WO2020247675A1 - Procédés pour atténuer une infection virale et pour traiter une lésion pulmonaire - Google Patents

Procédés pour atténuer une infection virale et pour traiter une lésion pulmonaire Download PDF

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WO2020247675A1
WO2020247675A1 PCT/US2020/036182 US2020036182W WO2020247675A1 WO 2020247675 A1 WO2020247675 A1 WO 2020247675A1 US 2020036182 W US2020036182 W US 2020036182W WO 2020247675 A1 WO2020247675 A1 WO 2020247675A1
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Marilyn GLASSBERG CSETE
Jodi GURNEY
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Spiritus Therapeutics, Inc.
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Definitions

  • the described invention generally relates to mesenchymal stem cell-derived extracellular vesicles, compositions thereof, and uses thereof for attenuating viral infection and for treating lung injury.
  • RV infections are an important cause of morbidity and, in some settings, of mortality.
  • One important feature of RV infections is the nonspecific nature of clinical signs and symptoms.
  • the epidemiology is characterized by annual outbreaks during the winter and spring seasons; some respiratory viruses, however some (e.g., PIVs), are prevalent throughout the year.
  • PIVs e.g., Proliferative virus
  • the Baltimore classification of viruses is based on the viral mechanism of mRNA production. Viral genomes may be single-stranded (ss) or double stranded (ds), RNA or DNA, and may or may not use reverse transcriptase. Additionally, single stranded RNA viruses may be either positive sense (+) or negative or antisense (-). This classification places viruses into seven groups, as shown in Table 1. [0004] Table 1: Baltimore classification of viruses
  • HCT recipients Human metapneumovirus (liMPV) can cause upper respiratory tract infection and fatal interstitial pneumonia in HCT recipients [Id., citing Dignan, F., C. et al. Parainfluenza type 3 infection post stem cell transplant: high prevalence but low mortality. J. Hosp. Infect. (2006) 63:452-458].
  • Adenovirus pneumonia can occur in HCT recipients, especially following in vivo or ex vivo T-cell depletion [Id., citing Flomenberg, P., J., et al. Increasing incidence of adenovirus disease in bone marrow transplant recipients. J. Infect. Dis. (1994) 169:775-781 ; Lion, T., et al.
  • Intestinal adenovirus infection with increasing viral load in stool is an early indicator of impending disseminated disease in children undergoing T-cell depleted allogeneic stem cell transplantation.
  • Respiratory viruses can cause major morbidity and also mortality in solid organ transplantation (SOT) recipients. Incident infection depends on virus exposure, but the disease severity appears to be different in different organ transplant settings.
  • liver and renal transplant recipients appear to be somewhat less affected [Id., citing Peigue-Lafeuille, HN, et al., Severe respiratory syncytial virus pneumonia in an adult renal transplant recipient: successful treatment with ribavirin. Scand. J. Infect. Dis. (1990) 22:87-89; Sinnott, J. T., et al. Respiratory syncytial virus pneumonia in a cardiac transplant recipient. J. Infect. Dis. (1988) 158:650-651, Wendt, C. H. Community respiratory viruses: organ transplant recipients. Am. J. Med. (1997) 102:31-36, 42-43].
  • Table 2 Classification of exemplary human respiratory viruses
  • RNA viruses of the Mononegavirales order contain single-stranded genomes that are negative sense.
  • This order is comprised of seven families including Orthomyxoviridae (which contains the influenza viruses) and Paramyxoviridae (which contains the parainfluenza viruses (PIVs), human respiratory syncytial virus [RSV], and human metapneumovirus [hMPV]).
  • PAVs parainfluenza viruses
  • RSV human respiratory syncytial virus
  • hMPV human metapneumovirus
  • the Picomaviridae are nonenveloped viruses with a single- stranded genome of positive polarity.
  • the rhinoviruses are the most important members of this family causing respiratory diseases in humans; other viruses in this group that can cause respiratory disease are enteroviruses (such as coxsackieviruses and numbered enteroviruses). Over 100 serotypes of rhinovirus have been identified. Viruses from the family Coronaviridae also contain positive-sense single-stranded RNA (ssRNA) but are enveloped.
  • ssRNA positive-sense single-stranded RNA
  • Human bocavirus a member of the Parvoviridae family, was discovered in 2005 in nasopharyngeal aspirates from children with respiratory tract infections. Although suspected, establishing its role as a respiratory pathogen has been difficult for several reasons. First, human bocavirus is not related to any known human respiratory pathogen. Second, it is commonly detected with other respiratory viruses, which have established pathogenic potential. Third, detection may simply reflect asymptomatic persistence or prolonged viral shedding, since other human parvoviruses also show' this capacity. It has been suggested that human bocavirus may be reactivated or produce a transient asymptomatic super-infection triggered by the presence of another replicating respiratory agent. [McNamara, PS, Van Doom HR, Respiratory viruses and atypical bacteria. In Manson’s Tropical Infectious Diseases (23 rd Ed.) (2014), 215-224].
