodiments, the chronic lung disease is due to chronic smoking or a severe viral infection. According to some embodiments the severe lung infection is due to a severe coronavirus infection. According to some embodiments, the dysregulated miRNAs expressed in the population of exosomes derived from subjects when compared to healthy controls are diagnostic of IPF. According to some embodiments the selective dysregulated miRNA expression can identify interstitial pulmonary fibrosis (IPF) at a stage before standard procedures (e.g., Ashcroft scoring and histology) demonstrate changes consistent with lung fibrosis.

[0386] According to some embodiments, the treating may modulate one or more of an injury, inflammation, an excess accumulation of extracellular matrix, cell senescence; or a pathway comprising fibrogenic signaling. According to some embodiments the treating of the progressive chronic lung disease may stabilize a parameter of lung function in the subject compared to an untreated control. According to some embodiments, the parameter of lung function is one or more of FEV1, FVC, or FEV1/FVC %. According to some embodiments the treating of the progressive chronic lung disease may improve a parameter of lung function in the subject compared to an untreated control.

[0387] According to some embodiments, the pathway comprises transforming growth factor (TGFβ) signaling. According to some embodiments, the pathway comprising fibrogenic signaling is one or more of a Smad pathway, a mitogen-activated protein kinase pathway, a phosphoinositide 3-kinase pathway; a canonical Wnt-β catenin pathway, a Notch signaling pathway.

Formulations of Active Agents for Treating the Staged Fibrotic Lung Disease

[0388] According to some embodiments, a composition for treating the staged fibrotic lung disease is a pharmaceutical composition comprising a therapeutic amount of an active agent and a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable carrier” is art recognized. It is used to mean any substantially non-toxic carrier conventionally useable for administration of pharmaceuticals in which the isolated EVs of the present invention will remain stable and bioavailable. The pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the mammal being treated. It further should maintain the stability and bioavailability of an active agent. The pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition. Exemplary carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin, which is incorporated herein by reference in its entirety. According to some embodiments, the pharmaceutically acceptable carrier is sterile and pyrogen-free water. According to some embodiments, the pharmaceutically acceptable carrier is Ringer's Lactate, sometimes known as lactated Ringer's solution.

[0389] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0390] Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, .alpha.-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0391] Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate alginates, calcium salicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, tragacanth, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, tale, magnesium stearate, water, and mineral oil. According to some embodiments, the pharmaceutically acceptable carrier comprises a pulmonary surfactant. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The compositions may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.

[0392] Specific modes of administration will depend on the indication. The selection of the specific route of administration and the dose regimen is to be adjusted or titrated by the clinician according to methods known to the clinician in order to obtain the optimal clinical response. The amount of active agent to be administered is that amount sufficient to provide the intended benefit of treatment. The dosage to be administered will depend on the characteristics of the subject being treated, e.g., the particular mammal or human treated, age, weight, health, types of concurrent treatment, if any, and frequency of treatments, and can be easily determined by one of skill in the art (e.g., by the clinician).

[0393] The local delivery of therapeutic amounts of a composition for the treatment of a lung injury or fibrotic lung disease can be by a variety of techniques that administer the compound at or near the targeted site. Examples of local delivery techniques are not intended to be limiting but to be illustrative of the techniques available. Examples include local delivery catheters, site specific carriers, implants, direct injection, or direct applications, such as topical application and, for the lungs, administration by inhalation. Local delivery by an implant describes the surgical placement of a matrix that contains the pharmaceutical agent into the affected site. The implanted matrix releases the pharmaceutical agent by diffusion, chemical reaction, or solvent activators.

[0394] Pharmaceutical formulations containing the active agents of the described invention and a suitable carrier can be solid dosage forms which include, but are not limited to, tablets, capsules, cachets, pellets, pills, powders and granules; topical dosage forms which include, but are not limited to, solutions, powders, fluid emulsions, fluid suspensions, semi-solids, ointments, pastes, creams, gels, jellies, and foams; and parenteral dosage forms which include, but are not limited to, solutions, suspensions, emulsions, and dry powder; comprising an effective amount of a polymer or copolymer of the described invention. It is also known in the art that the active ingredients can be contained in such formulations with pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, preservatives and the like. The means and methods for administration are known in the art and an artisan can refer to various pharmacologic references for guidance. For example, Modern Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., New York (1980) can be consulted.

[0395] The compositions for use according to the described invention relates to all routes of administration including intramuscular, subcutaneous, sublingual, intravenous, intraperitoneal, intranasal, intratracheal, topical, intradermal, intramucosal, intracavernous, intrarectal, into a sinus, gastrointestinal, intraductal, intrathecal, intraventricular, intrapulmonary, into an abscess, intraarticular, subpericardial, into an axilla, into the pleural space, intradermal, intrabuccal, transmucosal, transdermal, via inhalation, via nebulizer, and via subcutaneous injection. Alternatively, the pharmaceutical composition may be introduced by various means into cells that are removed from the individual. Such means include, for example, microprojectile bombardment, via liposomes or via other nanoparticle device.

[0396] The pharmaceutical compositions can be formulated for parenteral administration, for example, by injection, such as by bolus injection or continuous infusion. The pharmaceutical compositions can be administered by continuous infusion subcutaneously over a predetermined period of time. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0397] For oral administration, the pharmaceutical compositions can be formulated readily by combining the active agent(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the actives of the disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, alter adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including, but not limited to, lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as, but not limited to, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragecanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as, but not limited to, the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0398] Dragee cores can be provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0399] Pharmaceutical preparations that can be used orally include, but are not limited to, push-fit capsules made of gelatin, as well as soft, scaled capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as, e.g., lactose, binders such as, e.g., starches, and/or lubricants such as, e.g., talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration.

[0400] For buccal administration, the compositions can take the form of, e.g., tablets or lozenges formulated in a conventional manner.

[0401] For administration by inhalation, the compositions for use according to the described invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0402] In addition to the formulations described previously, the compositions for use according to the described invention can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.

[0403] Depot injections can be administered at about 1 to about 6 months or longer intervals. Thus, for example, the compositions can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0404] Pharmaceutical compositions also can comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as, e.g., polyethylene glycols.

[0405] For parenteral administration, a pharmaceutical composition can be, for example, formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques.

[0406] Examples of such active therapeutic agents include one or more immunomodulators, analgesics, anti-inflammatory agents, anti-fibrotic agents, anti-viral agents, proton pump inhibitors, or oxygen therapy.

[0407] Examples of immunomodulators include corticosteroids, for example, prednisone, azathioprine, mycophenolate, mycophenolate mofetil, colchicine, and interferon-gamma 1b.

[0408] Examples of analgesics include capsaisin, codeine, hydrocodone, lidocaine, oxycodone, methadone, resiniferatoxin, hydromorphone, morphine, and fentanyl.

[0409] Examples of anti-inflammatory agents include aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sulindac, and tolmetin.

[0410] Examples of anti-fibrotic agents are nintedanib and pirfenidone.

[0411] Examples of proton pump inhibitors are omeprazole, lansoprazole, dexlansoprazole, esomeprazole, pantoprazole, rabeprazole, and ilaprazole.

[0412] Examples of anti-viral agents include, for example, acyclovir, gancidovir, foscarnet; ribavirin; amantadine, azidodeoxythymidine/zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, and delavirdine.

