DIAGNOSTIC AND THERAPEUTIC USES OF COMPOSITIONS COMPRISING PURIFIED, ENRICHED POTENT EXOSOMES CONTAINING DISEASE-BASED AND THERAPY BASED SIGNATURE CARGO
20220296645 · 2022-09-22
Assignee
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61K35/51
HUMAN NECESSITIES
A61K9/0073
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
A61K9/1271
HUMAN NECESSITIES
C12Q2600/112
CHEMISTRY; METALLURGY
C12N5/0665
CHEMISTRY; METALLURGY
C12N2320/32
CHEMISTRY; METALLURGY
A61K9/0019
HUMAN NECESSITIES
C12N2320/35
CHEMISTRY; METALLURGY
A61K35/28
HUMAN NECESSITIES
C12Q1/6883
CHEMISTRY; METALLURGY
A61K9/5068
HUMAN NECESSITIES
A01N1/0226
HUMAN NECESSITIES
International classification
A61K35/28
HUMAN NECESSITIES
A61K35/51
HUMAN NECESSITIES
A61K9/50
HUMAN NECESSITIES
A61P17/02
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The present disclosure provides a composition containing a purified and enriched population of potent exosomes derived from extracellular vesicles derived from mesenchymal stem cells (MSCs), a method for diagnosing a human subject aged over 50 years with an age-related chronic disease characterized by disease related dysfunction and optimally treating the subject, and a method for reprogramming a donated organ or tissue comprising a fibrotic disposition including treating the donated organ or tissue with a composition comprising purified enriched population of potent exosomes derived from extracellular vesicles derived from MSCs of a normal healthy subject.
Claims
1. A composition comprising a purified and enriched population of potent exosomes derived from extracellular vesicles derived from mesenchymal stem cells (MSCs), wherein a. the exosomes comprise an identity signature comprising expression of three or more biomarkers selected from CD9, CD63, CD81, or Tsg101+; b. the exosomes comprise total protein of about 1 mg; c. the exosomes comprise total RNA content greater than 20 μg; d. the exosomes comprise a cargo comprising a therapeutic signature of one or more, miRNAs selected from miR-29a, miR-10a, miR-34a, miR-125, miR-181a, miR-181c, miR Let-7a, miR-Let-7b, miR-7d, miR-146a, miR-145, miR-21, miR-101, and miR-199; and e. size of the exosomes is about 90-110 nm, inclusive; wherein lot to lot and batch to batch variability relative to prior lots of the composition is below a significance level of ≤0.05; and wherein expression of the miRNA cargo can be configured to treat an age-related chronic disease.
2. The composition of claim 1, wherein the MSCs are derived from a tissue or a body fluid of a human subject.
3. The composition of claim 2, wherein (a) the MSCs are derived from placental tissue, adipose tissue, umbilical cord tissue, lung tissue, heart tissue, or dental pulp; or (b) the mesenchymal stem cells are derived from hone marrow of normal healthy subjects aged 21-40 years old; or (c) the body fluid is blood, amniotic fluid or urine.
4. The composition of claim 2, wherein identity of the MSCs is confirmed by a signature comprising CD29, CD44, and CD105.
5. The composition of claim 3, wherein the MSCs derived from placental tissue are derived from one or more of chorionic membrane (CM), chorionic trophoblast without villi (CT-V), chorionic villi (CV), or decidua (DC).
6. The composition of claim 3, wherein the blood is umbilical cord blood or peripheral blood.
7. The composition of claim 1, wherein the cargo comprises a potency signature of expression of one or more, two or more, three or more, four or more, or five or more of angiopoietin 2 (Ang-2), fibroblast growth factor (FGF), hepatic growth factor (HGF), interleukin 8 (IL-8), a tissue inhibitor of metalloproteinases (TIMP), vasculoendothelial growth factor (VEGF), platelet derived growth factor (PDGF), or tumor necrosis factor alpha (TNFα).
8. The composition of claim 7, wherein the tissue inhibitor of metalloproteinases is TIMP1, TIMP2, or TIMP1 and TIMP2.
9. The composition of claim 1, wherein the composition is a pharmaceutical composition comprising a therapeutic amount of the purified, enriched potent exosomes and a pharmaceutically acceptable carrier.
10. The composition of claim 1, wherein the exosomes are derived from at least 1×10.sup.12 EVs comprising exosomes per isolation.
11. The composition of claim 9, wherein the pharmaceutical composition is formulated for administration by inhalation or for intravenous administration.
12. The composition of claim 9, wherein a therapeutic amount of the purified, enriched potent exosomes comprises at least 1×10.sup.9 exosomes.
13. The composition of claim 1, wherein the cargo comprising the therapeutic signature a. is configured so as to modulate one or more of the injury, the inflammation, an excess accumulation of extracellular matrix, cell senescence; or b. is configured so as to modulate a pathway comprising fibrogenic signaling; or c. is configured so as to slow or reverse progression of an age-related chronic lung disease; or d. is configured to reprogram a tissue affected by an age-related chronic disease.
14. The composition of claim 13, wherein the age-related chronic disease if left untreated comprises one or more of a progressive injury, progressive inflammation, progressive fibrosis or a combination thereof.
15. The composition of claim 13 wherein the pathway comprises transforming growth factor (TGFβ) signaling.
16. The composition of 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, or a Notch signaling pathway.
17. The composition of claim 13, wherein the tissue is lung tissue, cardiac tissue, renal tissue, hepatic tissue, skin, pancreatic tissue, eye tissue, joint tissue, bone marrow, brain tissue, intestinal tissue, peritoneal tissue, retroperitoneal tissue, nerve tissue, spinal tissue, or skeletal muscle.
18. The composition of claim 13, wherein the age-related chronic disease is a chronic lung disease, chronic inflammation and immune dysfunction, mitochondrial dysfunction, organ transplantation dysfunction, organ resuscitation and rejuvenation, a viral infection, neuropathic pain; neurofibrosis, neurodegeneration, connective tissue dysfunction, musculoskeletal repair, dysfunction of the gut microbiome, or age-related decline.
19. The composition of claim 18, wherein the chronic lung disease is a fibrotic lung disease.
20. The composition of claim 18, wherein the chronic lung disease is due to chronic smoking or a severe viral infection.
21. The composition of claim 20, wherein the severe lung infection is due to a severe coronavirus infection.
22. The composition of claim 13, wherein the age-related chronic lung disease comprises reduced forced vital capacity compared to a normal healthy control.
23. The composition of claim 13, wherein the treatment results in stabilization or improvement of forced vital capacity in a subject compared to an untreated control.
24. The composition according to claim 23, wherein the subject is human.
25. A method for diagnosing a human subject aged over 50 years with an age-related chronic disease characterized by disease related dysfunction and optimally treating the subject, comprising a. diagnosing a stage of the age-related chronic disease by: isolating a population of extracellular vesicles (EVs) comprising exosomes derived from mesenchymal stem cells from a biological sample of the subject and from a normal healthy control aged 21-40 years; wherein the EVs comprise an identity signature of three or more biomarkers selected from CD9, CD63, CD81, or Tsg101+; purifying and enriching exosomes from the EVs from the subject and from the normal healthy control; measuring a level of expression of each of a plurality of miRNAs in the exosomes from the subject and from the normal healthy control; determining that expression of the one or more miRNAs in the EVs from the subject is dysregulated compared to the healthy control; and identifying the subject as one that can benefit therapeutically from being treated for the age-related chronic disease; b. treating the age-related chronic disease by administering to the subject a composition comprising a population of purified enriched potent exosomes with an appropriate therapeutic signature derived from the normal healthy subject, wherein i. the exosomes comprise an identity signature of three or more biomarkers selected from CD9, CD63, CD81, or Tsg101+; ii. the exosomes comprise total protein of about 1 mg; iii. the exosomes comprise total RNA content greater than 20 μg; iv. the exosomes comprise a cargo comprising a therapeutic signature of one or more, two or more, three or more, four or more, or five or more miRNAs selected from miR-29a, miR-10a, miR-34a, miR-125, miR-181a, miR-181c, miR-Let-7a, miR-Let-7b, miR-Let-7d, miR-146a, miR-145, miR-21, miR-101, and miR-199; v. size of the exosomes is 90-110 nm, inclusive; wherein lot to lot and batch to batch variability relative to prior lots of the composition is below a significance level of ≤0.05; and c. managing the age related chronic disease by modulating the dysfunction.
26. The method of claim 25, wherein identity of the MSCs is confirmed by expression of a biomarker signature comprising CD29, CD44, and CD105.
27. The method of claim 25, wherein the exosome cargo comprises a potency signature comprising expression of one or more two or more, three or more, four or more, or five or more of angiopoietin 2 (Ang-2), fibroblast growth factor (FGF), hepatic growth factor (HGF), interleukin 8 (IL-8), a tissue inhibitor of metalloproteinases (TIMP), vasculoendothelial growth factor (VEGF), platelet derived growth factor (PDGF), or tumor necrosis factor alpha (TNFα).
28. The method of claim 27, wherein the tissue inhibitor of metalloproteinases is TIMP1, TIMP2, or TIMP1 and TIMP2.
29. The method of claim 25, wherein the composition is a pharmaceutical composition comprising a therapeutic amount of the purified, enriched potent exosomes and a pharmaceutically acceptable carrier.
30. The method of claim 25, further comprising purifying the exosomes from at least 1×10.sup.12 EVs comprising exosomes per isolation.
31. The method of claim 25 wherein the administering is by inhalation or for intravenous administration.
32. The method of claim 25, wherein a therapeutic amount of exosomes comprises at least 1×10.sup.9 exosomes.
33. The method of claim 25, wherein the age-related chronic disease if left untreated comprises one or more of a progressive injury, progressive inflammation, progressive fibrosis or a combination thereof.
34. The method of claim 25, wherein the cargo comprising the therapeutic signature a. modulates one or more of the injury, the inflammation, an excess accumulation of extracellular matrix, cell senescence; or b. modulates a pathway comprising fibrogenic signaling; or c. reprograms a tissue affected by the age-related chronic disease; or d. a combination thereof.
35. The method of claim 34 wherein the pathway comprises transforming growth factor (TGFβ) signaling.
36. The method of claim 34, 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, or a Notch signaling pathway.
37. The method of claim 34, wherein the tissue is lung tissue, cardiac tissue, renal tissue, hepatic tissue, skin, pancreatic tissue, eye tissue, joint tissue, bone marrow, brain tissue, intestinal tissue, peritoneal tissue, retroperitoneal tissue, nerve tissue, spinal tissue, or skeletal muscle.
38. The method of claim 25, wherein the age-related chronic disease is a chronic lung disease, chronic inflammation and immune dysfunction, mitochondrial dysfunction, organ transplantation dysfunction; fibrotic disposition of a donor organ, rejection of a donor organ; graft failure; ex vivo lung perfusion dysfunction, musculoskeletal disorders, neurodegeneration, gut dysbiosis or microbiome dysfunction, or age-related decline in health.
39. The method of claim 38, wherein the chronic lung disease is a fibrotic lung disease.
40. The method of claim 38, wherein the chronic lung disease is due to chronic smoking or a severe viral infection.
41. The method of claim 40, wherein the severe lung infection is due to a severe coronavirus infection.
42. The method of claim 38, wherein the age-related chronic lung disease comprises reduced forced vital capacity compared to a normal healthy control.
43. The method of claim 38, wherein the treating is effective to stabilize or improve forced vital capacity in the subject compared to an untreated control.
44. A method for reprogramming a donated organ or tissue comprising a fibrotic disposition comprising a. treating the donated organ or tissue with a composition comprising a purified, enriched population of potent exosomes derived from extracellular vesicles derived from mesenchymal stem cells (MSCs) of a normal healthy subject, wherein i. the exosomes comprise an identity signature of three or more biomarkers selected from CD9, CD63, CD81, or Tsg101+; ii. the exosomes comprise total protein of about 1 mg; iii. the exosomes comprise total RNA content greater than 20 μg; iv. the exosomes comprise a cargo comprising a therapeutic signature including attributes of age, gender, estrogen receptor function and status, environmental impact/stressors, donor cell or tissue type, health of the donor organ or tissue, genomics of the donor cell or tissue; and v. size of the exosomes is 90-110 nm, inclusive; wherein lot to lot and batch to batch variability relative to prior lots of the composition is below a significance level of ≤0.05; and b. rejuvenating or resuscitating the organ or tissue.
45. The method of claim 44, wherein identity of the MSCs is confirmed by expression of a biomarker signature comprising CD29, CD44, and CD105.
46. The method of claim 44, wherein the purified, enriched population of potent exosomes derived from extracellular vesicles derived mesenchymal stem cells (MSCs) of a normal healthy subject is derived from a tissue or a body fluid of a human subject.
47. The method of claim 46, wherein (a) the tissue is placental tissue, adipose tissue, umbilical cord tissue, lung tissue, heart tissue, or dental pulp; or (b) the tissue is bone marrow of normal healthy subjects aged 21-40 years old; or (c) the body fluid is blood, amniotic fluid or urine.
48. The method of claim 47, wherein the MSCs derived from placental tissue are derived from one or more of chorionic membrane (CM), chorionic trophoblast without villi (CT-V), chorionic villi (CV), or decidua (DC).
49. The method of claim 46, wherein the blood is umbilical cord blood or peripheral blood.
50. The method of claim 44, wherein the cargo comprises a potency signature of one or more two or more, three or more, four or more, or five or more of angiopoietin 2 (Ang-2), fibroblast growth factor (FGF), hepatic growth factor (HGF), interleukin 8 (IL-8), a tissue inhibitor of metalloproteinases (TIMP), vasculoendothelial growth factor (VEGF), platelet derived growth factor (PDGF), or tumor necrosis factor alpha (TNFα).
51. The method of claim 50, wherein the tissue inhibitor of metalloproteinases is TIMP1, TIMP2, or TIMP1 and TIMP2.
52. The method of claim 44, wherein the composition is a pharmaceutical composition comprising a therapeutic amount of the purified, enriched potent exosomes and a pharmaceutically acceptable carrier.
53. The method of claim 44, further comprising purifying the exosomes from at least 1×10.sup.12 EVs comprising exosomes per isolation.
54. The method of claim 44, wherein a therapeutic amount of exosomes comprises at least 1×10.sup.9 exosomes.
55. The method of claim 44, wherein the therapeutic signature comprises one or more, two or more, three or more, four or more, or five or more miRNAs selected from miR-29a, miR-10a, miR-34a, miR-125, miR-181a, miR-181c, miR-Let-7a, miR-Let-7b, miR-Let-7d, miR-146a, miR-145, miR-21, miR-101, and miR-199.
56. The method of claim 44, wherein the organ or tissue that comprises the fibrotic disposition if left untreated comprises one or more of a progressive injury, progressive inflammation, progressive fibrosis or a combination thereof.
57. The method of claim 44, wherein the cargo comprising the therapeutic signature a. modulates one or more of the injury, the inflammation, an excess accumulation of extracellular matrix, cell senescence; or b. modulates a pathway comprising fibrogenic signaling; or c. reprograms a tissue affected by the age-related chronic disease; or d. a combination thereof.
58. The method of claim 57 wherein the pathway comprises transforming growth factor (TGFβ) signaling.
59. The method of claim 57, 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, or a Notch signaling pathway.
60. The method of claim 57, wherein the tissue is lung tissue, cardiac tissue, renal tissue, hepatic tissue, skin, pancreatic tissue, eye tissue, joint tissue, bone marrow, brain tissue, intestinal tissue, peritoneal tissue, retroperitoneal tissue, nerve tissue, spinal tissue, or skeletal muscle.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0245] 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.
[0246]
[0247]
[0248]
[0249]
[0250]
[0251]
[0252]
[0253]
[0254]
[0255]
[0256]
[0257]
[0258]
[0259]
[0260]
[0261]
[0262]
[0263]
[0264]
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0265] 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%.
[0266] 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).
[0267] 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.
[0268] 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.
[0269] “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.
[0270] 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.
[0271] The term “amniotic stem cells” as used herein refers to pluripotent stem cells, multipotent stem cells, and progenitor cells derived from amniotic membrane, which can give rise to a limited number of cell types in vitro and/or in vivo under an appropriate condition, and expressly includes both amniotic epithelial cells and amniotic stromal cells.
[0272] 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.
[0273] 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.
[0274] Antigen presenting cells. T cells recognize antigens on the surface of other cells and mediate their functions by interacting with, and altering, the behavior of antigen-presenting cells (APCs). There are three main types of antigen-presenting cells in peripheral lymphoid organs that can activate T cells: dendritic cells, macrophages and B cells. The most potent of these are the dendritic cells, whose only function is to present foreign antigens to T cells. Immature dendritic cells are located in tissues throughout the body, including the skin, gut, and respiratory tract. When they encounter invading microbes at these sites, they endocytose the pathogens and their products, and carry them via the lymph to local lymph nodes or gut associated lymphoid organs. The encounter with a pathogen induces the dendritic cell to mature from an antigen-capturing cell to an antigen-presenting cell (APC) that can activate T cells. APCs display three types of protein molecules on their surface that have a role in activating a T cell to become an effector cell: (1) MHC proteins, which present foreign antigen to the T cell receptor; (2) costimulatory proteins which bind to complementary receptors on the T cell surface; and (3) cell-cell adhesion molecules, which enable a T cell to bind to the antigen-presenting cell (APC) for long enough to become activated. (“Chapter 24: The adaptive immune system,” Molecular Biology of the Cell, Alberts, B. et al., Garland Science, N Y, 2002).
[0275] 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].
[0276] The term “aquaporins” as used herein refers to water-specific membrane channel proteins. Aquaporin 5 (AQPS) 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 AQPS 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 AQPS in submucosal glands of subjects with COPD. [Id., citing Id, citing Wang, K. et al. Acta Pharmacol. Sin. (2007) 28 (8): 1166-74]
[0277] 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).
[0278] 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].
[0279] 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.
[0280] 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).
[0281] 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.].
[0282] 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.
[0283] The term “cargo profile” as used herein refers to measurements of cargo components that characterize a population of extracellular vesicles
[0284] The term “caveolins (Cays)” 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). Cays 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.
[0285] The term “chorion” as used herein refers to the outer fetal membrane that surrounds the amnion, the embryo, and other membranes and entities in the womb. A spongy layer of loosely arranged collagen fibers separates the amniotic and chorionic mesoderm. The chorionic membrane consists of mesodermal and trophoblastic regions. Chorionic and amniotic mesoderm are similar in composition. A large and incomplete basal lamina separates the chorionic mesoderm from the extravillous trophoblast cells. The latter, similar to trophoblast cells present in the basal plate, are dispersed within the fibrinoid layer and express immunohistochemical markers of proliferation. The Langhans fibrinoid layer usually increases during pregnancy and is composed of two different types of fibrinoid: a matrix type on the inner side (more compact) and a fibrin type on the outer side (more reticulate). At the edge of the placenta and in the basal plate, the trophoblast interdigitates extensively with the decidua (Cunningham, F. et al., The placenta and fetal membranes, Williams Obstetrics, 20th ed. Appleton and Lange, 1997, 95-125; Benirschke, K. and Kaufmann, P. Pathology of the human placenta. New York, Springer-Verlag, 2000, 42-46, 116, 281-297). The chorion, which interfaces maternal tissues, consists of four layers. These are, from within outward: (F) the cellular layer, a thin layer consisting of an interlacing fibroblast network, which is frequently imperfect or completely absent; (G) a reticular layer, which consists of a reticular network, the fibers of which tend to be parallel, along with a few fibroblasts and many Hofbauer cells; (H) a pseudo-basement membrane, which is a layer of dense connective tissue firmly adherent to the reticular layer above, and which sends anchoring and branching fibers down into the trophoblast; and (I) a trophoblast layer, which is the deepest layer of the chorion consisting of from two to 10 layers of trophoblast cells in contact, on their deeper aspect, with maternal decidua. This layer contains the chorionic villi (Bourne, G L, Am. J. Obstet. & Gynec. (1960) 79 (6): 1070-73).
[0286] “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.
[0287] 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).
[0288] The term “CD29” as used herein refers to integrin (31.
[0289] 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).
[0290] 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.
[0291] 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).
[0292] 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).
[0293] 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).
[0294] 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.
[0295] 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].
[0296] 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].
[0297] 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 12th leading cause of morbidity. By 2030, it is expected to be the 4th leading cause of death worldwide, representing over 4.5 million deaths annually.
[0298] 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.
[0299] 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.
[0300] As used herein, the term “derived from” is meant to encompass any method for receiving, obtaining, or modifying something from a source of origin.
[0301] 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.
[0302] 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.
[0303] 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.
[0304] 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.
[0305] 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.
[0306] 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).
[0307] 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).
[0308] 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; Théry, 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 Géminard, 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-O (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].
[0309] 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).
[0310] 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.
[0311] 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.
[0312] 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.
[0313] 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.
[0314] The term “clinical efficacy” as used herein refers to the therapeutic effectiveness of a drug or therapeutic in humans using appropriate outcome measures.
[0315] 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.
[0316] 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 μm) 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 μm), 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).
[0317] The term “forced vital capacity” 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.
[0318] 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.
[0319] 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)
[0320] 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.
[0321] 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.
[0322] 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].
[0323] 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.
[0324] 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.
[0325] 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].
[0326] 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.
[0327] The term “tumor necrosis factor-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].
[0328] 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.
[0329] 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.
[0330] 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 disease or process.
[0331] Hydroxyproline assay. Collagen content is assessed by quantifying hydroxyproline, an amino acid present in appreciable quantities in collagen.
[0332] 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.
[0333] 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.
[0334] 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.
[0335] 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.
[0336] 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 (ILCs, 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.
[0337] The term “infuse” and its other grammatical forms as used herein refers to introduction of a fluid other than blood into a vein.
[0338] 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).
[0339] 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.
[0340] 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%.
[0341] 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.
[0342] 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.
[0343] 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.
[0344] 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.
[0345] 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.
[0346] 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).
[0347] 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.
[0348] 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.
[0349] 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, P P, 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 (JNK). 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].
[0350] 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.
[0351] 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 R C A, 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.
[0352] 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).
[0353] 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. 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)].
[0354] It has been reported that MSCs from different placental layers have different proliferation rates and differentiation potentials [Choio, Y S et al. “Different characteristics of mesenchymal stem cells isolated from different layers of full term placenta.” PLoS One (2017) 12 (2): e0172642], i.e., MSCs from chorionic membrane (CM), chorionic trophoblast without villi (CT-V), chorionic villi (CV), and decidua (DC) had better population doubling time and multi-lineage differentiation potentials compared to those from other layers [See also, Wu, M. et al. “Comparison Of The Biological Characteristics Of Mesenchymal Stem Cells Derived From The Human Placenta And Umbilical Cord.” Scientific Repts (2018) 8: 5014, comparing MSCs derived from amniotic membrane (AM), chorionic plate (CP), decidua parietalis (CP) and umbilical cord (UC)].
[0355] 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.
[0356] The term “microRNA” (or “miRNA” or “miR”) as used herein refers to a class of small, 18- to 28-nucleotide-long, noncoding RNA molecules.
[0357] The term “modulate” as used herein means to regulate, alter, adapt, or adjust to a certain measure or proportion.
[0358] The term “neuropathic” as used herein refers to relating to any disorder affecting the nervous system. The term “neuropathic pain” as used herein refers to pain derived from injury to the peripheral nervous system (e.g., peripheral nerves) or the CNS, which may result from major surgeries, e.g., amputation and thoracotomy, diabetic neuropathy, viral infection, chemotherapy, spinal cord injury, stoke, etc. Neuropathic pain is often characterized by spontaneous pain, described as shooting, lancinating, or bringing pain, and also by evoked pain, such as hyperalgesia (increased responsiveness to noxious stimuli) to mechanical and thermal stimuli. Mechanical allodynia, meaning painful responses to normally innocuous tactile stimuli may be the most distinct symptom of neuropathic pain. There are at least two phases of neuropathic pain in animal models: an early phase (first several days) when neuropathic pain is developed, and late phase (from a week to months and even years) when neuropathic pain is maintained. Animal model experiments have shown that MMP-9 induces early-phase neuropathic pain by activating IL-1β and microglia in the early phase. (Ji. R-R et al, US Neurology, Touch Briefings (2008) 71-74). MMP-2 inhibition experiments showed that MMP-2 contributes to late-phase neuropathic pain development by activating IL-1β and astrocytes in the late phase. [Id.] Apart from their pathological roles, MMP-9 and MMP-2 also play a physiological roles in regulating development and regeneration; depending on whether functional or dysfunctional remodeling occurs, the result might be recovery or the induction of aberrant neuronal circuits. (Ji, R-R et al, Trends Pharmacol. Sci. (2009) 30 (7): 336-40). Using a rat adjuvant-induced arthritis model, it was shown that the Chinese medicine crocin may alleviate neuropathic pain in AIA rats by inhibiting the expression of pain-related molecules through the Wnt5α/β-catenin pathway. Wang, J-F et al. Neural Plasticity (2020) 4297483. Although it was long known that crocin can effectively alleviate pain sensitization in rat pain models, its mechanism was unknown. Crocin significantly increased the mechanical thresholds of adjuvant-induced arthritis in rats, suggesting that crocin can alleviate neuropathic pain. Crocin significantly decreased the levels of pain-related factors and glial activation. Foxy5, activator of Wnt5a, inhibited these effects of crocin in AIA rats. In addition, intrathecal injection of a Wnt5a inhibitor significantly decreased hyperalgesia in AIA rats.
[0359] The term “nerve” as used herein refers to a whitish fiber or bundle of fibers that transmits impulses of sensation to the brain or spinal cord, and impulses from the brain or spinal cord to the muscles and organs.
[0360] The term “nervous system” as used herein refers to the network of nerve cells and fibers which transmits nerve impulses between parts of the body. The central nervous system (CNS) is that part of the nervous system that consists of the brain and spinal cord. It is one of the two major divisions of the nervous system. The other is the peripheral nervous system (PNS) which is outside the brain and spinal cord. The peripheral nervous system (PNS) connects the central nervous system (CNS) to sensory organs (such as the eye and ear), other organs of the body, muscles, blood vessels and glands. The peripheral nerves include the 12 cranial nerves, the spinal nerves and roots, and the autonomic nerves of the autonomic nervous system (ANS), meaning the part of the nervous system responsible for control of the bodily functions not consciously directed, such as breathing, the heartbeat, and digestive processes.
[0361] 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, P J, (2016) Pharmacol. Rev. 68: 788-815]. The molecular pathways involved in NF-κB activation include several kinases. The classic (canonical) pathway for inflammatory stimuli and infections to activate NF-κB signaling involve the IKK (inhibitor of κB 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 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 (NIK) 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].
[0362] 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 (D111, 3, and 4) [Id., citing Sharma, S. et al. Curr. Opin. Nephrol Hypertens. (2011) 20: 56-61; Bray, S J. 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 CBFI/RBP-Jκ, SU(H), Lag1 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].
[0363] The term “organ” as used herein refers to a differentiated structure consisting of cells and tissues and performing some specific function in an organism.
[0364] 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].
[0365] As used herein, the term “paracrine signaling” refers to short range cell-cell communication via secreted signal molecules that act on adjacent cells.
[0366] 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.
[0367] 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.
[0368] The term “particulate” as used herein refers to fine particles of solid or liquid matter suspended in a gas or liquid.
[0369] The term “pathogen” as used herein refers to a causative agent of disease. It includes, without limitation, viruses, bacteria, fungi and parasites.
[0370] The term “pathogenesis” as used herein refers to the pathologic, physiologic or biochemical mechanism resulting in the development of a disease.
[0371] 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.
[0372] 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.
[0373] “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).
[0374] 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).
[0375] Activation of growth factor receptor protein tyrosine kinases results in autophosphorylation on tyrosine residues. P13K 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.
[0376] 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, 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).
[0377] 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).
[0378] 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, GβL, 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, GβL, Sin1, PRRS/Protor-1, and DEPTOR. mTORC2 promotes cellular survival by activating Akt, regulates cytoskeletal dynamics by activating PKCα, and controls ion transport and growth via SGK1 phosphorylation. Aberrant mTOR signaling is involved in many disease states
[0379] 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, E B. 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}.
[0380] 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.
[0381] The term “potency” and its various grammatical forms as used herein, refers to power or strength of a formulation.
[0382] 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 [0383] where ΔV=change in lung volume, and ΔP=change in airway pressure
[0384] This total compliance may be related to lung compliance and thoracic (chest wall) compliance by the following relation: [0385] where CT=total compliance (e.g., 100 mL/cm H2O) [0386] CL=lung compliance (e.g., 200 mL/cm H2O) [0387] CTh=thoracic compliance (e.g., 200 mL/cm H2O)
[0388] The values shown in parentheses are some typical normal adult values that can be used for modeling purposes (Id.). 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.
[0389] 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.
[0390] 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.
[0391] The term “progression” as used herein refers in medicine to the course of a disease as it becomes worse or spreads in the body.
[0392] 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.
[0393] The term “reactive oxygen species” as used herein refers to oxygen-containing reactive species. It is a collective term to include superoxide (O2.square-solid.—), hydrogen peroxide (H2O2), hydroxyl radical (OH.square-solid.), singlet oxygen (1O2), peroxyl radical (LOO.square-solid.), alkoxyl radical (LO.square-solid.), 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]
[0394] 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.
[0395] 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.
[0396] 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.
[0397] 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].
[0398] 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.
[0399] The term “reverse” as used herein refers to turning backward or in an opposite direction.
[0400] 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. A, Murphy K. & Weaver, C. Eds. Garland Science, New York (2017) at 110-111].
[0401] The term “signature” as used herein refers to a specific and complex combination of biomarkers that reflect a biological state.
[0402] 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 p21.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).
[0403] 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, L P. 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, L P. Genes Dev. (2006) 20: 2913-21; Yamamoto, H. et al. Mol. Endocrinol. (2007) 21: 1745-55].
[0404] 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].
[0405] The term “slow” as used herein refers to holding back progress or development.
[0406] 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.
[0407] 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.
[0408] The term “solvate” as used herein refers to a complex formed by the attachment of solvent molecules to that of a solute.
[0409] 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).
[0410] 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.
[0411] 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.
[0412] 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.
[0413] 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).
[0414] 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.
[0415] 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.
[0416] 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.
[0417] 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.
[0418] 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.
[0419] 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.
[0420] 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.
[0421] 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, Abet 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: TIMP-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. TIMP-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.
[0422] 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.
[0423] 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.
[0424] 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.
[0425] 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.
[0426] 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, R A. 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].
[0427] 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/β 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, T R. Nat. Med. (2012) 18: 1028-40; Lam, A P and Gottardi, C J. 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, C J. 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}.
[0428] 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.
[0429] 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.
[0430] 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
[0431] 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.
[0432] 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 II: Inflammation
[0433] 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.
[0434] The early recruitment of eosinophils, neutrophils, lymphocytes, and macrophages providing inflammatory cytokines and chemokines can contribute to local TGF-13 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.
[0435] 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.
[0436] 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
[0437] 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.
[0438] 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.
[0439] 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.
[0440] 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).
[0441] Collectively, the degree of inflammation, angiogenesis, and amount of extracellular matrix deposition all contribute to ultimate development of a fibrotic lesion.
Embodiments
Compositions
[0442] According to one aspect, the present disclosure provides a composition comprising an isolated population of extracellular vesicles (EVs) comprising a purified, enriched population of potent exosomes derived from mesenchymal stem cells (MSCs), wherein
[0443] (a) the EVs comprise an identity signature of three or more biomarkers selected from CD9, CD63, CD81, or Tsg101+;
[0444] (b) the EVs compri se total protein of about 1 mg;
[0445] (c) the EVs comprise total RNA content greater than 20 μg;
[0446] (d) the exosomes comprise a cargo comprising a therapeutic signature of one or more miRNAs selected from miR-29a, miR-10a, miR-34a, miR-125, miR-181a, miR-181c, miR-Let-7a, miR-Let-7b, miR-Let-7d, miR-146a, miR-145, miR-21, miR-101, and miR-199; and
[0447] (e) size of the exosomes is 50-130 nm, inclusive;
[0448] wherein lot to lot and batch to batch variability relative to prior lots of the composition is below a significance level of ≤0.05; and wherein expression of the miRNA cargo is configured to treat an age-related chronic disease.
[0449] According to some embodiments, extracellular vesicles are derived from a biological sample. According to some embodiments, the source of MSCs is a tissue autologous to the recipient subject. According to some embodiments, the source of the MSCs is a tissue allogeneic to the recipient subject. According to some embodiments, the tissue is mammalian. According to some embodiments, the tissue is human. According to some embodiments, the source of the MSCs is placental tissue obtained from one or more areas, including both material and fetal tissue, e.g., chorionic membrane (CM), chorionic trophoblast without villi (CT-V), chorionic villi (CV), or decidua (DC). According to some embodiments, the source of MSCs is adipose tissue. According to some embodiments, the adipose tissue is subcutaneous white adipose tissue. According to some embodiments, the source of MSCs is bone marrow, umbilical cord tissue, lung tissue, heart tissue, or dental pulp. According to some embodiments, the source of the MSCs is a body fluid. According to some embodiments, the body fluid is peripheral blood, umbilical cord blood, urine, or amniotic fluid.