  • Measles is caused by Rubeola vims, which belongs to the Paramyxovirus family. It is a major cause of lower respiratory tract infection, accounting for 6-21% of its morbidity and 8-50% of its mortality. Radiographic evidence of pneumonia is common, also in clinically uncomplicated measles.
  • the effects of the vims on the respiratory tract can be direct (giant cell pneumonitis), in any part of the respiratory tract, or indirect. The latter includes the suppressive effects of the vims on the host immune system, stores of vitamin A and overall nutritional status, all of which may lead to an increased risk of super-infection with other (viral or bacterial) pathogens.
  • Measles pneumonitis can be especially severe in immunocompromised patients. [Id.]
  • HPS Hantavirus pulmonary syndrome
  • Herpesvirus Herpesviridae
  • Herpesviruses are a member of the family Herpesviridae . They are double-stranded DNA viruses. Varicella pneumonitis can occur as a severe complication of chickenpox or in the absence of classical symptoms. It occurs more commonly in adults (an estimated 1:400) and can be life threatening it occurs during pregnancy or in immunocompromised patients. Although relatively rare, radiographical abnormalities of the lungs without respiratory symptoms are reported in more than 15% of adults with chicken pox. Pneumonitis can also rarely occur as a complication of cytomegalovirus or Epstein-Barr virus mononucleosis. [Id.) Influenza (Myoviridae)
  • influenza virus A There are three genera of influenza viruses: influenza virus A, B, and C.
  • the influenza viruses, especially influenza virus A are the most variable of the respiratory viruses.
  • Influenza A viruses are subtyped based on their two surface antigens: hemagglutinin (HA; H1-H16) and neuraminidase (NA; N1-N9), which are responsible for host receptor binding/cell entry and cleavage of the HA-receptor complex to release newly formed viruses, respectively.
  • Aquatic birds are the natural reservoir of influenza A viruses, harboring all possible subtypes. (Id.)
  • Both influenza vims A and B exhibit antigenic drift. This phenomenon occurs when the surface antigens of the virus gradually change, progressively and directionally, to escape immunological pressure from the host species. Yearly epidemics of influenza vims A and B are caused worldwide by these drift variants, and contribute to mortality (an estimate 250- 500,000 every year) in the elderly, and in those with pre-existing conditions, such as chronic cardiopulmonary or renal disease; diabetes, immunosuppression, or severe anemia).
  • Sporadic dead-end human infections of animal (especially avian) viruses are known to occur and have caused concern regarding pandemic potential. Highly pathogenic H5N1 viruses were first detected in birds in 1996 in China. In 2003, the vims re-emerged in China.
  • Coronavimses a large family of single-stranded RNA viruses, can infect a wide variety of animals, including humans, causing respiratory, enteric, hepatic and neurological diseases.
  • RNA viruses a large family of single-stranded RNA viruses
  • Yin, Y., Wunderink, RG, Respirology (2016) 23 (2): 130-37 citing Weiss, SR, Leibowitz, IL, Coronavims pathogenesis. Adv. Virus Res. (2011) 81: 85-164].
  • Human coronavimses which were considered to be relatively harmless respiratory pathogens in the past, have now received worldwide attention as important pathogens in respiratory tract infection.
  • CoVs are further divided into four genera: alpha-, beta-, gamma- and delta-coronavims.
  • CoVs cause mainly respiratory tract infections.
  • HCoVs human coronavimses
  • SARS-CoV severe acute respiratory syndrome-CoV
  • MERS-CoV Middle East respiratory syndrome-CoV
  • Spike proteins (S proteins) of HCoVs are a surface-located trimeric glycoprotein consisting of two subunits: the N-terminal SI subunit and the C-terminal S2 subunit.
  • the SI subunit specializes in recognizing and binding to the host cell receptor while the S2 region is responsible for membrane fusion.
  • ACE2 the receptor for SARS-CoV and HCoV-NL63 [Id., citing Weiss, SR,
  • Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavims. Nature (2003) 426: 450-54; Hofmann, H. et al., Proc. Natl Acad. Sci. USA (2005) 102: 7988-93] is a surface molecule localized on arterial and venous endothelial cells, arterial smooth muscle cells, epithelia of the small intestine and the respiratory tract.
  • ACE2 In the respiratory tract, ACE2 is expressed on the epithelial cells of alveoli, trachea, and bronchi, bronchial serous glands, and alveolar monocytes and macrophages. ACE2 is a homologue of the ACE protein, and both are key enzymes of the renin-angiotensin system.35 ACE2 plays a protective role in lung failure and its counterpart ACE promoting lung edema and impaired lung function. [Id., citing Imai, Y., et al. Cir. J. (2010) 74: 405-10]. Downregulation of ACE2, as occurs during SARS-CoV infection, is believed to contribute to pathological changes in the lung.
  • HCoV-NL63 also employs the SARS receptor for cellular entry [Id., citing Hofmann, H. et al., Proc. Natl Acad. Sci. USA (2005) 102: 7988- 93] despite their markedly different pathogenicity and disease courses. This finding suggests that receptor usage may not be the only factor that determines the severity of HCoV infection.