[0413] According to the foregoing embodiments, the pharmaceutical composition may be administered once, for a limited period of time or as a maintenance therapy over an extended period of time, for example until the condition is ameliorated, cured or for the life of the subject. A limited period of time may be for 1 week, 2 weeks, 3 weeks, 4 weeks and up to one year, including any period of time between such values, including endpoints. According to some embodiments, the pharmaceutical composition may be administered for about 1 day, for about 3 days, for about 1 week, for about 10 days, for about 2 weeks, for about 18 days, for about 3 weeks, or for any range between any of these values, including endpoints. According to some embodiments, the pharmaceutical composition may be administered for more than one year, for about 2 years, for about 3 years, for about 4 years, or longer.

[0414] According to the foregoing embodiments, the composition or pharmaceutical composition may be administered once daily, twice daily, three times daily, four times daily or more.

[0415] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0416] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials have been described. All publications mentioned herein are incorporated herein by reference to disclose and described the methods and/or materials in connection with which the publications are cited.

[0417] It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural references unless the context clearly dictates otherwise.

[0418] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application and each is incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

EXAMPLES

[0419] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1

[0420] Abstract. Extracellular vesicles incorporate microRNAs (miRNA) and other cell-specific components that may convey disease to target cells. Alterations of miRNA expression have been shown in lung tissue, sputum and sera of individuals with idiopathic pulmonary fibrosis (IPF). We designed this study to test whether transfer of urine and/or tissue derived exosomal miRNAs from individuals with IPF carry cargo that can promote fibrosis. Exosomes were isolated from urine (U-IPFexo, n=16), lung tissue myofibroblasts (MF-IPFexo), and serum from individuals with IPF (n=8) and age/sex-matched controls without lung disease (n=10). We analyzed micro-RNA expression of isolated exosomes and their in vivo bio-distribution. We investigated the effect on ex vivo skin wound healing and in in vivo mouse lung models. U-IPFexo or MF-IPFexo expressed miR let-7d, miR-29a-5p, miR 181b-3p and miR-199a-3p consistent with previous reports of miRNA expression obtained from lung tissue/serum from patients with IPF. In vivo bio-distribution experiments detected bioluminescent exosomes in the lung of normal C57B16 mice within 5 minutes after intravenous infusion, followed by distribution to other organs irrespective of exosome source. When these exosomes were labeled with gold nanoparticles and imaged by transmission electron microscopy, they were visualized in alveolar epithelial type I and type II cells in lung tissue sections. Treatment of human and mouse lung punches obtained from control, non-fibrotic lungs, with either U-IPFexo or MF-IPFexo produced a fibrotic phenotype. A fibrotic phenotype was also induced in a human e vivo skin model and in in vivo lung models. Our results provide evidence that exosomes isolated from individuals with IPF contain pro-fibrotic miRNA and have the ability to promote a pro-fibrotic pathogenic response in normal and injured tissues.

Materials and Methods

[0421] Study Design: This study aimed to determine whether urine-derived exosomes from individuals with IPF (U-IPFexo) carry disease provoking cargo. An overview of the experimental details and design is shown in FIG. 13.

[0422] Urine and blood collection: Random urine samples were collected (between 10 am-2 pm) from either control individuals or individuals seen in the Interstitial Lung Disease (ILD, subjects with IPF ages 55-79; subjects with non-CF bronchiectasis ages 73-86; and subjects with asthma ages 36-61) clinic at the University of Miami Hospital. Written informed consent was obtained from the participants with an approved Institutional Review Board protocol (IRB #20060249) at the University of Miami Miller School of Medicine. Control urines were obtained from subjects that did not have underlying kidney, heart or lung comorbidities and had normal albumin creatinine ratios sampled in an aliquot of each urine. Urine was processed within 3 hours of collection and spun at 3000×g for 15 mins to remove sediment; supernatant was aliquoted at 10 mls/tube. Patients with known kidney disease or diabetes were excluded from the study and a small aliquot of urine was used to measure urine albumin and creatinine to exclude samples with albuminuria (Creatinine LiquiColor Test, Stanbio Laboratory Boerne, Tex. and albumin Elisa, Bethyl laboratories, Montgomery, Tex.). Tubes were frozen at −80° C. until exosome isolation. Blood was collected during the same clinic visit as urine collection. Blood was left to clot for up to an hour and serum separated after centrifugation at ˜2500 RPM for 15 mins. Serum was aliquoted and frozen at −80° C. until exosome isolation. Samples were selected for inclusion in the present study after review of imaging confirming a definitive UIP pattern. Lung function (forced vital capacity (FVC), VEV1, FEV1:FVC %) and diffusion capacity (DLCO) performed at several facilities were obtained from subjects' medical records obtained closest to the timing of the sample was also reviewed as shown in Table 1.

[0423] Cell culture: Myofibroblasts isolated from individuals with IPF and fibroblasts isolated from control lungs were propagated and characterized as previously described. Two IPF and one control cell isolate was purchased from Lonza (catalog number CC7231 and CC2512, MSCBM, Lonza Inc., Walkersville, Md.) Cells (passages 2 or 3) were grown until 80% confluence in 30 T175 flasks in Lonza media (MSCBM, Lonza Inc., Walkersville, Md.). At the time of collection, media was aspirated and each flask washed 3 times with PBS to remove serum and serum proteins. After serum free medium was added back to each flask for an additional 48 hours, the supernatant was collected and exosomes isolated and characterized (See Zen Bio, NC method below).

[0424] Exosome Isolation (conditioned tissue culture medium, urine), characterization and RNA isolation: A 4° C. sample was centrifuged at 3,000×g for 20 min at room temperature in a swinging bucket rotor to remove large cells and debris. The clarified supernatant was collected and ultracentrifuged at 100,000×g for 2 hours, in a fixed angle rotor at 4° C., to pellet exosomes. The exosome pellet was resuspended in a minimum volume of DPBS (approximately 120 uL/ultracentrifugation tube). Exosomes were characterized using a Thermo NanoDrop spectrophotometer for protein determination and approximate RNA concentration by direct absorbance; exosomes were not lysed, stained, or RNA extracted prior to taking these measurements. Particle diameter and concentration was assessed by tunable resistive pulse sensing (TRPS; (qNano, Izon Science Ltd) using a NP150 nanopore membrane at a 47 mm stretch. The concentration of particles was standardized using multi-pressure calibration with carboxylated polystyrene beads of a defined size (nm diameter) and at a defined concentration (particles/mL).

[0425] For RNA isolation the Norgen Preserved Blood RNA Purification Kit I was used according to manufacturer's directions. Briefly, for every 100 μl of exosome preparation, 300 μL of lysis solution was added. After a short vortex, 400 μL of 95-100% ethanol was added. 600 μl of the lysate containing ethanol was loaded onto the column and centrifuged for 1 minute at >3,500×g (˜6,000 RPM). The column was washed three times with wash solution and centrifuged each time for 1 minute (14,000×g). The column was spun for an additional two minutes in order to thoroughly dry the resin and the column contents eluded with 50 μl of Elution Solution by centrifuging for 2 minutes at 200×g (˜2,000 RPM), followed by 1 minute at 14,000×g.