[0450] Placenta. Structure and development. The placenta and associated extraembryonic membranes are formed from the zygote at the start of each pregnancy, and thus have the same genetic composition as the fetus. The two principal tissue sources are the trophectoderm that forms the wall of the blastocyst, and the underlying extraembryonic mesoderm. The trophectoderm differentiates into trophoblast, which in turn forms the epithelial covering of the placenta and also gives rise to the subpopulation of invasive extravillous trophoblast cells. The extraembryonic mesoderm forms the stromal core of the placenta, from which originate the fibroblasts, vascular network and resident macrophage population. [Burton, G J, Fowden, A L. Philos. Trans. R. Soc. Lond. B. Biol. Sci. (2015) 370 (1663): 20140066].
[0451] The surfaces of the mature placenta are the chorionic plate that faces the fetus and to which the umbilical cord is attached, and the basal plate that abuts the maternal endometrium. Between these plates is a cavity, the intervillous space, into which 30-40 elaborately branched fetal villous trees project. Each villous tree arises from a stem villus attached to the deep surface of the chorionic plate, and branches repeatedly to create a globular lobule 1-3 cm in diameter. The centre of a lobule is located over the opening of a maternal spiral artery through the basal plate. Maternal blood released at these openings percolates between the villous branches before draining into openings of the uterine veins and exiting the placenta. Each lobule thus represents an independent maternal-fetal exchange unit.
[0452] The final branches of the villous trees are the terminal villi. These present a surface area of 12-14 m.sup.2 at term, and are richly vascularized by a fetal capillary network. The capillaries display local dilations, referred to as sinusoids, which bring the endothelium into close approximation to the covering of trophoblast. This is locally thinned, and the diffusion distance between the maternal and fetal circulations may be reduced to as little at 2-3 μm. The morphological resemblance of these structures, termed vasculosyncytial membranes, to the alveoli of the lung has led to the assumption that they are the principal sites of maternal-fetal exchange. Terminal villi are formed primarily from 20 weeks of gestation onwards, and elaboration of the villous trees continues until term [Id., citing Simpson, R A et al. Placenta (1992) 13: 501-512].
[0453] The epithelial covering of the villous tree is the syncytiotrophoblast, a true multinucleated syncytium that presents no intercellular clefts to the intervillous space. This arrangement may assist in preventing the vertical transmission of pathogens from the maternal blood [Id., citing Robbins, J R et al. PLoS Pathog. (2010) 6: e1000732], but may also facilitate regional specializations of the syncytiotrophoblast. Because of its location, the syncytiotrophoblast is involved in many of the functions of the placenta, such as the synthesis and secretion of large quantities of steroid and peptide hormones, protection against xenobiotics and active transport. Hence, it has a high metabolic rate, and accounts for approximately 40% of the total oxygen consumption of the feto-placental unit [Id., citing Carter, A M (2000) Placenta (2000) 21 (Suppl. A): S31-37]. Interposing such an active tissue between the maternal and fetal circulations potentially reduces the oxygen available for the fetus, and so the syncytiotrophoblast shows regional variations in thickness around the villous surface, being very thin and devoid of organelles at the site of vasculosyncytial membranes and thicker over non-vascular parts of the villous surface. Having no lateral cell boundaries may facilitate flow of the syncytioplasm, and so help to optimize oxygen supply to the fetus [Id., citing Burton, G J, Tham, S W. J. Dev. Physiol. (1992) 18: 43-47].
[0454] The syncytiotrophoblast is a highly polarized epithelium, bearing a dense covering of microvilli on its apical border. The projections provide a surface amplification factor of 5-7× for insertion of receptor and transporter proteins. At the base of each microvillus is a clathrin-coated pit, which is capable of forming a coated vesicle for the transport of macromolecules across the syncytiotrophoblast [Id., citing Ockleford, C D, Whyte, a. J. Cell Si. (1977) 25: 293-312].
[0455] The syncytiotrophoblast is a terminally differentiated tissue, and its expansion during pregnancy is achieved by the fusion and incorporation of underlying mononuclear progenitor cytotrophoblast cells that rest on the underlying basement membrane. Fusion is a complex event that is still not fully understood, but involves exit of the progenitor from the cell cycle, the formation of gap junctions with the syncytiotrophoblast, externalization of phosphatidylserine and the expression of two endogenous retroviral proteins that entered the primate genome 25 and more than 40 million years ago [Id., citing Gauster, M., Huppertz, B. (2010) Placenta 31: 82-88; Rote, N S et al. Placenta (2010) 31: 89-96].
Placental Tissue Matrix
[0456] The placenta is considered one of the most important sources of stem cells, and has been studied extensively. It fulfills two main desiderata of cell therapy: a source of a high as possible number of cells and the use of non-invasive methods for their harvesting. Their high immunological tolerance supports their use as an adequate source in cell therapy (Mihu, C. et al., 2008, Romanian Journal of Morphology and Embryology, 2008, 49(4):441-446).
[0457] The fetal adnexa is composed of the placenta, fetal membranes, and umbilical cord. The term placenta is discoid in shape with a diameter of 15-20 cm and a thickness of 2-3 cm. The fetal membranes, amnion and chorion, which enclose the fetus in the amniotic cavity, and the endometrial decidua extend from the margins of the chorionic disc. The chorionic plate is a multilayered structure that faces the amniotic cavity. It consists of two different structures: the amniotic membrane (composed of epithelium, compact layer, amniotic mesoderm, and spongy layer) and the chorion (composed of mesenchyme and a region of extravillous proliferating trophoblast cells interposed in varying amounts of Langhans fibrinoid, either covered or not by syncytiotrophoblast).
[0458] Villi originate from the chorionic plate and anchor the placenta through the trophoblast of the basal plate and maternal endometrium. From the maternal side, protrusions of the basal plate within the chorionic villi produce the placental septa, which divide the parenchyma into irregular cotyledons (Parolini, O. et al., 2008, Stem Cell, 2008, 26:300-311).
[0459] Some villi anchor the placenta to the basal plate, whereas others terminate freely in the intervillous space. Chorionic villi present with different functions and structure. In the term placenta, the stem villi show an inner core of fetal vessels with a distinct muscular wall and connective tissue consisting of fibroblasts, myofibroblasts, and dispersed tissue macrophages (Hofbauer cells). Mature intermediate villi and term villi are composed of capillary vessels and thin mesenchyme. A basement membrane separates the stromal core from an uninterrupted multinucleated layer, called the syncytiotrophoblast. Between the syncytiotrophoblast and its basement membrane are single or aggregated Langhans cytotrophoblastic cells, commonly called cytotrophoblast cells (Parolini, O. et al., 2008, Stem Cell, 2008, 26:300-311).
[0460] Four regions of fetal placenta can be distinguished: an amniotic epithelial region, an amniotic mesenchymal region, a chorionic mesenchymal region, and a chorionic trophoblastic region.
Amniotic Membrane
[0461] Fetal membranes continue from the edge of the placenta and enclose the amniotic fluid and the fetus. The amnion is a thin, avascular membrane composed of an inner epithelial layer and an outer layer of connective tissue that, and is contiguous, over the umbilical cord, with the fetal skin. The amniotic epithelium (AE) is an uninterrupted, single layer of flat, cuboidal and columnar epithelial cells in contact with amniotic fluid. It is attached to a distinct basal lamina that is, in turn, connected to the amniotic mesoderm (AM). In the amniotic mesoderm closest to the epithelium, an acellular compact layer is distinguishable, composed of collagens I and III and fibronectin. Deeper in the AM, a network of dispersed fibroblast-like mesenchymal cells and rare macrophages are observed. It has been reported that the mesenchymal layer of amnion indeed contains two subfractions, one having a mesenchymal phenotype, also known as amniotic mesenchymal stromal cells, and the second containing monocyte-like cells.
Chorionic Membrane
[0462] A spongy layer of loosely arranged collagen fibers separates the amniotic and chorionic mesoderm. The chorionic membrane (chorion leave) consists of mesodermal and trophoblastic regions. Chorionic and amniotic mesoderm are similar in composition. A large and incomplete basal lamina separates the chorionic mesoderm from the extravillous trophoblast cells. The latter, similar to trophoblast cells present in the basal plate, are dispersed within the fibrinoid layer and express immunohistochemical markers of proliferation. The Langhans fibrinoid layer usually increases during pregnancy and is composed of two different types of fibrinoid: a matrix type on the inner side (more compact) and a fibrin type on the outer side (more reticulate). At the edge of the placenta and in the basal plate, the trophoblast interdigitates extensively with the decidua (Cunningham, F. et al., The placenta and fetal membranes, Williams Obstetrics, 20th ed. Appleton and Lange, 1997, 95-125; Benirschke, K. and Kaufmann, P. Pathology of the human placenta. New York, Springer-Verlag, 2000, 42-46, 116, 281-297).
Amnion-Derived Stem Cells
[0463] The amniotic membrane itself contains multipotent cells that are able to differentiate in the various layers. Studies have reported their potential in neural and glial cells, cardiac repair and also hepatocyte cells. Studies have shown that human amniotic epithelial cells express stem cell markers and have the ability to differentiate toward all three germ layers. These properties, the ease of isolation of the cells, and the availability of placenta, make amniotic membrane a useful and noncontroversial source of cells for transplantation and regenerative medicine.
[0464] Amniotic epithelial cells can be isolated from the amniotic membrane by several methods that are known in the art. According to one such method, the amniotic membrane is stripped from the underlying chorion and digested with trypsin or other digestive enzymes. The isolated cells readily attach to plastic or basement membrane-coated culture dishes. Culture is established commonly in a simple medium such as Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5%-10% serum and epidermal growth factor (EGF), in which the cells proliferate robustly and display typical cuboidal epithelial morphology. Normally, 2-6 passages are possible before proliferation ceases. Amniotic epithelial cells do not proliferate well at low densities.
[0465] Amniotic membrane contains epithelial cells with different surface markers, suggesting some heterogeneity of phenotype. Immediately after isolation, human amniotic epithelial cells express very low levels of human leukocyte antigen (HLA)-A, B, C; however, by passage 2, significant levels are observed. Additional cell surface antigens on human amniotic epithelial cells include, but are not limited to, ATP-binding cassette transporter G2 (ABCG2/BCRP), CD9, CD24, E-cadherin, integrins α6 and 1, c-met (hepatocyte growth factor receptor), stage-specific embryonic antigens (SSEAs) 3 and 4, and tumor rejection antigens 1-60 and 1-81. Surface markers thought to be absent on human amniotic epithelial cells include SSEA-1, CD34, and CD133, whereas other markers, such as CD117 (c-kit) and CCR4 (CC chemokine receptor), are either negative or may be expressed on some cells at very low levels. Although initial cell isolates express very low levels of CD90 (Thy-1), the expression of this antigen increases rapidly in culture (Miki, T. et al., Stem Cells, 2005, 23: 1549-1559; Miki, T. et al., Stem Cells, 2006, 2: 133-142).
[0466] In addition to surface markers, human amniotic epithelial cells express molecular markers of pluripotent stem cells, including octamer-binding protein 4 (OCT-4) SRY-related HMG-box gene 2 (SOX-2), and Nanog (Miki, T. et al., Stem Cells, 2005, 23: 1549-1559). Previous studies also have shown that human amnion cells in xenogeneic, chimeric aggregates, which contain mouse embryonic stem cells, can differentiate into all three germ layers and that cultured human amniotic epithelial cells express neural and glial markers, and can synthesize and release acetylcholine, cateholamines, and dopamine. Hepatic differentiation of human amniotic epithelial cells also has been reported. Studies have reported that cultured human amniotic epithelial cells produce albumin and α-fetroprotein and that albumin and α-fetroprotein-positive hepatocyte-like cells could be identified integrated into hepatic parenchyma following transplantation of human amniotic epithelial cells into the livers of severe combined immunodeficiency (SCID) mice. The hepatic potential of human amniotic epithelial cells was confirmed and extended, whereby in addition to albumin and α-fetroprotein production, other hepatic functions, such as glycogen storage and expression of liver-enriched transcription factors, such as hepatocyte nuclear factor (HNF) 3γ and HNF4α, CCAAT/enhancer-binding protein (CEBP α and β), and several of the drug metabolizing genes (cytochrome P450) were demonstrated. The wide range of hepatic genes and functions identified in human amniotic epithelial cells has suggested that these cells may be useful for liver-directed cell therapy (Parolini, O. et al., 2008, Stem Cell, 2008, 26:300-311).
[0467] Differentiation of human amniotic epithelial cells to another endodermal tissue, pancreas, also has been reported. For example, it was shown that human amniotic epithelial cells cultured for 2-4 weeks in the presence of nicotinamide to induce pancreatic differentiation, expressed insulin. Subsequent transplantation of the insulin-expressing human amniotic epithelial cells corrected the hyperglycemia of streptozotocin-induced diabetic mice. In the same setting, human amniotic mesenchymal stromal cells were ineffective, suggesting that human amniotic epithelial cells, but not human amniotic mesenchymal stromal cells, were capable of acquiring β-cell fate (Parolini, O. et al., 2008, Stem Cell, 2008, 26:300-311).
Mesenchymal Stromal Cells from Amnion and Chorion: hAMSC and hCMSC
[0468] Human amniotic mesenchymal cells (hAMSC) and human chorionic mesenchymal cells (hCMSC) are thought to be derived from extraembryonic mesoderm. hAMSC and hCMSC can be isolated from first-, second-, and third-trimester mesoderm of amnion and chorion, respectively. For hAMSC, isolations are usually performed with term amnion dissected from the deflected part of the fetal membranes to minimize the presence of maternal cells. For example, homogenous hAMSC populations can be obtained by a two-step procedure, whereby: minced amnion tissue is treated with trypsin to remove hAEC and the remaining mesenchymal cells are then released by digestion (e.g., with collagenase or collagenase and DNase). The yield from term amnion is about 1 million hAMSC and 10-fold more hAEC per gram of tissue (Casey, M. and MacDonald P., Biol Reprod, 1996, 55: 1253-1260).
[0469] hCMSCs are isolated from both first- and third-trimester chorion after mechanical and enzymatic removal of the trophoblastic layer with dispase. Chorionic mesodermal tissue is then digested (e.g., with collagenase or collagenase plus DNase). Mesenchymal cells also have been isolated from chorionic fetal villi through explant culture, although maternal contamination is more likely (Zhang, X., et al., Biochem Biophys Res Commun, 2006, 340: 944-952; Soncini, M. et al., J Tissue Eng Regen Med, 2007, 1:296-305; Zhang et al., Biochem Biophys Res Commun, 2006, 351: 853-859).
[0470] The surface marker profile of cultured hAMSC and hCMSC, and mesenchymal stromal cells (MSC) from adult bone marrow are similar. All express typical mesenchymal markers (Table 12) but are negative for hematopoietic (CD34 and CD45) and monocytic markers (CD14). Surface expression of SSEA-3 and SSEA-4 and RNA for OCT-4 has been reported (Wei J. et al., Cell Transplant, 2003, 12: 545-552; Wolbank, S. et al., Tissue Eng, 2007, 13: 1173-1183; Alviano, F. et al., BMC Dev Biol, 2007, 7: 11; Zhao, P. et al, Transplantation, 2005, 79: 528-535). Both first- and third trimester hAMSC and hCMSC express low levels of HLA-A, B, C but not HLA-DR, indicating an immunoprivileged status (Portmann-Lanz, C. et al, Am J Obstet Gynecol, 2006, 194: 664-673; Wolbank, S. et al., Tissue Eng, 2007, 13: 1173-1183).
[0471] Table 12 provides surface antigen expression profile at passages 2-4 for amniotic mesenchymal stromal and human chorionic mesenchymal stromal stem cells.
TABLE-US-00002 TABLE 12 Specific surface antigen expression for amniotic mesenchymal stromal cells and human chorionic mesenchymal stromal cells Positive (≥95%) Negative (≤2%) CD90 CD45 CD73 CD34 CD105 HLA-DR
[0472] Both hAMSCs and hCMSCs differentiate toward “classic” mesodermal lineages (osteogenic, chondrogenic, and adipogenic) and differentiation of hAMSC to all three germ layers-ectoderm (neural), mesoderm (skeletal muscle, cardiomyocytic and endothelial), and endoderm (pancreatic) was reported (Int′Anker, P. et al., Stem Cells, 2004, 22: 1338-1345; Portmann-Lanz, C. et al, Am J Obstet Gynecol, 2006, 194: 664-673; Wolbank, S. et al., Tissue Eng, 2007, 13: 1173-1183; Soncini, M. et al., J Tissue Eng Regen Med, 2007, 1:296-305; Alviano, F., BMC Dev Biol, 2007, 7: 11).
[0473] Human amniotic and chorionic cells successfully and persistently engraft in multiple organs and tissues in vivo. Human chimerism detection in brain, lung, bone marrow, thymus, spleen, kidney, and liver after either intraperitoneal or intravenous transplantation of human amnion and chorion cells into neonatal swine and rats was indeed indicative of an active migration consistent with the expression of adhesion and migration molecules (L-selectin, VLA-5, CD29, and P-selectin ligand 1), as well as cellular matrix proteinase (MMP-2 and MMP-9) (Bailo, M. et al., Transplantation, 2004, 78:1439-1448).
Umbilical Cord
[0474] Two types of umbilical stem cells can be found, namely hematopoietic stem cells (UC-HS) and mesenchymal stem cells, which in turn can be found in umbilical cord blood (UC-MS) or in Wharton's jelly (UC-MM). The blood of the umbilical cord has long been in the focus of attention of researchers as an important source of stem cells for transplantation, for several reasons: (1) it contains a higher number of primitive hematopoietic stem cells (HSC) per volume unit, which proliferate more rapidly, than bone marrow; (2) there is a lower risk of rejection after transplantation; (3) transplantation does not require a perfect HLA antigen match (unlike in the case of bone marrow); (4) UC blood has already been successfully used in the treatment of inborn metabolic errors; and (5) there is no need for a new technology for collection and storage of the mononuclear cells from UC blood, since such methods are long established.
[0475] Umbilical cord (UC) vessels and the surrounding mesenchyma (including the connective tissue known as Wharton's jelly) derive from the embryonic and/or extraembryonic mesodermis. Thus, these tissues, as well as the primitive germ cells, are differentiated from the proximal epiblast, at the time of formation of the primitive line of the embryo, containing MSC and even some cells with pluripotent potential. The UC matrix material is speculated to be derived from a primitive mesenchyma, which is in a transition state towards the adult bone marrow mesenchyma (Mihu, C. et al., 2008, Romanian Journal of Morphology and Embryology, 2008, 49(4):441-446).
[0476] The blood from the placenta and the umbilical cord is relatively easy to collect in usual blood donation bags, which contain anticoagulant substances. Mononuclear cells are separated by centrifugation on Ficoll gradient, from which the two stem cell populations will be separated: (1) hematopoietic stem cells (HSC), which express certain characteristic markers (CD34, CD133); and (2) mesenchymal stem cells (MSC) that adhere to the culture surface under certain conditions (e.g., modified McCoy medium and lining of vessels with Fetal Bovine Serum (FBS) or Fetal Calf Serum (FCS)). (Munn, D. et al., Science, 1998, 281: 1191-1193; Munn, D. et al., J Exp Med, 1999, 189: 1363-1372). Umbilical cord blood MSCs (UC-MS) can produce cytokines, which facilitate grafting in the donor and in vitro HSC survival compared to bone marrow MSC. (Zhang, X et al., Biochem Biophys Res Commun, 2006, 351: 853-859).
[0477] MSCs from the umbilical cord matrix (UC-MM) are obtained by different culture methods depending on the source of cells, e.g., MSCs from the connective matrix, from subendothelial cells from the umbilical vein or even from whole umbilical cord explant. They are generally well cultured in DMEM medium, supplemented with various nutritional and growth factors; in certain cases prior treatment of vessels with hyaluronic acid has proved beneficial (Baban, B. et al., J Reprod Immunol, 2004, 61: 67-77).
Bone Marrow
[0478] Human bone marrow can be obtained from the iliac crest of patients after having obtained their written consent. BM is collected aseptically into K2EDTA tubes. The buffy coat is isolated by centrifugation (450×g, 10 min), suspended in 1.5 mL PBS, and used for culture. The separated buffy coat is layered onto equal volume of Ficoll (GE Health Care, USA) and centrifuged (400× g, 20 min). Cells at the interface are removed, and washed twice in sterile PBS.
[0479] Human bone marrow progenitor cells are cultured on tissue treated culture plates in DMEM medium supplemented with 10% FBS and penicillin/streptomycin (50 U/mL and 50 mg/mL, respectively). The plates are maintained at 37° C. in a humidified atmosphere containing 5% CO.sub.2 for 48 h. To exchange the medium, the plates are washed with PBS in order to remove non-adhered cells and the medium is replaced. The remaining cells have a heterogeneous fibroblastic-like appearance and exhibit colony formation. The cultures can be maintained for an additional week with one medium exchange.
Adipose Tissue
[0480] In comparison to BM-MSC, MSC from adipose tissue, the adipose-derived stromal/stem cells (ASCs), occur at a 100-1000-fold higher frequency within adipose tissue on a volume basis (Aust L, et al., Cytotherapy. 2004; 6(1): 7-14). Harvesting adipose tissue is also minimally invasive and less painful than bone marrow tissue. Conventional enzymatic methods, using enzymes such as collagenase, trypsin, or dispase, are widely used for MSC isolation from adipose tissue. Although the isolation techniques for adipose tissue-derived cells are rather diverse, they follow a certain standard procedure. Differences lie mainly in numbers of washing steps, enzyme concentrations, centrifugation parameters, erythrocyte lysis methods as well as in filtration, and eventually culture conditions (Oberbauer E, et al., Cell Regen (Lond). 2015; 4: 7, citing Zuk P A, et al., Mol Biol Cell. 2002; 13(12): 4279-95; Gimble J, Guilak F. Cytotherapy. 2003; 5(5): 362-9; Carvalho P P, et al., Tissue Eng Part C Meth. 2013; 19(6): 473-8).
[0481] An exemplary protocol for isolating MSCs from adipose tissue includes the steps of obtaining adipose tissue by surgical resection or lipoaspiration; washing the tissue 3-5 times for 5 minutes in PBS each wash, discarding the lower phase until clear; adding collagenase and incubating 1-4 hr at 37° C. on a shaker; adding 10% FBS to neutralize the collagenase; centrifuging the digested fat at 800× g for 10 min; aspirating floating adipocytes, lipids and liquid, leaving the stromal vascular fraction (SVF) pellet; resuspending the SVF pellet in 160 mM NH4C1 and incubating for 10 minutes at room temperature; centrifuging at 400×g for 10 min at room temperature; layering cells on Percoll or Histopaque gradient; centrifuging at 1000×g for 30 minutes at room temperature; washing cells twice with PBS and centrifuging at 400×g for 10 min between each wash; resuspending the cell pellet in PBS and filtering cells through a 100 μM nylon mesh; passing the cells through a 400 μM nylon mesh; centrifuging at 400×g for 10 minutes; resuspending the cell pellet in 40% FBS/DMEM culture medium and plating the cells. The plastic-adherent cell fraction, including ASCs, can be obtained after passaging or cryopreservation or further cultivated for expansion for a more homogeneous ASC population (Id.).
[0482] An exemplary protocol for expansion and subculture of human MSCs includes the following steps: Precoating a tissue culture vessel with 5 μg/mL of PRIME-XV MatrIS F or PRIME-XV Human Fibronectin for 3 hr at room temperature or overnight at 2-8° C.; prewarming PRIME-XV MSC Expansion SFM to 37° C. for no more than 30 min; removing spent media from T-75 flask culture and gently rinsing cells once with 10 mL of PBS for each T-75 flask; adding 3 mL of room temperature TrupLE™ Express to each T-75 flask, and tilting the flask in all directions to disperse the TrypLE™ Express evenly over the cells; incubating the cells at 37° C., 5% CO.sub.2 to allow the cells to detach; adding 5 mL of PRIME-XV MSC expansion SFM to the flask and dispersing the cells by pipetting the media over the entire growing surface of the flask; transferring the contents to a 15 mL conical tube; centrifuging the cells at 400×g for 5 min and aspirating the supernatant; resuspending the cell pellet in a small amount of pre-warmed PRIME-XV MSC Expansion SFM and counting the cells; resuspending 4.5-5.0×10.sup.5 cells into 20 mL of the pre-warmed PRIME-XV MSC Expansion SFM for each pre-coated T-75 flask; gently aspirating off PRIME-XV attachment substrate solution from the flask and slowly adding the cell suspension to a T-75 flask; and incubating the cells at 37° C., 5% CO.sub.2. Spent media is removed and discarded and the cells fed with pre-warmed PRIME-XV MSC Expansion SFM every two days.
Dental Pulp
[0483] Similar to adipose tissue, generating stem cells from dental pulp is a relatively noninvasive and noncontroversial process. Deciduous teeth may be sterilized, and the dental pulp tissue separated from the pulp chamber and root canal, revealed by cutting around the cementoenamel junction using sterilized dental burs (Tsai A I, et al., Biomed Res Int. 2017: 2851906). After separation, the dental pulp may be isolated using, for example, a barbed broach or a sharp excavator (Id.). MSCs may be isolated enzymatically or non-enzymatically as described above for adipose tissue.
Lung or Heart Tissue
[0484] MSCs may be cultured from tissue biopsies or transplanted tissues. A study in heart transplant patients demonstrated that MSCs present in transplanted hearts were all of donor origin (Hoogduijn M J, et al., Am J Transplant. 2009 January; 9(1): 222-30). No MSCs of recipient origin were found, even not many years after transplantation. Similar data were found in lung transplant patients (Lama V N, et al., J Clin Invest. 2007 April; 117(4): 989-96). These data suggest that MSCs do not migrate between tissues, not even under inflammatory conditions as found in transplanted organs (Eggenholfer E, et al., Front Immunol. 2014; 5: 148).
[0485] For the isolation of lung or heart tissue-derived MSCs, tissues are minced into pieces and digested with a culture medium containing 0.2% collagenase (Wako) at 37° C. for 30 min. The collagenase is removed by washing twice with 1×PBS. The cell suspension is filtered through a cell strainer (40-μm) and collected in a 50-ml tube. Red blood cells are removed by incubating cells in 1×RBC lysis buffer (BioLegend) for 5 min at room temperature. Then, 2×10.sup.7 cells are seeded onto a collagen I-coated, 10-cm dish using MesenCult medium containing 1× MesenPure and 10 nM of a Rock inhibitor. MSCs may be cultured for up to three passages to reduce any artefacts potentially introduced by long-term culture.
Blood
[0486] Umbilical cord blood MSCs are obtained from 40 mL of UCB with citrate phosphate dextrose (Sigma-Aldrich, St. Louis, Mo.) as anticoagulant, and centrifuged through Ficoll-Paque (1.077 g/cm3) according to the manufacturer's instructions. MSC fractions are washed with PBS, counted using trypan blue exclusion staining and plated onto fibronectin-coated tissue culture flasks (Becton Dickinson) in MSC expansion medium (Iscove modified Dulbecco medium (IMDM, Life Technologies) and 20% FBS supplemented with 10 ng/mL recombinant human bFGF (Peprotech, Rocky Hill N.J.), 100 U penicillin, 100 U streptomycin and 2 mM L-Glutamine (Life Technologies/Gibco). Cells are allowed to adhere overnight and nonadherent cells washed out with medium changes.
[0487] In an exemplary protocol for obtaining MSCs from whole blood, a diluted mixture of PBS and peripheral blood is layered in a 50 ml centrifuge tube on top of Ficoll-Paque, and centrifuged at 400×g for 30-40 minutes at 20° C. in a swinging-bucket rotor without break. The upperlayer is aspirated, leaving the mononuclear cell layer (lymphocytes, monocytes and thrombocytes) undisturbed at the interface. The mononuclear cell layer is carefully transferred into a new 50 ml centrifuge tube. Cells are washed with PBS (pH 7.2) containing 2 mM EDTA, centrifuged at 300×g for 10 min at room temperature and the supernatant discarded. For removal of platelets, the cell pellet is resuspended in 50 mL buffer and centrifuged at 200×g for 10-15 minutes at room temperature. The supernatant containing the platelets is removed. This step is repeated. The cell pellet is resuspended in DMEM, 20% FBS and 1% antibiotic-antimycotic. Cultures are maintained at 37° C. in a humidified atmosphere containing 5% CO.sub.2. Suspended cells are discarded after 5-7 days of culture and adherent cells left to grow on the flask surface. Culture medium is changed every 3 days.
Amniotic Fluid
[0488] Amniotic fluid is formed at 2 weeks after fertilization in the amniotic cavity of early gestation (Kim E Y, et al., BMB Rep. 2014 March; 47(3): 135-140). Amniotic fluid keeps the fetus safe and supports organ development. The first progenitor cells derived from amniotic fluid was reported in 1993 by Torricelli et al. (Ital J Anat Embryol. 1993 April-June; 98(2): 119-26). Many studies have identified amniotic fluid (AF) as a source of MSCs. These AF-MSCs express the pluripotent marker Oct-4 in almost 90% of the active condition, and they also have multiple differentiation capacity like amniotic membrane MSCs (Tsai M S, et al., Hum Reprod. 2004 June; 19(6): 1450-6; De Coppi P, et al., Nature Biotechnol. 2007 January; 25(1): 100-6). AF is also routinely used to perform the standard evaluation of karyotyping, and genetic and molecular tested for diagnostic purposes. After prenatal diagnostic testing, AF cells can be used as a source of fetal progenitor cells or otherwise discarded (Prusa A R, et al., Med Sci Monit. 2002 November; 8(11): RA253-7). Use of these cells could minimize ethical objections, have a high renewal activity, and maintain genetic stability (Kim E Y, et al., BMB Rep. 2014 March; 47(3): 135-140). AF-MSCs are easily isolated and offer advantages of nontumorigenicity and low immunogenic activity. (Id.).
[0489] Amniotic fluid samples are obtained by amniocentesis performed between 16 and 20 weeks of gestation for fetal karyotyping. A two-stage culture protocol can be used for isolating MSCs from amniotic fluid (Tsai M S, et al., Hum Reprod. 2004 June; 19(6): 1450-6). For culturing amniocytes (first stage), primary in situ cultures are set up in tissue culture-grade dishes using Chang medium (Irvine Scientific, Santa Ana, Calif.). Metaphase selection and colony definition is based on the basic requirements for prenatal cytogenetic diagnosis in amniocytes (Moertel C A, et al., 1992; Prenat Diagn 12, 671-683). For culturing MSCs (second stage), non-adhering amniotic fluid cells in the supernatant medium are collected on the fifth day after the primary amniocytes culture and kept until completion of fetal chromosome analysis. The cells are then centrifuged and plated in 5 ml of α-modified minimum essential medium (α-MEM; Gibco-BRL) supplemented with 20% fetal bovine serum (FBS; Hyclone, Logan, Utah) and 4 ng/ml basic fibroblast growth factor (bFGF; R&D systems, Minneapolis, Minn.) in a 25 cm.sup.2 flask and incubated at 37° C. with 5% humidified CO.sub.2 for MSC culture. Similar to MSCs from umbilical cord blood and first-trimester fetal tissues, surface antigens such as SH3, SH4, CD29, CD44 and HLA-A,B,C (MHC class I) may be found, and CD10, CD11b, CD14, CD34, CD117, HLA-DR,DP,DQ (MHC class II) and EMA are absent (Tsai M S, et al., Hum Reprod. 2004 June; 19(6): 1450-6; Pittenger M F, et al., Science 284, 143-7; Colter D C, et al., Proc Natl Acad Sci USA 98, 78415; Young H Y, et al., Anat Rec 264, 51-62).
[0490] According to some embodiments, to characterize the adherent MSCs, osteoblastic differentiation is induced by culturing confluent human MSCs for 3 weeks in osteoblastic differentiation media (all from Sigma) and after three weeks, the cells are stained by Alizarin. To induce adipocyte differentiation, confluent MSCs are cultured 1 to 3 weeks in differentiation medium, and lipid droplet staining is carried out by S Red Oil (Sigma).
[0491] According to some embodiments, flow cytometry can be used to characterize cell markers expressed on the surface of the isolated MSCs. According to some embodiments, the phenotype of the adherent MSCs is CD73+, CD90+, CD105+, CD34-, CD45-.
[0492] According to some embodiments, the MSCs are derived from a biological sample, wherein the biological sample is a tissue or a body fluid of a human subject. According to some embodiments, the tissue is placental tissue, adipose tissue, umbilical cord tissue, lung tissue, heart tissue or dental pulp; or bone marrow of normal healthy subjects aged 21-40 years old; or the body fluid is blood, amniotic fluid or urine. According to some embodiments, the MSCs derived from placental tissue are derived from one or more of chorionic membrane (CM), chorionic trophoblast without villi (CT-V), chorionic villi (CV), or decidua (DC). According to some embodiments, the blood is umbilical cord blood or peripheral blood.
[0493] According to some embodiments, extracellular vesicles derived from a biological sample, e.g., human bone marrow, will be isolated from bone marrow-derived mesenchymal stem cells of an adult healthy donor (21-40 years old) who will have met strict donor acceptance criteria including current and past medical history in addition to passing highly sensitive nucleic acid testing for hepatitis B, hepatitis C, and HIV.
[0494] According to some embodiments, the MSCs are obtained from a human subject. According to some embodiments, identity of the hMSCs can be quickly confirmed by their expression of at least three cell surface markers selected from CD29, CD44, CD90, CD73, and CD105.