  • DPP4 Dipeptidyl peptidase 4
  • CD26 the receptor for DPP4
  • MERS-CoV [Id., citing Meyerholz, DK et al. Am. J. Pathol. (2016) 186: 78-86] is a multifunctional cell-surface protein widely expressed on epithelial cells in kidney, small intestine, liver and prostate and on activated leukocytes.
  • DPP4 is expressed in the upper respiratory tract epithelium of camels. [Id., citing Widagdo, W. et al. J. Vir. (2016) 90: 4838-42]. In the human respiratory tract, DPP4 is mainly expressed in alveoli rather than the nasal cavity or conducting airways. [Id., citing Meyerholz, DK et al. Am. J. Pathol.
  • DPP4 is a key factor in the activation of T cells and immune response costimulatory signals in T cells, which could indicate a possible manipulation of the host immune system. [Id., citing Boonacker, E., Van Noorden, CJ. Eur. J. Cell Biol. (2003) 82: 53-73]
  • IFN interferon
  • IFN-a interferon-a
  • IFN-b interferon-b
  • IFN-g interferon-a
  • IFN-b interferon-b
  • IFN-g interferon-g
  • Delayed recognition is critical for HCoVs to survive and replicate in the host.
  • MERS-CoV Similar to SARS-CoV, MERS-CoV also fails to induce IFNs prior to 12 h, with the exception of IFN-a5. Lau et al. serially measured mRNA levels of eight cytokine genes up to 30 h post-infection in Calu-3 cells infected with MERS-CoV and SARS-CoV. [Id., citing Lau, SK et al. J. Gen. Virol. (2013) 94: 2679-90].
  • Calu-3 cells infected by MERS-CoV showed marked induction of the proinflammatory cytokines IL-Ib, IL-6 and IL-8 at 30 h but lack of production of the innate antiviral cytokines tumour necrosis factor (TNF)-a, IFN-b and IEN-g-induced protein- 10, compared with SARS-CoV.
  • TNF tumour necrosis factor
  • IFN-b IFN-b
  • IEN-g-induced protein- 10 compared with SARS-CoV.
  • Both SARS and MERS present with a spectrum of disease severity ranging from flu-like symptoms to acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • Acute kidney injury is a significant characteristic of both SARS and
  • MERS patients MERS patients.
  • SARS patients One study reported that 6.7% of SARS patients had acute renal impairment and 84.6% had proteinuria. [Id., citing Chu, KH et al. Kidney Int. (2005) 67: 98-705]. AKI is much more common in MERS patients, occurring in up to 43%. [Id., citing Saad, M. et al. Int. J. Infect. Dis. (2014). 29: 301-6].
  • thrombocytopenia and lymphopenia were common in both SARS [Id., citing Peiris, JS, et al, Lancet (2003) 361: 1767-72; Booth,, CM et al, JAMA (2003) 289: 2801-9] and MERS patients. [Id., citing Assiri, A. et al. Lancet Infect. Dis. (2013) 13: 752-61; Arabi, YM, et al, Ann. Intern. Med. (2014) 160: 387-97] Thrombocytopenia and lymphopenia may be predictive of fatal outcome in MERS -Co V patients. [Id., citing Arabi, YM, et al, Ann. Intern. Med. (2014) 160: 387-97]. Other laboratory findings included elevated CK, lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase levels.
  • Air-space opacities are the main radiographical feature in SARS patients. [Id., citing Booth, CM, et al. JAMA (2003) 289: 2801-9; Wong, KT, et al. Radiology (2003) 228: 401-6]. In one retrospective study, initial chest radiographs were abnormal in 108 of 138 (78.3%) of SARS patients and all showed air-space opacities. [Id., citing Wong, KT, et 1. Radiology (2003) 228: 401-6]. Of these 108 patients, 59 had unilateral focal involvement while 49 had either unilateral multifocal or bilateral involvement. Lower lung zone (64.8%) and right lung (75.9%) were more commonly involved.
  • vasopressor therapy was proposed to be an independent risk factor for death in the ICU. [Id., citing Almekhlafi, GA et al. Crit. Care (2016) 20: 123].
  • MERS demonstrated a higher case fatality rate than SARS.
  • Differences in host factors, such as age and underlying diseases, [Id., citing Chan, KS, et al. Respirology (2003) 8-(Suppl): S36-40); Choi, WS et al. Infect. Chemother. (2016) 48: 118-26] may explain some differences.
  • differential cell line susceptibility, viral replication efficiency, ability to inhibit IFN production and receptor characteristics may also be responsible for the difference in the outcome of SARS-CoV and MERS-CoV infection.
  • HCoVs infection is available. Symptomatic and supportive treatment is the mainstay of therapy for patients infected by HCoVs.
  • a number of agents show effectiveness in vitro and/or in animal models and may improve the outcome in patients.
  • the most commonly prescribed antiviral regimens in the clinical settings are ribavirin, IFNs and lopinavir/ritonavir.
  • Ribavirin a nucleoside analogue, has a wide spectrum of antiviral activity by inhibiting viral RNA synthesis and mRNA capping.
  • IFNs are important for host defense against viruses.