[0426] Lung Punch: To test the effect of exosomes on fibrotic pathways, we used ex vivo lung punches obtained from the lungs of 17-18-month-old male C57BL/6 mice (FIG. 10A). Naïve exosomes (40 ug) and exosomes labeled with nanoparticles were injected around the punch followed by collection four days later. Punches were embedded, cut and stained with trichrome. The normal histology of the punch was preserved as shown by light and electron microscopic examination after PBS injection (FIG. 10B and FIG. 10C).

[0427] Transmission Electron Microscopy: For conventional transmission electron microscopy (TEM), exosome pellets were placed in a droplet of 2.5% glutaraldehyde in PBS buffer at pH 7.2 and fixed overnight at 4° C. The samples were rinsed in PBS (3 times, 10 minutes each) and post-fixed in 1% osmium tetroxide for 60 minutes at room temperature. The samples were then embedded in 10% gelatin, fixed in glutaraldehyde at 4° C. and cut into several blocks (smaller than 1 mm.sup.3). The samples were dehydrated for 10 minutes per step in increasing concentrations of alcohol (30%, 50%, 70%, 90%, 95% and 100%×3). Next, pure alcohol was replaced with propylene oxide, and the specimens were infiltrated with increasing concentrations (25%, 50%, 75% and 100%) of Quetol-812 epoxy resin mixed with propylene oxide for a minimum of 3 hours per step. The samples were embedded in pure, fresh Quetol-812 epoxy resin and polymerized at 35° C. for 12 hours, 45° C. for 12 hours, and 60° C. for 24 hours. Ultrathin sections (100 nm) were cut using a Leica UC6 ultramicrotome and post-stained, first with uranyl acetate for 10 minutes and then with lead citrate for 5 minutes at room temperature, prior to observation using the FEI Tecnai T20 transmission electron microscope (Hillsboro, Tex., USA) operated at 120 kV.

[0428] Punch TEM: Individual punches were fixed in 2% glutaraldehyde in 0.05M phosphate buffer and 100 mM sucrose, post-fixed overnight in 1% osmium tetroxide in 0.1M phosphate buffer, dehydrated through a series of cold graded ethanol, and embedded in a mixture of EM-bed/Araldite (Electron Microscopy Sciences). 1 μm thick sections were stained with Richardson's stain for observation under a light microscope. 100 nM sections were cut on a Leica UC7 ultramicrotome and stained with uranyl acetate and lead citrate. The grids were viewed t 80 kV in a JEOL JEM-1400 transmission electron microscope and images captured by an AMT BioSprint 12 digital camera.

[0429] Biodistribution: Mice were fed an alfalfa-free diet to prevent auto fluorescence of tissues (Perkin-Elmer personal communication). Exosomes were labeled with ExoGlow™-Vivo EV Labeling Kit (System Biosciences, Palo Alto, Calif.). Labeled exosomes or PBS containing exosomes were injected via tail vein into 17-18 month-old C57BL/6 mice. Mice were anesthetized with isoflurane and imaged using an IVIS Spectrum In Vivo Imaging System (PerkinElmer). The mice were imaged at 5, 10, 15, 30, 60, 90 mins, 2, 4, 6, and 24 hours as well as at 2, 3 and 19 days post injection. Imaging data was analyzed using Living Image Software (Perkin Elmer). Control experiments were conducted with dye alone.

[0430] Gold nanoparticle labelling of exosomes: Gold nanoparticles (nanospheres) modified with branched polyethylenimine (BPEI) of 10 nm size were used (nanoComposix, co). The original concentration of gold nanoparticles purchased was 1 mg/mL (5.7E+13 particles/mL). We performed experiments to optimize the ratio between the exosome particles and the modified gold nanoparticles with the final prep containing 10*8 exosomes: 0.001 mg of the modified gold nanoparticles. Briefly, gold nanosuspensions were sonicated prior to the experiment. The concentration of gold nanoparticles was adjusted based on the concentration of each exosome prep and the desired volume for injection. The mixture was vortexed and then placed in a thermomixer (Eppendorf ThermoMixer F1.5) at 37° C. and spun at 300 rpm. After 3 hours, the mixture was vortexed, allowed to sit at room temperature for 15 mins, and placed at 4° C. until the next day.

[0431] Transmission electron microscopy: For conventional transmission electron microscopy (TEM), exosome pellets were placed in a droplet of 2.5% glutaraldehyde in PBS buffer at pH 7.2 and fixed overnight at 4 C. The samples were rinsed in PBS (3 times, 10 minutes each) and post-fixed in 1% osmium tetroxide for 60 minutes at room temperature. The samples then were embedded in 10% gelatin, fixed in glutaraldehyde at 4 C and cut into several block (smaller than 1 mm.sup.3). The samples were dehydrated for 10 minutes per step in increasing concentrations of alcohol (30%, 50%, 70%, 90%, 95%, and 100%×3). Next, pure alcohol was replaced with propylene oxide, and the specimens were infiltrated with increasing concentrations (25%, 50%, 75% and 100%) of Quetol-812 epoxy resin mixed with propylene oxide for a minimum of 3 hours per step. The samples were embedded in pure, fresh Quetol-812 epoxy resin and polymerized at 35 C for 12 hours, 45 C for 12 hours, and 60 C for 24 hours. Ultrathin sections (100 nm) were cut using a Leica UC6 ultramicrotome and post-stained, first with uranyl acetate for 10 minutes and then with lead citrate for 5 minutes at room temperature, prior to observation using a JEOL JEM-1400 transmission electron microscope operated at 120 kV.

[0432] Ex Vivo lung punches: Cannulated lungs from 17-18 month-old male mice were filled with warm low melting point agarose (3%, Sigma-Aldrich, St. Louis, Mo.). Lung segments were cooled on ice for 30 min to allow solidification of the agarose, punched to a diameter of 4 mm using a biopsy punch (Acuderm) and transferred to an air-liquid interface with phenol red free minimal essential media (MEM) without serum. Human urine-derived or cell derived exosomes were injected into the lung punch with 29G needles in a volume of 100 μl. The 100 ul volume of injection was divided equally into four injection sites on the punch. Punches were incubated at 37° C. in a humidified atmosphere of 5% CO.sub.2 for 4 days. Parallel punches (injected and naïve) from the same experiment were prepared for histology, immunofluorescence staining, RNA and protein purification. The normal histology of the punch was preserved after injection with PBS at four sites. For human lung, we injected a bronchial branch with 2% agarose and performed punch experiments as described above. Selected punches were injected with nanoparticle containing exosomes for EM experiments and collected within 24 hours of injection. A series of punches were collected that did not receive any injections or were injected with nanoparticles alone. Four days after injection punches were fixed in 10% formalin (Sigma-Aldrich), processed for paraffin embedding and stained with trichrome to assess collagen content.

[0433] Punch TEM: Individual punches were fixed in 2% glutaraldehyde in 0.05M phosphate buffer and 100 mM sucrose, post-fixed overnight in 1% osmium tetroxide in 0.1 M phosphate buffer, dehydrated through a series of cold graded ethanol, and embedded in a mixture of EM-bed/Araldite (Electron Microscopy Sciences). 1 μm thick sections were stained with Richardson's stain for observation under a light microscope. 100 nm sections were cut on a Leica UC7 ultramicrotome and stained with uranyl acetate and lead citrate. The grids were viewed at 80 kV in a JEOL JEM-1400 transmission electron microscope and images captured by an AMT BioSprint 12 digital camera.