[0495] An exemplary protocol is as follows. Human mesenchymal stem cells used to generate the extracellular vesicle (EV) product originate from a single donor bone marrow source of adherent stromal progenitor cells expanded under GTP/cGMP compliant conditions. In sort, the MSC product is expanded in a 3D bioreactor until 80% confluence. Expansion media is then washed with PBS and replaced with serum-free DMEM. Conditioned media is collected from the waste outlet of the bioreactor and subjected to ultracentrifugation for exosome precipitation. This collection procedure is performed daily for up to 96 hours. EVs are isolated by differential filtration through nylon membrane filters of defined pore size. A first filtration through a large pore size retains cellular fragments and debris; a subsequent filtration through a smaller pore size retains EV and purifies them from smaller size contaminants. Tangential flow filtration (TFF) is utilized to concentrate the fluid containing the EVs. 0.22 μfiltration is first completed, followed by TFF with pore size of either 100 kD molecular weight cut off (MWCO) or 300 kD MWCO is used for concentration and diafiltration. EVs are further purified using size exclusion chromatography by loading the concentrated clarified conditioned media on a PBS-equilibrated Chroma S-200 column (Clontech) using a 70 nm pore size, eluting with PBBS and collecting final bulk drug substance. This is sterile filtered and provided in Dulbeccos' Phosphate Buffered Saline (DPBS) without calcium or magnesium added to sterile vials at concentration of ≥80 billion particles per vial, capped packaged, frozen at −80° C.
[0496] The population of exosomes resulting from this isolation process is a purified enriched population of potent exosomes. Primary exosome product characterization analysis includes, without limitation, nanoparticle size and concentration analysis; flow cytometry analysis of exosome specific protein markers (CD63, CD9), 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, NH4+, Na+, K+, Ca++, pH, pCO.sub.2, pO.sub.2 and osmolality.
[0497] The product comprising the purified enriched population of potent exosomes will be tested for sterility and is manufactured in FDA-registered facilities that meet Current Good Manufacturing Practices (cGMP). The exosome product will be filtered [pore size] and purified prior to filling, and all containers will be visually inspected prior to cryopreservation to ensure normal appearance, intact container closure, and the absence of visible extraneous particulate matter. Product will be released for clinical use only if all safety, identity, quality, and purity specifications are met and all applicable GMPs are appropriately followed as determined by Zen-Bio Quality Assurance.
[0498] According to some embodiments, the purified enriched population of potent exosomes comprises a cargo. According to some embodiments the cargo comprises a potency signature including expression of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more of angiopoietin 2 (Ang-2), fibroblast growth factor (FGF), hepatic growth factor (HGF), interleukin 8 (IL-8), a tissue inhibitor of metalloproteinases (TIMP), vasculoendothelial growth factor (VEGF), platelet derived growth factor (PDGF), or tumor necrosis factor alpha (TNFα). According to some embodiments, the tissue inhibitor of metalloproteinases is TIMP1, TIMP2, or TIMP1 and TIMP2.
[0499] According to some embodiments, per isolation process, the composition comprises at least 1×10.sup.12 EVs comprising exosomes of which at least 1×10.sup.9 are exosomes]
[0500] According to some embodiments, a therapeutic amount of a purified, enriched population of potent exosomes comprises at least 1×10.sup.9 exosomes.
[0501] According to some embodiments, the cargo of the exosomes comprising the therapeutic signature: is configured so as to modulate one or more of the injury, the inflammation, an excess accumulation of extracellular matrix, cell senescence; or is configured so as to modulate a pathway comprising fibrogenic signaling; or is configured so as to slow or reverse progression of the age-related chronic lung disease; or is configured to reprogram a tissue affected by an age-related chronic disease.
[0502] According to some embodiments, the age-related chronic disease if left untreated comprises one or more of a progressive injury, progressive inflammation, progressive fibrosis or a combination thereof.
[0503] 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
[0504] According to some embodiments, the tissue is lung tissue, cardiac tissue, renal tissue, hepatic tissue, skin, pancreatic tissue, eye tissue, joint tissue, bone marrow, brain tissue, intestinal tissue, peritoneal tissue, retroperitoneal tissue, nerve tissue, spinal tissue, or skeletal muscle.
[0505] According to some embodiments, the-age-related chronic disease comprises a chronic lung disease, a chronic inflammation and immune dysfunction, a mitochondrial dysfunction, organ transplantation dysfunction, organ resuscitation and rejuvenation, a viral infection, neuropathic pain; neurofibrosis, neurodegeneration, connective tissue dysfunction, musculoskeletal repair, dysfunction of the gut microbiome, or an age-related decline in overall health.
[0506] Microbiome Dysfunction
[0507] According to some embodiments, a composition of the present disclosure comprising a population of purified enriched potent exosomes with an appropriate therapeutic signature of MSC-derived EVs isolated from a normal healthy subject may modulate microbiome dysfunction.
[0508] The human microbiota consists of 10-100 trillion symbiotic microbial cells harbored by each person, primarily bacteria in the gut, while the human microbiome consists of the genes these cells harbor. [Ursell, L K et al., “Defining the human microbiome.” Nutr. Rev. (2012) 70 (Suppl. 1) S38-S44).] These microbial cells, and their genetic material, live with humans from birth, and every individual has a unique mix of species. This relationship is important for nutrition, immunity and effects on the brain and behavior, and has been implicated in a number of diseases where the disease is caused by a disturbance in the normal balance of microbes or where the disturbance is another downstream consequence of the disease. The interaction between the human microbiota and the environment is dynamic, meaning that microbial communities are constantly being transferred between surfaces, and that a dynamic interaction exists between environmental microbiota and different human body sites. There is increasing evidence that individuals actually share a core microbiota, with vastly different sets of microbial species yielded very similar functional molecular interactions, reactions and relation networks for metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems referred to as KEGG pathways.
[0509] There is accumulating evidence that a cross-talk between the gut and lungs exists. The lungs of healthy individuals harbor Fusobacterium, Haemophilus, Prevotella, Streptococcus, and Veillonella as main genera, which are relatively small in size when compared to the enteric microbiota [Ahlawat, S. et al. Virus Res. (2020) 286: 198103, citing He, et al., Y. et al. Crit. Rev. Microbiol. (2017) 43 (1): 81-95]. The emergence and maintenance of lung microbiota is governed by the equilibrium between microbial migration from the upper respiratory tract and microbial removal by the host defense systems, with small contribution from the multiplication of native microbes. Even in small concentrations, the airway microbiome is believed to be crucial to host immunity, such that an imbalance between microbial immigration and removal predisposes its host to the progression and exacerbations of respiratory diseases. [Id., citing He, et al., Y. et al. Crit. Rev. Microbiol. (2017) 43 (1): 81-95; Wypych, T P et al. Nat. Immunol. (2019) 20 (10): 1279-90]. Alterations in the lung microbial community including airways also affects the composition of the intestinal microbiota, and respiratory viral infections can alter the intestinal microbiome, where the intestinal microbiome determines the adaptive immune responses against the respiratory pathogens and is necessary for priming the innate immune responses against the pulmonary infection. Indeed, during respiratory viral infections, the level of macrophage response to the respiratory viruses depends on the presence of intestinal microbes. [Id., citing Hanada, S. et al. Front. Immunol. (2018) 9: 2640].
[0510] Gut microbiota composition and diversity is affected by many factors, including by aging. An apparent age-related gut microbiota imbalance has been described, featuring an altered microbial diversity, a lower abundance of probiotic strains (e.g., Difidobacteria), and a reduced number of species producing butyrate, a short chain fatty acid (SCFA) that plays important metabolic functions and has a major role in maintaining the integrity of intestinal epithelium. [Mangiola, F. et al. Eur. Rev. Med. Pharmacol. Sci. 2018] 22: 7404-13].
[0511] The integrity of the commensal microbiota can be disturbed by invading viruses, which cause dysbiosis (meaning a general imbalance of gut microbiota) in the host and further influence virus infectivity. [Li, N. et al. Frontiers Immunol. (2019) 10: 1551]. It has been shown that RNA viruses, such as poliovirus, benefit from the delivery of various viral genomes into a single target cell, thereby allowing the recombination of multiple virus genomes; this process potentiates those viral progeny with enhanced environmental fitness [Id., citing Aguilera, E R et al. M. Bio. (2017) 8: 16; Chen, Y H et al. Cell (2015) 160: 619-30; Combe, M. et al. Cell Host Microbe (2015) 18: 424-32]. Some viruses (e.g., poliovirus, reovirus) use commensal microbiota components to enhance viral stability and increase infectivity. There is also likely a link between commensal microbiota and the lytic reactivation of viruses. In addition to fostering the generation of immunoregulatory Treg cells and thereby suppress anti-viral immunity, the commensal microbiota also have been reported to directly skew antiviral immunity by suppressing the activation of effector immune cells and by inhibiting the production of various inflammatory cytokines that are pivotal for virus elimination, thus creating a more favorable environment for viral infection [Id., citing Baldridge, M T et al. Science (2015) 347: 266-9].
[0512] The commensal microbiota also can prevent viral infection. For example, Enterococcus faecium can prevent infection by influenza viruses upon direct absorptive trapping of these viruses. [Id., citing Wang, X Y et al. PLoS One (2013) 8: e53043]. An extracellular matrix binding protein produced by Staphylococcus epidermidis, a Gram-positive bacterium that lives in the human nasal cavity as a commensal, can stably bind to influenza virus and thus block further infection [Id., citing Chen, H W et al. Sci. Rep. (2016) 6: 27870]. The replication of herpes simplex virus-2 (HSV-2) can also be suppressed by commensal microbiota metabolites; lactic acid, a major end product of the carbohydrate fermentation of all Lactobacillus species, can strongly inactivate HSV-2 in the vaginal mucosa by maintaining an acidic pH in the local environment. [Id., citing Tuyama, C G e t al. J. Infect. Dis. (2006) 194: 795-803]. Commensal microbiota also exert their antiviral activity through cell wall associated bacterial components [Id., citing Mastromarino, P. et al. Anaerobe (2011) 17: 334-6].
[0513] Supporting studies have shown that intact healthy commensal microbiota help maintain robust antiviral immunity, while microbiota disruption increases viral infectivity due to the impaired capacity of the immune system to limit viral infection. For example Clostridium orbiscindens, a human-associated gut microbe produces desaminotyrosine to prime the amplification loop of type I IFN signaling, thereby mediating protection against influenza infection. [Id, citing Steed, A L et al. Science (2017) 357: 498-502]. It has been shown that during respiratory influenza virus infection, antibiotic exposure led to a defective generation of virus-specific CD4 and CD8 T cells and antibodies due to an impaired inflammasome-dependent migration of APCs from the lung to the draining lymph nodes. [Id., citing Ichinohe, T. et al. Proc. Natl Acad. Sci. USA (2011) 108: 5354-9].
[0514] Studies also suggest an important role of virus infection in inducing microbiota dysbiosis. This is true for HIV/SIV infection, influenza virus infection, HBV or hepatitis C virus infection (HCV) and norovirus infection. For example, microbial diversity in saliva of HIV patients was significantly reduced compared to healthy controls, accompanied by increased abundance of potentially pathogenic Megasphaera, Camplyobacter, Veillonella and Prevotella species, and decreased commensal Veillonella and Streptococcus species [Id., citing Li, Y. et al. J. Clin. Microbiol. (2014) 52: 1400-11; Dang, A T et al. BMC Microbiol. (2012) 12 (152): 95, 96]. In addition, fungal communities in HIV infected and uninfected individuals differed significantly. [Id., citing Mukherjee, P K et al. PLoS Pathog. (2014) 10: e1003996]. In bronchoalveolar lavage fluids, although there were no significant differences among the microbial composition in HIV-infected and uninfected subjects, specific metabolic profiles were associated with bacterial organisms that potentially play a role in the pathogenesis of pneumonia in HIV infected patients. [Id., citing Cribbs, S K et al. Microbiome (2016) 4: 3]. Mechanistically, specific immune suppression by HIV is partly responsible for the enrichment of certain potentially pathogenic bacteria.
[0515] Several studies have shown that influenza virus infection can result in decreased colonization by healthy bacteria and increased abundance of potentially pathogenic microbiota. There is a consensus that influenza virus infection alters the commensal microbiota of the host, causing corresponding disruptions of the microbiota-host homeostasis, which largely accounts for the mechanisms by which infections are established. Several lines of data have highlighted that the modulation of immune responses by influenza contributes to the dysbiosis of the gut. [Id., citing Wang, J. et al. J. Exp. Med. (2014) 211: 2397-410; Deriu, E. et al. PLoS Pathog. (2016) 12: e1005572].
[0516] Although SARS-CoV-2 primarily causes lung infection through binding of ACE2 receptors present on alveolar epithelial cells, SARS-CoV-2 RNA has been found in the feces of infected patients. It is known that intestinal epithelial cells, particularly the enterocytes of the small intestine, also express ACE2 receptors. [Dhar, D. and Mohanty, A. Virus Res. (2020) 198018]. Poor outcomes of COVID-19 infection have been observed in elderly patients, particularly those with pre-existing cardiovascular, metabolic and renal disorders, in whom the disease severity and mortality rate are considerably higher. [Viana, S D et al. Ageing Research Reviews (2020) 62: 101123, citing Du, R H et al. Eur. Respir. J. (2020) 55; Li, B et al Clin. Res. Cardio. (2020) 109: 531-38; Mehra, M R et al N. Eng. J. Med. (2020) 382 (26): e102; Roncon, L. et al J. Clin. Virol. (2020) 127: 104354; Shi, Q. et al. Diabetes Care (2020) 43 (70: 1382-91; Siordia, Jr., JA. J. Clin. Virol. (2020) 127: 104357; Team, CC-R. MMWR Morb. Mortal. Wkly Rep. (2020) 69: 343-46; Wang, X. et al. Research (Wash D.C.) (2020) 2402961; Zhou, F. et al. Lancet (2020) 395: 1054-62).
[0517] ACE2 is the receptor for SARS-CoV2-entry in human cells. [Id., citing Hoffmann, M. et al. Cell (2020) 181: 271-80]. There is a constitutive ACE2 expression within the luminal surface of differentiated small intestinal epithelial cells and colonic crypt cells. [Id, citing Harmer, D. et al. FEBS Lett. (2002) 532: 107-10], where ACE2 functions as the chaperone for membrane trafficking of the amino acid transporter B° AT1, which mediates the uptake of neutral amino acids into intestinal cells in a sodium dependent manner, even in the absence of collectrin [Id., citing Camargo, S M et al. Gastroenterology (2009) 136: 872-82; Kowalczuk, S. et al. FASEB J. (2008) 22: 2880-87]. ACE2 shares around 50% homology with collectrin, a type I transmembrane protein that regulates the transporter of neutral amino acids in the kidney (Id., citing Zhang, H. et al. J. Biol. Chem. (2001) 276: 17132-9). It has been suggested that a B° AT1/ACE2 complex in the intestinal epithelium regulates gut microbiota composition and function, with important repercussions on local and systemic immune responses against pathogenic agents, e.g., viruses. A dysbiotic condition (decreased microbial diversity and richness, and impaired Firmicutes to Bacteroides ratio) has been reported in the elderly and in age-related cardiovascular, metabolic, renal and pulmonary conditions. In an ACE2/γ-Akita mouse model, gut barrier disruption and exacerbated diabetes-induced dysbiosis have been described. [Id., citing Duan, Y. et al. Cir. Res. (2019) 125: 969-88]. Some patients with COVID-19 have presented with intestinal microbial dysbiosis. It therefore has been hypothesized that pre-existing disorders displaying an altered gut microbiome may worsen SARS-CoV-2 infection due to a loss of ACE2 integrity/functionality in the gut.
[0518] In a pilot study of 15 patients with COVID-19, persistent alterations in the fecal microbiome during the time of hospitalization were found compared with controls. Fecal microbiota alterations were associated with fecal levels of levels of SARS-CoV-2 and COVID-19 severity. Over the course of hospitalization, bacterial species which downregulate expression of ACE2 in murine gut correlated inversely with SARS-CoV-2 load in fecal samples. [Zuo, T. et al. Gastroenterology (2020) 159: 944-5].
Mitochondrial Dysfunction
[0519] According to some embodiments, a composition of the present disclosure comprising a purified enriched population of potent exosomes comprising an appropriate therapeutic signature may modulate mitochondrial dysfunction. Mitochondrial dysfunction has been defined as changes in gene expression of mitochondrial markers [Montgomery, M K. Biology (Basel) (2019) 8 (2): 33, citing Heilbaronnn, L K et al. J. Clin. Endocrinol. Metab. (2007) 92: 1467-73; Mootha, V K et al. Nat. Rev. Genet. (2003) 34: 267-73], protein content, or enzymatic activities of mitochondrial proteins [Id., citing Heilbronn, L K et al. J. Clin. Endocrinol. Metab. (2007) 92: 1467-73; Ritov, V B, Diabetes (2005) 54: 8-14], changes in mitochondrial size and shape [Id., citing Rritov, V B et al. Diabetes (2005) 54: 8-14; Kelly, D E et al. Diabetes (2002) 51: 2944-2950], as well as functional assessment of mitochondrial oxidative capacity [Id., citing Kelley, D E et al. Am. J. Physiol. (1999) 277: E1130-41] and ROS generation [Id., citing Anderson, E J et al. J. Clin. Invest. (2009) 119: 573-81]. Mitochondrial defects are present in the heart in individuals with insulin resistance and diabetes [Id., citing Montaigne, D. et al. Circulation (2014) 130: 554-64; Dhalla, N S et al. Heart Fail. Rev. (2014) 19: 87-99; Mackenzie, R M et al. Clin. Sci. (2013) 124: 403-411; Croston, T L et al. Am. J. Physiol. Heart Cir. Physiol. (2014) 307: H54-H651 as well as in rodent models [Id., citing Marciniak, C. et al. Cardiovasc. Diabetol. (2014) 13: 118; Pham, T. et al. Am. J. Physiol. Cell Physiol. (2014) 307: C499-0507; Hicks, S. et al. Am. J. Physiol. Heart Cir. Physiol. (2013) 304: H903-H915; Yan, W. et al. Basic Res. Cardiol (2013) 108: 329; Luo, M. et al. J. Clin. Invest. (2013) 123: 1262-74; Vazquez, E J et al. Cardiovasc. Res. (2015) 107: 453-65]. Alterations in mitochondrial energy metabolism are a common feature of various forms of heart disease [Id., citing Lopaschuk, G D, et al. Physiol Rev. (2010) 90: 207-258; Fillmore, N. et al. Br. J. Pharmacol.]. diabetic cardiomyopathies (ventricular dysfunction in patients with diabetes mellitus) are associated with dysregulated oxidative substrate selection. In humans and rodents, obesity and insulin resistance are associated with increased myocardial fatty acid uptake and fatty acid oxidation [Id., citing Szczepaniak, L S et al. Magn. Reson. Med. (2003) 49: 417-23; Peterson, L. et al. Circulation (2004) 109: 2191-96], with a simultaneous decrease in glucose oxidation [Id., citing Mazumder, P K et al. Diabetes (2004) 53: 2366-74]. As fatty acid oxidation produces less ATP (per mol oxygen consumed), this scenario makes the heart less energy efficient [Id., citing Peterson, L. et al. Circulation (2004) 109: 2191-96]. In addition, increased fatty acid supply to the heart is also associated with oxidative stress [Ansley, D M and Wang, B. J. Pathol. (2013) 229: 232-41] and accumulation of bioactive lipid intermediates that directly interfere with insulin signaling [Id., citing Zhang, L. et al. Cardiovasc. Res. (2011) 89: 148-56], and lead to a greater decline in heart function with age [Id., citing Kuramoto, K. et al. J. Biol. Chem. (2012) 287: 23852-63]. Exosomes containing increased content of mitochondrial lipids, proteins and nucleic acids were found to be released from adipose tissue of obese diabetic and obese non-diabetic rats [Id., citing Lee, J. et al. Protein J. (2015) 34: 220-35], from pulmonary cells after cigarette smoke injury [Id., citing Szczesny, B. et al. Sci. Rep. (2018) 8: 914], and are found in plasma upon infection with the human T-lymphotropic retrovirus type 1 (HTLV-1) [Id., citing Jeannin, P. et al. Sci. Rep. (2018) 8: 5170] and in circulating exosomes of breast cancer patients [Id., citing Kannan, A. e t al. Clin. Cancer Res. (2016) 22: 3348-60] Very little is known about exosomal transfer of mitochondrial cargo in the presence of mitochondrial dysfunction during the development of insulin resistance and T2D.
Neurodegeneration:
[0520] According to some embodiments, a composition of the present disclosure comprising a purified, enriched population of potent exosomes comprising an appropriate therapeutic signature may improve cognitive outcome after repetitive mild traumatic brain injury (rmTBI), may reduce symptoms of neurologic diseases comprising spread of toxic proteins within the nervous system; or may reduce neurological manifestations of a severe viral infection with a neurotropic virus, e.g., SARS-CoV-2.
[0521] miR-124-3p level in microglial exosomes from injured brain has been reported to be significantly altered in the acute, sub-acute, and chronic phases after repetitive mild traumatic brain injury (rmTBI). In in vitro experiments, microglial exosomes with upregulated miR-124-3p alleviated neurodegeneration in repetitive scratch-injured neurons. The effects were exerted by miR-124-3p targeting Rela, an inhibitory transcription factor of ApoE that promotes the β-amyloid proteolytic breakdown, thereby inhibiting β-amyloid abnormalities. In mice with rmTBI, the intravenously injected microglial exosomes were taken up by neurons in injured brain. miR-124-3p in the exosomes also was transferred into hippocampal neurons and alleviated neurodegeneration by targeting the Rela/ApoE signaling pathway. [Ge, X. Mol. Ther. (2020) 28 (20): 503-22].
[0522] It has been suggested that EVs may be involved in the spread of toxic proteins within the nervous system in a number of neurologic diseases, such as Alzheimer's disease, Parkinson's disease, prion diseases, multiple sclerosis, brain tumor, and schizophrenia. [Tsilioni, I. et al. Clinical-Therapeutics (2014) 36 (6): 882-88, citing Vella, L J et al. Eur. Biophys. J. (2008) 37: 323-32; Banigan, M G et al. PLoS One (2013) 8: e48814; Vingtdeux, V. et al. J. Biol. Chem. (2007) 282: 18197-205; Guest, W C et al. J. Toxicol. Environ. Health. A (2011) 74: 1433-59; Saenz-Cuesta, M. et al. Front. Cell Neurosci. (2014) 8: 100; Gonda, D D et al. Neurosurgery (2013) 72: 501-10]. In all of these diseases, exosomes are involved in the spread of “toxic” proteins that are mutated or misfolded and serve as templates for the formation of disease-producing oligomers. [Id., citing Vella, L J et al. Eur. Biophys. J. (2008) 37: 323-32; Fruhbeis, C. et al. Front. Physiol. (2012) 3: 119]. Without being limited by theory, neurons may try to dispose of these proteins by processing them through the endosomal pathway, which leads wither to degradation from lysosomes or to incorporation into MVBs and release into the extracellular space as exosomes.
SARS-CoV-2
[0523] Neurological investigations and isolation of SARS-CoV-2 from cerebrospinal fluid indicate that SARS-CoV-2 is a neurotropic virus and causes multiple neurological manifestations. [Ahmed, M U et al. Frontiers Neurology (2020) 11: 518]. A retrospective case series study involving 214 patients, of which 78 (36.4%) had some neurological symptoms, in Wuhan, China showed that patients with severe COVID-19 develop more neurological symptoms, such as acute cerebrovascular accidents, altered level of consciousness, and skeletal muscle damage compared to those with mild infection. [Id., citing Mao, L. et al. JAMA Neurol. (2020) e201127]. ACE2 receptors are expressed on glial tissues, neurons and brain vasculature, which make them a target for the attack by SARS-CoV-2. [Id., citing Turner, A J et al. Trends Pharmacol. Sci. (2004) 25: 291-4]. The presence of the virus in neuronal and vascular endothelial cells in frontal tissues was detected on an autopsy of a confirmed COVID-19 patient [Id., citing Paniz-Mondolfi, A. et al. J. Med. Virol. (2020) doi: 10.1002/jmv.25915].
[0524] The presence of the virus in general circulation may enable virus entry into cerebral circulation, where sluggish blood movement in microvessels enables interaction of the viral spike protein with ACE2 receptors of capillary endothelium [Id., citing Baig, A M et al. ACS Chem. Neurosci. (2020) 11: 995-8]. This interaction subsequently leads to viral budding from capillary endothelium; resultant damage to the endothelial lining favors viral entry into the brain, where viral interaction with ACE2 receptors expressed over neurons can result in damage to the neurons without substantial inflammation, which has been described [Id., citing Wrapp, D. et al. Science (2020) 367: 1260-3]. Endothelial damage in cerebral capillaries with resulting bleeding alone can have fatal consequences in COVID-19 patients. The avid binding of the virus to the ACE2 receptors also may result in their destruction via unknown mechanisms, leading to hemorrhage in the brain. Since ACE2 is a cardio-cerebral vascular protecting factor, its damage may cause a leak of the virus in the CNS [Id., citing Turner, A J et al. Trends Pharmacol. Sci. (2004) 25: 291-4]. Neurotropic viruses also can reach the CNS by anterograde and retrograde transport with the help of motor proteins kinesins and dynein via sensory and motor nerve endings [Id., citing Swanson, P A 2d and McGavern, DB. Curr. Opin. Virol. (2015) 11: 44-54], especially via afferent nerve endings of the vagus nerve from the lungs [Id., citing Li, Y C et al. J. Med. Virol. (2020) 92: 552-5]. SARS-CoV-2 also can cause gastrointestinal tract infection and spread to the CNS via enteric nerve and sympathetic afferents. [Id, citing Wong, S H et al. J. Gastroenterol. Hepatol. (2020) 35: 744-8]. In addition, cytokine storms characterized by increased levels of inflammatory cytokines and activities of T lymphocytes, macrophages and endothelial cells can also cause neuronal damage; the release of IL-6 can cause vascular leakage and activation of complement and coagulation cascades [Abdullahi, A. et al. Front. Neurology (2020) 11: 687, citing Mehta, P et al. Lancet (2020) 395: 1033-4].
[0525] The spectrum of neurological manifestations of COVID-19 documented by case reports includes encephalitis, anosmia (loss of the sense of smell)/hyposmia (meaning reduced ability to smell), viral meningitis, post-infectious acute disseminated encephalomyelitis; post-infectious brainstem encephalitis, Guillain Barre syndrome, and acute cerebrovascular disease. The prevalence of neurological and musculoskeletal manifestations of COVID-19 was 35% for smell impairment, 33% for taste impairment, 19% for myalgia, 12% for headache, 10% for back pain, 3% for acute cerebrovascular disease, and 2% for impaired consciousness. [Abdullahi, A. et al. Front. Neurology (2020) 11: 687].
Pain Conditions
[0526] According to some embodiments, a composition comprising a purified, enriched population of potent exosomes with a distinct therapeutic signature that addresses specific inflammatory and/or fibrotic pathways and/or cellular senescence may treat pain conditions.
[0527] There is some support in the literature for this use. Overexpression of MiR-21-5p supports a pro-inflammatory phenotype of macrophages attracted on the site of damage, while intrathecal delivery of a miR-21-5p antagomir appears to avoid an extension of inflammatory condition and neuropathic hypersensitivity onset. [D′Agnelli, S. et al. Mol. Pain (2020) 16: PMC7493250, citing Simeoli, R. Nat. Commun. (2017) 8: 1778]. A proteome characterization of exosomes from mouse spared nerve injury (SNI) model suggested the cargo sorting of vesicular proteins as a crucial step in mediating signaling mechanisms underlying neuropathic pain and evidenced unique patterns of proteins. In particular, significant upregulation of complement component 5a (C5a) and Intercellular Adhesion Molecule 1 are detected in exosomes from SNI model compared to sham control [Id., citing Jean-Toussaint, R. et al. J. Proteomics (2020) 211: 103540]. The involvement of exosomes in neuropathic pain is also underlined by many studies on complex regional pain syndrome (CRPS). It is a chronic neuropathic pain disorder, disabling for sensory, motor, and autonomic dysfunctions as well as of allodynia, hyperalgesia, dystonia, and tremors [Id., citing Bruehl, S. BMJ (2015) 351: h2730]. A different miRNA-exosomal profile between responders and non-responders to treatment in CRPS patients has been identified, suggesting a potential tool to prior identify a subgroup of patients with higher possibility to have benefit by that specific treatment, in this case from plasmapheresis [Id., citing Ramanathan, S. et al. J. Transl. Med. (2019) 17: 81] In a mouse model of CRPS, the mechanism of action of macrophage-derived exosomes and their cargo has been investigated. A decrement of thermal hyperalgesia following a single injection of macrophage-derived exosomes has been found, suggesting a potential immunoprotective role. In the same study, serum-derived exosomes from CRPS patients were analyzed and 127 miRNAs were significantly different comparing CRPS exosomes with control-derived exosomes. Among them, three miRNAs (miR-21-3p, miR-146a, and miR-146b), known to be involved in the control of over activation of innate immune response, are over expressed in both murine and human model [Id., citing McDonald, M K et al. Pain (2014) 155: 1527-39].
Solid Organ Transplantation.
[0528] In view of their anti-fibrotic and pro-angiogenic effects, a composition comprising a purified, enriched population of potent exosomes with an appropriate therapeutic signature may improve graft survival by, for example, reducing ischemia reperfusion injury, by improving recovery from acute damage, and by modulating tolerance towards the graft.
[0529] According to some embodiments, a pharmaceutical composition comprising a purified, enriched population of potent exosomes with a distinct therapeutic signature may be helpful for preserving donor organs when administered before transplant in the context of hypothermic or normothermic perfusion machines. According to some embodiments, addition of EVs to the perfusion solution may improve donor organ viability and functionality.
[0530] There is some support for this notion in the literature. For example, perfusion solution containing EVs has shown some success in organ preconditioning. One report demonstrated that EVs released by MSCs, delivered in the perfusate during organ cold perfusion (4 h), preserved and protected kidney function. Histological and genetic analyses on EV-treated kidneys revealed upregulation of enzymes involved in energy metabolism and reduction of global ischemic damage. [Id., citing Gregorini, M. et al. J. Cell Mol. Med. (2017) 21: 3381-93] Another report demonstrated that EVs isolated from the venal perfusate of rats subjected to remote ischemia preconditioning ameliorated renal function when injected into another animal with ischemia-reperfusion injury (IRI). To explore the underlying mechanism, the authors tested in the same IRI model in vivo the effect of EVs released by human proximal tubular cells cultured in hypoxia, supporting the hypothesis that remote ischemia preconditioning activates a repairing program into tubular cells by the release of pro-regenerative EVs [Id., citing Zhang, G. et al. Biomed. Pharmacother. (2017) 90: 473-478]. The use of EVs released by stem cells to improve the viability of pre-transplant livers was recently assessed in a model of ex vivo rat liver NMP. EVs isolated from human liver stem cells (HLSC-EVs) were added to the perfusate 15 min after the initiation of normothermic machine perfusion (NMP) and administered for 4 h within the perfusate. The results showed that HLSC-EVs limited the progression of ischemic injury, with a significant reduction of the levels of aspartate aminotransferase and alanine aminotransferase and a decrease of histological damage compared with results of NMP alone [Id., citing Rigo, F. et al. Transplantation (2018) 102: e205-e210].
[0531] Bone marrow MSC-EVs have been shown to recapitulate the therapeutic effects of the cells against acute GVHD [Id., citing Selmani, Z. et al. Cells (2008) 26: 212-22]. A systemic infusion of MSC-EVs in mice with acute GVHD was associated with the suppression of CD4+ and CD8+ T cells and with the preservation of circulating naive T cells, possibly due to the unique microRNA profiles of MSC-EVs. The analysis on microRNA cargo in MSC-EVs identified that their target genes were involved in regulation of the cell cycle, T-cell receptor signaling, and GVHD
[0532] In testing the effect of EVs in a rat model of IRI after DCD renal transplantation, MSC-EVs derived from Wharton's jelly, intravenously injected after renal transplantation, were shown to mitigate renal damage, and improve survival and function. In particular, MSC-EVs were shown to reduce cell apoptosis and inflammation, to stimulate HGF production, and subsequently to alleviate fibrosis [Id., citing Wu, X. et al. J. Cell Biochem. (2018) 119: 1879-88].
[0533] According to some embodiments, the chronic lung disease is a fibrotic lung disease. 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 age-related chronic lung disease comprises reduced forced vital capacity compared to a normal healthy control. According to some embodiments, the treatment results in stabilization or improvement of forced vital capacity in a subject compared to an untreated control.
Method for Diagnosis and Treatment
[0534] According to another aspect, the present disclosure provides a method for diagnosing a human subject aged over 50 years with an age-related chronic disease characterized by disease related dysfunction and optimally treating the subject, comprising
[0535] (a) diagnosing a stage of the age-related chronic disease by: isolating a population of extracellular vesicles (EVs) from mesenchymal stem cells derived from a biological sample obtained from the subject and from a normal healthy control aged 21-40, inclusive; purifying and enriching a purified enriched population of potent exosomes derived from the mesenchymal stem cells from the subject and the normal healthy control; wherein the purified, enriched exosomes comprise an identity signature comprising expression three or more biomarkers selected from CD9, CD63, CD81, or Tsg101+; measuring a level of expression of each of a plurality of miRNAs in the purified enriched exosomes from the subject and from the normal healthy control; determining that expression of the one or more miRNAs in the purified, enriched population of exosomes from the subject is dysregulated compared to the healthy control; and identifying the subject as one that can benefit therapeutically from being treated for the age-related chronic disease; and
[0536] (b) treating the age-related chronic disease by administering a composition comprising the population of purified enriched potent exosomes comprising an appropriate therapeutic signature derived from the normal healthy subject, wherein the exosomes comprise an identity signature of three or more biomarkers selected from CD9, CD63, CD81, or Tsg101+; the exosomes comprise total protein of about 1 mg; the exosomes comprise total RNA content greater than 20 μg; the exosomes comprise a cargo comprising a therapeutic signature of one or more, two or more, three or more, four or more, or five or more miRNAs selected from miRNA-29a, miRNA-10a, miRNA-34a, miRNA-125, miRNA-181a, miRNA-181c, miRNA-Let-7a, miRNA-Let-7b, miRNA-Let-7d, miRNA-146a, miRNA-145, miRNA-21, miRNA-101, and miRNA-199; and size of the exosomes is 90-110 nm, inclusive; wherein lot to lot and batch to batch variability relative to prior lots of the composition is below a significance level of ≤0.05; and
[0537] (c) managing the age related chronic disease by reducing or slowing the dysfunction.