  • IFNs are important for host defense against viruses.
  • IFN products were effective in inhibiting both SARS-CoV and MRES-CoV, with best antiviral activity seen with IFN-pib.
  • IFN-pib IFN-pib.
  • Previous studies had shown a positive impact of various IFNs on aspects of treatment of SARS and MERS patients, such as a better oxygen saturation and rapid resolution of inflammation, but no effect on more significant outcomes like hospital stay and long-term survival.
  • citing Al-Tawfiq IA et al. Inti. J. Infect. Dis. (2014) 20: 42-6; Omrani, AS et al. Lancet Infect. Dis. (2014) 14: 1090-95; Loutfy, MR et al. JAMA (2003) 290: 3222-28].
  • Lopinavir and ritonavir are protease inhibitors that may inhibit the 3C-like protease of MERS-CoV and modulate apoptosis in human cells.
  • Addition of lopinavir/ritonavir to ribavirin was associated with improved clinical outcome compared with ribavirin alone in SARS patients.
  • lopinavir only showed suboptimal 50% effective cytotoxic concentration (EC50) against MERS-CoV in vitro, [Id., citing Chan, JF et al. J. Infect.
  • MPA Mycophenolic acid
  • Corticosteroids were used extensively during the SARS outbreak, generally in combination with ribavirin. Lessons from SARS showed that corticosteroid treatment was associated with a higher subsequent plasma viral load [Id., citing Lee, N. et al., J. Clin. Virol. (2004) 31: 304-9] with increased complications.
  • COVID- 19 can present as an asymptomatic carrier state, acute respiratory disease, and pneumonia.
  • Adults represent the population with the highest infection rate; however, neonates, children, and elderly patients can also be infected by SARS-CoV-2.
  • nosocomial infection of hospitalized patients and healthcare workers, and viral transmission from asymptomatic carriers are possible.
  • the most common finding on chest imaging among patients with pneumonia was ground-glass opacity with bilateral involvement. Severe cases are more likely to be older patients with underlying comorbidities compared to mild cases. Indeed, age and disease severity may be correlated with the outcomes of COVID- 19. [Id].
  • the human immune system is 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.
  • the innate arm of the immune system is a nonspecific fast response to pathogens that is predominantly responsible for an initial inflammatory response via a number of soluble factors, including the complement system and the chemokine/cytokine system; and a number of specialized cell types, including mast cells, macrophages, dendritic cells (DCs), and natural killer cells (NKs).
  • soluble factors including the complement system and the chemokine/cytokine system
  • NKs natural killer cells
  • the adaptive arm involves 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.
  • these immune responses are initiated by an encounter between an individual and a foreign substance, e.g., an infectious microorganism.
  • the infected individual rapidly responds with both a humoral immune response with the production of antibody molecules specific for the antigenic determinants/epitopes of the immunogen, and a cell mediated immune response with the expansion and differentiation of antigen- specific regulatory and effector T-lymphocytes, including cells that produce cytokines and killer T cells, capable of lysing infected cells.
  • ALI acute lung injury
  • ARDS acute respiratory distress syndrome
  • ARDS are syndromes of acute respiratory failure that result from acute pulmonary edema and inflammation.
  • ALLARDS is a cause of acute respiratory failure that develops in patients of all ages from a variety of clinical disorders, including sepsis (pulmonary and nonpulmonary), pneumonia (bacterial, viral, and fungal), aspiration of gastric and oropharyngeal contents, major trauma, and several other clinical disorders, including severe acute pancreatitis, drug over dose, and blood products [Ware, L. and Matthay, M., N Engl J Med, 342:1334-1349, 2000]. Most patients require assisted ventilation with positive pressure.
  • the primary physiologic abnormalities are severe arterial hypoxemia as well as a marked increase in minute ventilation secondary to a sharp increase in pulmonary dead space fraction.
  • Patients with ALLARDS develop protein-rich pulmonary edema resulting from exudation of fluid into the interstitial and airspace compartments of the lung secondary to increased permeability of the barrier. Additional pathologic changes indicate that the mechanisms involved in lung edema are complex and that edema is only one of the pathophysiologic events in ALLARDS.
  • One physiologic consequence is a significant decrease in lung compliance that results in an increased work of breathing [Nuckton T. et al, N Engl J Med, 346:1281-1286, 2002], one of the reasons why assisted ventilation is required to support most patients.
  • MV ventilator-associated lung injury
  • HV T high tidal volumes
  • MV mechanical ventilation
  • ACE2 has an opposing function to ACE and protects against lung injury.
  • the protective effect of ACE2 seems to result partially from the coversion of angiotensin II by ACE2 to angiotensin 1-7, thereby reducing angiotensin II binding to the cell membrane receptors ATlaR (angiotensin II type la receptor) and AT2R (angiotensin II type 2 receptor). It is believed that angiotensin II binding to ATlaR will stimulate lung injury, whereas binding to AT2R reduces lung injury.
  • Kuba et al propose that binding of SARS- CoV to ACE2 downregulates ACE2, thus leaving angiotensin II unmodified, allowing it to continue to bind to the ATlaR to aggravate the lung injury and produce lung edema. [Kuba, K. et al., Nat. Med. (2005) 11: 875-79].