[0434] Immunofluorescence staining of lung punches: Formalin fixed paraffin embedded punches were processed as previously described for tissue. Fluorescent staining was performed using α-SMA (Abcam, Cambridge, Mass.) or anti-prosurfactant protein C (SPC, Abcam) and DAPI (Vector, Burlingame Ca).

[0435] Real-Time PCR: Total RNA was extracted from lung tissue homogenates. Amplification and measurement of target RNA was performed on the Step 1 real time PCR system as previously described. α.sub.v-integrin, collagen type I and TGF-β were measured using RNA extracted from lung punches or lung tissue. TGF-β and IGF-1 mRNA expression was measured using RNA extracted from exosomes. The TaqMan rRNA control reagents kit (Life Technologies) was used to detect 18S rRNA gene, an endogenous control, and samples were normalized to the 18S transcript content as previously described. For miRNA analyses, cDNA was generated using qScript™ microDNA cDNA Synthesis Kit (Quanta Biosciences, Beverly, Mass.) according to manufacturer's instructions. Amplification of all miRNAs was performed using specific primers, let-7d, miR-29a-5p, miR-34a-5p, miR-142-3p, miR-199a-3p, and miR-181b (IDT, Coralville, Iowa) using Real-Time SYBR Green qRT-PCR Amplification kit (Quanta Biosciences, Beverly, Mass.). U6 expression was used as a control for miRNA analyses, and relative expression was calculated using the comparative C(T) method.

[0436] Western Blot: Punches or lung pieces were homogenized and western analysis was performed as previously described using the Invitrogen mini-cell gel surelock cell module Xcell II vertical surelock box (Thermofisher, Waltham, Mass.). For CD63, Hsp70, pAKT, AKT, c-Jun, Caveolin-1, ERα and β-actin, 5 to 25 μg of protein lysate was fractionated on 10% polyacrylamide gels. Immunoreactive bands were determined by exposing nitrocellulose blots to a chemiluminescence solution (Denville Scientific Inc.; Metuchen, N.J.) followed by exposure to Amersham Hyperfilm ECL (GE Healthcare Limited; Buckinghamshire, UK). To determine the relative amounts of protein densitometry Image J version 1.48v (National Institutes of Health; Bethesda, Md.) was utilized. All values from western blots were standardized to the corresponding β-actin band prior to comparative analyses.

MMP Activity:

[0437] MMP activity was assessed in lung punches and lung tissue using a previously described method. Briefly, Novex® 10% zymogram gels (Life Technologies) were incubated for 24 hours in a gelatinase solution, which allows the determination of total proteolytic MMP activities without interference from associated tissue inhibitors. Relative MMP activity was determined by densitometry using Image J (NIH).

[0438] Ex vivo human skin wound model to evaluate functional effects of urine-derived exosomes: Human skin samples were obtained from healthy subjects following panniculectomy (abdominal skin; median age 44 years old). Informed consent was obtained per the requirements of the Institutional Review Board at the University of Miami (IRB protocol #20070922). Under sterile conditions, subcutaneous fat was trimmed from skin prior to generating wounds. A 3 mm punch (Acuderm) was used to make wounds in the epidermis through the reticular dermis and 3 mm discs of eipidermis were excised. Skin discs (8 mm), with the 3 mm epidermal wound in the middle, were excised using a 6 mm biopsy punch (Acuderm). Wounded skin specimens were immediately transferred to anir-liquid interface with DMEM medium (BioWhittaker) supplemented with antibiotics-antimycotics and 10% fetal bovine serum (Gemini Bio—Products). The skin samples were incubated at 37 C in a humidified atmosphere of 5% CO2 for 4 days. Tissues were fixed in 10% formalin (Sigma-Aldrich), processed for paraffin embedding and stained with hematoxylin and eosin to follow the rate of healing.

Animal Model:

[0439] 17-18 month-old male C57BL/6 mice obtained from Jackson Laboratories were housed under specific pathogen-free conditions with food and water ad libitum. All experiments and procedures were approved by the Institutional Animal Care and Use Committee at the Leonard M. Miller School of Medicine at the University of Miami (Miami, Fla.), a facility accredited by the American Association for the Accreditation of Laboratory Animal Care. Following treatments all mice were housed one per cage until sacrifice. Sample size was based on our published data (72). Starting with 8-10 mice per group we have >90% statistical power to detect a Cohen's effect size d>1.75 standard deviations.

[0440] Bleomycin (“Bleo”) administration: After the administration of anesthesia, Bleomycin sulfate (Sigma-Aldrich Corp; St. Louis, Mo.) dissolved in 50 μl sterile saline at 2.5 U per kg of bodyweight was administered by direct intratracheal instillation via intubation. Control mice received 50 μl of sterile saline using the same method. Mice were weighed and sacrificed at 21 days post-Bleo administration.

Exosome Injections and Time Course:

[0441] Urine-derived or cell-derived exosomes were thawed in a 37° C. water bath and washed in PBS to remove the cell freezing solution immediately prior to injection. Twenty-four hours following Bleo administration, each animal received 100 μl either PBS (control) or 40 ug of exosomes in 100 μl of PBS by tail vein injection over a one minute period. This amount was calculated based on the amount of exosomes derived from 10.sup.5 cells (number of cells utilized in whole cell experiments). Some mice received Bleo+vehicle injection. Treatments were assigned by simple randomization and the technician was blinded to the treatments. Dose response experiments were conducted with 20 and 40 ug of exosomes. A separate set of naïve mice received the same exosome injections and were sacrificed at 21 days post injection.

Marine Lung Tissue Analysis/Immunohistochemistry:

[0442] Left lung lobes were harvested for protein, zymography, and mRNA analysis. For morphometry and histology studies, right lung lobes were inflated with 10% neutral buffered formalin (NBF) under 25 cm H.sub.2O pressure. The lungs were postfixed by immersion in 10% NBF for 24 hours and then transferred to PBS at 4° C. Samples were paraffin-embedded and 4 m sections were obtained for hematoxylin-eosin and Masson's Trichrome staining, Ashcroft scoring:

[0443] Pulmonary fibrosis was assessed by a pulmonary pathologist blinded to the experimental groups using the semi-quantitative Ashcroft method on Masson's Trichrome-stained slides at 20× magnification. Individual fields were assessed by systematically moving over a 32-square grid; each field was assessed for fibrosis severity and assigned a score on a scale of 0 (normal lung) to 8 (total fibrosis of the field) and an average was obtained for each slide.

[0444] [Collagen content assessment by hydroxyproline content: Hydroxyproline content was determined according to the manufacturer's instructions (Hydroxyproline Assay Kit; Sigma-Aldrich, St. Louis, Mo.). Briefly, 2 mg lung fragments were weighed and homogenized in 100 μl of distilled water. An equal volume of 10 N HCl was added to the samples before drying at 49° C. for 3 hours. 50 μl of sample was loaded in the plate and incubated overnight at 37° C. A hydroxyproline standard curve was prepared according to a standard solution (0-1 ug/well). Hydroxyproline content was read at 557 nm, using the SoftMax Pro Software (Molecular Devices Corp; Sunnyvale, Calif.).