[0538] According to some embodiments, identity of the MSCs from which the purified, enriched potent exosomes are isolated is confirmed by a signature comprising expression of CD29, CD44, and CD105. According to some embodiments, the cargo comprises a potency signature of expression of one or more, two or more, three or more, four or more, or five or more of angiopoietin 2 (Ang-2), fibroblast growth factor (FGF), hepatic growth factor (HGF), interleukin 8 (IL-8), a tissue inhibitor of metalloproteinases (TIMP), vasculoendothelial growth factor (VEGF), platelet derived growth factor (PDGF), or tumor necrosis factor alpha (TNFα). According to some embodiments, the tissue inhibitor of metalloproteinases is TIMP1, TIMP2, or TIMP1 and TIMP2.
[0539] According to some embodiments, the composition is a pharmaceutical composition comprising a therapeutic amount of the purified enriched population of exosomes and a pharmaceutically acceptable carrier. According to some embodiments, per isolation process, there are at least 1×10.sup.9 EVs comprising the purified enriched population of exosomes. According to some embodiments, a therapeutic amount of exosomes comprises at least 1×10.sup.9 exosomes. According to some embodiments, the administering is by inhalation or by intravenous administration.
[0540] According to some embodiments, the age-related chronic disease if left untreated comprises one or more of a progressive injury, progressive inflammation, progressive fibrosis or a combination thereof.
[0541] According to some embodiments, the therapeutic signature of the exosome cargo may modulate one or more of an injury, inflammation, an excess accumulation of extracellular matrix, cell senescence; or a pathway comprising fibrogenic signaling; or may reprograms a tissue affected by the age-related chronic disease. 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. According to some embodiments, the tissue is lung tissue, cardiac tissue, renal tissue, hepatic tissue, skin, pancreatic tissue, eye tissue, joint tissue, bone marrow, brain tissue, intestinal tissue, peritoneal tissue, retroperitoneal tissue, nerve tissue, spinal tissue, or skeletal muscle.
[0542] According to some embodiments the age-related chronic disease is a progressive chronic lung disease, chronic inflammation and immune dysfunction, mitochondrial dysfunction, organ transplantation dysfunction; fibrotic disposition of a donor organ, rejection of a donor organ; graft failure; ex vivo lung perfusion dysfunction, musculoskeletal disorders, neurodegeneration, gut dysbiosis or microbiome dysfunction, or age-related decline. According to some embodiments the progressive chronic lung disease is a fibrotic lung disease. 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 age-related progressive chronic lung disease comprises reduced forced vital capacity compared to a normal healthy control. According to some embodiments the treating of the progressive chronic lung disease is effective to stabilize or improve forced vital capacity in the subject compared to an untreated control.
Microbiome Dysfunction
[0543] According to some embodiments, a composition of the present disclosure comprising a population of purified enriched potent exosomes with an appropriate therapeutic signature of MSC-derived EVs isolated from a normal healthy subject may modulate microbiome dysfunction.
[0544] The human microbiota consists of 10-100 trillion symbiotic microbial cells harbored by each person, primarily bacteria in the gut, while the human microbiome consists of the genes these cells harbor. [Ursell, L K et al., “Defining the human microbiome.” Nutr. Rev. (2012) 70 (Suppl. 1) S38-S44).] These microbial cells, and their genetic material, live with humans from birth, and every individual has a unique mix of species. This relationship is important for nutrition, immunity and effects on the brain and behavior, and has been implicated in a number of diseases where the disease is caused by a disturbance in the normal balance of microbes or where the disturbance is another downstream consequence of the disease. The interaction between the human microbiota and the environment is dynamic, meaning that microbial communities are constantly being transferred between surfaces, and that a dynamic interaction exists between environmental microbiota and different human body sites. There is increasing evidence that individuals actually share a core microbiota, with vastly different sets of microbial species yielded very similar functional molecular interactions, reactions and relation networks for metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems referred to as KEGG pathways.
[0545] There is accumulating evidence that a cross-talk between the gut and lungs exists. The lungs of healthy individuals harbor Fusobacterium, Haemophilus, Prevotella, Streptococcus, and Veillonella as main genera, which are relatively small in size when compared to the enteric microbiota [Ahlawat, S. et al. Virus Res. (2020) 286: 198103, citing He, et al., Y. et al. Crit. Rev. Microbiol. (2017) 43 (1): 81-95]. The emergence and maintenance of lung microbiota is governed by the equilibrium between microbial migration from the upper respiratory tract and microbial removal by the host defense systems, with small contribution from the multiplication of native microbes. Even in small concentrations, the airway microbiome is believed to be crucial to host immunity, such that an imbalance between microbial immigration and removal predisposes its host to the progression and exacerbations of respiratory diseases. [Id., citing He, et al., Y. et al. Crit. Rev. Microbiol. (2017) 43 (1): 81-95; Wypych, T P et al. Nat. Immunol. (2019) 20 (10): 1279-90]. Alterations in the lung microbial community including airways also affects the composition of the intestinal microbiota, and respiratory viral infections can alter the intestinal microbiome, where the intestinal microbiome determines the adaptive immune responses against the respiratory pathogens and is necessary for priming the innate immune responses against the pulmonary infection. Indeed, during respiratory viral infections, the level of macrophage response to the respiratory viruses depends on the presence of intestinal microbes. [Id., citing Hanada, S. et al. Front. Immunol. (2018) 9: 2640].
[0546] Gut microbiota composition and diversity is affected by many factors, including by aging. An apparent age-related gut microbiota imbalance has been described, featuring an altered microbial diversity, a lower abundance of probiotic strains (e.g., Difidobacteria), and a reduced number of species producing butyrate, a short chain fatty acid (SCFA) that plays important metabolic functions and has a major role in maintaining the integrity of intestinal epithelium. [Mangiola, F. et al. Eur. Rev. Med. Pharmacol. Sci. 2018] 22: 7404-13].
[0547] The integrity of the commensal microbiota can be disturbed by invading viruses, which cause dysbiosis (meaning a general imbalance of gut microbiota) in the host and further influence virus infectivity. [Li, N. et al. Frontiers Immunol. (2019) 10: 1551]. It has been shown that RNA viruses, such as poliovirus, benefit from the delivery of various viral genomes into a single target cell, thereby allowing the recombination of multiple virus genomes; this process potentiates those viral progeny with enhanced environmental fitness [Id., citing Aguilera, E R et al. M. Bio. (2017) 8: 16; Chen, Y H et al. Cell (2015) 160: 619-30; Combe, M. et al. Cell Host Microbe (2015) 18: 424-32]. Some viruses (e.g., poliovirus, reovirus) use commensal microbiota components to enhance viral stability and increase infectivity. There is also likely a link between commensal microbiota and the lytic reactivation of viruses. In addition to fostering the generation of immunoregulatory Treg cells and thereby suppress anti-viral immunity, the commensal microbiota also have been reported to directly skew antiviral immunity by suppressing the activation of effector immune cells and by inhibiting the production of various inflammatory cytokines that are pivotal for virus elimination, thus creating a more favorable environment for viral infection [Id., citing Baldridge, M T et al. Science (2015) 347: 266-9].
[0548] The commensal microbiota also can prevent viral infection. For example, Enterococcus faecium can prevent infection by influenza viruses upon direct absorptive trapping of these viruses. [Id., citing Wang, X Y et al. PLoS One (2013) 8: e53043]. An extracellular matrix binding protein produced by Staphylococcus epidermidis, a Gram-positive bacterium that lives in the human nasal cavity as a commensal, can stably bind to influenza virus and thus block further infection [Id., citing Chen, H W et al. Sci. Rep. (2016) 6: 27870]. The replication of herpes simplex virus-2 (HSV-2) can also be suppressed by commensal microbiota metabolites; lactic acid, a major end product of the carbohydrate fermentation of all Lactobacillus species, can strongly inactivate HSV-2 in the vaginal mucosa by maintaining an acidic pH in the local environment. [Id., citing Tuyama, C G e t al. J. Infect. Dis. (2006) 194: 795-803]. Commensal microbiota also exert their antiviral activity through cell wall associated bacterial components [Id., citing Mastromarino, P. et al. Anaerobe (2011) 17: 334-6].
[0549] Supporting studies have shown that intact healthy commensal microbiota help maintain robust antiviral immunity, while microbiota disruption increases viral infectivity due to the impaired capacity of the immune system to limit viral infection. For example Clostridium orbiscindens, a human-associated gut microbe produces desaminotyrosine to prime the amplification loop of type I IFN signaling, thereby mediating protection against influenza infection. [Id, citing Steed, A L et al. Science (2017) 357: 498-502]. It has been shown that during respiratory influenza virus infection, antibiotic exposure led to a defective generation of virus-specific CD4 and CD8 T cells and antibodies due to an impaired inflammasome-dependent migration of APCs from the lung to the draining lymph nodes. [Id., citing Ichinohe, T. et al. Proc. Natl Acad. Sci. USA (2011) 108: 5354-9].
[0550] Studies also suggest an important role of virus infection in inducing microbiota dysbiosis. This is true for HIV/SIV infection, influenza virus infection, HBV or hepatitis C virus infection (HCV) and norovirus infection. For example, microbial diversity in saliva of HIV patients was significantly reduced compared to healthy controls, accompanied by increased abundance of potentially pathogenic Megasphaera, Camplyobacter, Veillonella and Prevotella species, and decreased commensal Veillonella and Streptococcus species [Id., citing Li, Y. et al. J. Clin. Microbiol. (2014) 52: 1400-11; Dang, A T et al. BMC Microbiol. (2012) 12 (152): 95, 96]. In addition, fungal communities in HIV infected and uninfected individuals differed significantly. [Id., citing Mukherjee, P K et al. PLoS Pathog. (2014) 10: e1003996]. In bronchoalveolar lavage fluids, although there were no significant differences among the microbial composition in HIV-infected and uninfected subjects, specific metabolic profiles were associated with bacterial organisms that potentially play a role in the pathogenesis of pneumonia in HIV infected patients. [Id., citing Cribbs, S K et al. Microbiome (2016) 4: 3]. Mechanistically, specific immune suppression by HIV is partly responsible for the enrichment of certain potentially pathogenic bacteria.
[0551] Several studies have shown that influenza virus infection can result in decreased colonization by healthy bacteria and increased abundance of potentially pathogenic microbiota. There is a consensus that influenza virus infection alters the commensal microbiota of the host, causing corresponding disruptions of the microbiota-host homeostasis, which largely accounts for the mechanisms by which infections are established. Several lines of data have highlighted that the modulation of immune responses by influenza contributes to the dysbiosis of the gut. [Id., citing Wang, J. et al. J. Exp. Med. (2014) 211: 2397-410; Deriu, E. et al. PLoS Pathog. (2016) 12: e1005572].
[0552] Although SARS-CoV-2 primarily causes lung infection through binding of ACE2 receptors present on alveolar epithelial cells, SARS-CoV-2 RNA has been found in the feces of infected patients. It is known that intestinal epithelial cells, particularly the enterocytes of the small intestine, also express ACE2 receptors. [Dhar, D. and Mohanty, A. Virus Res. (2020) 198018]. Poor outcomes of COVID-19 infection have been observed in elderly patients, particularly those with pre-existing cardiovascular, metabolic and renal disorders, in whom the disease severity and mortality rate are considerably higher. [Viana, S D et al. Ageing Research Reviews (2020) 62: 101123, citing Du, R H et al. Eur. Respir. J. (2020) 55; Li, B et al Clin. Res. Cardio. (2020) 109: 531-38; Mehra, M R et al N. Eng. J. Med. (2020) 382 (26): e102; Roncon, L. et al J. Clin. Virol. (2020) 127: 104354; Shi, Q. et al. Diabetes Care (2020) 43 (70: 1382-91; Siordia, Jr., JA. J. Clin. Virol. (2020) 127: 104357; Team, CC-R. MMWR Morb. Mortal. Wkly Rep. (2020) 69: 343-46; Wang, X. et al. Research (Wash D.C.) (2020) 2402961; Zhou, F. et al. Lancet (2020) 395: 1054-62].
[0553] ACE2 is the receptor for SARS-CoV2-entry in human cells. [Id., citing Hoffmann, M. et al. Cell (2020) 181: 271-80]. There is a constitutive ACE2 expression within the luminal surface of differentiated small intestinal epithelial cells and colonic crypt cells. [Id, citing Harmer, D. et al. FEBS Lett. (2002) 532: 107-10], where ACE2 functions as the chaperone for membrane trafficking of the amino acid transporter B° AT1, which mediates the uptake of neutral amino acids into intestinal cells in a sodium dependent manner, even in the absence of collectrin [Id., citing Camargo, S M et al. Gastroenterology (2009) 136: 872-82; Kowalczuk, S. et al. FASEB J. (2008) 22: 2880-87]. ACE2 shares around 50% homology with collectrin, a type I transmembrane protein that regulates the transporter of neutral amino acids in the kidney (Id., citing Zhang, H. et al. J. Biol. Chem. (2001) 276: 17132-9). It has been suggested that a B° AT1/ACE2 complex in the intestinal epithelium regulates gut microbiota composition and function, with important repercussions on local and systemic immune responses against pathogenic agents, e.g., viruses. A dysbiotic condition (decreased microbial diversity and richness, and impaired Firmicutes to Bacteroides ratio) has been reported in the elderly and in age-related cardiovascular, metabolic, renal and pulmonary conditions. In an ACE2/γ-Akita mouse model, gut barrier disruption and exacerbated diabetes-induced dysbiosis have been described. [Id., citing Duan, Y. et al. Cir. Res. (2019) 125: 969-88]. Some patients with COVID-19 have presented with intestinal microbial dysbiosis. It therefore has been hypothesized that pre-existing disorders displaying an altered gut microbiome may worsen SARS-CoV-2 infection due to a loss of ACE2 integrity/functionality in the gut.
[0554] In a pilot study of 15 patients with COVID-19, persistent alterations in the fecal microbiome during the time of hospitalization were found compared with controls. Fecal microbiota alterations were associated with fecal levels of levels of SARS-CoV-2 and COVID-19 severity. Over the course of hospitalization, bacterial species which downregulate expression of ACE2 in murine gut correlated inversely with SARS-CoV-2 load in fecal samples. [Zuo, T. et al. Gastroenterology (2020) 159: 944-5].
Mitochondrial Dysfunction
[0555] According to some embodiments, a composition of the present disclosure comprising an appropriate therapeutic signature of a population of purified enriched potent exosomes isolated from a normal healthy subject may modulate mitochondrial dysfunction. Mitochondrial dysfunction has been defined as changes in gene expression of mitochondrial markers [Montgomery, M K. Biology (Basel) (2019) 8 (2): 33, citing 18,19], protein content, or enzymatic activities of mitochondrial proteins [Id., citing Heilbronn, L K et al. J. Clin. Endocrinol. Metab. (2007) 92: 1467-73; Ritov, V B, Diabetes (2005) 54: 8-14], changes in mitochondrial size and shape [Id., citing Rritov, V B et al. Diabetes (2005) 54: 8-14; Kelly, D E et al. Diabetes (2002) 51: 2944-2950], as well as functional assessment of mitochondrial oxidative capacity [Id., citing Kelley, D E et al. Am. J. Physiol. (1999) 277: E1130-41] and ROS generation [Id., citing Anderson, E J et al. J. Clin. Invest. (2009) 119: 573-81]. Mitochondrial defects are present in the heart in individuals with insulin resistance and diabetes [Id., citing Montaigne, D. et al. Circulation (2014) 130: 554-64; Dhalla, N S et al. Heart Fail. Rev. (2014) 19: 87-99; Mackenzie, R M et al. Clin. Sci. (2013) 124: 403-411; Croston, T L et al. Am. J. Physiol. Heart Cir. Physiol. (2014) 307: H54-H65] as well as in rodent models [Id., citing Marciniak, C. et al. Cardiovasc. Diabetol. (2014) 13: 118; Pham, T. et al. Am. J. Physiol. Cell Physiol. (2014) 307: C499-0507; Hicks, S. et al. Am. J. Physiol. Heart Cir. Physiol. (2013) 304: H903-H915; Yan, W. et al. Basic Res. Cardiol. (2013) 108: 329; Luo, M. et al. J. Clin. Invest. (2013) 123: 1262-74; Vazquez, E J et al. Cardiovasc. Res. (2015) 107: 453-65]. Alterations in mitochondrial energy metabolism are a common feature of various forms of heart disease [Id., citing Lopaschuk, G D, et al. Physiol Rev. (2010) 90: 207-258; Fillmore, N. et al. Br. J. Pharmacol.]. diabetic cardiomyopathies (ventricular dysfunction in patients with diabetes mellitus) are associated with dysregulated oxidative substrate selection. In humans and rodents, obesity and insulin resistance are associated with increased myocardial fatty acid uptake and fatty acid oxidation [Id., citing Szczepaniak, L S et al. Magn. Reson. Med. (2003) 49: 417-23; Peterson, L. et al. Circulation (2004) 109: 2191-96], with a simultaneous decrease in glucose oxidation [Id., citing Mazumder, P K et al. Diabetes (2004) 53: 2366-74]. As fatty acid oxidation produces less ATP (per mol oxygen consumed), this scenario makes the heart less energy efficient [Id., citing Peterson, L. et al. Circulation (2004) 109: 2191-96]. In addition, increased fatty acid supply to the heart is also associated with oxidative stress [Ansley, D M and Wang, B. J. Pathol. (2013) 229: 232-41] and accumulation of bioactive lipid intermediates that directly interfere with insulin signaling [Id., citing Zhang, L. et al. Cardiovasc. Res. (2011) 89: 148-56], and lead to a greater decline in heart function with age [Id., citing Kuramoto, K. et al. J. Biol. Chem. (2012) 287: 23852-63]. Exosomes containing increased content of mitochondrial lipids, proteins and nucleic acids were found to be released from adipose tissue of obese diabetic and obese non-diabetic rats [Id., citing Lee, J. et al. Protein J. (2015) 34: 220-35], from pulmonary cells after cigarette smoke injury [Id., citing Szczesny, B. et al. Sci. Rep. (2018) 8: 914], and are found in plasma upon infection with the human T-lymphotropic retrovirus type 1 (HTLV-1) [Id., citing Jeannin, P. et al. Sci. Rep. (2018) 8: 5170] and in circulating exosomes of breast cancer patients [Id., citing Kannan, A. e t al. Clin. Cancer Res. (2016) 22: 3348-60] Very little is known about exosomal transfer of mitochondrial cargo in the presence of mitochondrial dysfunction during the development of insulin resistance and type 2 diabetes (T2D).
Neurodegeneration:
[0556] According to some embodiments, a composition of the present disclosure comprising a population of purified enriched potent exosomes with an appropriate therapeutic signature may improve cognitive outcome after repetitive mild traumatic brain injury (rmTBI), in neurologic diseases comprising spread of toxic proteins within the nervous system; or neurological manifestations of a severe viral infection with a neurotropic virus, e.g., SARS-CoV-2.
[0557] miR-124-3p level in microglial exosomes from injured brain has been reported to be significantly altered in the acute, sub-acute, and chronic phases after repetitive mild traumatic brain injury (rmTBI). In in vitro experiments, microglial exosomes with upregulated miR-124-3p alleviated neurodegeneration in repetitive scratch-injured neurons. The effects were exerted by miR-124-3p targeting Rela, an inhibitory transcription factor of ApoE that promotes the β-amyloid proteolytic breakdown, thereby inhibiting β-amyloid abnormalities. In mice with rmTBI, the intravenously injected microglial exosomes were taken up by neurons in injured brain. miR-124-3p in the exosomes also was transferred into hippocampal neurons and alleviated neurodegeneration by targeting the Rela/ApoE signaling pathway. [Ge, X. Mol. Ther. (2020) 28 (20): 503-22].
[0558] It has been suggested that EVs may be involved in the spread of toxic proteins within the nervous system in a number of neurologic diseases, such as Alzheimer's disease, Parkinson's disease, prion diseases, multiple sclerosis, brain tumor, and schizophrenia. [Tsilioni, I. et al. Clinical-Therapeutics (2014) 36 (6): 882-88, citing Vella, L J et al. Eur. Biophys. J. (2008) 37: 323-32; Banigan, M G et al. PLoS One (2013) 8: e48814; Vingtdeux, V. et al. J. Biol. Chem. (2007) 282: 18197-205; Guest, W C et al. J. Toxicol. Environ. Health. A (2011) 74: 1433-59; Saenz-Cuesta, M. et al. Front. Cell Neurosci. (2014) 8: 100; Gonda, D D et al. Neurosurgery (2013) 72: 501-10]. In all of these diseases, exosomes are involved in the spread of “toxic” proteins that are mutated or misfolded and serve as templates for the formation of disease-producing oligomers. [Id., citing Vella, L J et al. Eur. Biophys. J. (2008) 37: 323-32; Fruhbeis, C. et al. Front. Physiol. (2012) 3: 119]. Without being limited by theory, neurons may try to dispose of these proteins by processing them through the endosomal pathway, which leads wither to degradation from lysosomes or to incorporation into MVBs and release into the extracellular space as exosomes.
SARS-CoV-2
[0559] According to some embodiments, a composition according to the present disclosure comprising a population of purified enriched potent exosomes with a distinct therapeutic signature may address neurological manifestations of infection by SARS-CoV-2.
[0560] Neurological investigations and isolation of SARS-CoV-2 from cerebrospinal fluid indicate that SARS-CoV-2 is a neurotropic virus and causes multiple neurological manifestations. [Ahmed, M U et al. Frontiers Neurology (2020) 11: 518]. A retrospective case series study involving 214 patients, of which 78 (36.4%) had some neurological symptoms, in Wuhan, China showed that patients with severe COVID-19 develop more neurological symptoms, such as acute cerebrovascular accidents, altered level of consciousness, and skeletal muscle damage compared to those with mild infection. [Id., citing Mao, L. et al. JAMA Neurol. (2020) e201127]. ACE2 receptors are expressed on glial tissues, neurons and brain vasculature, which make them a target for the attack by SARS-CoV-2. [Id., citing Turner, A J et al. Trends Pharmacol. Sci. (2004) 25: 291-4]. The presence of the virus in neuronal and vascular endothelial cells in frontal tissues was detected on an autopsy of a confirmed COVID-19 patient [Id., citing Paniz-Mondolfi, A. et al. J. Med. Virol. (2020) doi: 10.1002/jmv.25915].
[0561] The presence of the virus in general circulation may enable virus entry into cerebral circulation, where sluggish blood movement in microvessels enables interaction of the viral spike protein with ACE2 receptors of capillary endothelium [Id., citing Baig, A M et al. ACS Chem. Neurosci. (2020) 11: 995-8]. This interaction subsequently leads to viral budding from capillary endothelium; resultant damage to the endothelial lining favors viral entry into the brain, where viral interaction with ACE2 receptors expressed over neurons can result in damage to the neurons without substantial inflammation, which has been described [Id., citing Wrapp, D. et al. Science (2020) 367: 1260-3]. Endothelial damage in cerebral capillaries with resulting bleeding alone can have fatal consequences in COVID-19 patients. The avid binding of the virus to the ACE2 receptors also may result in their destruction via unknown mechanisms, leading to hemorrhage in the brain. Since ACE2 is a cardio-cerebral vascular protecting factor, its damage may cause a leak of the virus in the CNS [Id., citing Turner, A J et al. Trends Pharmacol. Sci. (2004) 25: 291-4]. Neurotropic viruses also can reach the CNS by anterograde and retrograde transport with the help of motor proteins kinesins and dynein via sensory and motor nerve endings [Id., citing Swanson, P A 2d and McGavern, DB. Curr. Opin. Virol. (2015) 11: 44-54], especially via afferent nerve endings of the vagus nerve from the lungs [Id., citing Li, Y C et al. J. Med. Virol. (2020) 92: 552-5]. SARS-CoV-2 also can cause gastrointestinal tract infection and spread to the CNS via enteric nerve and sympathetic afferents. [Id, citing Wong, S H et al. J. Gastroenterol. Hepatol. (2020) 35: 744-8]. In addition, cytokine storms characterized by increased levels of inflammatory cytokines and activities of T lymphocytes, macrophages and endothelial cell can also cause neuronal damage; the release of IL-6 can cause vascular leakage and activation of complement and coagulation cascades [Abdullahi, A. et al. Front. Neurology (2020) 11: 687, citing Mehta, P et al. Lancet (2020) 395: 1033-4].
[0562] The spectrum of neurological manifestations of COVID-19 documented by case reports includes encephalitis, anosmia (loss of the sense of smell)/hyposmia (meaning reduced ability to smell), viral meningitis, post-infectious acute disseminated encephalomyelitis; post-infectious brainstem encephalitis, Guillain Barre syndrome, and acute cerebrovascular disease. The prevalence of neurological and musculoskeletal manifestations of COVID-19 was 35% for smell impairment, 33% for taste impairment, 19% for myalgia, 12% for headache, 10% for back pain, 3% for acute cerebrovascular disease, and 2% for impaired consciousness. [Abdullahi, A. et al. Front. Neurology (2020) 11: 687].
Pain Conditions
[0563] According to some embodiments, a composition of the present disclosure comprising a population of purified enriched potent exosomes with an appropriate therapeutic signature isolated from a normal healthy subject may treat pain conditions.
[0564] Overexpression of MiR-21-5p supports a pro-inflammatory phenotype of macrophages attracted on the site of damage, while intrathecal delivery of a miR-21-5p antagomir appears to avoid an extension of inflammatory condition and neuropathic hypersensitivity onset. [D′Agnelli, S. et al. Mol. Pain (2020) 16: PMC7493250, citing Simeoli, R. Nat. Commun. (2017) 8: 1778]. A proteome characterization of exosomes from mouse spared nerve injury (SNI) model suggested the cargo sorting of vesicular proteins as a crucial step in mediating signaling mechanisms underlying neuropathic pain and evidenced unique patterns of proteins. In particular, significant upregulation of complement component 5a (C5a) and Intercellular Adhesion Molecule 1 are detected in exosomes from SNI model compared to sham control [Id., citing Jean-Toussaint, R. et al. J. Proteomics (2020) 211: 103540]. The involvement of exosomes in neuropathic pain is also underlined by many studies on complex regional pain syndrome (CRPS). It is a chronic neuropathic pain disorder, disabling for sensory, motor, and autonomic dysfunctions as well as of allodynia, hyperalgesia, dystonia, and tremors [Id., citing Bruehl, S. BMJ (2015) 351: h2730]. A different miRNA-exosomal profile between responders and non-responders to treatment in CRPS patients has been identified, suggesting a potential tool to prior identify a subgroup of patients with higher possibility to have benefit by that specific treatment, in this case from plasmapheresis [Id., citing Ramanathan, S. et al. J. Transl. Med. (2019) 17: 81] In a mouse model of CRPS, the mechanism of action of macrophage-derived exosomes and their cargo has been investigated. A decrement of thermal hyperalgesia following a single injection of macrophage-derived exosomes has been found, suggesting a potential immunoprotective role. In the same study, serum-derived exosomes from CRPS patients were analyzed and 127 miRNAs were significantly different comparing CRPS exosomes with control-derived exosomes. Among them, three miRNAs (miR-21-3p, miR-146a, and miR-146b), known to be involved in the control of over activation of innate immune response, are over expressed in both murine and human model [Id., citing McDonald, M K et al. Pain (2014) 155: 1527-39].
Solid Organ Transplantation.
[0565] In view of their anti-fibrotic and pro-angiogenic effects, a composition of the present disclosure comprising a population of purified enriched exosomes with an appropriate therapeutic signature isolated from a normal healthy subject may improve graft survival by, for example, reducing ischemia reperfusion injury, improving recovery from acute damage, and modulate tolerance towards the graft.
[0566] According to some embodiments, a composition of the present disclosure comprising a population of purified enriched potent exosomes with an appropriate therapeutic signature derived from EV-derived MSCs of a normal healthy subject may be helpful for preserving donor organs when administered before transplant in the context of hypothermic or normothermic perfusion machines. According to some embodiments, addition of the composition to the perfusion solution may improve donor organ viability and functionality.
[0567] Perfusion solutions containing EVs have shown some success in organ preconditioning. One report demonstrated that EVs released by MSCs, delivered in the perfusate during organ cold perfusion (4 h), preserved and protected kidney function. Histological and genetic analyses on EV-treated kidneys revealed upregulation of enzymes involved in energy metabolism and reduction of global ischemic damage. [Id., citing Gregorini, M. et al. J. Cell Mol. Med. (2017) 21: 3381-93] Another report demonstrated that EVs isolated from the venal perfusate of rats subjected to remote ischemia preconditioning ameliorated renal function when injected into another animal with ischemia-reperfusion injury (IRI). To explore the underlying mechanism, the authors tested in the same IRI model in vivo the effect of EVs released by human proximal tubular cells cultured in hypoxia, supporting the hypothesis that remote ischemia preconditioning activates a repairing program into tubular cells by the release of pro-regenerative EVs [Id., citing Zhang, G. et al. Biomed. Pharmacother. (2017) 90: 473-478]. The use of EVs released by stem cells to improve the viability of pre-transplant livers was recently assessed in a model of ex vivo rat liver NMP. EVs isolated from human liver stem cells (HLSC-EVs) were added to the perfusate 15 min after the initiation of normothermic machine perfusion (NMP) and administered for 4 h within the perfusate. The results showed that HLSC-EVs limited the progression of ischemic injury, with a significant reduction of the levels of aspartate aminotransferase and alanine aminotransferase and a decrease of histological damage compared with results of NMP alone [Id., citing Rigo, F. et al. Transplantation (2018) 102: e205-e210].
[0568] Bone marrow MSC-EVs have been shown to recapitulate the therapeutic effects of the cells against acute GVHD [Id., citing Selmani, Z. et al. Cells (2008) 26: 212-22]. A systemic infusion of MSC-EVs in mice with acute GVHD was associated with the suppression of CD4+ and CD8+ T cells and with the preservation of circulating naive T cells, possibly due to the unique microRNA profiles of MSC-EVs. The analysis on microRNA cargo in MSC-EVs identified that their target genes were involved in regulation of the cell cycle, T-cell receptor signaling, and GVHD
[0569] In testing the effect of EVs in a rat model of IRI after DCD renal transplantation, MSC-EVs derived from Wharton's jelly, intravenously injected after renal transplantation, were shown to mitigate renal damage, and improve survival and function. In particular, MSC-EVs were shown to reduce cell apoptosis and inflammation, to stimulate HGF production, and subsequently to alleviate fibrosis [Id., citing Wu, X. et al. J. Cell Biochem. (2018) 119: 1879-88].
Method for Reprogramming a Donated Organ or Tissue Comprising a Fibrotic Disposition
[0570] According to another aspect, the present disclosure provides a method for reprogramming a donated organ or tissue comprising a fibrotic disposition comprising
[0571] (a) treating the donated organ or tissue ex vivo with a composition of the present disclosure comprising a population of purified enriched potent exosomes derived from extracellular vesicles (EVs) derived from mesenchymal stem cells of a normal healthy subject comprising purified, enriched potent exosomes with an appropriate therapeutic signature, wherein the EVs comprise an identity signature of expression of three or more biomarkers selected from CD9, CD63, CD81, or Tsg101+; the exosomes comprise total protein of about 1 mg; the exosomes comprise total RNA content greater than 20 μg; the exosomes comprise a cargo comprising a therapeutic signature including attributes of age, gender, estrogen receptor function and status, environmental impact/stressors, donor cell or tissue type, health of the donor organ or tissue, genomics of the donor cell or tissue, size of the exosomes is 50-130 nm, inclusive; wherein lot to lot and batch to batch variability relative to prior lots of the composition is below a significance level of ≤0.05; and
[0572] (b) reducing or slowing dysfunction of the organ or tissue.
[0573] According to some embodiments, the purified, enriched population of potent exosomes derived from extracellular vesicles derived mesenchymal stem cells (MSCs) of a normal healthy subject is derived from a tissue or a body fluid of a human subject. According to some embodiments, the tissue is placental tissue, adipose tissue, umbilical cord tissue, lung tissue, heart tissue, or dental pulp; or wherein the tissue is bone marrow of normal healthy subjects aged 21-40 years old; or the body fluid is blood, amniotic fluid or urine. According to some embodiments, the MSCs derived from placental tissue are derived from one or more of chorionic membrane (CM), chorionic trophoblast without villi (CT-V), chorionic villi (CV), or decidua (DC). According to some embodiments, the blood is umbilical cord blood or peripheral blood.