  • Age-dependent differences in host defense and the pulmonary renin-angiotensin system are responsible for observed differences in epidemiology of ARDS between children and adults.
  • higher levels of markers involved in the neutrophil response e.g., levels of myeloperoxidase, interleukin (IL-6), IL-10 and p-selectin were higher with increasing age, whereas ICAM-1 was higher in neonates.
  • IL-6 interleukin
  • ICAM-1 was higher in neonates.
  • age-dependent differences in the RAS system are responsible for observed differences in epidemiology of ARDS between children and adults
  • age was not associated with changes in the pulmonary renin-angiotensin system (RAS); indeed, no differences in activity of ACE and ACE2 were seen.
  • EGFR plays a key role in the host response to SARS-CoV, and also may be implicated in lung disease induced by other highly pathogenic respiratory viruses. [Venkataraman et al. J. Virool. (2017) 91 (12): e00182-17]. Using mouse models of SARS- CoV pathogenesis, the wound repair pathway, controlled by the epidermal growth factor receptor (EGFR), was shown to be critical to recovery from SARS-CoV-induced tissue damage.
  • EGFR epidermal growth factor receptor
  • mice with constitutively active EGFR they found that SARS-CoV infection causes enhanced lung disease.
  • SARS-CoV infection causes enhanced lung disease.
  • the EGFR ligands amphiregulin and heparin-binding EGF-like growth factor (HB-EGF) are upregulated, and exogenous addition of these ligands during infection leads to enhanced lung disease and altered wound healing dynamics.
  • HB-EGF heparin-binding EGF-like growth factor
  • Fibrosis represents the formation or development of excess fibrous connective tissue in an organ or tissue, which is formed as a consequence of the normal or abnormal/reactive wound healing response leading to a scar. Although the fibrogenic response may have adaptive features in the short term, when it progresses over a prolonged period of time, parenchymal scarring and ultimately cellular dysfunction and organ failure ensue (Rockey DC et al., N Engl J Med. 2015 Mar 19; 372(12): 1138-49).
  • Fibrosis is characterized by, for example, without limitation, an aberrant deposition of an extracellular matrix protein, an aberrant promotion of fibroblast proliferation, an aberrant induction of differentiation of a population of fibroblasts into a population of myofibroblasts, an aberrant promotion of attachment of myofibroblasts to an extracellular matrix, or a combination thereof.
  • the fibrinogenic response There are four major phases of the fibrinogenic response.
  • First is initiation of the response, driven by primary injury to the organ.
  • the second phase is the activation of effector cells, and the third phase is the elaboration of extracellular matrix, both of which overlap with the fourth phase, during which the dynamic deposition (and insufficient resorption) of extracellular matrix promotes progression to fibrosis and ultimately to end- organ failure (Id.).
  • Acute and chronic inflammation often trigger fibrosis (Id.). Inflammation leads to injury of resident epithelial cells and often endothelial cells, resulting in enhanced release of inflammatory mediators, including cytokines, chemokines, and others. This process leads to the recruitment of a wide range of inflammatory cells, including lymphocytes, polymorphonuclear leukocytes, eosinophils, basophils, mast cells, and macrophages. These inflammatory cells elicit the activation of effector cells which drive the fibrogenic process (Id., citing Wynn TA. Nat Rev Immunol 2004; 4: 583-94).
  • macrophages can play a prominent role in interstitial fibrosis, often driven by the TGF-b pathway (Id., citing Meng XM, et al. Nat Rev Nephrol 2014; 10: 493-503).
  • some inflammatory cells may be protective.
  • certain populations of macrophages phagocytose apoptotic cells that promote the fibrogenic process and activate matrix-degrading metalloproteases (Id., citing Ramachandran P, Iredale JP. J Hepatol 2012; 56: 1417-9).
  • Fibroblasts and myofibroblasts have been identified as key fibrosis effectors in many organs, and as such are responsible for the synthesis of extracellular matrix proteins (Id., citing Hinz B, et al. Am J Pathol 2007; 170: 1807-16).
  • the matrix proteins that compose the fibrotic scar consist predominantly of interstitial collagens (types I and III), cellular fibronectin, basement-membrane proteins such as laminin, and other, less abundant elements.
  • myofibroblasts which by definition are cells that express smooth-muscle proteins, including actin (ACTA2), are contractile (Id., citing Rockey DC, et al. J Clin Invest 1993; 92: 1795-804). The contraction of these cells contributes to the distortion of parenchymal architecture, which can promote disease pathogenesis and tissue failure.
  • myofibroblasts also contribute to the normal wound healing process by contracting the edges of the wound and synthesizing and depositing extracellular matrix components (Hinz B. Curr Res Transl Med. 2016 Oct - Dec; 64(4): 171-177; Darby IA, et al. Clin Cosmet Investig Dermatol. 2014; 7: 301-311).