[0445] Statistical analysis: Mean and SEM were determined using GraphPad Prism 9.0 (GraphPad Software, San Diego, Calif.). All values are expressed as mean±SEM. Differences between experimental groups were assessed by using Kruskal-Wallis test and Mann-Whitney test for single comparisons. Given limited sample sizes in some experiments, data were determined to be normally distributed using Kolmogorov-Smirnov test and tested by one-way analysis of variance (ANOVA) and Tukey multiple comparison. Results were considered statistically significant at P<0.05. ANOVA was also used to analyse rate of epithelialization among treatment groups; p<0.05 was considered significant.

Results

[0446] Exosome isolation and characterization: Urine samples were collected from individuals diagnosed with IPF (A group) or non-CF bronchiectasis (B group) or asthma (C group, Table 1) and normal age and sex-matched controls (Table 2, IRB #20060249). Control urines were obtained from subjects that did not have underlying kidney, heart, or lung comorbidities and had normal albumin creatinine ratios sampled in an aliquot of each urine. IPF and control exosomes were isolated from myofibroblasts and fibroblasts (Table 3). Human urine, blood and cell derived exosome isolation were performed by Zen-Bio Inc. (Durham, N.C.). Exosome size from urine, blood and cells was approximately 30-150 um. EM performed on isolated exosomes (FIG. 1A) confirmed a size and shape previously reported for exosomes (23). The presence of exosome markers CD1, CD9 and CD63 was determined by MACSPlex analysis and CD63 (FIG. 1B) was confirmed by Western analysis. Other exosome markers present were CD9 and CD81.

TABLE-US-00002 TABLE 1 Male IPF (A group), non-CF bronchiectasis (B group) or asthma (C group) urine-derived exosomes. Age of Subject subject at FEV1 FVC FEV/FVC FEV/FVC DLCO (% Number collection Ethnicity (liters) (liters) (%) (predicted) reference) A4 72 Caucasian 2.93 3.75 78 77 11.8 (47) A6 79 Caucasian 2.36 2.91 81 67 16.2 (101) A26 69 Hispanic 1.67 1.98 84 72 NT A35 69 Hispanic 1.71 2.22 70 73 NT A37 69 Hispanic 2.7 3.21 84 77 10.5 (44) A62 67 Hispanic 1.94 2.33 82 77 8.7 (33) A74 75 Hispanic 2.48 2.77 80 74 9.9 (49) A77 55 Hispanic 1.14 1.27 90 78 6.7 (32) A80 70 Caucasian 2.09 2.62 78.9 87.49 11.8 (48) A83 68 Hispanic 2.39 2.69 86 75 10.5 (49) A84 67 Caucasian 1.78 2.12 84 84 14.1 (57) A88 66 Hispanic 1.36 1.39 98 75 2.6 (11) A90 67 Caucasian 2.48 2.85 88.9 74 12.8 (45) A103 72 Hispanic 1.95 2.64 74 75 13.4 (59) A104 76 Caucasian 2.95 3.27 90 65 14.6 (58) A105 62 Hispanic 1.47 1.69 87 78 11.7 (41) B1 73 Caucasian 1.04 2.06 50 79.91 9.6 (48) B10 70 Hispanic 1.33 2.89 46 74 21.29 (69.5) B13 86 Hispanic 2.54 3.59 70.7 74 23.08 (76.78) C1 36 Hispanic 3.67 4.75 77.16 82.89 31.49 (92) C4 61 Caucasian 3.38 4.72 71.65 79.09 27.77 (96) C7 44 Caucasian 3.08 4.57 67.3 70 27.83 (96) NT = not tested.

TABLE-US-00003 TABLE 2 Male control (D group) urine derived exosomes (Age of subject at collection). No evidence of documented lung disease or abnormal pulmonary function tests (PFTs). Age of subject at Subject number collection Ethnicity C8 66 Caucasian D9 70 Caucasian D12 77 Hispanic D28 77 Caucasian D31 55 Hispanic D32 73 Caucasian D38 72 Hispanic D41 65 Caucasian D50 75 Hispanic D101 67 Caucasian

TABLE-US-00004 TABLE 3 Myofibroblast and control fibroblast-derived exosomes (age of subject at collection) Male myofibroblasts IPF Male fibroblast Control (Age of subject at collection) (Age of subject at collection) 1 (52).sup.  5 (70) 2 (83).sup.  6 (69) 3 973) .sup. 7 967) 4 974)

[0447] Determination of miRNAs expressed in urine- and serum-derived exosomes: to our knowledge, determination of the cargo of U-IPFexo has not previously been reported. U-IPFexo relative expression analysis revealed dysregulated expression of miR-let-7D, miR-199, miR-29 and miR-181 (FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D) compared to urine-derived control exosomes. We compared serum-derived exosome miRNA to urine-derived exosomal miRNA from the same patient and found no difference in relative expression of selected miRNAs (FIG. 2E). These selected miRNAs have also been shown to be altered in lung tissue and serum from individuals with IPF (Cassanova, N. et al. Translational Research (2021) 228: 1-12; Omote, N. and Sauler, M. Frontiers in Medicine (2020) 7 (908); Ortiz-Quinntero, B. et al. Cells (2020)). These data suggest that cargo carried in urine exosomes from individuals with IPF may parallel cargo of serum-derived exosomes in the same individual.

[0448] Detection of expression of mRNA from exosomes: Isolated exosomes were subject to PCR to detect potential mRNA expression that is reflective of fibrotic pathways. As expected in pure exosome preparations (Batagov, A O and Kurochkin, IV (20013) Biology Direct (2013) 8: 12), we did not detect mRNA expression of TGFβ, IGF or ER subtypes from normal or diseased exosomes.

[0449] Tracking exosomes reveals a rapid systemic distribution: We assessed exosome signal and time course in 17-18 month-old male C57BL/6 mice using an in vivo bioluminescent imaging system. Regardless of source (U-IPFexo or control exosomes), exosomes were located the lung within 5 minutes post-tail vein injection. Images were taken at 5 min, 15 min 30 min, 60 min, 90 min and 2, 4, 6, 24 and 48 hours (FIG. 3) in all preparations. No adverse events were noted in mice followed for 20 days. All mice injected with U-IPFexo had a higher lung signal over time compared to mice injected with urine-derived age and sex-matched controls (FIG. 9A), suggesting that these exosomes may be retained longer in the lung than control exosomes. A set of mice sacrificed at 30 min or 48 hours to quantify signals in heart/lung, spleen, liver and kidney (FIG. 9B) showed that the lung signal evident at 30 min was not evident at 48 hours as seen by IVIS suggesting clearance of the exosomes. Mice injected with PBS had no signal (FIG. 9C).

[0450] Transmission Electron Microscopy (TEM) reveals uptake of exosomes into alveolar epithelial cells (AEC): Exosomes were labeled with gold nanoparticles at varying ratios and processed for EM to determine the optimum ratio of nanoparticles to exosomes. Utilizing TEM, exosomes labeled with nanoparticles were visualized in mouse lung punches (FIG. 10) injected with urine-derived control exosomes (FIG. 4A, FIG. 4B, FIG. 4C) or U-IPFexo (FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, FIG. 411). FIG. 10A, FIG. 10B, FIG. 10C and FIG. 10D show histology and trichrome staining of lung punches from C57BL6 mice. Lung punches from control lungs shown in tissue culture dish (FIG. 10A) have normal histology (FIG. 10B, Trichrome staining 10×mag) and structure by TEM (FIG. 10C, 500 mag). Histology of non-injected lung punch (FIG. 10D). TEM revealed increased collagen bands in those punches injected with U-IPFexo (FIG. 411), Collagen bands were not as evident in the control punches as compared with U-IPF samples due to sampling limitation, however we observed collagen bundles in both normal and U-IPF treated samples. Lung structure was preserved in both normal and U-IPF treated samples.