[0574] According to some embodiments, identity of the MSCs is confirmed by a marker signature comprising CD29, CD44, and CD105. According to some embodiments, the exosome cargo comprises a potency signature comprising expression of one or more, two or more, three or more, four or more, or five or more of angiopoietin 2 (Ang-2), fibroblast growth factor (FGF), hepatic growth factor (HGF), interleukin 8 (IL-8), a tissue inhibitor of metalloproteinases (TIMP), vasculoendothelial growth factor (VEGF), platelet derived growth factor (PDGF), or tumor necrosis factor alpha (TNFα). According to some embodiments, the tissue inhibitor of metalloproteinases is TIMP1, TIMP2, or TIMP1 and TIMP2.
[0575] According to some embodiments, the composition is a pharmaceutical composition comprising a therapeutic amount of the composition and a pharmaceutically acceptable carrier. According to some embodiments, the composition comprises at least 1×10.sup.12 EVs comprising exosomes per isolation. According to some embodiments, a therapeutic amount of exosomes comprises at least 1×10.sup.9 exosomes.
[0576] According to some embodiments, the therapeutic signature comprises one or more two or more, three or more, four or more, or five or more miRNAs selected from miR-29a, miR-10a, miR-34a, miR-125, miR-181a, miR-181c, miR-Let-7a, miR-Let-7b, miR-Let7d, miR-146a, miR-145, miR-21, miR-101, and miR-199.
[0577] According to some embodiments, the organ or tissue if left untreated comprises one or more of a progressive injury, progressive inflammation, progressive fibrosis or a combination thereof. According to some embodiments, the exosome cargo comprising the therapeutic signature modulates one or more of the injury, the inflammation, an excess accumulation of extracellular matrix, cell senescence; or modulates a pathway comprising fibrogenic signaling; or reprograms a tissue affected by the age-related chronic disease. 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, or a Notch signaling pathway According to some embodiments, the tissue is lung tissue, cardiac tissue, renal tissue, hepatic tissue, skin, pancreatic tissue, eye tissue, joint tissue, bone marrow, brain tissue, intestinal tissue, peritoneal tissue, retroperitoneal tissue, nerve tissue, spinal tissue, or skeletal muscle tissue.
Formulations
[0578] According to some embodiments, the composition is a pharmaceutical composition comprising a therapeutic amount of the purified enriched exosomes comprising a cargo comprising a therapeutic signature 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.
[0579] 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.
[0580] 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.
[0581] 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.
[0582] 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).
[0583] 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.
[0584] 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.
[0585] The pharmaceutical compositions of the described invention 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.
[0586] 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.
[0587] 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.
[0588] 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.
[0589] For buccal administration, the compositions can take the form of, e.g., tablets or lozenges formulated in a conventional manner.
[0590] 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.
[0591] In addition to the formulations described previously, the compositions of 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.
[0592] 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.
[0593] Pharmaceutical compositions comprising any one or plurality of the active agents disclosed herein 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.
[0594] 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.
[0595] 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.
[0596] According to some embodiments, the pharmaceutical compositions of the claimed invention comprises one or more therapeutic agent other than the EVs as described. Examples of such additional active therapeutic agents include one or more immunomodulators, analgesics, anti-inflammatory agents, anti-fibrotic agents, proton pump inhibitors, or oxygen therapy.
[0597] Examples of immunomodulators include corticosteroids, for example, prednisone, azathioprine, mycophenolate, mycophenolate mofetil, colchicine, and interferon-gamma 1b.
[0598] Examples of analgesics include capsaisin, codeine, hydrocodone, lidocaine, oxycodone, methadone, resiniferatoxin, hydromorphone, morphine, and fentanyl.
[0599] 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.
[0600] Examples of anti-fibrotic agents are nintedanib and pirfenidone.
[0601] Examples of proton pump inhibitors are omeprazole, lansoprazole, dexlansoprazole, esomeprazole, pantoprazole, rabeprazole, and ilaprazole.
[0602] 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.
[0603] 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.
[0604] 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.
[0605] 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.
[0606] 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.
[0607] 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
Example 1
[0608] Age-related lung disease and inflammation. Age-related changes in the lung tissue result in impaired cellular protective mechanisms that predispose to chronic respiratory diseases (3). Age-related changes in innate and adaptive immune responses may further predispose aging lungs to development and progression of chronic lung disease (7). As the percentage of persons over 65 years old increases worldwide (5.87% in 1985, 7.28% in 2005, 8.28% in 2015, and 16.5% in 2019) (https://www.statista.com/statistics/457822/share-of-old-age-population-in-the-total-us-population/), so does the prevalence of lung disease: whereas globally COPD is reported in 200 per 10,000 patients under the age of 45, there are 1,200 cases reported per 10,000 patients over the age of 65. The predominance of cases in the older age group is also reported in patients with IPF. An estimated 200,000 Americans currently suffer from age-associated fibrotic lung disease (8-10). Approximately 50,000 adults aged 60 and over are diagnosed annually with IPF. Overall, there is an almost five-fold increase in incidence of IPF and COPD based solely on age (11).
[0609] Inflammation, induced by cigarette smoke, is a common risk factor for patients with IPF and COPD (12). Decreased cellular repair, increased production of pro-inflammatory cytokines, fibroblasts that acquire a senescent phenotype with decreased numbers of alveolar epithelial type 2 cells (AEC2) characterize the histology of COPD lung tissue (3). In interstitial lung diseases like IPF, the activity of a fibro-proliferative phenotype in fibroblasts (myofibroblasts) and ongoing epithelial apoptosis are mediated by factors that promote alveolar damage (2-15). Older lungs are characterized by diminished/dysregulated responses of alveolar macrophages to pathogenic stimuli, and their impaired ability to remove damaged neutrophils and resolve inflammation and tissue damage (16-18). Reprogramming the age-related inflammatory phenotype of the lung and other organs with exosome therapy could have major benefits in the aging population.
[0610] Exosomal microRNAs; their age and sex-related dimorphism. Important and incompletely resolved issues center on the age and sex of exosome donors, and it is not clear what characterizes an ideal profile of therapeutically “effective” exosomes. With regard to age, multiple studies, including our published and preliminary data, show that the cumulative effect of age-related cellular damage is responsible for reduced MSC/exosome functionality (39-42). Age-related miRNA profile changes during normal aging (43) could explain reduced functionality of old MSCs/exosomes and most likely compromise their potential therapeutic effects (44). Our proposed comparative studies of adult and old exosomes will be critical in that regard, aiming to identify the “effective” signature of therapeutic exosomes for human age-related lung diseases.
[0611] Similarly, sex-related dimorphic effects on the human MSC exosome secretome are not well studied. Sex-related differences arise at the molecular level due to differential gene expression (45, 46), differential alternative transcripts (47) and/or epigenetic modifications (48). Transcriptional differences are a consequence of the genetic sex and are not strictly dependent on gonadal hormones. A recent meta-analysis of microarrays comparing hMSC of male and female donors highlighted chromosomal segments and genes differently expressed by sex (49). There were twenty genes over or under expressed in male vs female samples that regulated inflammation and cell communication pathways.
[0612] MitomiRs, Immflamma-miRs and Senescence-associated (SA)-miRs Age-related diseases share a common inflammatory phenotype. It remains incompletely understood whether the inflammatory mediators originate from senescent cells with the altered secretome (senescence-associated secretory phenotype, SASP), from increased barrier permeability (e.g. gut or lung) for microbial products that stimulate inflammation, from accumulated proinflammatory immune cells or a combination of these and other sources. Aged cells can further contribute to the inflammatory/senescent phenotype through production and delivery of SA-miRs, mitomiRs, and inflamma-miRs. A signature group of miRNAs including increased miR-146a, and decreased miR-181a, -181c have been suggested to define aging and belong to all three groups of miRNAs (50). We will specifically test whether these or other miRs mark inefficient vs efficient EV therapy. Our preliminary data (
[0613] Therapeutic advantage of exosomes over other aging interventions. As of 2020 about 567 distinct chemical substances are noted to increase lifespan in non-disease models (https://genomics.senescence.info/drugs/). Multiple treatments have been investigated in rodent models with some disparity in results most likely due to strain/genetic differences. Some compounds, such as polyphenols, occur naturally and act as senolytics to modulate oxidative damage, inflammation and cell senescence (51, 52). Two natural metabolites, the NAD precursors nicotinamide riboside (NCT02950441) and nicotinamide mononucleotide (53) are also being investigated. Other drugs are being repurposed to test senotherapeutic ability (54, 55), including the TAME study to test the anti-hyperglycemic agent, metformin, on 65- to 80-year-old individuals without diabetes who are at high risk for the development of chronic diseases of aging (NCT02432287). Clinical trials with rapamycin (Sirolimus), an mTOR inhibitor, are also ongoing, however one completed trial (NCT02874924) showed no impact upon immune, cognitive, and functional consequences in healthy aging subjects compared to placebo controls. Overall, there are few clinical trials using compounds that are able to target multiple indications. To this end, exosome therapy offers a major advantage with powerful miRNA and anti-inflammatory cytokine delivery that have multitargeted potential. Indeed, pre-clinical studies suggest that methods of regulating microRNAs altered in aging and age-related diseases could restore a healthy aging phenotype (12, 56)
Target Product Profile
[0614] MSCs and their exosomes may modify multiple pathways simultaneously, lending support for the proposed use of exosomes as potential therapeutic agents that “seek and repair” multiple targets associated with the age-related disease phenotypes in multiple organs. To date, many potential geroscience therapies fall short in this regard. With a defined therapeutic exosome signature, our pleiotropic therapy will seek to treat age-related chronic lung disease by targeting the associated inflammation and immune aging thereby improving lung function through both repair and potential restoration (and, if successful, in follow-up studies, other tissues). Our preliminary data using in vivo mouse models provides evidence for this approach, as it demonstrates age- and sex-related differences in exosomes' functional impact on lung tissue which we believe correlate or will correlate to their differential miRNA cargos. Pre-clinical studies suggest that methods of regulating microRNAs altered in age-related diseases and senescence could restore a healthy aging phenotype (12, 56). To this end, exosome therapy with a defined pleiotropic signature may offer a major advantage.
[0615] Exosomes have several advantages over MSCs as potential therapeutic agents. Exosomes easily enter the systemic circulation and are encapsulated. This process protects their cargo from unfavorable conditions, such as changes in pH or degradation in vivo. Moreover, whereas infused MSCs quickly diminish post-infusion, it may be that delivery of MSC-derived exosomes can achieve a higher circulating “dose” that lasts longer. Tumor formation risk should be lower or nonexistent with exosomes, which are not self-replicating. They can be stably stored for several months at −20° C. or −80° C., without cryo-preservatives, remaining biologically active. This cell-free “cell therapy 2.0” can help to circumvent complicated handling issues of biological therapeutics containing viable cells.
[0616] To the extent that exosomes derived from MSCs can be used as an instrument for cell-free regenerative medicine, much will depend on the quality, reproducibility, and potency of their production, in the same manner that these parameters dictate the development of cell-based MSC therapies. As detailed in Phases I & II, we will carefully address all these issues in detail, bearing in mind that the MSC exosome's contents are not static, but rather a product of the MSC tissue of origin, its activities, and the immediate intercellular environment of the MSCs. Careful attention to detail in selection of the MSC donor source, as well as the characterization and production of MSC exosomes is therefore needed to provide a new therapeutic paradigm for cell-free MSC-based therapies with decreased risk. Production and standardization issues are as follows:
[0617] A major bottleneck in the advancement of exosome-based therapy into the clinic is the development of high scale, reproducible and efficient production of clinical-grade efficacious exosomes. This requires sterile generation of exosomes with therapeutic payloads, produced in sufficient amounts for clinical testing, without batch-to-batch variation that could compromise efficacy. Development and optimization of production methods, including methods for isolation and storage of exosome formulations, are required for realizing exosome-based therapeutics. In addition, improvement of therapeutic potential and delivery efficiency of exosomes would be highly therapeutically relevant.
[0618] A significant challenge for the use of exosomes for scientific and medical applications is the isolation process, as exosomes can be found in various complex biological matrices that are composed of a plethora of nanosized biomaterials that overlap in size and density. Common isolation techniques for the separation of EVs from other fluid components are ultracentrifugation (UC), density gradients (DG), and size exclusion chromatography (SEC). However, these methods can be limited by the input volume, time consumption, and exosome yield.
[0619] UC protocols are able to process relatively large volumes, but they result in low recovery rates, have time-consuming centrifugation steps, frequently damage the exosome structure, and cause the coprecipitation of contaminants.
[0620] DG and SEC usually result in EV formulations with high purity; however, these protocols are time-consuming, result in poor yields, and require small volumes of starting material for processing.
[0621] Clinical translation of the above-mentioned methods is also challenging due to sterility and scale-up requirements for clinical-grade manufacturing.
[0622] Improved techniques that enable exosome concentration in a scalable, reproducible, and sterile manner, are necessary for future clinical translation.
[0623] Following “the process is the product” principle is useful for translational research during development of complex biological therapeutics in a very early stage of manufacturing and even during early clinical safety and proof-of-concept testing. This strategy helps bridge the initial gaps in knowledge regarding the active substance and mechanism or modes of action (MOA) responsible for a certain therapeutic activity until proof of mechanisms and therapeutic activity are identified. Our data using in vivo mouse models indicates age- and sex-related differences in exosomes' functional impact on lung tissue, accompanied by differential miRNA cargos. Developing a miRNA signature for “effective” exosome donors will be undertaken by comparing outcomes/pathways of “adult” compared to “old” exosomes; we will further compare donor cargo with the high vs. low potential therapeutic efficacy using predefined endpoints. Success will be defined by the determination of differentially enriched miRNA cargos in young vs. old and male vs. female human exosomes. Correlative analysis of miRNA cargos with functional efficacy in protecting lung tissues in a murine inflammation model will validate our target product profile. These studies will lead to the preparation of an exosome candidate suitable for entry into focused preclinical drug development with input from the FDA.
[0624] Recognizing the increasing potential for their clinical utilization, the imperative to optimize an exosome isolation method for maximum yield, purity and assay reproducibility has been addressed. We have partnered with Zen-Bio to address the exosome productivity challenges.
[0625] As a result, a consistent and validated manufacturing protocol for the production of MSC exosomes has been established, which utilizes a fed-batch process for large-scale expansion of MSCs and a protocol for exosome production in suspension bioreactors that is scalable to 15 L and yields consistent extracellular vesicles.
[0626] A cornerstone to today's challenges related to therapeutic exosome development has been industrializing exosome platforms to make them more productive.
[0627] cGMP lots of defined signature exosomes prepared with our manufacturing partner will be synthesized and Investigational New Drug (IND)-enabling studies will be conducted in mouse and ex vivo human models to further test the potency of our signature. Purified GMP investigational product, TPP Exosome Therapeutic, will show the typical nanoparticle tracking analysis profile and expression of the main exosome markers (CD9/CD63/CD81/Tsg101). The GMP manufacturing method utilized will guarantee high exosome yield and consistent removal of contaminating proteins. The resulting GMP product, will be tested for safety, purity, identity, and potency in vitro, showing functional immunomodulatory and inflammation resolution activity.
[0628] The standardized production method and testing strategy for large-scale manufacturing of GMP TPP Exosome Therapeutic opens new perspectives for reliable human therapeutic applications including age-related chronic respiratory disease as well as targeted age- and organ-specific inflammation.
[0629] Target product profile—defined exosomal signature. The present disclosure will exploit the age and sex related decline that occurs in MSC/exosome functionality involving the local and systemic environment of aging tissues/cells, mitochondria! dysfunction, and increased oxidative stress to define our optimal expected exosomal signature summarized in Table 2.
TABLE-US-00003 TABLE 2 Expression of exosome derived microRNAs that might be altered during aging. Expected expression of proposed Female Male Female Male “signature” young young aging aging miRNAs exosomes exosomes exosomes exosomes miR-10a (cell ↓ ↓ ↑↑ ↑ senescence MSCs (57, 58)) miR-34a ↓↓ ↓ ↑↑ ↑ (inflammation) miR-125 ↓ ↓ (inflamma-miR (59, 60) miR-146a (immune ↑↑ ↑↑ ↓ ↓ response (57) miR-181a ↓ ↓ (inflamma-miR (61) miR-181c ↓ ↓ (inflamma-miR(62) miR-Let-7b(SA- ↓ ↓ mitomiR)Complex I, IV and V(50, 59) * indicates known to be regulated by change in estrogen status of post-menopausal women.
[0630] The large-scale manufacturing of exosomes will be demonstrated using a GMP-compliant process with well-defined criteria for the release of the product for human use. These criteria include the size distribution of the exosome preparation, ascertained by NanoSight; flow cytometry analyses of defined markers on exosomes; and potency assays. The proposed study will provide the first quantitative POC analysis of safety and efficacy using what appears to be the most therapeutically relevant, most potent and practical MSC-derived product (Exosomes) to have entered the clinic. Success of these studies will provide basis for pivotal trials.
[0631] The bone marrow derived exosome product of interest in this study will be manufactured as follows. The TPP Exosome Therapeutic will be isolated from Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs) of an adult (21-40 year old) healthy donor who will have met a strict donor acceptance criteria including current and past medical history in addition to passing highly sensitive nucleic acid testing for hepatitis B, hepatitis C, and HIV. The exosomes will be tested for sterility and have been manufactured in FDA-registered facilities that meet Current Good Manufacturing Practices (cGMP). (See Phase II below).
Manufacturing and Quality Control
[0632] Donor and Cell Source—Human Mesenchymal Stem Cells used to generate the TPP Exosome Therapeutic will originate from a single donor bone marrow source of adherent stromal progenitor cells that were expanded under GTP/cGMP compliant conditions and controls to generate a Master Cell Bank for Phase II studies. We will file a Master File at the FDA detailing donor selection, testing, qualification, master cell bank production and cell characterization for identity and purity. These cells will be characterized for expansion potential, cell surface marker expression, and differentiation potential into adipocytes, osteocytes, and chondrocytes (63). In addition, functional analyses for angiogenic cytokine secretion and inducible Indoleamine-pyrrole 2,3-dioxygenase (IDO) activity will be performed and data reported
[0633] Current Good Manufacturing Practice (cGMP) compliant processes and testing will be implemented from cell donor selection to final distribution. Details of the Master Cell Bank and growth media will be filed with the FDA. Inspections and testing will be in place throughout manufacturing process. These will include required monitoring of equipment and environment, monitoring of pH, glucose, and lactate content of cell medium, visual monitoring of cell density, nanoparticle tracking analysis to establish final particle count and size distribution, and USP<71> sterility testing of final product. Product tracking controls and cold supply chain monitoring protocols are in place. We will audit key suppliers, manufacturer, and testing organizations to ensure compliance.
[0634] The exosome product will be filtered and purified prior to filling, and all containers will be visually inspected prior to cryopreservation to ensure normal appearance, intact container closure, and the absence of visible extraneous particulate matter. Product will be released for clinical use only if all safety, identity, quality, and purity specifications are met and all applicable GMPs are appropriately followed as determined by Zen-Bio Quality Assurance. These include assurance of a sterile product by in-process and release testing for bacteriology, mycology, mycoplasma, endotoxin, and nonspecific viral adventitious agents. In addition, Quality control tests will be performed for each lot of GMP exosome product. The vesicles will be characterized by their protein concentration, estimated defined RNA signature concentration, particle concentration/and particle size to ensure there is an acceptable concentration, viability, and purity. Stability testing will also be conducted to verify both frozen storage and post-thaw stability. The final cell suspension of TPP Exosome Therapeutic Thawed/Diluted product will be released for administration based on assessment of Exosome dose (0.8-10″), mycoplasma testing (must be negative), Endotoxin (<1.65 EU/ml), FLOW Cytometric assessment of exosome identity surface markers (CD9+/CD63+/CD81+/Tsg 101+), defined mRNA signature and 14-day sterility (must be negative) immediately prior to transfer to the final container (60 ml cryobag). The product will be placed in a validated transport container fitted with the continuous temperature monitoring device.
[0635] Final product: TPP Exosome Therapeutic re-suspended in sterile Saline.
[0636] Exosome target product properties and outcomes are listed in Table 3.
TABLE-US-00004 TABLE 3 Target Product Profile Product Minimum acceptable Ideal Properties result Result Primary Chronic lung disease Age-sex related chronic indication lung diseases Patient Adults with age-related Adults and children with population chronic lung disease lung disease Treatment Chronic Short term leading to duration long term improvement Delivery mode IV IV, Injectable, inhalation Dosage Form Exosomes suspended Suspension or in sterile saline lyophilized Regimen The preclinical studies One or two doses suggest that at least 1 dose will be needed Efficacy Stabilization or Improvement of FVC improvement of FVC to above normal range Risks/Side Bronchitis-Clinically None Effects significant bronchitis was not reported in AETHER trial
[0637] Overall, interventions that can modulate (even partially) several of the aging hallmarks need to be studied to provide new insight into druggable targets against age-related diseases. In that context, exosomes fulfill the criteria as promising candidate therapeutics for chronic age-related diseases. The described plan for manufacturing, characterizing, standardizing, quality-controlling and delivering exosomes is intended to fully and thoroughly assess their anti-geronic therapeutic potential.
[0638] Preliminary data suggest why adult (21-40 years old) donor exosomes are superior to exosomes derived from older (>65 years old, and likely >55 years old, based on our preliminary data) donors and may provide new ways to functionally modulate/reverse adverse effects of aging. There are three novel conceptual aspects of this work:
[0639] (i) We will, for the first time, dissect how sex-related differences in the signature of male vs. female bone-marrow-derived exosomes could lead to enhanced therapeutic options for treatment of age-related diseases.
[0640] (ii) We will test the hypothesis that age-induced miRNA changes in donor exosomes may provide a signature to optimize efficacy of donor exosome selection.
[0641] (iii) We will dissect and identify some of the components lacking in old donor exosomes that lead to loss of their therapeutic effects, paving the way for potential supplementation of such components in future trials.
[0642] Rigor and Reproducibility: Age-related studies will use adult (21-40 years old) donors, compared to older (65 years old and older) donors, unless indicated otherwise. This definition will be used across the proposal. For in vivo experiments, as outlined in Vertebrate Animals, the number of mice in each group will be selected based on power calculations to achieve at least 80% power to detect a 20% difference in outcome at p<0.05. Experiments will be repeated at least twice. All ex vivo (3D lung punches) assays will be performed with a minimum of 3 biological replicates/sex, three technical triplicates, and at least three independent experiments. Data will be analyzed by investigators blinded to the experimental group.
Characterization of Age-and Sex-Related Differences in Exosome Function and Cargo
[0643] Rationale: Our data using in vivo mouse models suggests that important age-and sex-related differences in exosomes has functional impact on lung tissue as well as in their miRNA cargos. Defining an miRNA signature for “effective” exosome donors will be undertaken by comparing outcomes following transfer of adult and older exosomes and correlating these outcomes to donor cargo so as to putatively define exosome preparations with the highest potential therapeutic efficacy.
[0644] Success Metrics: Correlative analysis of miRNA cargos with functional efficacy protecting lung tissues in a murine bleomycin lung injury model. These studies will lead to the preparation of an exosome candidate suitable for entry into focused preclinical drug development with input from the FDA.
miRNA Identification and In Vivo Efficacy Milestones Must be Satisfied Prior to Transitioning to Phase II.
Preliminary Results
[0645] An age-related increase in oxidative stress may erode MSC function (64, 65) potentially reducing their therapeutic efficacy and altering their exosome signature.
[0646]
[0647]
[0648] Old human ASCs displayed increased mitochondrial activity as shown by mito tracker Green and Red (
[0649] Both catalase protein expression (
TABLE-US-00005 TABLE 4 Human (female) SOD2 mRNA Catalase mRNA ASC (age) expression expression 29 yrs #1 6643 3741 29 yrs #2 17460 2332 23 yrs 4528 2035 80 yrs 76 593 66 yrs 6 14 63 yrs 334 427 59 yrs 75 205 58 yrs 363 498
[0650] Reversing the aging-related phenotype in old human ASCs (>58 years old) leads to repair of age-related lung fibrosis and improvement in wound healing: Aberrant antioxidant defense may be important in the pathogenesis of age-related lung diseases including pulmonary fibrosis and COPD (66, 67). Our published and preliminary studies show that adult ASCs prevent or repair fibrotic lung tissue in a model of pulmonary fibrosis; in contrast, old human ASCs were unable to do so (39). We investigated whether the age-related differences in antioxidant mRNA and protein expression observed in a separate batch of ASCs (see Table 4 and
[0651] Antioxidant dysregulation also contributed to decreased wound healing ability in old ASCs, as demonstrated by the fact that the catalase knock-in “normalized” wound healing. Young human skin punch wounds were obtained and 5×10.sup.5 human ASCs were injected radially. After daily media changes on wounds for four days, wounds were embedded and stained with hematoxylin and eosin (
[0652] Taken together, decreasing catalase expression in adult ASCs to comparable levels of antioxidant expression in old ASCs, reduces the effectiveness of the adult ASCs in repair of bleomycin-induced lung injury and ex-vivo wounds.
[0653] The above results support our strategy to understand the phenotype and cargo of old ASCs as the first step in determining a therapeutic signature for their exosomes.
[0654] Cigarette smoke (CS) exposure induces inflammation in multiple organs. CS exposure increases oxidative stress in the lung, kidney and skin of female mice. Cigarette smoke is the most common factor associated with COPD, a disease that is at least six-fold higher in individuals over the ages of >55, and the third leading cause of death in the U.S. (www.lung.org). Our published data showed that the combination of smoking and estrogen deficiency in old female mice contributed to an emphysematous phenotype in the lung, glomerulosclerosis in the kidney and decreased wound healing in the skin (68-70). In the lung we noted that CS induced a decrease in collagen content, macrophage number, and respiratory chain complex-1 protein. Lung sections revealed patchy areas of subpleuretic fibrosis and increased number of inflammatory cells with perivascular edema and additional hyperplasia of columnar epithelium.
[0655] CS also resulted in an increase in matrix metalloproteinase-2 (MMP) activity and total number of apoptotic cells in lung tissue. In the same mouse used to study the lung, CS induced MMP dysregulation of kidney and skin, as well as other dysregulated pathways, suggesting that this model may be useful to test our preferred donor signature across different organs and chronic age-related pathologies. In addition, the CS model may also be useful to determine the importance of our signature for multiple age-related conditions since miRNAs are readily influenced by environmental chemicals found in CS. Thus, the lungs of rodents exposed to CS demonstrate down-regulation of miRNAs (71, 72). Schembri et al. found that most of the differentially expressed miRNAs in the human bronchial airway epithelium were down-regulated in smokers and were inversely correlated with their predicted targets (73). Graff et al. noted that a decrease in global miRNA expression was more pronounced in heavy smokers, suggesting that the magnitude of miRNA repression is related to the extent of smoking history (74). These data raise the possibility that CS may modulate the aging phenotype in part though changes in mesenchymal stem cells/exosomes. We will utilize this model in our studies (Phase II) to obtain a generalizable proof-of principle of anti-geronic therapeutic effect of exosomes.
[0656] Exosomes as an off-the-shelf therapy. It is generally accepted that membrane-bound and soluble factors play a major role in the therapeutic function of MSCs, and that membrane-bound vesicles (exosomes) are the main vectors for MSC paracrine effects. Therefore, in preparation for Phase I and the entire project, we isolated and characterized ASC-derived exosomes (isolation performed by Zen-Bio Inc., our manufacturing partner). Exosome size was approximately 30-150 μm. Transmission electron microscopy (TEM) was performed on isolated exosomes (
[0657] Biodistribution of ASC-derived exosomes: We assessed the time course of biodistribution of young and old human ASC exosomes injected into 16-month-old male C57/BL/6 mice using an in vivo bioluminescent imaging system. Exosomes were located in the lung within 5 minutes post-tail vein injection. Images were taken at 5 min, 30 min, 60 min, 90 min and 2, 4, 6 and 24 hours (
[0658] Exosomes derived from ASCs enter alveolar epithelial cells (AEC). To visualize whether exosomes were present after injection into 3D lung punches, we utilized TEM. TEM revealed exosomes labeled with gold nanoparticles in alveolar (AEC) type I and AEC type II cells (red arrows,
[0659] Similar to our previous studies (39), adult human ASC exosome (35 year-old male) infusion prevented bleomycin-induced fibrosis while old human ASC exosomes (65 year-old male) did not (
[0660] Immunofluorescent staining with SP1, a marker of alveolar type 2 cells, revealed an increase in positive cells (yellow color) compared to plasmalyte control or old exosomes (
[0661] Exosomes isolated from old male human ASCs downregulate microRNAs found at high levels in adult ASCs. We hypothesized that analysis of differentially expressed miRNAs and their targets in human old donor ASCs could elucidate pathways relevant to loss of therapeutic potential. miRNA was extracted from adult and old human ASC exosomes using the ExoQuick and miRNAeasy kits. The results of the human miRNA Exiqon array indicated 25 miRNAs were either up or downregulated with a greater than five-fold difference. Target genes were derived from the miRWalk database (78). We found that senescent (SA) related miR-146a and -181a were upregulated in old exosomes, while another SAmiR-10a, as well as mitomiRs-125 and let-7b were decreased. These data were confirmed by qPCR and suggest that aging exosomes may carry cargo mirroring or promoting the aging phenotype. Based on these data we expect that adult exosome cargo potentially contribute to the repair process in age-related diseases.
[0662] Is exosome cargo different in males and females? The postmenopausal estrogen deficient state sustains the development of inflamma-aging and regulates multiple pathways differently than in aging males (79). 118 miRNAs are located on the human X chromosome but only 4 are present on the Y chromosome. In addition, 15% of the miRNA encoded by the X chromosome are able to escape X-inactivation (17, 80). These data suggest that the cargo delivered by the exosomes may exhibit sexual dimorphism.
[0663] Experimental design: Based on our preliminary and published data, we will isolate exosomes in collaboration with our partners at Zen-Bio. We will use 5 male and female adult MSCs (21-40 years of age) to isolate exosomes from bone marrow derived MSCs (available from Rooster, AlICells, and Lonza). The old MSCs (preferably >65 years of age, n=3/sex) will be isolated from healthy bone marrow donors (see letter from Dr. Ulrickson). We also wanted to avoid any postmenopausal effects in 45-60 year-old females. The identification of miRNA expression profiles will be performed by next-generation sequencing with sequencing-by-synthesis technology; this will allow a dynamic range of detection (from very low to highly abundant RNAs) and measurement of relatively limited differences in expression between samples. Using RNA derived from human exosomes (200 ng), miRNA-seq libraries will be prepared using the Illumina TruSeq Small RNA Sample Kit and sequenced with Illumina platform MiSeq (MiSeq Reagent Kit v3). Bowtie (version 0.12.5) will be used to perform a stepwise alignment of fastq files to Illumina databases. Differentially expressed miRNAs will be identified using Bioconductor's limma package. Data will also be validated by qPCR experiments. Primary data will be analyzed using bioinformatics pipeline software (NIH) http://genboree.org/theCommons/projects/exrnatools may2014/wiki/Small %2ORNAseq %20Pipeline.
[0664] Once we have compiled a list of validated miRNAs, we will confirm the relevance of this “miR signature set”. We will transfect exosomes with a mimic or inhibitor of relevant miRNAs (81) and inject into-22 month-old C57BL/6 male and female mice (equivalent to -61 to 65-year-old males and females) and determine inflammatory and fibrosis endpoints. We will choose one donor (out of 5) with the desired signature to test in duplicate and compare the outcomes of mice receiving “desired” signature exosomes with outcomes from mice receiving old exosomes. Due to the short timeline of phase I we will only test our research exosomes in the old bleomycin lung injury model (Table 5). This model has a well-established inflammatory phase at day 7 (82) and replicates several of the specific pathogenic molecular changes associated with IPF (76). BLM (2.0 Ukg/BW in 50p1 saline) or 50p1 of sterile saline (controls) will be administered by direct intratracheal instillation via intubation as in our preliminary experiments above. 1010 particles of either male or female exosomes will be injected in the tail vein at day 7 post-BLM, a time that injury has occurred in both young and old mice (39, 82). At sacrifice, (day 21 post-BLM) lungs will be inflated and perfused.
TABLE-US-00006 TABLE 5 Human Male Male Female Female Male Male Female Female exosome + + + + + + + + isolate Exo vehicle (V) Exo V Exo V Exo V Total Mouse BLM BLM BLM BLM Saline Saline Saline Saline treatment 21-40 yr 8 8 8 8 8 8 8 8 64 female exo >50 yr 8 8 8 8 8 8 8 8 64 female exo 21-40 yr 8 8 8 8 8 8 8 8 64 male exo >50 yr 8 8 8 8 8 8 8 8 64 male exo
[0665] Assessments: At sacrifice, lungs will be inflated, perfused, and studied as follows (39): 1) Histology (assessed by a pulmonary pathologist blinded to the groups) and immunohistochemistry of the lung (epithelial lung markers; SPC and AQ5, aSMC-actin and endothelial lung marker) performed; 2) Bio-Plex Pro Mouse Cytokine 23-plex Assay that includes IL-113, IL-6, IL-10 and TNF, as well as anti-inflammatory markers. Using this as a screening tool we will also measure sTNFR1 and 2 and CRP (83), because together with IL-6 these markers provide the most accurate measures of inflammation that correlate to all-cause morbidity and mortality. Sensitivity of IL-6 measurement in a multiplex could also be insufficient and in that case we will use a high-sensitivity IL-6 ELISA kit (LEGEND MAX™ Mouse IL-6 ELISA, BioLegend, Inc. Kit) for all measurements. We will also assess quantitative measures of fibrotic disease (Ashcroft, collagen types I and III expression and hydroxyproline) (39), as well as associated molecular markers and 3) downstream pathways of inflammation and aging (e.g. avintegrin, matrix metalloproteinases (MMP), AKT phosphorylation). We will measure Nrf-2 activation which has been shown to protect against the oxidative stress seen in cigarette smoke-induced emphysema. In addition, pertinent to age-related diseases, the NAD+-dependent deacetylase and anti-inflammatory factor SIRT1 is suppressed in aging (84). SIRT1 deacetylates Foxo3a, which enhances stress resistance through several antioxidants including MnSOD, and catalase (85). Furthermore, experiments in which SIRT1 is either reduced or overexpressed show that it modulates senescence of both BM-derived MSCs and ASCs (86). Therefore we will analyze lungs to assess SIRT1 expression as well as antioxidant expression (Table 6).