  • TGF-b cascade which plays a major role in fibrosis, involves the binding of a ligand to a serine-threonine kinase type II receptor that recruits and phosphorylates a type I receptor. This type I receptor subsequently phosphorylates SMADs, which function as downstream effectors, typically by modulating target gene expression.
  • TGF-b which is a potent stimulator of the synthesis of extracellular matrix proteins in most fibrogenic cells, and is synthesized and secreted by inflammatory cells and by effector cells, thereby functioning in both an autocrine and paracrine fashion (Id.).
  • PDGF Platelet-derived growth factor
  • CTGF vasoactive peptide systems
  • angiotensin II and endothelin-1 play important roles
  • vasoactive systems endothelin plays a role in fibrosis in virtually all organ systems, acting through G- protein-coupled endothelin-A or endothelin-B cell-surface receptors or both (Id., citing Khimji AK, Rockey DC. Cell Signal 2010; 22: 1615-25).
  • angiogenic pathways may be important in fibrosis (Id., citing Johnson A, DiPietro LA. FASEB J 2013; 27: 3893- 901).
  • Integrins which link extracellular matrix to cells, are considered critical in the pathogenesis of fibrosis (Id., citing Levine D, et al. Am J Pathol 2000; 156: 1927-35; Henderson NC, et al. Nat Med 2013; 19: 1617-24).
  • Pulmonary fibrosis occurs in association with a wide range of diseases, including scleroderma (systemic sclerosis), sarcoidosis, and infection, and as a result of environmental exposures (e.g., silica dust or asbestos), but in most patients it is idiopathic and progressive.
  • Pulmonary fibrosis is characterized by parenchymal honeycombing (meaning the characteristic appearance of variably sized cysts in a background of densely scarred lung tissue. Microscopically, enlarged airspaces surrounded by fibrosis with hyperplastic or bronchiolar type epithelium are present.
  • IPF Idiopathic Pulmonary Fibrosis
  • Idiopathic Pulmonary fibrosis also known as cryptogenic fibrosing alveolitis, CFA, or Idiopathic Fibrosing Interstitial Pneumonia
  • IPF interstitial pneumonia
  • uncertain etiology that occurs primarily in older adults, is limited to the lungs, and is associated with the radiologic and histological pattern of usual interstitial pneumonia (UIP) (Raghu G. et al, Am J Respir Crit Care Med., 183(6): 788-824, 2011; Thannickal, V. et al, Proc Am Thorac Soc., 3(4): 350- 356, 2006).
  • IPF afflicts nearly 130,000 patients in the United States, with approximately
  • IPF can be described as accumulation of activated myofibroblasts (or mesenchymal cells) in fibroblastic foci (Thannickal, V. et al., Proc Am Thorac Soc., 3(4): 350-356, 2006). Impaired apoptosis of myofibroblasts may result in a persistent and dysregulated repair process that culminates in tissue fibrosis. Arguably, inflammation also plays a critical role in IPF, perhaps through cyclic acute stimulation of fibroblasts. These findings point to potential targets for therapeutic intervention.
  • the heart undergoes extensive structural and functional remodeling (meaning a group of molecular, cellular and interstitial changes that manifest clinically as changes in size, mass, geometry and function of the heart) in response to injury, central to which is the hypertrophy (meaning an increase in size of the individual muscle cells without changing their total number) of cardiac myocytes, with excessive deposition of extracellular matrix (Rockey DC et al, N Engl J Med. 2015 Mar 19; 372(12): 1138-49, citing Hill JA, Olson EN. N Engl J Med 2008; 358: 1370-80).
  • Myocardial fibrosis is commonly categorized as one of two types: reactive fibrosis or replacement fibrosis. Reactive fibrosis occurs in perivascular spaces (meaning the fluid-filled space that surrounds a blood vessel or organ) and corresponds to similar fibrogenic responses in other tissues; replacement fibrosis occurs at the site of myocyte loss.
  • fibrosis is attributed to cardiac fibroblasts, the most abundant cell type in the myocardium, the middle muscular layer of the heart wall. These cells are derived from fibroblasts that are native to the myocardium, from circulating fibroblasts, and from fibroblasts that emerge from epithelial-to-mesenchymal transition (Id., citing Zeisberg EM, et al. Nat Med 2007; 13: 952-61; Moore-Morris T, et al. J Clin Invest 2014; 124: 2921-34).
  • Cardiac fibrosis contributes to both systolic and diastolic dysfunction and to perturbations of electrical excitation; it also disrupts repolarization (Id., citing Spinale FG. Physiol Rev 2007; 87: 1285-342). Proarrhythmic effects (meaning worsening of existing arrhythmias) are the most prominent. Collagenous septa in the failing heart contribute to arrhythmogenesis by inducing a discontinuous slowing of conduction (Id., citing Spach MS, Boineau JP. Pacing Clin Electrophysiol 1997; 20: 397-413).
  • microreentrant circuits meaning small areas of continuous circulating electricity in which an impulse reenters and repetitively excites a region of the heart; reentrant circuits are the basis of most clinical arrhythmias. Smith W, Hood M. Arrhythmias, in Cardiothoracic Critical Care, 2007) occurs as a result of the heterogeneous spatial distribution of fibrosis and the triggering of activity caused by the depolarization of myocytes by electrically coupled myofibroblasts (Rockey DC et al., N Engl J Med. 2015 Mar 19; 372(12): 1138-49, citing Tanaka K, et al. Circ Res 2007; 101: 839-47; Miragoli M, et al. Circ Res 2007; 101: 755-8).