[0451] Immunofluorescent staining of punches revealed decreased surfactant protein C positive cells (AEC II) after treatment with IPF derived exosomes (FIG. 5C) compared to treatment with control exosomes (FIG. 5B). Taken together these data suggest that exosomes are taken up by the alveolar cells and that IPFexo may promote disease by inhibiting the reparative AEC II cells.

[0452] U-IPFexo stimulate a fibrotic pathway response in human lung punches ex vivo: To determine the effects of exosomes on control human lung tissue, we performed ex vivo punch experiments utilizing lungs deemed unsuitable for transplant due to trauma (n=2; FIG. 6). α.sub.v-integrin and collagen type I mRNA increased after injection with MF-IPFexo compared to control (FIG. 6A). Consistent with our published data in the lungs of mice with Bleo-injury and lung tissue from individuals with IPF (27, 28), Cav-1 protein decreased (FIG. 6B) and ERα protein expression increased (FIG. 6C) after U-IPFexo were injected into human lung punches. We conducted additional experiments using lung tissue obtained from 18 month-old male C57BL/6 mice (n=3 technical replicates/individual exosome isolate). Markers for fibrosis (αv-integrin, collagen type I mRNA expression) and other downstream fibrotic pathways (c-Jun, AKT expression and MMP-9 activity) were only activated by the U-IPFexo and not control exo or PBS alone (FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H). We found a 2.5 fold increase in TGFβ mRNA expression in punches that were injected with IPF exosomes compared to those injected with PBS or control exosome injections (p<0.05). Similar data were obtained from analysis of punches injected with MF-IPFexo (FIG. 6J, FIG. 6K, FIG. 6L). mRNA expression of αv-integrin and collagen type I mRNA were increased in punches injected with MF-IPFexo compared with punches injected with age and sex-matched control fibroblast exosomes (FIG. 6J, FIG. 6K). ERα protein expression shown to be upregulated in the lung tissue and myofibroblasts isolated from individuals with IPF (27) was increased after injection of U-IPFexo (1.4 fold increase over control, p=0.05) and MF-IPFexo (>3 fold) into lung punches (FIG. 6F, FIG. 6L) compared to control exosomes. Finally, increased c-Jun expression was noted after injection of IPF exosomes but not after injection with control exosomes. We conclude that U-IPFexo stimulate a fibrotic pathway response similar to that stimulated by MF-IPFexo.

[0453] U-IPFexo impair tissue repair of human skin ex vivo: We used the human ex vivo skin wound healing assay to test whether would impair closure of wounds. U-IPFexo treatment delayed wound healing mirroring our previous data showing that mice who develop pulmonary fibrosis after treatment with Bleo also demonstrate a delay in wound healing (28). We found that U-IPFexo decreased wound closure compared to control exosomes or PBS (FIG. 7A and FIG. 7B, 27±6.3% vs. 14±0.7%, vs 95%, n=3 technical replicates from 2-3 biological exosome isolates/group), suggesting that exosomes carry a disease phenotype that can impair tissue repair. As expected, due to age of control exosomes, we noted less effective wound healing compared to the PBS control.

[0454] U-IPFexo regulate miRNA expression and MMP-9 activity: We measured miRNAs in exosome treated lung punches to determine whether expression changes found in the punches correlated with data published from lung tissue/cells isolated from individuals with IPF (25). We found decreased expression of miR-29 and miR-let-7d in punches treated with U-IPFexo (Table 4) or MF-IPFexo. MiR-199 was upregulated in those punches injected with U-IPFexo, but remained unchanged by MF-IPFexo. This was surprising since we and others have previously shown an increase in miR-199 expression in IPF lung tissue and cells and that increased miR-199 correlates with downregulated Cav-1 expression (29, 30). Additional miRNAs were assessed that were reported dysregulated in tissues, serum or sputum of individuals with IPF. Punch expression of miR-34a, and miR-142 was regulated in a manner similar to expression found in isolated exosomes after treatment with either U-IPFexo or MF-IPFexo, supporting the relevance of the cargo carried in urine of individuals with IPF (31).

TABLE-US-00005 TABLE 4 Table 4 Punch miRNA expression (% PBS) Let-7d miR-29 miR-181b miR-199 miR-34a miR-142 Urine exosomes (n = 6-8/group) PBS 100 ± 0.5 100 ± 0.3 100 ± 0.2 100 ± 0.1 100 ± .3.1  100 ± 0.2 Control exo  74 ± 6.7  96 ± 13.3 137 ± 18  92 ± 5  88 ± 15 .sup.  115 ± 15  Urine-IPFexo   .sup. 32 ± 6.5@      55 ± 12.1+*& .sup. 52 ± 17+  169 ± 18+& 131 ± 10+*+  .sup. 103 ± 18.sup.NS Fibroblast exosomes (n = 3-4/group) PBS 100 ± 0.1 100 ± 0.3 100 ± 0.5 100 ± 0.5 100 ± 0.2 .sup.   100 ± 0.2 Control exo 114 ± 5.sup.  110.8 ± 9.2.sup.  110 ± 8.2 53 ± 5  101 ± 16.5.sup.   78 ± 14 MF-IPFexo   .sup. 57 ± 5.9@    56 ± 7.9*&  .sup.  42 ± 12.7@  .sup. 115 ± 14.sup.NS .sup. 63 ± 4.6.sup.NS  .sup. 47 ± 12.sup.NS +P = 0.05 compared to PBS, @<0.01 compared to PBS and control exosomes, *P < 0.05 compared to control exosomes, &P < 0.01 compared to control exosomes, P values were calculated by Mann-Whitney U test. NS = not significant

[0455] U-IPFexo impact lung fibrosis in vivo: Analysis of lung tissue obtained from Bleo treated mice sacrificed at day 21 showed a higher Ashcroft score (FIG. 8M) and collagen content (FIG. 8N) in those mice receiving U-IPFexo compared to control exosomes or Bleo alone. αV-integrin (FIG. 8O) mRNA expression was higher in lungs of mice that received U-IPFexo compared to Bleo alone. We noted higher TGFβ mRNA expression in the lung tissue of mice injected with U-IPFexo compared to Bleo treatment alone (p>0.001, 23.8±3.9 vs 11.66±0.6). Lungs obtained from mice treated with urine exosomes from either asthmatics or subjects with bronchiectasis also exhibited increased TGFβ mRNA expression. MMP-2 was induced 2.5-fold by U-IPFexo (p<0.05) compared to Bleo treatment, control exo or other exo. When Bleo injury was followed by injection of U-IPFexo, fibrosis was more homogeneous and widespread compared to Bleo treatment alone. Mice treated with IPF exosomes lost 35±2.6% BW compared to mice treated with Bleo (29±2.6% of BW; p=0.05 compared to IPF) or mice receiving control exo (19±3.2% of BW; p<0.05 compared to Bleo, p<0.01 compared to IPF). Bleo-treated Mice that received urine derived urine derived exosomes from individuals with non-CF bronchiectasis lost more weight (39±5.7% BW; p<0.05) than Bleo treated mice. The Bleo treated mice receiving urine derived exosomes from individuals with asthma lost a similar amount of weight as the Bleo treated mice (27±3.5% of BW). We also found that miR let-7d, and -142 expression decreased while miRNA miR-34a, and -199 were increased in lung tissue from Bleo mice treated with U-IPFexo (Table 5) but not urine exosomes derived from either asthmatics or subjects with bronchiectasis (Table 5). Although Ashcroft scoring and histology did not demonstrate changes consistent with fibrosis in lung tissue collected from naïve animals treated with PBS or treated

[0456] with U-IPFexo, increased collagen content (FIG. 11), dysregulated miR-34a, miR-199, miR29, and miR-142 expression (Table 5) were detected in lung tissue obtained from these mice.