[0666] Statistics. All murine studies will employ an n=8 animals per cohort with independent male and female cohorts for all studies, time points and strains. Data will be evaluated by ANOVA for comparisons between two or more groups followed by post-hoc Tukey's multiple comparison tests. Results will be reported as the mean±standard deviation or standard error while significance will be defined by p values <0.05. Power for the primary outcome—reduction of fibrosis—is >80% to detect a 20% or larger difference at p<0.05. Data will be analyzed by investigators blinded to experimental group.
[0667] Success metrics/Alternative outcomes/Potential difficulties: Success metrics and go/no-go decisions are summarized in Table 6.
TABLE-US-00007 TABLE 6 Readouts of lung and milestones Aims Milestones Month Compare 1. Produce exosomes— 1-3 (go/no go) Adult and project is a go if enough Old Male and exosomes produced can treat Female ≈. 100 mice (5 × 10.sup.12) exosomes 2. Exosomes biophysical 2-3 (go/no go) characterization—project is a go if >90% of EVs are 50- 150 um in size and have positive CD63, CD90 3. Exosomes biophysical 4-6 (descriptive characterization—project is milestone, will be ago if >90% of EVs are 50- go/no-go together 150 um in size and have with milestones positive CD63, CD90; # 2, 4 & 5 Test exosomes 4. Therapeutic effects of 5-10 (this milestone, in the old adult and old exosomes from like milestones 3 bleomycin lung male and female donors. To and 5, are all injury model be considered efficacious, playing into a go/ exosome will have to rescue no-go decision to 90% of mice from identify optimal bleomycin-induced death by signature and guide fibrosis, and to reduce selection of donors) fibrosis by 80% as judged by (Ashcroft score, collagen content and related fibrotic inducing pathways) 5. Using siRNA or knock-in 8-12 (data from 3&4 transfections, identify will help guide these exosome signatures and experiments, that will molecules that are themselves be go/no- therapeutic. Criteria from #4 go decision makers) for therapeutic efficacy will apply. project is a go if we have identified at least 3 molecules from adult exosomes that provide superior efficacy to old exosomes if up- or down- regulated, and can be used for exosome donor selection″
[0668] First, because our preliminary data primarily used exosomes from ASCs and we propose to generate exosomes from bone marrow MSCs, we will ascertain that our exosome preparations have reproducible effects, and that their aging also recapitulates our prior results. The production of exosomes from an individual cell line is not static, therefore we will test at least two exosome preparations from the same donor. We expect to find a signature similar to Table 2 as noted in the TPP section above. Exosomes from estrogen replete (adult) females may have a different profile than old female exosomes since normal estrogens/estrogen receptor function protects against excess cellular oxidative stress, regulates SIRT1, mitochondria! function and microRNAs (87, 88). Although the adult female signature may be different than the adult male signature based on our preliminary data, we anticipate that adult (young) exosomes, regardless of sex, will have anti-inflammatory and antifibrotic effects. Our experiments will ascertain whether the extent of these changes are sex-dependent. If not sex dependent, then the number of available donors for MSC/exosome production would increase since historically, most clinical trials have utilized adult male donors predominately. We realize that obtaining bone marrow from healthy donors over 65 may be a challenge therefore we may have to lower our donor age to 55-60 and document the length of time that the female donors have been in menopause (89). We recognize that the proposed “therapeutic” signature may not be exhaustive, but our sequencing and bioinformatics should be informative. In either case, the signature will guide and better define criteria for donor selection.
[0669] We do not expect significant obstacles to our approach to optimize the beneficial exosome signature by exosome transfections using knock-in or knock-down miRNAs. Our manufacturing partner, Zen-Bio, Inc. will take the lead with these experiments since they have extensive experience in this field. It is possible that we may be unable to completely alter the signature, however, as we have shown in our prior publication, inhibiting or activating a single miRNA has a dramatic effect on targeted protein expression and relevant downstream pathways, including an ultimate therapeutic effect (81).
[0670] We do not expect any off-target effects since our biodistribution data suggest that exosomes are quickly cleared in mouse organs roughly 24 hours following administration, except for the liver. However, after 20 day follow-up of the mice, no adverse events were noted.
Example 2. PHASE II AIM 2
[0671] A. Perform cGMP isolation of exosomes and B. conduct IND-enabling studies. We will pursue two complementary sets of approaches utilizing cGMP exosomes: in vivo animal studies and ex vivo human lung punches from male and female donors >65 years old.
[0672] 1. Preliminary Data and Tools for Phase II: CT scans verify lung injury. Baseline chest pCT prior to bleomycin administration demonstrated well-aerated lungs without evidence of pulmonary edema or increased tissue density (
[0673] In preliminary experiments, we have injected adult and old ASC-derived exosomes into lung punches. These punches were obtained from C57BL/6 mice 10 days post-bleomycin treatment, at the peak of inflammation. Adult exosomes were effective in interrupting bleomycin-induced fibrotic pathways while old exosomes were not. Bleomycin-installation increased baseline lung matrix metalloproteinase-2 (MMP-2) activity in the punches (
[0674] 2. Experimental Design and Methods:
[0675] We will produce cGMP lots of defined exosomes prepared with our manufacturing partner and conduct Investigational New Drug (IND)-enabling studies in mouse and ex vivo human models to further test the potency of our signature. To date, a bottleneck in the advancement of exosome therapy into the clinic has been the development of high quality, reproducible, and efficient production of clinical grade exosomes. Our collaborator Zen-Bio, Inc. has now developed such a pipeline, as shown in section 2a.
[0676] a. Manufacturing: Zen-Bio routinely isolates EVs containing exosomes from mesenchymal stem cells using a hollow-fiber cartridge containing bioreactor as previously described (95). The bioreactor is amenable to culturing any cell type, although adherent cells are preferred. To prepare the bioreactor, a total of 1×10.sup.9 cells are seeded onto the cartridge. After seeding, the separate cell cartridge is filled with 40 mL of serum-free medium for cell maintenance and EV production, and growth medium is continually circulated through the bioreactor, with medium exchanges every 3-5 days. Cell metabolism is monitored by measuring the glucose level in the circulating medium; when levels drop 50% the circulating medium is changed. Bioreactor cultures have been maintained in this manner for up to three months while the MSCs retain their International Society for Cellular Therapy (ISCT)-mandated MSC characteristics (63).
[0677] EVs are collected every 3-5 days after the bioreactor culture is established. The conditioned serum-free medium will be removed from the cell cartridge and subjected to sterile filtration through a 0.22 μm filter, followed by tangential flow filtration (TFF) to concentrate the EVs and reduce the overall volume to 1 mL. The concentrated EVs are then subjected to size exclusion chromatography (SEC) over a 70 nm porous resin particle column. Each column is standardized, quality assured and certified to ISO 13485 standard (Medical Devices) making it perfect for clinical testing. Fractions are collected from the column and are analyzed for EVs using Nanoparticle Tracking Analysis (NTA). Peak fractions are identified and pooled for use and further analysis.
[0678] A typical yield from 40 mL of conditioned medium has been 3.6±0.6×10.sup.11 particles, and this yield can generate up to 1×10.sup.13 EVs per cartridge over the life of the bioreactor run.
[0679] b. EV physical characterization and surface marker expression: We will characterize the EV preparations for average particle size and concentration using ZetaView® BASIC NTA—Nanoparticle Tracking Video Microscope PMX-120. Protein concentration, DNA concentration, and RNA concentration will be determined by a Nanodrop ND-1000 spectrophotometer. EVs are then characterized for CD9, 63, and 81 expression to confirm the presence of exosomes. Exosome-specific protein markers will be determined by high throughput flow cytometry analysis using the MACSPIex Exosome Kit and MACSQuant analyzer to show the necessary presence of CD9, CD63 and CD81. Human foreskin fibroblast derived exosomes will be used as a control since fibroblasts and MSCs have a similar morphology and surface marker expression (96).
[0680] Success metrics and alternative approaches for EV production: Success criteria are based on Zen-Bio's historical read-outs of EV properties and results as follows: particle yield should be at least 1×10.sup.9 per isolation; particle diameter should be consistent, to include exosomes (30-150 nm); total protein should be ≈1 mg; total RNA content should be >20 pg; and exosome and specific biomarkers (CD9, 63, and 81) must be present by 90%. Should any of the lots fail to produce exosomes that pass our quality control metrics, the lots will be stored for future analysis but not subjected to cell-based testing. In addition, as outlined in Phase_milestones (Table 6), we will utilize exosome preparations that meet our desired donor signature.
[0681] c. EV Transcriptome/miRNAome: In parallel with EV surface protein expression described above, we will analyze the miRNA using the MacsPlex miRNA Kit (Miltenyi). We will monitor the relative abundance of 39 miRNAs in a high through put manner. This will allow us to determine consistency within EV batches, to assess population composition, and to assess batch-to-batch and lot-to-lot consistency. Each sample is tested in triplicate using independent experiments on different days to ensure data quality. Lot-to-lot and batch-to-batch consistency is determined using ANOVA and/or t-tests depending on the comparison and differences between measures are considered significant with a p value ≤0.05.
[0682] Using this methodology, we have analyzed multiple lots of EVs and detected elevated levels of several miRNAs related to our desired therapeutic properties and exosomal signature.
TABLE-US-00008 SET1000 PRODUCT RELEASE TESTS AND SPECIFICATIONS Test/Reference Specification Control EV Quality 80-100 Billion EVs Particle yield should be at least 1 × 10.sup.9 per isolation EV Size 50-130 nm Mean of 165 ± 50 nm and mode of 120 ± 20 nm; p value ≤0.05 MSC Identity CD28, CD44, CD105 p value ≤0.05 Exosome Identity CD9, CD63, p value ≤0.05 CD81, Tsg101+ Exosome Potency Ang-2, FGF, HGF, Total protein should be IL-8, TIMP-1, TIMP-2, about 1 mg; p value ≤0.05 VEGF, PDGF, TNF-α Therapeutic miRNA: Total RNA content Signature 29a/10a/34a/125/181a/ should be >20 ug; 181c/Let-7b/146a/ p value ≤0.05 199/153/101/199 Sterility USP <71> Sterile, no evidence of CFR 610.12 microbial growth Mycoplasma Negative
[0683] 3. In Vivo Models:
[0684] To translate our preclinical findings from Phase 1 into the clinic, we will test cGMP-grade compliant young donor bone marrow-derived MSC exosomes. We will evaluate the exosome signature from 3 adult donors and choose one donor/group (male and female based on the data from Phase 1) for subsequent experiments based on an anti-aging signature. We will test duplicate exosome preps from the same donor to assure that this signature is reproducible. Controls will utilize an old exosome preparation that does not have a “therapeutic” signature. We will utilize a variety of models and deliver 5×10.sup.10 exosomes in 100 μl of PBS by tail vein injection in mice. The control mice will receive tail vein injection of 100 μl of PBS.
[0685] To expand our in vivo models and test generalized anti-geronic activity of exosomes, we elected the cigarette smoke (CS) model and the pneumonia model since the large majority of patients with IPF and COPD have a past history of CS exposure (97, 98), and because older adults are highly vulnerable to pneumonia. Although CS exposure confers long-term risk of many diseases even decades after cessation, these effects are not well understood (99) CS exposed lung tissue removed from rodents and humans have subsets of dysregulated miRNAs (71, 74, 99). We propose to expose mice to 6 months of CS and determine the ability of exosomes to repair the lung though delivery of miRNA that may be dysregulated in the lung post CS. As a model of viral pneumonia, we will include the SARS-COV-2 infection model as an important lung injury model fundamentally different from both bleomycin and CS. Specifically, in the case of COVID-19 infection, autopsy data supports adequate expansion of the lungs despite an inability to oxygenate hemoglobin in contrast to the lungs of patients with COPD and IPF. Mechanisms of COVID-19 lung injury remain incompletely understood as the prevalence of patients with chronic lung disease (“Ionghaulers”) increases. Therefore, addressing whether exosomes can protect against this type of lung injury has the potential to fundamentally broaden the antigeronic spectrum of exosome therapeutic indications.
[0686] 2. Experimental Design and Methods:
[0687] We will produce cGMP lots of defined exosomes prepared with our manufacturing partner and conduct Investigational New Drug (IND)-enabling studies in mouse and ex vivo human models to further test the potency of our signature. To date, a bottleneck in the advancement of exosome therapy into the clinic has been the development of high quality, reproducible, and efficient production of clinical grade exosomes. Our collaborator Zen-Bio, Inc. has now developed such a pipeline, as shown in section 2a.
[0688] Manufacturing: Zen-Bio routinely isolates EVs containing exosomes from mesenchymal stem cells using a hollow-fiber cartridge containing bioreactor as previously described (95). The bioreactor is amenable to culturing any cell type, although adherent cells are preferred. To prepare the bioreactor, a total of 1×10.sup.9 cells are seeded onto the cartridge. After seeding, the separate cell cartridge is filled with 40 mL of serum-free medium for cell maintenance and EV production, and growth medium is continually circulated through the bioreactor, with medium exchanges every 3-5 days. Cell metabolism is monitored by measuring the glucose level in the circulating medium; when levels drop 50% the circulating medium is changed. Bioreactor cultures have been maintained in this manner for up to three months while the MSCs retain their International Society for Cellular Therapy (ISCT)-mandated MSC characteristics (63).
[0689] EVs are collected every 3-5 days after the bioreactor culture is established. The conditioned serum-free medium will be removed from the cell cartridge and subjected to sterile filtration through a 0.22 μm filter, followed by tangential flow filtration (TFF) to concentrate the EVs and reduce the overall volume to 1 mL. The concentrated EVs are then subjected to size exclusion chromatography (SEC) over a 70 nm porous resin particle column. Each column is standardized, quality assured and certified to ISO 13485 standard (Medical Devices) making it perfect for clinical testing. Fractions are collected from the column and are analyzed for EVs using Nanoparticle Tracking Analysis (NTA). Peak fractions are identified and pooled for use and further analysis.
[0690] A typical yield from 40 mL of conditioned medium has been 3.6±0.6×10.sup.11 particles, and this yield can generate up to 1×10.sup.13 EVs per cartridge over the life of the bioreactor run.
[0691] EV physical characterization and surface marker expression: We will characterize the EV preparations for average particle size and concentration using ZetaView® BASIC NTA—Nanoparticle Tracking Video Microscope PMX-120. Protein concentration, DNA concentration, and RNA concentration will be determined by a Nanodrop ND-1000 spectrophotometer. EVs are then characterized for CD9, 63, and 81 expression to confirm the presence of exosomes. Exosome-specific protein markers will be determined by high throughput flow cytometry analysis using the MACSPIex Exosome Kit and MACSQuant analyzer to show the necessary presence of CD9, CD63 and CD81. Human foreskin fibroblast derived exosomes will be used as a control since fibroblasts and MSCs have a similar morphology and surface marker expression (96).
[0692] Success metrics and alternative approaches for EV production: Success criteria are based on Zen-Bio's historical read-outs of EV properties and results as follows: particle yield should be at least 1×109 per isolation; particle diameter should be consistent, to include exosomes (30-150 nm); total protein should be ≈1 mg; total RNA content should be >20 pg; and exosome and specific biomarkers (CD9, 63, and 81) must be present by 90%. Should any of the lots fail to produce exosomes that pass our quality control metrics, the lots will be stored for future analysis but not subjected to cell-based testing. In addition, as outlined in Phase 1 milestones (Table 6), utilize exosome preparations that meet our desired donor signature.
[0693] EV Transcriptome/miRNAome: In parallel with EV surface protein expression described above, we will analyze the miRNA using the MacsPlex miRNA Kit (Miltenyi). We will monitor the relative abundance of 26 miRNAs in a high through put manner. This will allow us to determine consistency within EV batches, to assess population composition, and to assess batch-to-batch and lot-to-lot consistency.
[0694] 3. In Vivo Models:
[0695] To translate our preclinical findings from Phase 1 into the clinic, we will test cGMP-grade compliant young donor bone marrow-derived MSC exosomes. We will evaluate the exosome signature from 3 adult donors and choose one donor/group (male and female based on the data from Phase 1) for subsequent experiments based on an anti-aging signature. We will test duplicate exosome preps from the same donor to assure that this signature is reproducible. Controls will utilize an old exosome preparation that does not have a “therapeutic” signature. We will utilize a variety of models and deliver 5×10.sup.10 exosomes in 100 μl of PBS by tail vein injection in mice. The control mice will receive tail vein injection of 100 μl of PBS.
[0696] To expand our in vivo models and test generalized anti-geronic activity of exosomes, we elected the cigarette smoke (CS) model and the pneumonia model since the large majority of patients with IPF and COPD have a past history of CS exposure (97, 98), and because older adults are highly vulnerable to pneumonia. Although CS exposure confers long-term risk of many diseases even decades after cessation, these effects are not well understood (99) CS exposed lung tissue removed from rodents and humans have subsets of dysregulated miRNAs (71, 74, 99). We propose to expose mice to 6 months of CS and determine the ability of exosomes to repair the lung though delivery of miRNA that may be dysregulated in the lung post CS. As a model of viral pneumonia, we will include the SARS-COV-2 infection model as an important lung injury model fundamentally different from both bleomycin and CS. Specifically, in the case of COVID-19 infection, autopsy data supports adequate expansion of the lungs despite an inability to oxygenate hemoglobin in contrast to the lungs of patients with COPD and IPF. Mechanisms of COVID-19 lung injury remain incompletely understood as the prevalence of patients with chronic lung disease (“Ionghaulers”) increases. Therefore, addressing whether exosomes can protect against this type of lung injury has the potential to fundamentally broaden the antigeronic spectrum of exosome therapeutic indications.
[0697] Smoking protocol: Mice will be exposed to 6 months of mainstream and sidestream cigarette smoke (CS) following the protocol previously published by our group (Teague smoke machine, 3 hours a day, 5 days a week) (68, 70). A similar protocol by Matulionis revealed the presence of fibrosis in male C57BL/6 mice that were exposed to CS, starting at 8-10 months of age (100). Initially, we will smoke 14-month-old mice and perform a time course with CT and sacrifice at 5 and 6 months post-CS to determine lung injury at these time points. Based on these data we will inject exosomes one month prior to sacrifice (
[0698] Success metrics: Since CS induces high levels of inflammation, we anticipate a reduction in inflammatory markers, at least in BAL, after exosome infusion by 20% or more. Not surprisingly, changes in antioxidant and SIRT1 expression have been reported in the lungs of CS exposed mice. Based on our preliminary data we expect that these and other proteins will be increased after treatment with exosomes. It is possible that we will not realize these metrics and will require additional dosing regimens which we are prepared to initiate.
TABLE-US-00009 TABLE 7 Male and female Vehicle exosome IV IV injection 5 or 6 CS injection one months post and Air one month month CS or room Male and 5 and 6 prior to prior to air sacrifice Female 14 month develop- develop- (either month old post ment ment depending on C57BL/6 CS or air of inflam- of inflam- preliminary Smoking mice analysis mation mation findings) CT scans CS Males CS Males Collect BAL; 4 months and and determine with sac Females Females inflammation (10 mice) 8/group + 8/group + markers, lung exosomes exosomes histology CT scans Room air Room air 6 months Males and Males and with sac Females Females (10 mice) 8/group + 8/group + exosomes exosomes Assess injury for timing of exosome injections SARS- D30 CoV-2 D0 D4 D7 D14 survival/ infection infection analysis analysis analysis analysis 24 adult & 4 mice/ 4 mice/ 4 mice/ 12 mice/group 24 old group group lung group lung survival; lung hACE- lung pathology, pathology, pathology, 20 Tg pathology, cytokines, cytokines, cytokines, mice + cytokines, viral titer, viral titer, viral titer, exosomes viral titer immune immune immune response response response 24 adult 4 mice/ 4 mice/ 4 mice/ 12 mice/group and 24 old group group lung group lung survival; lung hACE2-Tg lung pathology, pathology, pathology, mice + pathology, cytokines, cytokines, cytokines, PBS cytokines, viral titer, viral titer, viral titer, viral titer immune immune immune response response response 12 2 mice/ 2 mice/ 2 mice/ 6 mice/group uninfected group group lung group lung survival; lung adult and lung pathology, pathology, pathology, old hACE- pathology, cytokines, cytokines, cytokines, 2 Tg cytokines, viral titer, viral titer, viral titer, controls viral titer immune response Male and female Vehicle exosome IV IV injection 5 or 6 CS injection one months post and Air one month month CS or room Male and 5 and 6 prior to prior to air sacrifice Female 14 month develop- develop- (either month old post ment ment depending on C57BL/6 CS or air of inflam- of inflam- preliminary Smoking mice analysis mation mation findings) immune immune response response
[0699] b. SARS-Cov-2 Protocol:
[0700] For the coronavirus infection, we will use adult (3-month old) and old (>18 month old) C57BL/6 male mice transgenically expressing the human ACE2 molecule under the control of the epithelial keratin 18 promoter (B6.Cg-Tg (K18-ACE2)2Prlmn/J, Jax #034860). This is necessary because mice are not susceptible to SARS-COV2 unless the human ACE2 molecule is present in their cells via transgenesis or transduction. The K18-hACE2 Tg mice have been breeding in the Nikolich lab since June 2020 and are also available from Jackson Laboratories and we do not foresee any problem with generating the numbers outlined below; old mice of this strain will be aged in the Nikolich lab mouse colony and will be used in the ABSL/3 suite of the Keating Building at UArizona. Female mice will be used for confirmation and sex-related difference studies once all parameters are established in male mice.
[0701] Mice will be infected with 10.sup.4 plaque-forming units of SARS-CoV2 strain WA-1/2020, which in the Nikolich lab produces 100% mortality within one week (see Table 8), with breathing difficulties and tachypnea observable from day 4 onwards (severe lethal COVID-19). 3 month old K18-hACE2 mice on a C57BL/6 background were infected intranasally with the indicated doses of the SARS-COV2 strain WA-1/2020; two days later 2 mice/group were sacrificed and virus titer in lung homogenates determined by plaque assay. Mortality was scored until day 21 and is shown as number of dead animals out of total, with the day of last death indicated. In Exp. #1, mice were also evaluated on d5 for breathing frequency and were found to have accelerated breathing.
TABLE-US-00010 TABLE 8 SARSCoV-2 infection in K18-hACE2 mice K18- D2 lung Mortality Sars2 hACE2 titers (PFU, Accelerated (died/total; Dose (mice n = 2/ breathing day of last Experiment (PFU) (#) group) (Y/N) death) #1 10.sup.4 6 10.sup.7-10.sup.8 Y 4/4 (d8) #2 10.sup.1 6 10.sup.5 NT 0/4 10.sup.2 6 ND NT 1/4 (d6) 10.sup.3 5 10.sup.5 NT 1/3 (d6) 10.sup.4 2 10.sup.7 NT NA
[0702] 24 and 72 h later, 5×10.sup.10 exosome particles will be injected and the animals assessed as follows: 1) We will determine survival over 30 days, weight, temperature and activity of treated and control animals; 2) We will measure lung virus titers (by plaque assays) in the lungs on d4, 7, and 14; 3) we will determine induction of type I interferon (via CXCL10 production) and of other cytokines and chemokines as described in Phase I (see Bleomycin model Assessment), with all additional considerations mentioned therein) (101, 102). Final selection of cytokines and chemokines will be guided by the observed findings in pilot experiments with mice, as well as those elevated in severe COVID-19 patients (101). Histology and immunohistochemistry of the lung will be assessed (surfactant protein C, and aquaporin 5, a-smooth muscle cell actin and CD31 as endothelial marker). Design of groups and numbers of mice are depicted in Table 7, lower half.
[0703] Success metrics: Extension of survival by at least 20% over the control group to 14 days and beyond, will be considered our smallest acceptable level of efficacy. A secondary endpoint will be reduction of virus titers by an order of magnitude or more. Virus in these mice spreads to other organs as well, and by the time of these experiments, we will map precisely how much the virus spreads in old vs. adult mice; to that effect, we may be able to also analyze whether exosomes can reduce viral replication and/or damage in/to other organs, most notably the brain, heart and kidney (plaque assays and gross pathology). The analysis of immune responses in these animals (antibody response as described in our published work (103); T cell responses after stimulation with combined peptide pools as in (104), and in our prior work (105-110) we will test whether injection of exosomes, due to its anti-inflammatory properties, may interfere with the immune responses in adult or old animals, an issue of obvious clinical importance in therapeutics.
[0704] Pitfalls and alternatives: Lethal infection may prove a very high bar and we may need to reduce the virus does to 10.sup.3 or below, although in that case we will have to perform additional pilot experiments to ascertain whether the extent of lung injury may be sufficient (as judged by expression of the same lung injury markers, surfactant protein C, and aquaporin 5, a-smooth muscle cell actin and CD31 as endothelial marker). At this dose, some mice die (see Table 8), which could be advantageous as we could aim for partial lethality improvement. Moreover, due to broad whole-body expression of hACE2 in the above transgenic mice, there could be virus pathology in other organs that kills the animal. As our alternative model, we will use infection of old and adult mice with the adeno-associated virus (AAV) encoding human ACE2 (AAV-hACE2), as described (104, 111). Fourteen days later, mice will be infected with SARS-CoV2, and treated with exosomes and examined as above. It is possible that are dose concentration may have to be modified, however based on our preliminary data using the bleomycin mouse model we do not anticipate any problems. If for any reason we encounter problems with the extent of injury or reproducibility in the SARS-CoV-2 infection model, we will use the well-established S. pneumoniae model, with the influenza virus A PR/34 as the second backup model.
[0705] c. Ex vivo human tissue: To further test our signature in human lung tissue, we will perform 3-D punch experiments using male and female lung derived from patients who smoked and deemed not suitable for transplant at our hospital, Banner-University Medical Center Phoenix Ariz., (see accompanying letter). We will cannulate and inject a bronchial branch with 2% agarose and performed punch experiments as described above. A series of punches will be collected that will receive vehicle injection. Four days after injection punches will be fixed in 10% formalin (Sigma—Aldrich), processed for paraffin embedding and stained with trichrome to assess collagen content. Parallel punches will be utilized to measure mRNA, miRNA expression, the inflammation panel and other relevant cytokines as outlined above (
[0706] Success metrics: Based on our ongoing experiments, exosomes alter relevant for inflammation and fibrosis pathways in human lung punches. We would expect that lung punches treated with exosomes should have at least a 50% reduction over baseline in the inflammation panel. Lung punches obtained from subjects that smoked will express dysregulated inflamma-Mirs and others discussed in Significance section 5. Exosomes should normalize these miRNAs somewhat.
[0707] Success metrics/Alternative outcomes/Potential difficulties: The environment can influence the release of cargo once exosomes are circulating. This will be addressed by repeating the experiments in Phase 11 with biological duplicates from the same donor and technical triplicates. We acknowledge the incomplete nature of the punch assays, omitting important components of the immune system, including macrophages and neutrophils. We also recognize that the conditions used in these experiments may be hypoxic. If we find that to be the case, we will utilize an incubator to regulate oxygen amounts during the four days of the experiment and compare outcomes. However, these studies are a complement to our pre-clinical mouse studies prior to submission to the FDA.
Example 3. IND Enabling Studies
[0708] There is a well-established pathway for pharmaceutical small molecule drug development and review by the FDA. However, developers of biological products, such as EVs, have to modify certain parts of this established pathway, specifically that which is related to ADME-Toxicity testing (96, 112). Based on experience (95, 113, 114), the guidance provided by a 2018 position paper on the minimal information for studies of extracellular vesicles (115), and current open-enrollment information for therapies using EVs and exosomes, our team will perform the IND-enabling studies as outlined in the timeline. These studies are critical to define the product and as such will all be carried out.
[0709] 1) EV Morphology—Size: A consistent particle size distribution between lots and batches is required as part of our release criteria and is necessary for further clinical development. Exosomes have been described as being 30-150 nm in diameter (116, 117). Microvesicles are larger than exosomes and are often described as being 100-300 nm in diameter. The degree of overlap in the sizes for these classes of EVs varies depending upon the technology used to make the measurement. Since we are measuring particle quantity and size, we can assign an exosome and microsome population percentage to each EV preparation, further defining our EV preparations into exosome and microsome composition, with a goal towards setting parameters for product release and purity. If EV size fractionation is suggested by the FDA prior to IND submission, these data will provide the necessary information to design effective EV size exclusion/recovery approaches. EV sizing and concentration characterizations will be performed on each sample in triplicate using each pore size membrane. Additionally, three independent analyses will be performed on different days. We will compare differences between the two sizing technologies using Student's t-tests. Success metrics: Particle yield should be at least 1×10.sup.9 per isolation; particle diameter should be consistent with a mean of 165±50 nm and a mode of 120±20 nm to include exosomes (30-150 nm). Significant variation from the above parameters at p<0.05 will be considered unacceptable and would signal a no-go decision for a particular batch of exosomes.
[0710] 2) EV Phenotype: We will continue using the MacsPlex Exosome Kit (Miltenyi) for analysis of 37 different surface proteins, including CD9, CD63, and CD81, which are exosome markers present in our EV preparations. We will monitor the relative abundance of these exosome markers in a high throughput manner across batches to assess population composition and assess batch-to-batch consistency for exosomes. Since these markers are detected using the capture antibody technology, the data generated for the additional surface proteins will reflect only those present on (captured) exosomes. We will analyze each EV lot across batches to determine the consistency of marker expression as part of our release criteria. Each analysis will be performed in triplicate through independent experiments on different days. Statistical differences between lots and batches will be determined using ANOVA and/or t-tests depending on the comparison. Success metrics: Exosome (CD9, CD63, CD81) and MSC-specific (CD29, CD44, CD105) biomarkers must be present in all lots, and variability between lots must remain below the significance level of p ≤0.05.
[0711] 3) EV miRNA content: Using the MacsPlex miRNA Kit (Miltenyi) we have analyzed 39 different micro-RNAs (miRNAs) that may be present in EV cargo. In parallel with EV surface protein expression described above, we will monitor the relative abundance of these miRNAs in a high through put manner. Each sample will be tested in triplicate using independent experiments on different days to ensure data quality. Lot-to-lot and batch-to-batch consistency will be determined using ANOVA and/or t-tests depending on the comparison and differences between measures will be considered significant with a p value ≤0.05. We expect consistent lot-to-lot and batch-to-batch miRNA content to include members of the let7, miR200, miR335 and miR145 families. Success metrics: For a lot to be considered consistent with production criteria, variability relative to prior lots must remain below the significance level of p≤0.05.
[0712] 4) Affinity: Information on how well the EVs bind to cell membranes is needed for subsequent pharmacokinetic studies, in addition to determining a dosage regimen for toxicity analysis. We will use our previously established lipophilic dye transfer assay in a time course of binding study (118). An aliquot of EVs will be labeled with Vybrant Dil cell labeling solution for 20 minutes at 37° C. and excess dye removed. Recipient lung cells will be seeded in triplicate at 70% confluence in 48 well plates and allowed to attach overnight. Growth medium will be removed and 25 million dye-loaded EVs added to recipient cells. Dye transfer from the labeled EVs to cultured recipient cells will be measured by flow cytometry after 1, 2, 4, 8, 12, 16, and 24 hours of incubation at 37° C. Controls will include dye-labeled EVs incubated with recipient cells at 4° C. (Negative control) and recipient cells alone (Background). Once the optimal incubation time is identified, an ascending dose of EVs (1-100 million) will be tested to determine the saturating dose of EVs and a potential EC50 value for competitive assays. An in vitro competitive assay will also be performed to investigate equilibrium binding at a fixed concentration of dye-labeled EVs (EC75) in the presence of increasing concentrations of unlabeled EV competitor. All assays will be performed in triplicate using at least two donor recipient cell lots of MSCs. Additionally, three independent experiments will be performed on different days to ensure the reproducibility of the response. Success metrics: Statistical differences between EVs derived from different MSC treatments will be analyzed by ANOVA and/or t-tests depending on the comparison and significance will be considered acceptable if p value is >0.05. Alternatively, these studies can also be extended to include EV miRNA labeling studies (ExoGlow™-RNA EV Labeling Kit, SBI) to assess EV miRNA cargo delivery to recipient cells.