  • Fibrotic scarring in the heart correlates strongly with an increased incidence of arrhythmias and sudden cardiac death (Id., citing Wu KC, et al. J Am Coll Cardiol 2008; 51: 2414-21). For example, a 3% increase in the extracellular volume fraction of fibrous tissue (measured by means of magnetic resonance imaging after the administration of gadolinium) is associated with a 50% increase in the risk of adverse cardiac events (Id., citing Wong TC, et al. Circulation 2012; 126: 1206-16).
  • the liver is made up of two lobes, each of which is made up of thousands of hexagonally- shaped lobules.
  • Each lobule is made up of numerous liver cells, called hepatocytes, that are cuboidal epithelial cells that line up in radiating rows and make up the majority of cells in the liver.
  • Hepatocytes perform most of the liver's functions— metabolism, storage, digestion, and bile production.
  • sinusoids which are small blood vessels lined by hepatocytes that diffuse oxygen and nutrients through their capillary walls into the liver cells.
  • the lobules are connected to small bile ducts that connect with larger ducts to ultimately form the hepatic duct.
  • Hepatic biliary cells which line the bile ducts, are targets of liver injury, but also orchestrate liver repair. They undergo extensive morphogenesis to form a complex network of intrahepatic biliary ducts. This network functions to drain the bile produced by hepatocytes to the intestine.
  • Hepatic stellate cells exist in the space between parenchymal cells and sinusoidal endothelial cells of the hepatic lobule and store 80% of retinoids in the whole body as retinyl palmitate in lipid droplets in the cytoplasm.
  • retinoid homeostasis In physiological conditions, these cells play pivotal roles in the regulation of retinoid homeostasis, which contributes to many diverse functions including vision, inflammatory/immune response, adipogenesis, cell differentiation, and insulin sensitivity.
  • pathological conditions such as liver fibrosis, hepatic stellate cells lose retinoids and synthesize a large amount of extracellular matrix (ECM) components including collagen, proteoglycan, and adhesive glycoproteins (Senoo H. Med Electron Microsc. 2004 Mar; 37(1): 3-15). Healthy sinusoidal endothelial cells maintain hepatic stellate cell quiescence, thus inhibiting their vasoconstrictive effect (Poisson J, et al. J Hepatol. 2017 Jan; 66(1): 212-227).
  • ECM extracellular matrix
  • Hepatic fibrosis typically results from an inflammatory process that affects hepatocytes or biliary cells. Inflammation leads to the activation of effector cells, which results in the deposition of extracellular matrix. Although a variety of effectors synthesize extracellular matrix in the liver, hepatic stellate cells appear to be the primary source of extracellular matrix. Abundant evidence suggests that the stellate cell is pericyte-like (pericytes are spatially isolated contractile cells on capillaries which control blood flow), undergoing a transformation into a myofibroblast in response to injury (Rockey DC et al., N Engl J Med. 2015 Mar 19; 372(12): 1138-49, citing Rockey DC, et al. J Clin Invest 1993; 92: 1795-804).
  • Kupffer cells the resident macrophages of the liver
  • bile-duct cells the resident macrophages of the liver
  • immune cells orchestrate the cellular and molecular response to injury (Id., citing Rockey DC. Clin Gastroenterol Hepatol 2013; 11(3): 224-31).
  • a pathway that appears to be unique to the liver involves toll-like receptor 4 (TLR4); TLR4 is activated on the surface of stellate cells by intestinal bacterial lipopolysaccharides derived from the gut (i.e., translocated bacteria), triggering cell activation and fibrogenesis and thereby linking fibrosis to the microbiome (Id., citing Seki E, et al.
  • TLR4 expression is associated with portal inflammation and fibrosis in patients with fatty liver disease (Id., citing Vespasiani-Gentilucci U, et al. Liver Int 2015; 35: 569-81).
  • hepatic fibrogenesis is cirrhosis, an ominous parenchymal lesion that underlies a wide range of devastating complications that have adverse effects on survival.
  • Portal hypertension meaning an increase in the pressure within the portal vein, which carries blood from the digestive organs to the liver
  • a devastating result of injury develops during the fibrogenic response after disruption of the normal interaction between sinusoidal endothelial cells and hepatic stellate cells; the resulting activation and contraction of pericyte-like stellate cells leads to sinusoidal constriction (sinusoidal capillaries are a special type of capillary that have a wide diameter) and increased intrahepatic resistance (meaning the resistance in the liver vascular bed to the flow that reaches the liver via the portal vein, which can be assessed experimentally, based on Ohm’s law, by measuring portal pressure changes when an increasing portal venous flow is applied).