[0457] Table 5 shows lung tissue microRNA expression/U6.

TABLE-US-00006 Lung Tissue microRNA Mir-199 Mir-34a Mir-142 expression/U6 miR-let-7d Mir-29 miR-181b fibrotic fibrotic antifibrotic PBS 0.18 ± 0.03 2.1 ± 0.4 ±0.26 ± 0.06  0.03 ± 0.001 0.07 ± 0.02  0.23 ± 0.016 Control U-exo   0.3 ± 0.01## 2.8 ± 0.7  0.3 ± 0.07 0.06 ± 0.03 0.09 ± 0.01 0.19 ± 0.03 U-IPF-exo 0.14 ± .002 .sup. 0.48 ± 0.1*& .015 ± 0.05  0.05 ± 0.005#  0.13 ± 0.03*  0.16 ± 0.02* Bleomycin 0.057 ± 0.009{circumflex over ( )}{circumflex over ( )} 0.17 ± 0.02 0.009 ± 0.001 0.19 ± 0.02 0.18 ± 0.05 0.23 ± 0.04 (Bleo) Bleo +  0.24 ± 0.05# 0.17 ± 0.02 0.008 ± 0.001 0.35 ± 0.11 0.18 ± 0.02 0.29 ± 0.05 Control U-exo Bleo +   0.10 ± 0.011*$${circumflex over ( )}{circumflex over ( )}  0.2 ± 0.17  0.004 ± 0.001#   1.68 ± 0.44#*$${circumflex over ( )}{circumflex over ( )}   0.43 ± 0.08#*$${circumflex over ( )}   0.16 ± 0.01&${circumflex over ( )} U-IPFexo Bleo + 0.17 ± 0.03 0.14 ± 0.03 ND 0.13 ± 0.35 0.06 ± 0.02  0.3 ± 0.07 U- bronchiectasis exo Bleo + 0.39 ± 0.1  0.11 ± 0.01 ND 0.06 ± 0.01 0.06 ± 0.02 0.29 ± 0.04 U-asthma exo *P < 0.05 compared to control exo, &P < 0.001 compared to control exosomes; #P < 0.05 compared to Bleo, ##P < 0.001 compared to Bleo, $P < 0.05, $$P < 0.01 compared to bronchiectasis exo {circumflex over ( )}P < 0.01 {circumflex over ( )}{circumflex over ( )}P < 0.001 compared to asthma exo. P values were calculated by one way analysis of variation (ANOVA) and Mann-Whitney U test. ND = not detected.

[0458] Discussion

[0459] EVs isolated from various tissues, cells and body fluids contain bioactive molecules such as proteins, lipids, and RNAs derived from the cell of origin (32), (33). Most recently, studies on exosomes have focused on miRNAs, small noncoding RNA molecules able to influence protein expression. Since exosomes act as an interface for cell-cell communication, we investigated the ability of “diseased” exosomes to promote fibrotic lung disease and impair wound healing. Our study is the first to report that U-IPFexo confer a disease phenotype in vivo and in ex vivo lung punches and impair ex vivo wound healing in mouse and human tissue. In a similar manner, MF-IPFexo activated markers of fibrosis in ex vivo lung punches compared to punches treated with lung fibroblast-derived exosomes isolated from the lung tissue of age and sex-matched individuals without fibrotic lung disease.

[0460] Changes in miRNAs have been implicated in gene expression associated with the development of IPF (15, 16, 18). We found dysregulated expression of miRNAs; let-7, miR-29a, miR-181b and miR-199 in the isolated U-IPFexo compared to control exosomes. These miRNAs are known to regulate expression of pro-fibrotic, inflammatory and ECM encoding genes (16, 19, 34-37) and found in lung, serum and sputum of individuals with IPF (15, 18, 19, 27, 38, 39).

[0461] Micro RNA Let-7 regulates ERα protein expression as well as the regulation of downstream fibrotic inducers including TGF-β cell signaling pathways (27). Dysregulation of miRNA let-7 contributes to endothelial/epithelial to mesenchymal transition (Endo/EMT) in the lung (18, 19), heart (37) and kidney (40), often leading to fibrosis. Njock et al. reported a positive correlation between diffusing capacity of the lungs for carbon monoxide/alveolar volume (DLCO/VA) and presence of let-7d (16). Dysregulation of miR-29 in the lung alters expression of MMP-2 and collagen1α1, while miR-199 regulates caveolin-1 (18, 28, 41-43). Rescue of miR-181b, an inhibitor of NF-κB targets, suppressed TGFβ in burns and in a Bleo model of IPF (44, 45). Increasing miR-181b expression mitigated TGF-β1 induced EMT in vitro and alleviated alveolar septal thickening and decreased collagen and MMP expression in vivo (45).

[0462] Collectively these data suggest the exosomes can deliver expression of dysregulated miRNAs and may promote a disease phenotype. When distinct regions were histologically analyzed from the same IPF lung and the histology labeled as minimal, moderate and end-stage disease severity divergent sets of genes and miRNA expression were dependent on the severity of diseased tissue (38). MicroRNA let-7, a critical marker in our studies, was associated with end-stage fibrotic lung disease. In a recent study, PBMC miRNA expression correlated with survival in individuals with IPF (24), supporting the disease conferring role of miRNA expression as a potential signature of disease. In the present study, we compared expression of miRNAs from urine-derived exosomes and serum-derived exosomes isolated from the same individuals with IPF. We acknowledge that the absence of differences in miRNA expression between urine and serum-derived exosomes may reflect our small sample size. These data warrant a larger study, that is currently ongoing.

[0463] To demonstrate delivery of exosomes to the lungs, we performed biodistribution experiments using U-IPFexo and urine exosomes from control subjects (without lung disease). The urine-derived exosomes were located in the lungs of 18-month old male mice within 5 minutes followed by a diminished signal at 24-48 hours; similar transit time was recorded whether the urine exosomes were prepared from individuals with IPF or controls. Additional studies showed that an exosome signal was no longer visualized in the mouse at 19 days post-injection. Our studies uses ExoGlow™-Vivo that employs an amine binding dye that emits in the near infrared (NIR) range instead of infrared lipophilic cyanine dye, DiIC18 (DiD) to label urine derived exosomes (5). Since the ExoGlow dye is non-lipophilic, it provides a more specific signal when used in vivo. Similar transit times have been reported using DiD to label bone marrow mesenchymal stem cell-derived exosomes showing delivery after intravenous infusion to the lung at 5 minutes and to the liver and spleen at 24 hours (46). Exosomes derived from breast cancer cells have a similar profile of biodistribution: lung, >liver>spleen, kidney>heart>bone marrow (47).