[0713] 5) Immune Response-in vitro: Previous in vitro and in vivo studies suggest that human MSCs do not induce an immune response, and in some studies higher passage human MSCs actually were immunosuppressive in mixed lymphocyte reactions (MLRs) (119, 120). In the typical MLR, an inactivated stimulator cell population is used to stimulate a naïve T-cell population from a different donor. These naïve T-cells then recognize the foreign cells and are activated and begin to proliferate (121). A one-way MLR using EVs as the stimulator population will be performed as previously described (122). Briefly, splenocytes from BALB/c mice will be isolated and seeded in a volume of 100 pL at a density of 1×10.sup.5 cells per well in 96-well round-bottom plates, in triplicate. An ascending dose of 30, 100, and 500 million EV particles from C57BL/6 mice will be added as a stimulator to obtain a 100 pL final volume. After 3 days of incubation, 1 pCi/well [.sup.3H] thymidine will be added overnight and thymidine incorporation measured using a [3-scintillation counter. Values will be compared to positive (phytohemagglutinin) and negative (vehicle alone) controls. Although we feel this is unlikely, an increase in [.sup.3H] thymidine counts for the experimental compared to the negative control will indicate a positive immune response. In addition to the murine MLR assay, we will test EVs for a similar response using human CD3+ T-cells freshly isolated at Zen-Bio (121). The one-way MLR will be performed using responder T-cells with the following stimulator cell populations that have been inactivated by treatment with mitomycin C: autologous PBMCs (from the T-cell donor, negative control) and allogeneic PBMCs (positive control). T-cells alone will also serve as a background control. Responder cells will be seeded in triplicate in a volume of 100 pL at a density of 1×10.sup.5 cells per well in 96-well round-bottom plates. Ascending doses of mitomycin-treated stimulator cells (1,000-20,000) or EVs (30, 100, and 500 million particles) will be added to a final volume of 200 pl. Proliferation will be determined after 72 hours using a chemiluminescent cell proliferation assay for BrDu incorporation (Roche) to assess active DNA synthesis and Cell Titer Blue for correlation with live cell numbers. All assays will be performed in triplicate using at least 5 different donor responder cell lots, and three independent experiments performed on different days to ensure the reproducibility of the response. Success metrics: Statistical analysis will be performed using student's t-test for comparing treatment groups with a p value ≤0.05 being considered as significant. Only the batches that are not significantly different in immunostimulatory capacity from negative control will be considered acceptable.
[0714] 6) Immune Response-in vivo: We anticipate that the in vitro MLR will be negative. As such, we will proceed with the in vivo rodent T-cell-dependent antibody response (TDAR) assay to complete the immune-response analysis. The TDAR assay is commonly used to assess new drug potential for immunotoxicity and immunosuppression, and is dependent on antigen uptake and presentation, B-cell activation with T-cell help, and antibody production. We will conduct these studies at Synchrony Labs using C57BL/6 mice according to previously described detailed pharmaceutical guidance to provide enough power to identify significant differences (123). Four groups of 20 mice (10 female and 10 male) will be injected subcutaneously with saline, 300 pg keyhole limpet hemocyanin, 1×10.sup.8 untreated MSC-derived EVs or 1×10.sup.8 optimally produced EVs from Aims 1 and 2. Injections of saline and EVs will be performed daily for 28 days as suggested by EPA guidelines for unknown entities; KLH will be injected at day 0 and again on day 14. Blood samples will be taken for analysis on Days 0, 5, 7, 10, 14, 19, 21, 24 and 28. We will assess the effect of EVs on the immune system by ELISA for total IgM, IgG subclass, IgA, and IgE in responses to EV challenge (124). Values will be compared to positive (KLH) and negative (saline) controls to determine the positive or negative immune response in vivo. Success metrics: Statistical analysis will be performed using GraphPad Prism software and analyzed by ANOVA with multiple and pair-wise comparisons and/or t-tests depending on the comparison, and significance will be considered as a p value ≤0.05. Only the batches that are not significantly different in immunostimulatory capacity from negative control will be considered acceptable
[0715] 7) Toxicity: To generate an initial assessment of EV toxicity, we will perform repeated dose 28-day and 90-day toxicity studies in mice with slight modifications to the OECD guidelines (125, 126). Male and female C57BL/6 mice will be used for these studies. Two groups of 30, 6-7 week old mice (15 male and 15 female) will be treated daily with a 100 pL subcutaneous injection of saline or 1×10.sup.8 optimal EVs determined from Aims 1 and 2. Animals will be observed daily for any overt signs of general toxicity including, changes in skin, fur, eyes, mucous membranes, unusual respiratory pattern, changes in response to handling, and repetitive or bizarre behaviors. Weekly measures of body weight, and food and water consumption will be recorded. On day 28, 10 animals from each group (5 male and 5 female) will have blood collected and be euthanized for post-mortem analyses. The remaining animals will continue to be treated until day 90 whereupon they will have blood collected and be euthanized for post-mortem analysis. Endpoint analyses for these studies consist of an evaluation of clinical observations (body weight, food and water consumption), blood cell analysis (red and white cell counts), blood chemistry (glucose, BUN, creatinine, total protein, ALP, AST), whole body gross necropsy, and histopathologic examination of heart, liver, lungs, spleen, kidneys, and brain. Pathology analytical services will be provided by Infinium Pathology Consultants. Success metrics: Toxicity endpoints will include increased mortality and unfavorable physiological and biochemical changes in the EV treated group compared to the control saline group. Statistical analysis will be performed using Student's t-test for comparing treatment groups with a p value ≤0.05 being considered as significant, and samples with significant toxicity will be considered unacceptable.
[0716] 8) Tumorigenicity: Unlike many regenerative medicine therapeutic products which contain live cells as the active component, EVs are non-replicating, thereby eliminating the possibility that EVs themselves will become malignantly transformed. As such, we will test whether optimized EVs facilitate tumor formation in immunocompromised mice as an initial assessment of tumorigenicity. Due to the strict requirements of handling and housing immunocompromised mice, we will contract out this study to Charles River Labs using their in-house protocols. Twenty athymic nude mice will be randomly sorted into two groups for 200 pL injections of saline or 1×10.sup.8 optimal EVs from Aims 1 and 2 into the subcutaneous dorsal flank. The mice will be monitored for survival and tumor growth 2-3 times a week for up to 8 weeks, measuring the length and width of the tumor using a slide caliper (127). Should tumors in these immunocompromised rodents grow to a diameter of 150-200 mm.sup.3, they will be removed from euthanized animals, weighed and measured for tumor size and volume, and preserve appropriately for histological investigation. As an alternative, we will consider transplanting EVs into the kidney capsule of mice to determine if EVs can generate a tumor in specific organ tissue (128). Success metrics: Statistical analysis will be performed using Student's t-test for comparing EV treated animals to the control group with a p value ≤0.05 being considered as significant; any thus defined significant tumorigenicity will be considered unacceptable.
[0717] 9) EV preservation and shelf-life: We have previously lyophilized EVs and stored them at room temperature for several weeks before reconstituting them for size analyses and in vivo studies. Throughout this process we did not observe any differences in particle size or in vivo efficacy compared to freshly isolated EVs. In order to provide a more thorough analysis of EV preservation, shelf-life and integrity, we will perform a time course study over 12 months and include endpoint assays of in vitro morphology, phenotype, miRNA content, affinity, and efficacy analysis in human primary cells (proliferation, migration and angiogenesis). Nine 1×108 aliquots of all EV lots prepared will be subjected to lyophilization and stored in the dark at room temperature in an air-tight desiccator. Parallel aliquot samples will include EVs suspended in PBS immediately after isolation and stored at −80, −20, and +4 degrees Celsius. Triplicate samples will be thawed or suspended in PBS at 3, 6 and 12 months and subjected to the battery of in vitro assays listed above. Statistical analysis will be performed using GraphPad Prism software and analyzed by ANOVA with multiple and pair-wise comparisons and/or t-tests depending on the comparison, and significance will be considered as a p value ≤0.05. Success metrics: Based on our previous results, we expect that the 4° C. stored EVs will lose efficacy and show degradation prior to all other storage conditions. Additionally, we expect that the −80° C. stored EVs will maintain their physical and biochemical properties for 12 months with less than 15% reduction in these characteristics. Batches that diminish in potency by more than 15% will be considered unacceptable. We expect that these in vitro assays will serve as in-process controls during product manufacturing and be useful in determining preservation conditions for optimal product shelf-life.
[0718] 10) Sterility and Bioburden: All of our human tissue from which we isolated MSCs is tested by a third-party using PCR to identify the presence of common viral pathogens (HIV I & II and hepatitis B & C) and tested internally for bacterial and fungal contamination. Any tissues found to be positive are discarded as well as any cells derived from those tissues. We also perform sterility testing during cell expansion prior to seeding the bioreactor, during bioreactor medium collection and post-EV isolation. As part of our pre-clinical development, we will subject our EV products to bioburden and sterility testing using Eurofins Scientific. EV products will be tested for sterility, mycoplasma, endotoxin, and bioburden according to their methodologies accepted by regulatory agencies. Success metrics: Any positive bioburden samples will be analyzed to identify contaminating organism(s). Any EV batch which is not sterile will be discarded. To date, we have not produced any EV lots that have tested positive for mycoplasma or endotoxin, but we will continue to monitor for these contaminants as we proceed through development. Triplicate blinded samples containing vehicle or optimal EVs from Aims 1 and 2 from at least 3 batches will be sent to Eurofins for analysis.
[0719] Timeline: Phase II is expected to take 24 months to complete. Our timeline allows for additional exosomes to be prepared as necessary.
REFERENCES FOR EXAMPLES 1-3
[0720] 2. W. Merkt, M. Bueno, A. L. Mora, D. Lagares, Senotherapeutics: Targeting senescence in idiopathic pulmonary fibrosis. Semin Cell Dev Biol 101, 104-110 (2020). [0721] 3. K. Ascher, S. J. Elliot, G. A. Rubio, M. K. Glassberg, Lung Diseases of the Elderly: Cellular Mechanisms. Clin Geriatr Med 33, 473-490 (2017). [0722] 4. D. M. E. Bowdish, The Aging Lung: Is Lung Health Good Health for Older Adults? Chest 155, 391-400 (2019). [0723] 5. D. Furman et al., Chronic inflammation in the etiology of disease across the life span. Nature Medicine 25, 1822-1832 (2019). [0724] 6. B. K. Kennedy et al., Geroscience: linking aging to chronic disease. Cell 159, 709-713 (2014). [0725] 7. S. Navarro, B. Driscoll, Regeneration of the Aging Lung: A Mini-Review. Gerontology 63, 270-280 (2017). [0726] 8. J. F. Cordier, V. Cottin, Neglected evidence in idiopathic pulmonary fibrosis: from history to earlier diagnosis. Eur Respir J 42, 916-923 (2013). [0727] 9. D. J. Lederer, F. J. Martinez, Idiopathic Pulmonary Fibrosis. N Engl J Med 379, 797-798 (2018). [0728] 10. L. Richeldi, H. R. Collard, M. G. Jones, Idiopathic pulmonary fibrosis. Lancet 389, 1941-1952 (2017). [0729] 11. S. Meiners, O. Eickelberg, M. Konigshoff, Hallmarks of the ageing lung. Eur Respir J 45, 807-827 (2015). [0730] 12. P. J. Barnes, Oxidative stress-based therapeutics in COPD. Redox Biology 33, 101544 (2020). [0731] 13. P. J. Barnes, J. Baker, L. E. Donnelly, Cellular Senescence as a Mechanism and Target in Chronic Lung Diseases. American journal of respiratory and critical care medicine 200, 556-564 (2019). [0732] 14. P. J. Barnes, Small airway fibrosis in COPD. The international journal of biochemistry & cell biology 116, 105598 (2019). [0733] 15. P. J. Barnes, Pulmonary Diseases and Ageing. Subcell Biochem 91, 45-74 (2019). [0734] 16. S. J. Cho, H. W. Stout-Delgado, Aging and Lung Disease. Annu Rev Physiol 82, 433-459 (2020). [0735] 17. U. Kulkarni et al., Excessive neutrophil levels in the lung underlie the age-associated increase in influenza mortality. Mucosal Immunol 12, 545-554 (2019). [0736] 18. C. K. Wong et al., Aging Impairs Alveolar Macrophage Phagocytosis and Increases Influenza-Induced Mortality in Mice. J Immunol 199, 1060-1068 (2017). [0737] 39. J. Tashiro et al., Therapeutic benefits of young, but not old, adipose-derived mesenchymal stem cells in a chronic mouse model of bleomycin-induced pulmonary fibrosis. Transl Res 166, 554-567 (2015). [0738] 40. J. Fafián-Labora et al., Influence of mesenchymal stem cell-derived extracellular vesicles in vitro and their role in ageing. Stem cell research & therapy 11, 13 (2020). [0739] 41. D. Gnani et al., An early-senescence state in aged mesenchymal stromal cells contributes to hematopoietic stem and progenitor cell clonogenic impairment through the activation of a pro-inflammatory program. Aging cell, e12933 (2019). [0740] 42. R. Vono, E. Jover Garcia, G. Spinetti, P. Madeddu, Oxidative Stress in Mesenchymal Stem Cell Senescence: Regulation by Coding and Noncoding RNAs. Antioxidants & redox signaling 29, 864-879 (2018). [0741] 43. F. Olivieri et al., Circulating miRNAs and miRNA shuttles as biomarkers: Perspective trajectories of healthy and unhealthy aging. Mechanisms of Ageing and Development 165, 162-170 (2017). [0742] 44. J. Boulestreau, M. Maumus, P. Rozier, C. Jorgensen, D. Noel, Mesenchymal Stem Cell Derived Extracellular Vesicles in Aging. Front Cell Dev Biol 8, 107 (2020). [0743] 45. J. L. Rinn, M. Snyder, Sexual dimorphism in mammalian gene expression. Trends in Genetics 21, 298-305 (2005). [0744] 46. T. Connallon, L. L. Knowles, Intergenomic conflict revealed by patterns of sex-biased gene expression. Trends in Genetics 21, 495-499 (2005). [0745] 47. L. M. McIntyre et al., Sex-specific expression of alternative transcripts in Drosophila. Genome Biology 7, R79 (2006). [0746] 48. A. Vigé, C. Gallou-Kabani, C. Junien, Sexual Dimorphism in Non-Mendelian Inheritance. Pediatric Research 63, 340-347 (2008). [0747] 49. E. Bianconi et al., Sex-Specific Transcriptome Differences in Human Adipose Mesenchymal Stem Cells. Genes (Basel) 11, (2020). [0748] 50. A. Giuliani et al., Mitochondrial (Dys) Function in Inflammaging: Do MitomiRs Influence the Energetic, Oxidative, and Inflammatory Status of Senescent Cells? Mediators Inflamm 2017, U.S. Pat. No. 2,309,034 (2017). [0749] 51. G. L. Russo et al., Mechanisms of aging and potential role of selected polyphenols in extending healthspan. Biochem Pharmacol 173, 113719 (2020). [0750] 52. F. Gurau et al., Anti-senescence compounds: A potential nutraceutical approach to healthy aging. Ageing Res Rev 46, 14-31 (2018). [0751] 53. K. Tsubota, The first human clinical study for NMN has started in Japan. NPJ Aging Mech Dis 2, 16021 (2016). [0752] 54. J. Campisi et al., From discoveries in ageing research to therapeutics for healthy ageing. Nature 571, 183-192 (2019). [0753] 55. M. Fuentealba et al., Using the drug-protein interactome to identify anti-ageing compounds for humans. PLOS Computational Biology 15, e1006639 (2019). [0754] 56. D. Zhang et al., Exosome-Mediated Small RNA Delivery: A Novel Therapeutic Approach for Inflammatory Lung Responses. Molecular Therapy 26, 2119-2130 (2018). [0755] 57. R. Kangas et al., Aging and serum exomiR content in women-effects of estrogenic hormone replacement therapy. Sci Rep 7, 42702 (2017). [0756] 58. J. Li et al., miR-10a restores human mesenchymal stem cell differentiation by repressing KLF4. Journal of cellular physiology 228, 2324-2336 (2013). [0757] 59. L. S. Huang et al., The Mitochondrial Cardiolipin Remodeling Enzyme Lysocardiolipin Acyltransferase (LYCAT) is a Novel Target in Pulmonary Fibrosis. American journal of respiratory and critical care medicine, (2014). [0758] 60. J. M. Yu et al., Age-related changes in mesenchymal stem cells derived from rhesus macaque bone marrow. Aging cell 10, 66-79 (2011). [0759] 61. L. Liu et al., MicroRNA-181a Regulates Local Immune Balance by Inhibiting Proliferation and Immunosuppressive Properties of Mesenchymal Stem Cells. STEM CELLS 30, 1756-1770 (2012). [0760] 62. K. Watanabe et al., Functional similarities of microRNAs across different types of tissue stem cells in aging. Inflammation and Regeneration 38, 9 (2018). [0761] 63. T. Squillaro, G. Peluso, U. Galderisi, Clinical Trials With Mesenchymal Stem Cells: An Update. Cell Transplant 25, 829-848 (2016). [0762] 64. A. Stolzing, E. Jones, D. McGonagle, A. Scutt, Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev 129, 163-173 (2008). [0763] 65. S. Bork et al., DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells. Aging cell 9, 54-63 (2010). [0764] 66. C. R. Kliment, T. D. Oury, Oxidative stress, extracellular matrix targets, and idiopathic pulmonary fibrosis. Free Radical Biology and Medicine 49, 707-717 (2010). [0765] 67. A. van der Vliet, Y. M. W. Janssen-Heininger, V. Anathy, Oxidative stress in chronic lung disease: From mitochondrial dysfunction to dysregulated redox signaling. Mol Aspects Med 63, 59-69 (2018). [0766] 68. M. K. Glassberg et al., Estrogen deficiency promotes cigarette smoke-induced changes in the extracellular matrix in the lungs of aging female mice. Transl Res 178, 107-117 (2016). [0767] 69. N. Kassira et al., Estrogen deficiency and tobacco smoke exposure promote matrix metalloproteinase-13 activation in skin of aging B6 mice. Ann Plast Surg 63, 318-322 (2009). [0768] 70. S. J. Elliot et al., Smoking induces glomerulosclerosis in aging estrogen-deficient mice through cross-talk between TGF-beta1 and IGF-I signaling pathways. Journal of the American Society of Nephrology:JASN 17, 3315-3324 (2006). [0769] 71. A. Izzotti et al., Downregulation of microRNA expression in the lungs of rats exposed to cigarette smoke. FASEB J 23, 806-812 (2009). [0770] 72. A. Izzotti, G. A. Calin, V. E. Steele, C. M. Croce, S. De Flora, Relationships of microRNA expression in mouse lung with age and exposure to cigarette smoke and light. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 23, 3243-3250 (2009). [0771] 73. F. Schembri et al., MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium. Proc Natl Acad Sci USA 106, 2319-2324 (2009). [0772] 74. J. W. Graff et al., Cigarette smoking decreases global microRNA expression in human alveolar macrophages. PloS one 7, e44066 (2012). [0773] 75. M. K. Glassberg et al., Allogeneic Human Mesenchymal Stem Cells in Patients With Idiopathic Pulmonary Fibrosis via Intravenous Delivery (AETHER): A Phase I Safety Clinical Trial. Chest 151, 971-981 (2017). [0774] 76. R. Peng et al., Bleomycin induces molecular changes directly relevant to idiopathic pulmonary fibrosis: a model for “active” disease. PloS one 8, e59348 (2013). [0775] 77. N. Srour, B. Thebaud, Mesenchymal Stromal Cells in Animal Bleomycin Pulmonary Fibrosis Models: A Systematic Review. Stem cells translational medicine, (2015). [0776] 78. H. Dweep, C. Sticht, P. Pandey, N. Gretz, miRWalk—database: prediction of possible miRNA binding sites by “walking” the genes of three genomes. Journal of biomedical informatics 44, 839-847 (2011). [0777] 79. A. Jusic et al., Approaching Sex Differences in Cardiovascular Non-Coding RNA Research. International journal of molecular sciences 21, 4890 (2020). [0778] 80. R. Song et al., Many X-linked microRNAs escape meiotic sex chromosome inactivation. Nat Genet 41, 488-493 (2009). [0779] 81. S. Elliot et al., MicroRNA let-7 Downregulates Ligand-Independent Estrogen Receptor-mediated Male-Predominant Pulmonary Fibrosis. American journal of respiratory and critical care medicine 200, 1246-1257 (2019). [0780] 82. G. Izbicki, M. J. Segel, T. G. Christensen, M. W. Conner, R. Breuer, Time course of bleomycin-induced lung fibrosis. Int J Exp Pathol 83, 111-119 (2002). [0781] 83. L. Ferrucci et al., The origins of age-related proinflammatory state. Blood 105, 2294-2299 (2005). [0782] 84. S.-i. Imai, L. Guarente, NAD+ and sirtuins in aging and disease. Trends in Cell Biology 24, 464-471 (2014). [0783] 85. A. Brunet et al., Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303, 2011-2015 (2004). [0784] 86. H. F. Yuan et al., SIRT1 is required for long-term growth of human mesenchymal stem cells. J Mol Med (Berl) 90, 389-400 (2012). [0785] 89. LEFT BLANK [0786] 90. K. Zscheppang et al., Human Pulmonary 3D Models For Translational Research. Biotechnol J 13, (2018). [0787] 91. H. N. Alsafadi et al., Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery. Am J Respir Cell Mol Biol 62, 681-691 (2020). [0788] 92. G. B. Fields, The Rebirth of Matrix Metalloproteinase Inhibitors: Moving Beyond the Dogma. Cells 8, (2019). [0789] 93. K. Ostridge et al., Relationship between pulmonary matrix metalloproteinases and quantitative CT markers of small airways disease and emphysema in COPD. Thorax 71, 126 (2016). [0790] 94. M. Provenzano et al., The Association of Matrix Metalloproteinases with Chronic Kidney Disease and Peripheral Vascular Disease: A Light at the End of the Tunnel? Biomolecules 10, (2020). [0791] 95. W. G. L. Whitford, J. W.; Cadwell, J. J. S., Continuous production of exosomes. Genetic Engineering and Biotechnology News 35, 34 (2015). [0792] 96. M. Mendt et al., Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight 3, (2018). [0793] 97. K. B. Baumgartner, J. M. Samet, C. A. Stidley, T. V. Colby, J. A. Waldron, [0794] Cigarette smoking: a risk factor for idiopathic pulmonary fibrosis. American journal of respiratory and critical care medicine 155, 242-248 (1997). [0795] 98. W. I. Choi et al., Risk factors for interstitial lung disease: a 9-year Nationwide population-based study. BMC Pulm Med 18, 96 (2018). [0796] 99. R. Joehanes et al., Epigenetic Signatures of Cigarette Smoking. Circulation: Cardiovascular Genetics 9, 436-447 (2016). [0797] 100. D. H. Matulionis, Chronic cigarette smoke inhalation and aging in mice: 1. Morphologic and functional lung abnormalities. Exp Lung Res 7, 237-256 (1984). [0798] 101. T. Takahashi et al., Sex differences in immune responses that underlie COVID-19 disease outcomes. Nature, (2020). [0799] 102. Y. Zhao et al., Longitudinal COVID-19 profiling associates IL-1RA and IL-10 with disease severity and RANTES with mild disease. JCI Insight 5, (2020). [0800] 103. T. J. Ripperger et al., Orthogonal SARS-CoV-2 Serological Assays Enable Surveillance of Low-Prevalence Communities and Reveal Durable Humoral Immunity. Immunity, (2020). [0801] 104. M. Hassert et al., mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2. bioRxiv, (2020). [0802] 105. J. L. Uhrlaub et al., Dysregulated TGF-beta Production Underlies the Age-Related Vulnerability to Chikungunya Virus. PLoS Pathog 12, e1005891 (2016). [0803] 106. J. L. Uhrlaub, M. J. Smithey, J. Nikolich-Zugich, Cutting Edge: The Aging Immune System Reveals the Biological Impact of Direct Antigen Presentation on CD8 T Cell Responses. J Immunol 199, 403-407 (2017). [0804] 107. J. D. Brien, J. L. Uhrlaub, A. Hirsch, C. A. Wiley, J. Nikolich-Zugich, Key role of T cell defects in age-related vulnerability to West Nile virus. J Exp Med 206, 2735-2745 (2009). [0805] 108. L. Cicin-Sain et al., Cytomegalovirus infection impairs immune responses and accentuates T-cell pool changes observed in mice with aging. PLoS Pathog 8, e1002849 (2012). [0806] 109. G. Li, M. J. Smithey, B. D. Rudd, J. Nikolich-Zugich, Age-associated alterations in CD8alpha+ dendritic cells impair CD8 T-cell expansion in response to an intracellular bacterium. Aging cell 11, 968-977 (2012). [0807] 110. M. J. Smithey, K. R. Renkema, B. D. Rudd, J. Nikolich-Zugich, Increased apoptosis, curtailed expansion and incomplete differentiation of CD8+ T cells combine to decrease clearance of L. monocytogenes in old mice. Eur J Immunol 41, 1352-1364 (2011). [0808] 111. B. Israelow et al., Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling. J Exp Med 217, (2020). [0809] 112. G. R. Willis, S. Kourembanas, S. A. Mitsialis, Toward Exosome-Based Therapeutics: Isolation, Heterogeneity, and Fit-for-Purpose Potency. Front Cardiovasc Med 4, 63 (2017). [0810] 113. J. Basu, J. W. Ludlow, Cell-based therapeutic products: potency assay development and application. Regen Med 9, 497-512 (2014). [0811] 114. J. Basu, J. W. Ludlow, Exosomes for repair, regeneration and rejuvenation. Expert Opin Biol Ther 16, 489-506 (2016). [0812] 115. C. Thery et al., Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7, 1535750 (2018). [0813] 116. R. A. Dragovic et al., Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine 7, 780-788 (2011). [0814] 117. E. van der Pol et al., Optical and non-optical methods for detection and characterization of microparticles and exosomes. J Thromb Haemost 8, 2596-2607 (2010). [0815] 118. M. C. Deregibus et al., Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood 110, 2440-2448 (2007). [0816] 119. K. McIntosh et al., The immunogenicity of human adipose-derived cells: temporal changes in vitro. Stem Cells 24, 1246-1253 (2006). [0817] 120. K. R. McIntosh et al., Immunogenicity of allogeneic adipose-derived stem cells in a rat spinal fusion model. Tissue Eng Part A 15, 2677-2686 (2009). [0818] 121. L. M. Muul et al., Measurement of proliferative responses of cultured lymphocytes. Curr Protoc Immunol Chapter 7, Unit? 10 (2011). [0819] 122. F. Djouad et al., Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood 102, 3837-3844 (2003). [0820] 123. H. Lebrec et al., The T-cell-dependent antibody response assay in nonclinical studies of pharmaceuticals and chemicals: study design, data analysis, interpretation. Regul Toxicol Pharmacol 69, 7-21 (2014). [0821] 124. K. Hata et al., Differential regulation of T-cell dependent and T-cell independent antibody responses through arginine methyltransferase PRMT1 in vivo. FEBS Lett 590, 1200-1210 (2016). [0822] 125. OECD, Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents. (2008). [0823] 126. OECD. (2018). [0824] 127. M. M. Tomayko, C. P. Reynolds, Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 24, 148-154 (1989).
Example 4. Sex Disparity in COVID-19 Viral Infection
Specific Aims:
[0825] In most countries, men are disproportionately dying of COVID infections (http://globalhealth5050.org/covid19; accessed Jun. 13, 2020), while the incidence and prevalence of infection is equal between men and women (1, 2). This is particularly evident in cohorts over 50 (http://globalhealth5050.org/covid19). Therefore biological (age, hormonal state, chromosome contribution, immune function, higher rate of comorbid conditions) and gender-related (lifestyle and socioeconomic status) factors in patients with COVID-19 need to be assessed to determine why men may have increased mortality. For example, levels of ACE2, the mechanism for viral infection, are generally higher in men in a study examining patients with cardiovascular disease. Initial investigations into sex differences have begun to dissect disparate responses to SARS-CoV2, with males showing higher plasma levels of certain chemokines (CCLS) and cytokines (IL-8&18), as well as increased induction of non-classical monocytes; by contrast, females have exhibited a more robust cell activation. Understanding how and why one or the other type of reactivity provides advantage or disadvantage in COVID-19 remains a critical task. This proposal has high impact since sex chromosome genes and/or sex hormone receptor activation could potentiate protective factors in females or harmful factors in males. Discovery of these mechanisms may guide new therapies that prevent or ameliorate COVID-19 lung disease in both sexes.
[0826] COVID-19 infection induces a lung inflammatory response that can progress to inflammation with cytokine storm. This can lead to acute lung injury (ALI), Acute Respiratory Distress Syndrome (ARDS), organ failure, and death. Therefore in this proposal we will focus on the lung, although we recognize that other organs including the heart, kidney, gastrointestinal tract, liver, pancreas, nervous system and skin are also viral targets. Sex-specific disease outcomes following virus infections are attributed to sex-dependent production of steroid hormones, different copy numbers of immune response X-linked genes, and the presence of disease susceptibility genes in males and females (3, 4). An ongoing clinical trial is testing whether transdermal estrogen (NCT04359329) may have a protective effect in males and females with COVID-19. Therefore, understanding the hormonal and chromosomal underpinnings of the documented sex disparities in COVID-19 is critical to the efforts to treat, promote earlier detection, and evaluate drugs for this formidable disease.
[0827] This disclosure hypothesizes that activation of gonadal hormone receptors and sex chromosome complement in alveolar cells (epithelial and endothelial) of aging lungs are integral components driving the pronounced sex disparity. To test this hypothesis we propose to isolate exosomes from the serum or plasma from age-matched and disease severity matched male and female patients with COVID-19 that had been collected and banked by at the University of Arizona.
[0828] We will utilize exosomes since cells employ exosomes to transfer regulatory factors that modulate the response of local and distant cells and systemic responses. In cells with active viral or bacterial infections, the exosome machinery can package pathogen-derived factors that alter the phenotype of the recipient cells (5, 6). We will isolate exosomes from adult and older male and female patients to investigate biological differences that may be related not only to sex but age. We will inject exosomes into lung punches. Our preliminary data show that naïve lung punches contain alveolar cell types important in COVID-19 infection. We also show that the normal lung phenotype can be converted to a diseased phenotype though the introduction of fibrotic cell-derived exosomes into the punch. This is mediated in part by exosome delivery of microRNAs which may differ between males and females. We will test the following specific aims
[0829] Aim 1: Determine the contribution of hormone receptor expression and subsequent downstream signaling in sex disparity of COVID-19-associated lung disease. Determine if sex, age or disease severity of COVID-19-derived exosomes influence sex hormone receptor expression, signaling or cytokine and chemokine response to SARS-COV2 differently in male and female control or GDX mouse lung punches.
[0830] Aim 2: Determine the contribution of sex chromosomes to differences in sex disparity in Covid-19 lung disease using the four core genotypes (FCG) mouse model. Compare lung punches derived from FCG mice that have the same type of gonads to determine if sex differences in COVID-19 are caused in part by genes encoded by the sex chromosomes which are inherently differently expressed in XX and XY lungs. Test whether these mice themselves are showing sex-determined differences in immune responses and in susceptibility to SARS-CoV2 using lung-restricted transduction of hACE2, followed by SARS-CoV2 infection. Investigate, in the same model, whether sex and age interact to pronounce the susceptibility.
[0831] These studies provide critical initial insights into the fundamental basis of sex-related differences in susceptibility to COVID-19 and will pave the way towards detailed mechanistic studies of this key topic.
[0832] Significance: The convergence of aging and gonadal hormones. Gonadal hormone production/activation declines during reproductive aging and has been linked to multiple age-associated diseases including cardiovascular disease (7, 8), diabetic kidney disease (9-12), prostate cancer (13), lung cancer (14), and other lung diseases (15, 16). Underlying differences between males and females may become more apparent with age-associated changes of gonadal hormones and their signaling due to either loss of protection and/or gain of harmful effects, or by the emergence of sex chromosome effects that may be suppressed by gonadal hormones. Our prior studies support a protective effect of estrogen in the lungs of aged female mice; E2 replacement partially restored the destruction of inter-alveolar septa in the lungs of the aged mice (15, 16). To date and to our knowledge there are no comparable studies in aged male mice. Recent population-based studies continue to suggest a protective effect of estrogens as menopause is associated with accelerated lung function decline (17). However changes of gonadal hormones in aged men are not as predictable as in women making comparable population studies in males more challenging (18).
[0833] Sex chromosomes and aging. Although gonadal sex is a major difference between men and women, it is not the only difference, and may not account for all differences in age-associated lung disease. Since the number of X chromosomes is different between the sexes, the presence or absence of the Y chromosome independently or in concert with gonadal hormones may promote or protect against age-associated lung disease. Approximately 15% of human X-linked genes escape silencing and are expressed from both X chromosomes in females, resulting in the higher expression in XX females as compared to XY males (19). Because the ratio of estrogens to androgens becomes more similar in males and females with age, over time the influence of gonadal hormones may be less important than the modulation of disease phenotypes by sex chromosomes (18, 20). This will be addressed in specific Aim (SA) 2.
[0834] Sex differences, viral infections, immunity and aging. As discussed, both above and below, issues of sex differences in response to viral infections, particularly in older adults and animals, have not been thoroughly dissected. Our collaboration between experts in sex genetics and hormone biology and its impact on lung physiology and leaders in antiviral immunity in older subjects is an ideal vehicle to address such gaps in knowledge
[0835] STRENGTHS/WEAKNESSES OF CITED STUDIES: Gonadal hormones/receptors and the human lung.
[0836] The human lung is a gonadal hormone target tissue (21). Gonadal hormones regulate normal lung development, physiology, and are implicated in several lung diseases including asthma, pulmonary fibrosis, and pulmonary hypertension in males and females (21, 22). AR and ER are present in the lung; however, their signaling remains poorly understood. Our published data support the idea that steroid hormone receptors participate in signaling pathways relevant to fibrosis. In SA1 the experiments will lead us to characterize mechanisms that target viral pathways stimulated by sex steroid hormone receptors (23)
[0837] Hormones and coronavirus infection: Both the Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe respiratory syndrome coronavirus 1 (SARS-CoV1) were found to infect and clinically affect more men than women (24). Similar data was derived from pre-clinical studies of SARS-CoV1 infection. Male mice were more susceptible to infection than female mice (25). The enhanced susceptibility of male mice to SARS-CoV1 correlated with a moderate increase in virus titer and extensive alveolar macrophages and neutrophil accumulation in the lungs (25). Gonadectomy had no effect on disease outcome in male mice, while oophorectomy or treating female mice with an estrogen receptor (ER) antagonist resulted in increased mortality to SARS-CoV infection (25). These findings suggest that estrogen signaling protects female mice from a lethal infection (25).
[0838] Sex chromosomes and viral infection: Sex chromosome genes and sex hormones, including estrogens, progesterone and androgens, contribute to the differential regulation of immune responses between the sexes. Women are functional mosaics for X-lined genes, and the X-chromosome contains a high density of immune-related genes; therefore, women generally mount stronger innate and adaptive immune responses than men (26). This results in faster clearance of pathogens and greater vaccine efficacy in females than in males but also contributes to their increased susceptibility to inflammatory and autoimmune diseases. With regard to SARS-CoV2, a recent study showed sex-related dichotomies between innate and adaptive responses in the blood and correlated them to clinical outcomes (27). Male subjects exhibited increased chemokine (CCLS) and cytokine (IL-8, IL-18) levels, along with increased activation of non-classical monocytes; females exhibited increased and sustained T cell activation, and poor T cell activation correlated to poor disease outcomes in males (27). The contribution of immunological response mounted in women as compared to men, particularly in the respiratory system, and its association with decreased mortality requires investigation.
[0839] Aging and viral infection: Immune responses to viruses are compromised in older adults at several levels, including both innate and adaptive immune responses (28, 29). Perhaps not surprisingly, that has held true in a few studies with coronaviruses, most notably with the mouse-adapted strains of SARS-CoV1 (30, 31). Our group has been at the forefront of characterization and mechanistic dissection of such defects (32-37) and we will apply that expertise to the studies herein. Of interest, relatively few studies have examined sex differences in antiviral responses and vulnerability to viruses in older humans and animals, and we will fill this gap in SA2 of this proposal.
[0840] Exosomes: Extracellular vesicles (EVs) are a heterogeneous group of bilayer membrane structures that, according to their size, shape, biogenesis, and composition, are classified into two major categories known as exosomes and micro-vesicles (MVs) (38, 39). Exosomes are particles of endosomal origin with sizes ranging from 30 to 150 nm in diameter and are generated from the internal budding of multivesicular bodies and released via exocytosis (40). MVs are exosome-like vesicles with a size ranging from 50 to 1000 nm in diameter that are released by the budding of the cell membrane (41). EVs are secreted by a variety of cell types such as mesenchymal stem cells (MSCs), T cells, B cells, and dendritic cells, and can be isolated from all biological body fluids including serum, blood, breast milk, urine, and semen (41-43). Due to their size, EVs are transported through blood and biological body fluids where they interact with target cells. EVs contain proteins, soluble factors, and microRNAs based on their cellular origin and exert biological action in target cells via both an endocrine effect on distant cells and a paracrine effect on adjacent cells (44).
[0841] EVs facilitate viral and bacterial pathogenesis through their cargo of microRNA: Viral and bacterial pathogens can subvert exosome functions to promote pathogen replication, survival, or pathology. Cells employ EVs to transfer regulatory factors that modulate the response of local and distant cells and systemic responses. In cells with active viral or bacterial infections, the exosome machinery can package pathogen-derived factors that alter the phenotype of the recipient cells (5, 6). The major functions of viral-miRNAs (carried by EVs) across divergent virus families have been broadly attributed to immune evasion, autoregulation of the viral life cycle and tumorigenesis (45). Dysregulation of miRNAs, including increased miR-155 expression, enhanced host innate immunity and promoted T cell differentiation in human T-cell lymphotropic virus type 1 (HTLV-1) infection (46). miRNAs dysregulated by influenza virus infection also exerted an effect on virus replication and host immune responses.
[0842] INNOVATION: To our knowledge, this is the first study to focus and determine mechanisms regarding sex disparity in an age-and sex-associated viral lung disease, COVID-19.
[0843] There are two novel aspects to this proposal:
[0844] Preliminary data in this proposal (Aim 1) suggest that disease phenotype can be conferred by exosomes. We also show that this is easily assessed in lung punches. We will utilize this combination to dissect the role of gonadal hormones and their respective receptors in discovery of sex disparities in COVID-19 mortality.
[0845] This proposal will utilize the FCG mouse model to investigate the contribution of sex chromosomes to viral lung disease. Results from the FCG mice experiments will lead to further studies to find the X or Y genes responsible for the sex chromosome effect, and to study how those gene(s) interact with sex steroid-sensitive pathways to affect disease. Thus, we are breaking down sex into its component parts, to be studied one by one, to build pathways of regulation of disease. We believe that results found in mice will be most easily translated to humans when specific X or Y genes, which are common to human and mouse sex chromosomes, are discovered in mice to generate hypotheses for their action in human. Examining sex effects in this way and in Aim 2 is innovative for the viral field and a rigorous next step toward understanding the increased male mortality in COVID-19.
Preliminary Data:
[0846] We have previously shown that exosomes can be isolated from diverse biofluids: Serum-derived and cell derived exosome isolation was performed by Zen Bio Inc (Research Triangle, N C). Exosome size from blood and cells was approximately 30-150 um. From 10 ml of serum we isolate between 10.sup.10-10.sup.12 particles of exosomes. This provides enough material from the same patient for the experiments outlined in SA1 and 2. EM was performed on isolated exosomes (
[0847] All aims in this proposal will utilize exosomes isolated from male and female patient serum. Patient samples will be matched for age and disease severity as outlined in Table 9, below and are available in the UA Biobank.
[0848] Exosomes derived from lungs of patients with and without lung disease enter alveolar epithelial cells. To visualize whether exosomes were present after injection into lung punches, we utilized Transmission electron microscopy (TEM). TEM revealed exosomes labeled with gold nanoparticles in alveolar (AEC) type I and type II cells (red arrows,
[0849] We have also previously shown that exosomes confer disease to lung punches via miRNAs. Lung punches from a healthy 15 month old male mouse were treated with exosomes derived from lung cells with and without fibrotic lung disease. As shown in
[0850] Statistical analysis for in vitro and in vivo studies will be as follows: data will be expressed as mean±SE. For single time point studies, unpaired Student's t test will be used for comparison between two groups, while 1-way ANOVA followed by Tukey post-hoc test will be used for multiple comparisons. For all studies statistical significance is defined as p<0.05. For in vivo experiments, as outlined in Vertebrate Animals, the number of mice in each group will be selected based on power calculations. All assays will be performed with a minimum of 3 biological replicates/sex, three technical triplicates, and at least three independent experiments. Data will be analyzed by investigators blinded to experimental group.
[0851] Research Strategy
[0852] Sex as a biological variable: We include equal numbers of each sex in every experiment with subset analyses. Age as a biological variable: Since the mortality of COVID-19 is higher in patients >50, we will compare exosomes from adult (21-40 years old) and older (>50) patients to evaluate age-related biological differences in males and females.
Aim 1: Determine the contribution of hormone receptor expression and subsequent downstream signaling in sex disparity of COVID-19 associated lung disease.
[0853] Rationale: Males are disproportionately dying of COVID-19 infections (http://globalhealth5050.org/covid19; accessed Jun. 13, 2020), while the incidence and prevalence of infection is equal between males and females (1, 2). This is particularly evident in cohorts over the age of 50. Gonadal hormones regulate normal lung development, physiology, and are implicated in several lung diseases including asthma, pulmonary fibrosis, and pulmonary hypertension in males and females (21, 22). In addition, since the lung is primarily affected by SARS-CoV2, in this aim we will investigate the effect of steroid hormone receptor signaling that may be stimulated differently in the male and female lung by exosomes derived from male and female patients with COVID-19 that will confer disease to the punch.
[0854] Experimental design: We will use lung punches from young adult (3-month-old C57BL6 male and female mice transgenic for human ACE2 molecule (B6.Cg-Tg (K18-ACE2).sub.2Prlmn/J, Jax #034860). This is necessary because mice are not susceptible to SARS-COV2 unless the human ACE2 molecule is present in their cells via transgenesis or transduction. The K18-hACE2 Tg mice are breeding in the Nikolich lab since June 2020 and we do not foresee any problem with generating the numbers outlined below. We will isolate exosomes from n=3 male and n=3 female patients diagnosed with COVID-19 from each of the following groups: 21-40 years old with severe (hospitalized to Intensive Care Unit— ICU) COVID-19— adult severe (AS); >50 years of age with severe COVID-19— older severe (OS); 21-40 years old with moderate/mild (not hospitalized) COVID-19— adult moderate (AM); >50 years of age with moderate/mild COVID-19— older moderate (OM), for a total of 24 serum samples, isolated within the first two weeks of infection. Exosomes will be extracted from each serum sample, and samples paired so that one pairwise comparison is made between one female and one male exosome sample within the same group (e.g. one male and one female AS (or OS, or AM or OM) donor will form the AS comparison set 1, (
TABLE-US-00011 TABLE 9 Experiment matrix. Note that each exosome group contained a total of 81 mice. Male Female M + GD M + GDX + F + GDX (M) (F) M + GD F + GD X + F + GD DHT + + Placebo Exosome type intact) intact X X DHT X + E2 Flutamide E2 + ICI control 21-40 years 9 9 9 9 9 9 9 9 9 (Adult) Severe (AS) Male and Female >50 years 9 9 9 9 9 9 9 9 9 (Older, O); Severe (OS), Male and Female 21-40 years 9 9 9 9 9 9 9 9 9 (adult) Moderate/mild; Female >50 years 9 9 9 9 9 9 9 9 9 (older, O); Moderate/mild; Male and Female
[0855] Because one mouse can give us approximately nine equivalent lung punches from each mouse, we will get both biological and technical replicates in each group (3 punches will be vehicle control). Groups of mice vs exosome sets are shown in Table 9. Our preliminary data show that 3D lung punch model (47) recapitulates cellular and tissue interplay in the lung (47, 48), recognizing that they lack immune recruitment and systemic perfusion. Lungs from 3-month-old mice will be instilled with warm agarose at the pressure of 25 cm H2O. The trachea will be ligated just caudal to the larynx and the contents of thorax removed as one unit to maintain airways and alveolar integrity. Lung segments will be cooled on ice for 30 min to allow solidification of the agarose. Using a biopsy punch device, punches (4 mm) (
[0856] Expected results/Alternative outcomes/Potential difficulties: We expect that exosomes from severely sick older males may modulate innate immune defenses of lung epithelia adversely, and that they will be either quantitatively or qualitatively worse for the host lower chemokine induction, increased inflammation (increased IL-6 and 8) compared to older females, younger males, and in particularly, young adult females. Clearly, divergent results will lead us to modify our hypotheses, but will nonetheless begin to establish the basis of sex differences in susceptibility to SARS-COV2 across ages. We recognize the difficulty we may have in “matching” male and female patient exosome preps since we do not have viral load of patients at the time of collection. However, because we will be studying the impact of exosomes on uniform exposure to viral infection of the punch (at a fixed virus infection in vitro), that will be of lesser concern. Although we determined the number of particles necessary to confer disease in vivo and in lung punches, it is possible we may have to perform a dose response with one set of exosome preps. To ensure we have enough exosomes for both SA1 and 2, we will isolate exosomes from at least 2 samples of serum/patient. We have also elected early times post infection (first 10 days) for exosome harvest, expecting that early changes set up the sex-related pathogenesis. Depending on initial results, this timeline may have to be adjusted subsequently. We have preliminary data that hormone receptors in lung punches are regulated by exosome treatment (
Aim 2: Determine the contribution of sex chromosomes to differences in sex disparity in COVID-19 lung disease using the four core genotypes (FCG) mouse model.
[0857] We will compare XX and XY mice, that have the same type of gonads, to determine if sex differences after exposure to COVID-19 derived exosomes are caused in part by genes encoded by the sex chromosomes, which are inherently differently expressed in XX and XY lungs.
[0858] Rationale: Because females appear to be less likely to die of COVID-19, relative to males, we ask what inherently sex-biased factors might protect females or harm males. Although most sex differences have been attributed to different effects of gonadal hormones, sex differences are increasingly found to be caused also by sex-biased effects of X or Y genes (51-53). Y genes affect only males, and numerous X genes are expressed inherently higher or lower in XX than XY because they escape X inactivation or are parentally imprinted (54). This may help explain sex differences in COVID-19 mortality as the X chromosome contains a high density of immune-related genes; therefore, women generally mount stronger innate and adaptive immune responses than men (26). The ACE2 gene is located on the X chromosome, which suggests that women might have higher ACE2 levels and thus be protected against more severe disease compared to men (55). We will test our hypothesis using the FCG mice. Because the sex of the gonads is no longer based on sex chromosome complement (XX vs. XY), The FCG model produces XX and XY gonadal males (XXM, XYM), and XX and XY gonadal females (XXF, XYF) (
[0859] Experimental Design: We have an active breeder colony of FCG mice (Table 10).
TABLE-US-00012 TABLE 10 Description of mouse for lung punches Description of mouse for lung punches Total mouse number XX Male 9 XY Male 9 XXY Female 9 XY Female 9 XX Male GDX 9 XY Male GDX 9 XX Female GDX 9 XY Female GDX 9
[0860] Because the FCG mice would not be susceptible to SARS-CoV2, before taking the biopsies, we will infect them with the adeno-associated virus (AAV) encoding human ACE2 (AAV-hACE2), as described in (27, 34). Four days later, punch biopsies would occur, infected with SARS-CoV2, and examined as in SA1. To confirm whether or not there is hormonal involvement, 3 month old mice will be GDX and lung punches performed. We will inject a set of male and female human COVID exosomes per mouse and test each set in punches from 3 mice. We will perform all assessments on the punches as described in SA 1. Time and resources-permitting, we will next use the exosomes that produce the greatest sex difference and test them in a full in vivo SARS-CoV2 infection model. Exosomes will be injected IV into XX and XY male and female mice day 4 after induction of hACE2 in their lungs (AAV-hACE2 virus). Animals will be infected with SARS-CoV2 6 h later, and their ability to clear the virus from the lungs (viral titers), weight, temperature, activity and 30-d mortality will be followed. This same group will be bled every 7 days to assess antibodies against spike RBD, S2 and neutralizing Ab, using our newly developed serological assays described in (56). A cross-sectional cohort of 6 mice/group will be sacrificed on d10 to evaluate T cell responses as in (56) and in our prior work (32-37)-we will use a 30-color spectral flow cytometry and stimulation with SARS-CoV2 overlapping peptide pools to detect and compare CD4 and CD8 T cell responses. These experiments will begin to address whether exosomes directly modulate SARS-CoV2 pathogenicity in mice in a sex-dependent manner, and whether that modulation may involve modulation of immunity. Histology and other assessments will be performed as described in SA1.
[0861] Expected outcomes and future studies: We may find no difference among groups that differ in sex chromosome complement with the same type of gonads (XXF=XYF; XXM=XYM), but that gonadal males have more severe outcome after male-derived exosomes than gonadal females (XXM>XXF; XYM>XYF). That outcome would cause us to focus exclusively on understanding the protective or harmful effects of gonadal hormones. Alternatively, we may find that XX mice have worse or better outcomes than XY independent of the gonadal sex (XXF< or >XYF; XXM< or >XYM), from which we would conclude that X or Y genes contribute to sex differences in viral outcomes. Because of the male predominance of in COVID-19 mortality, we might expect that XY mice fare worse than XX. However, in other disease models such as obesity and ischemic cardiovascular disease (51, 52, 57), the effects of gonadal hormones counteract the effects of sex chromosome complement. For example, gonadal males weigh more than gonadal females, but XX weigh more than XY. Thus, sex differences in hormones and chromosomes can reduce the effects of each other. We believe that results found in mice will be most easily translated to humans when specific X or Y genes, which are common to human and mouse sex chromosomes, are discovered in mice to formulate hypotheses for their action in human. These genes and their downstream pathways may become targets for novel therapies.
[0862] Timeline: SA1 experiments will begin immediately and will proceed for 18-20 months. In vitro part of SA2 will also start immediately and proceed for two years; as the in vivo part of SA2 builds on initial results from SA1 as well as the in vitro results from SA2, it will start as soon as the first data from these two experimental sets become available, most likely 4-6 months from the start of the project, and will continue to the end of Y2.
[0863] Long-term goals and perspectives: Our unique and transdisciplinary collaboration on the above experimental plan will yield new and important insights to begin to unravel the impact of sex hormones and genes on epithelial lung physiology, innate cellular defenses in alveolar epithelium and, if possible, whole animal immune defense and antiviral resistance. Results from these experiments will allow us to generate refined, specific hypotheses and will pave the way to in-depth, mechanistic and whole animal studies of sex, aging, hormonal signaling and immunity in response to the NIAID PAR-20-178 or similar initiatives.
REFERENCES CITED
[0864] 1. Gebhard C, Regitz-Zagrosek V, Neuhauser H K, Morgan R, Klein S L. Impact of sex and gender on COVID-19 outcomes in Europe. Biol Sex Differ 2020; 11: 29. [0865] 2. Guan W J, Ni Z Y, Hu Y, Liang W H, Ou C Q, He J X, Liu L, Shan H, Lei C L, Hui D S C, Du B, Li L J, Zeng G, Yuen K Y, Chen R C, Tang C L, Wang T, Chen P Y, Xiang J, Li S Y, Wang J L, Liang Z J, Peng Y X, Wei L, Liu Y, Hu Y H, Peng P, Wang J M, Liu J Y, Chen Z, Li G, Zheng Z J, Qiu S Q, Luo J, Ye C J, Zhu S Y, Zhong N S, China Medical Treatment Expert Group for C. [0866] Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med 2020; 382: 1708-1720. [0867] 3. Klein S L, Flanagan K L. Sex differences in immune responses. Nat Rev Immunol 2016; 16: 626-638. [0868] 4. Robinson D P, Huber S A, Moussawi M, Roberts B, Teuscher C, Watkins R, Arnold ΔP, Klein S L. Sex chromosome complement contributes to sex differences in coxsackievirus B3 but not influenza A virus pathogenesis. Biol Sex Differ 2011; 2: 8. [0869] 5. Anderson M R, Kashanchi F, Jacobson S. Exosomes in Viral Disease. Neurotherapeutics 2016; 13: 535-546. [0870] 6. Schorey J S, Harding C V. Extracellular vesicles and infectious diseases: new complexity to an old story. J Clin Invest 2016; 126: 1181-1189. [0871] 7. Umetani M, Domoto H, Gormley A K, Yuhanna I S, Cummins C L, Javitt N B, Korach K S, Shaul P W, Mangelsdorf D J. 27-Hydroxycholesterol is an endogenous SERM that inhibits the cardiovascular effects of estrogen. Nat Med 2007; 13: 1185-1192. [0872] 8. Umetani M, Ghosh P, Ishikawa T, Umetani J, Ahmed M, Mineo C, Shaul P W. The cholesterol metabolite 27-hydroxycholesterol promotes atherosclerosis via proinflammatory processes mediated by estrogen receptor alpha. Cell Metab 2014; 20: 172-182. [0873] 9. Doublier S, Lupia E, Catanuto P, Elliot S J. Estrogens and progression of diabetic kidney damage. Curr Diabetes Rev 2011; 7: 28-34. [0874] 10. Elliot S J, Karl M, Berho M, Potier M, Zheng F, Leclercq B, Striker G E, Striker U. Estrogen deficiency accelerates progression of glomerulosclerosis in susceptible mice. Am J Pathol 2003; 162: 1441-1448. [0875] 11. Elliot S J, Karl M, Berho M, Xia X, Pereria-Simon S, Espinosa-Heidmann D, Striker G E. Smoking induces glomerulosclerosis in aging estrogen-deficient mice through cross-talk between TGF-beta1 and IGF-I signaling pathways. J Am Soc Nephrol 2006; 17: 3315-3324. [0876] 12. Karl M, Berho M, Pignac-Kobinger J, Striker G E, Elliot S J. Differential effects of continuous and intermittent 17beta-estradiol replacement and tamoxifen therapy on the prevention of glomerulosclerosis: modulation of the mesangial cell phenotype in vivo. Am J Pathol 2006; 169: 351-361. [0877] 13. Nelson A W, Tilley W D, Neal D E, Carroll J S. Estrogen receptor beta in prostate cancer: friend or foe? Endocr Relat Cancer 2014; 21: T219-234. [0878] 14. Siegfried J M, Stabile L P. Estrongenic steroid hormones in lung cancer. Semin Oncol 2014; 41: 5-16. [0879] 15. Glassberg M K, Catanuto P, Shahzeidi S, Aliniazee M, Lilo S, Rubio G A, Elliot S J. Estrogen deficiency promotes cigarette smoke-induced changes in the extracellular matrix in the lungs of aging female mice. Transl Res 2016; 178: 107-117. [0880] 16. Glassberg M K, Choi R, Manzoli V, Shahzeidi S, Rauschkolb P, Voswinckel R, Aliniazee M, Xia X, Elliot S J. 17beta-estradiol replacement reverses age-related lung disease in estrogen-deficient C57BL/6J mice. Endocrinology 2014; 155: 441-448. [0881] 17. Triebner K, Matulonga B, Johannessen A, Suske S, Benediktsdottir B, Demoly P, Dharmage S C, Franklin K A, Garcia-Aymerich J, Gullon Blanco J A, Heinrich J, Holm M, Jarvis D, Jogi R, Lindberg E, Moratalla Rovira J M, Muniozguren Agirre N, Pin I, Probst-Hensch N, Puggini L, Raherison C, Sanchez-Ramos J L, Schlunssen V, Sunyer J, Svanes C, Hustad S, Leynaert B, Gomez Real F. Menopause Is Associated with Accelerated Lung Function Decline. Am J Respir Crit Care Med 2017; 195: 1058-1065. [0882] 18. Vermeulen A, Kaufman J M, Goemaere S, van Pottelberg I. Estradiol in elderly men. Aging Male 2002; 5: 98-102. [0883] 19. Libert C, Dejager L, Pinheiro I. The X chromosome in immune functions: when a chromosome makes the difference. Nat Rev Immunol 2010; 10: 594-604. [0884] 20. Zheng H Y, Li Y, Dai W, Wei C D, Sun K S, Tong Y Q. Imbalance of testosterone/estradiol promotes male CHD development. Biomed Mater Eng 2012; 22: 179-185. [0885] 21. Sathish V, Martin Y N, Prakash Y S. Sex steroid signaling: implications for lung diseases. Pharmacol Ther 2015; 150: 94-108. [0886] 22. Sathish V; Prakash Y. Sex differences in pulmonary anatomy and physiology: Implications for health and disease. Sex differences in physiology; 2016. p. 89-106. [0887] 23. Elliot S, Periera-Simon S, Xia X, Catanuto P, Rubio G, Shahzeidi S, El Salem F, Shapiro J, Briegel K, Korach K S, Glassberg M K. MicroRNA let-7 Downregulates Ligand-Independent Estrogen Receptor-mediated Male-Predominant Pulmonary Fibrosis. Am J Respir Crit Care Med 2019; 200: 1246-1257. [0888] 24. Alghamdi I G, Hussain, I I, Almalki S S, Alghamdi M S, Alghamdi M M, El-Sheemy M A. The pattern of Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive epidemiological analysis of data from the Saudi Ministry of Health. Int J Gen Med 2014; 7: 417-423. [0889] 25. Channappanavar R, Fett C, Mack M, Ten Eyck P P, Meyerholz D K, Perlman S. Sex-Based Differences in Susceptibility to Severe Acute Respiratory Syndrome Coronavirus Infection. J Immunol 2017; 198: 4046-4053. [0890] 26. Schurz H, Salie M, Tromp G, Hoal E G, Kinnear C J, Moller M. The X chromosome and sex-specific effects in infectious disease susceptibility. Hum Genomics 2019; 13: 2. [0891] 27. Takahashi T, Wong P, Ellingson M, Lucas C, Klein J, Israelow B, Silva J, Oh J, Mao T, Tokuyama M, Lu P, Venkataraman A, Park A, Liu F, Meir A, Sun J, Wang E, Wyllie A L, Vogels C B F, Earnest R, Lapidus S, Ott I, Moore A, Casanovas A, Dela Cruz C, Fournier J, [0892] Odio C, Farhadian S, Grubaugh N, Schulz W, Ko A, Ring A, Omer S, Iwasaki A. Sex differences in immune responses to SARS-CoV-2 that underlie disease outcomes. medRxiv 2020: 2020.2006.2006.20123414. [0893] 28. Montgomery R R, Shaw A C. Paradoxical changes in innate immunity in aging: recent progress and new directions. J Leukoc Biol 2015; 98: 937-943. [0894] 29. Nikolich-Zugich J. The twilight of immunity: emerging concepts in aging of the immune system. Nat Immunol 2018; 19: 10-19. [0895] 30. Rockx B, Baas T, Zornetzer G A, Haagmans B, Sheahan T, Frieman M, Dyer M D, Teal T H, Proll S, van den Brand J, Baric R, Katze M G. Early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection. J Virol 2009; 83: 7062-7074. [0896] 31. Sheahan T, Whitmore A, Long K, Ferris M, Rockx B, Funkhouser W, Donaldson E, Gralinski L, Collier M, Heise M, Davis N, Johnston R, Baric R S. Successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus. J Virol 2011; 85: 217-230. [0897] 32. Brien J D, Uhrlaub J L, Hirsch A, Wiley C A, Nikolich-Zugich J. Key role of T cell defects in age-related vulnerability to West Nile virus. J Exp Med 2009; 206: 2735-2745. [0898] 33. Cicin-Sain L, Brien J D, Uhrlaub J L, Drabig A, Marandu T F, Nikolich-Zugich J. Cytomegalovirus infection impairs immune responses and accentuates T-cell pool changes observed in mice with aging. PLoS Pathog 2012; 8: e1002849. [0899] 34. Li G, Smithey M J, Rudd B D, Nikolich-Zugich J. Age-associated alterations in CD8alpha+dendritic cells impair CD8 T-cell expansion in response to an intracellular bacterium. Aging Cell 2012; 11: 968-977. [0900] 35. Smithey M J, Renkema K R, Rudd B D, Nikolich-Zugich J. Increased apoptosis, curtailed expansion and incomplete differentiation of CD8+ T cells combine to decrease clearance of L. monocytogenes in old mice. Eur J Immunol 2011; 41: 1352-1364. [0901] 36. Uhrlaub J L, Pulko V, DeFilippis V R, Broeckel R, Streblow D N, Coleman G D, Park B S, Lindo J F, Vickers I, Anzinger J J, Nikolich-Zugich J. Dysregulated TGF-beta Production Underlies the Age-Related Vulnerability to Chikungunya Virus. PLoS Pathog 2016; 12: e1005891. [0902] 37. Uhrlaub J L, Smithey M J, Nikolich-Zugich J. Cutting Edge: The Aging Immune System Reveals the Biological Impact of Direct Antigen Presentation on CD8 T Cell Responses. J Immunol 2017; 199: 403-407. [0903] 38. Batsali A K, Georgopoulou A, Mavroudi I, Matheakakis A, Pontikoglou C G, Papadaki H A. The Role of Bone Marrow Mesenchymal Stem Cell Derived Extracellular Vesicles (MSC-EVs) in Normal and Abnormal Hematopoiesis and Their Therapeutic Potential. J Clin Med 2020; 9. [0904] 39. Dilsiz N. Role of exosomes and exosomal microRNAs in cancer. Future Sci O A 2020; 6: FSO465. [0905] 40. Rani S, Ryan A E, Griffin M D, Ritter T. Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications. Mol Ther 2015; 23: 812-823. [0906] 41. Giebel B, Kordelas L, Borger V. Clinical potential of mesenchymal stem/stromal cell-derived extracellular vesicles. Stem Cell Investig 2017; 4: 84-84. [0907] 42. Colombo M, Raposo G, Théry C. Biogenesis, Secretion, and Intercellular Interactions of Exosomes and Other Extracellular Vesicles. Annual Review of Cell and Developmental Biology 2014; 30: 255-289. [0908] 43. Keller S, Ridinger J, Rupp A-K, Janssen J W G, Altevogt P. Body fluid derived exosomes as a novel template for clinical diagnostics. J Transl Med 2011; 9: 86-86. [0909] 44. Lou G, Chen Z, Zheng M, Liu Y. Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases. Experimental & Molecular Medicine 2017; 49: e346-e346. [0910] 45. Mishra R, Kumar A, Ingle H, Kumar H. The Interplay Between Viral-Derived miRNAs and Host Immunity During Infection. Frontiers in Immunology 2020; 10. [0911] 46. Bellon M, Lepelletier Y, Hermine O, Nicot C. Deregulation of microRNA involved in hematopoiesis and the immune response in HTLV-I adult T-cell leukemia. Blood 2009; 113: 4914-4917. [0912] 47. Zscheppang K, Berg J, Hedtrich S, Verheyen L, Wagner D E, Suttorp N, Hippenstiel S, Hocke A C. Human Pulmonary 3D Models For Translational Research. Biotechnol J 2018; 13. [0913] 48. Alsafadi H N, Uhl F E, Pineda R H, Bailey K E, Rojas M, Wagner D E, Konigshoff M. Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery. Am J Respir Cell Mol Biol 2020; 62: 681-691. [0914] 49. Zhao Y, Qin L, Zhang P, Li K, Liang L, Sun J, Xu B, Dai Y, Li X, Zhang C, Peng Y, Feng Y, Li A, Hu Z, Xiang H, Ogg G, Ho L-P, McMichael A, Jin R, Knight J C, Dong T, Zhang Y. Longitudinal COVID-19 profiling associates IL-IRA and IL-10 with disease severity and RANTES with mild disease. JCI Insight 2020; 5: e139834. [0915] 50. Bulynko Y A, O'Malley B W. Nuclear receptor coactivators: structural and functional biochemistry. Biochemistry 2011; 50: 313-328. [0916] 51. Arnold ΔP, Cassis L A, Eghbali M, Reue K, Sandberg K. Sex Hormones and Sex Chromosomes Cause Sex Differences in the Development of Cardiovascular Diseases. Arterioscler Thromb Vasc Biol 2017; 37: 746-756. [0917] 52. Chen X, McClusky R, Chen J, Beaven S W, Tontonoz P, Arnold A P, Reue K. The number of x chromosomes causes sex differences in adiposity in mice. PLoS Genet 2012; 8: e1002709. [0918] 53. Smith-Bouvier D L, Divekar A A, Sasidhar M, Du S, Tiwari-Woodruff S K, King J K, Arnold A P, Singh R R, Voskuhl R R. A role for sex chromosome complement in the female bias in autoimmune disease. J Exp Med 2008; 205: 1099-1108. [0919] 54. Arnold ΔP. A general theory of sexual differentiation. J Neurosci Res 2017; 95: 291-300. [0920] 55. Bhatia K, Zimmerman M A, Sullivan J C. Sex Differences in Angiotensin-Converting Enzyme Modulation of Ang (1-7) Levels in Normotensive W K Y Rats. American Journal of Hypertension 2013; 26: 591-598. [0921] 56. Rippinger T W, M.; Wong, R.; Uhrlaub, J. L.; Castaneda, Y.; Pizzato, H.; Thompson, M. R.; Bradshaw, C.; Erickson, H. L.; Weinkauf, C. C; Bime, C.; Know, K.; Bixby, B.; Dake, M. D.; Parthasarthy, S.; Edwards, T.; Kaplan, M. E.; Scott, S. J.; Sprissler, R.; Nikolich-Zuglich, P; Bhattacharya, D. Detection, prevalence and duration of humoral responses to SARS-CoV2 under conditions of limited population exposure (In press). [0922] 57. Li J, Chen X, McClusky R, Ruiz-Sundstrom M, Itoh Y, Umar S, Arnold ΔP, Eghbali M. The number of X chromosomes influences protection from cardiac ischaemia/reperfusion injury in mice: one X is better than two. Cardiovasc Res 2014; 102: 375-384.
Example 5. Production of Exosomes in a Large-Scale Bioreactor Platform
[0923] Process development runs in which exosome products have been successfully produced and tested using a large scale, bioreactor method have been completed. In short, MSC products are expanded in the 3D Bioreactor until 80% confluence. Expansion media is then washed with PBS and replaced with serum-free DMEM. Conditioned media is collected from the waste outlet of the bioreactor and subjected to ultracentrifugation for exosome precipitation. This collection procedure is performed daily for up to 96 hours. Nanosight nanoparticle tracking analysis reveals the product to have a mean particle size of 101.2±7.5 nm, which is the expected size of exosomes. The protocol consistently produces high concentrations and total particle yields suitable for multiple clinical doses.
[0924]
TABLE-US-00013 TABLE 11 Flow cytometry analysis of three independent batches CD63 CD9 CD105 Batch #1 92.2% 75.5% 0.37% Batch #2 94.4% 89.7% 1.33% Batch #3 90.9% 61.4% 1.50%
[0925]
[0926] 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.