[0464] To study cellular and tissue interplay in the lung (48, 49) we evaluated how the exosomes integrated in ex vivo lung punches by loading exosomes with nanoparticles and performing TEM (50, 51). We found that the punch architecture was preserved after injection of exosomes and that the urine-derived exosomes were integrated into AEC I or AEC II cells. Our studies do not distinguish whether homing of the “diseased” exosomes favored a specific cell type compared to injection with control exosomes. These extensive studies are ongoing in our laboratory.

[0465] Further studies using ex vivo lung punches from human non-fibrotic lung tissue and naïve 18-month old male mice noted minimal effect on the histology of the lung punch after treatment with PBS or urine-derived exosomes isolated from age and sex-matched controls without lung disease. We found that treatment with U-IPFexo increased expression of fibrotic markers and pathways (αv-integrin, collagen type I and TGFβ mRNA expression, c-Jun protein expression and MMP-9 and AKT activation). Additionally, there was a notable decrease in SPC positive cells in punches (AEC II cells) receiving U- or MF-IPFexo. Since AEC II are regarded as the progenitor population of the alveolus responsible for injury repair and homeostatic maintenance, these data suggest that IPFexo may be inhibiting the repair mechanism.

[0466] We have previously reported an increase of ERα protein expression in cells and tissue isolated from lungs of male individuals with IPF (27). Studies suggest that activation of ERα may be profibrotic in multiple organs including the lung (52, 53). In the current investigation we found at least ˜1.4-fold increase in ERα protein expression in human and mouse punches treated with U-IPFexo or MF-IPFexo. Punches injected with MF-IPFexo also induced other components of fibrotic pathways consistent with data derived from punches injected with U-IPFexo. Furthermore, U-IPFexo impaired the wound healing response, supporting our hypothesis that exosome cargo delivered signals impairing wound repair in skin. This suggests that the systemic feature of IPF exosomes may predispose impaired tissue healing.

[0467] We recognize that under our experimental conditions, lung punches lack immune recruitment, systemic perfusion and are under potentially hypoxic conditions. Therefore, we sought to determine if these exosomes would confer lung injury in vivo under normoxic conditions in old male C57BL/6 mice. We injected U-IPFexo into naive 18-month-old C57BL/6 mice and collected the lungs 21 days post-exosome treatment. Although there was no change in Ashcroft score, we noted increased collagen content (measured by hydroxyproline) and evidence of inflammatory changes, including increased macrophages in lung tissue from mice treated with U-IPFexo compared to tissue from mice treated with control exosomes. Another group of mice were treated with Bleo. Ashcroft score, collagen content, αV-integrin and TGFβ mRNA expression increased in the lungs of mice treated with Bleo+U-IPFexo compared to mice treated with Bleo+vehicle and Bleo+control exosomes, suggesting that U-IPFexo escalated fibrotic pathways induced by Bleo in old male mice. There was no increase of TGFβ in lungs of mice treated with non-fibrotic exo. As reviewed, TGFB is ubiquitous in chronic inflammatory lung diseases including COPD, IPF and asthma and may contribute to an immune-suppressed state (Thomas, B J et al. Am. J. Respir. Cell Mol. Biol. (2016) 55(6): 759-66).

[0468] We also found an expected increase in MMP-2 activity in the lungs of U-IPFexo treated mice, as MMP-2 increases migration and invasiveness of lung myofibroblasts during Bleo-induced pulmonary fibrosis (54). This was in contrast to urine derived control-exosomes or exosomes from urine of individuals with bronchiectasis or asthma. BW, historically shown to decrease with Bleo treatment (55) was decreased in mice receiving Bleo+U-IPFexo compared to Bleo alone or Bleo+control exosomes. Loss of BW is associated with worse survival in individuals with IPF (21, 22). Our studies also found similar changes in miRNA expression as reported in prior studies from patients with IPF (14-18, 25-27, 31, 41, 56-58).

[0469] Our study demonstrates that U-IPFexo preparations contain disease invoking cargo. Two recent studies by Martin-Medina et al (59) and Parimon et al. (17) showed that antifibrotic microRNAs (miR 144-3p, miR144-3p, miR 34-5p, miR 503-5) packaged into EVs regulate epithelial plasticity and potential pulmonary fibrosis and reported an increase in the number of EV in BALF (bronchoalveolar lavage fluid) from Bleo-treated mice as well as from patients with IPF that function as carriers for signaling mediators, such as WNT5A. Both studies reported that isolated EV preparations consisted predominantly of exosomes, although microvesicles were present, unlike our preparations that did not contain microvesicles. Neither study investigated urine-derived exosomes. In the described study, lung tissue isolated from naïve or Bleo-treated mice injected with U-IPFexo also exhibited the same changes in miRNA expression as found in ex vivo lung punches. The present study illustrates the correlation between changes in mRNA and protein expression (TGFβ, MMPs) and dysregulated miRNA expression suggesting that a miRNA signature could function as a potential biomarker for fibrotic lung disease. Reproducible biomarkers would enable early diagnosis and non-invasive detection methods are currently under investigation for multiple diseases (60).

[0470] We further analyzed lungs from Bleo mice that were treated with exosomes derived from subjects with asthma or bronchiectasis, non-fibrotic lung diseases. We did not find an increase in the fibrotic markers studied and in fact noted a decrease in collagen content and integrin in those lungs analyzed. We recognize that this small set of mice will need to be expanded in future experiments and also noted that the subjects with asthma were not age matched to the control or IPF subjects. However, the decreased expression of miR-34a and miR-199, fibrotic inducing microRNA highlight the differences in cargo content between exosomes from various lung diseases (61, 62).

[0471] In an effort to detect expression of other potential fibrotic pathways, we analyzed the urine-exosomes for mRNA expression of TGFβ, IGF, and Cav-1 (63-65). Because exosomes contain non-coding and other small coding RNAs, derived from transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA) (66-69), we attempted to characterize the more easily studied expression of mRNA and mRNA fragments (70, 71). As expected, we found no evidence of mRNA expression for our selected targets (supplement). However, the presence of 3′ UTR mRNA fragments which could bind miRNA and allow for RNA transcription to proceed in target cells ultimately altering the phenotype of otherwise healthy cells has been reported (71).

[0472] Our study provides the first evidence of circulating cargo found in U-IPFexo that confers disease in two target organs (lungs and skin) and suggests a potential mechanism for initiation and/or progression of disease. Urine-derived exosomes potentially represent a novel way to identify biomarkers for lung and fibrotic diseases. Our data further suggest that increased miRNA profiling of urine-derived exosomes as biomarkers may lead to noninvasive assessments for earlier diagnosis. Overlap of miRNA expression yielding a fibrotic profile to produce a personalized panel ofurine-derived miRNAs or fibro miRs to detect IPF and other lung and fibrotic diseases may be a diagnostic and prognostic tool.

[0473] While the present invention has been described with reference to the specific embodiments thereof it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adopt a particular situation, material, composition of matter, process, process step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

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