  • the intrarenal renin-angiotensin-aldosterone axis (a signaling pathway that regulates the body’s blood pressure by homeostatic control of arterial pressure, tissue perfusion, and extracellular volume) is particularly important in hypertension-induced fibrosis (Id., citing Mezzano SA, et al. Hypertension 2001; 38: 635-8).
  • the kidney’s unique cellular architecture consists of the glomeruli (meaning a tuft formed of capillary loops at the beginning of each nephiric tubule in the kidney; this tuft with its capsule (Bowman’s capsule) constitutes the Malpighian body), tubules (meaning the portion that extends from the Bowman capsule in the kidney cortex (meaning the outer part of the kidney between the renal capsule and the renal medulla) into the kidney medulla (meaning the innermost part of the kidney), interstitium (meaning the intratubular, exxtraglomerular, extravascular space of the kidney), and capillaries. Damage at any of these sites triggers the deposition of extracellular matrix (Id., citing Burchfield JS, et al.
  • Glomeruli and podocytes (highly specialized cells of the kidney glomerulus that wrap around capillaries and that neighbor cells of the Bowman's capsule, see Reiser J and Altintas MM. Podocytes.
  • Podocytes cooperate with mesangial cells (contractile cells that constitute the central stalk of the glomerulus) to support the structure and function of the glomerulus (see, e.g., Pavenstadt, H, Am. J. Physiol. Renal Physol. (2000) 278 (2): F173-F179).
  • Mesangial cells have characteristics of a modified smooth muscle cell, but also are capable of generation of prostaglandins and mediators of inflammation; production and breakdown of basement membrane and other biomatrix material; synthesis of cytokines, and uptake of macromolecules, including immune complexes (see Schlndorff D., FASEB J. (1987) 1(4): 272-81).
  • Glomerular fibrosis regardless of the cause, diminishes renal blood flow, which leads to hypoxia and the activation of hypoxia- inducible factor 1, a dimeric protein complex that plays an integral role in the body's response to low oxygen concentrations, or hypoxia, which in turn triggers nephron collapse and fibrotic replacement by means of rarefaction (meaning a decrease in the capillary density) (Id., citing Seki E, et al. Nat Med 2007; 13: 1324-32).
  • the renal interstitium and capillaries contribute substantially to tubulointerstitial disease, as peritubular pericytes migrate into the interstitium, where they are transformed into myofibroblasts (Id., citing Fouts DE, et al. J Hepatol 2012; 56: 1283-92).
  • renal fibrosis leads to loss of function and organ failure.
  • Homeostasis can be maintained with a glomerular filtration rate as low as approximately 10% of the normal rate.
  • anemia develops and the regulation of electrolyte balance and pH is disrupted (Id.).
  • Ionizing radiation induces damage not only in rapidly proliferating tumor cells but also in normal tissue in the radiation field.
  • a significant contributor to patient morbidity is radiation-induced fibrosis (RIF), which may occur in the skin and subcutaneous tissue, lungs, gastrointestinal and genitourinary tracts, as well as any other organs in the treatment field.
  • RIF radiation-induced fibrosis
  • Radiation injury triggers inflammation and ultimately stimulates transdifferentiation of fibroblasts into myofibroblasts. In addition to their excessive proliferation, these myofibroblasts produce excess collagen and other extracellular matrix (ECM) components, which is compounded by a reduction in remodeling enzymes.
  • ECM extracellular matrix
  • RIF usually occurs 4-12 months after radiation therapy and progresses over several years. It affects almost every part of the body that is exposed to radiation. The clinical presentation depends on the type of tissue exposed to irradiation. In general, RIF may manifest as skin induration and thickening, muscle shortening and atrophy, limited joint mobility, lymphedema, mucosal fibrosis, ulceration, fistula, hollow organ stenosis, and pain (Id., citing Dorr W, Hendry JH. Radiother Oncol J Eur Soc Ther Radiol Oncol. 2001; 61: 223-231).
  • An initial injury incites an acute response that leads to inflammation, followed by fibroblast recruitment and activation with extracellular matrix deposition.
  • Radiation is energy in the form of waves or high-speed particles.
  • the term“ionizing” indicates that said energy is strong enough to displace bound electrons.
  • Ionizing radiation refers to three types of emissions— alpha, beta, and gamma— with therapeutic radiation being predominantly gamma (Id., citing Harrison JD, Stather JW. J Anat. 1996; 189(Pt 3): 521-530).
  • Radiation injury results from two primary mechanisms: direct DNA damage and the generation of reactive oxygen species (ROS) (Id., citing Travis EL. Semin Radiat Oncol. 2001 Jul; 11(3): 184-96).
  • ROS reactive oxygen species
  • Reactive nitrogen species also likely play a role in radiation injury, as treatment with the inducible nitric oxide synthase (iNOS) inhibitor, L- nitroarginine methyl ester (L-NAME), prevented acute lung injury in rats (Id., citing Khan MA, et al. Radiother Oncol J Eur Soc Ther Radiol Oncol. 2003; 66: 95-102). Free radicals damage all components of cells, including proteins, nucleic acids, and lipids (Id., citing Terasaki Y, et al. Am J Physiol Lung Cell Mol Physiol. 2011; 301: L415-L426; Zhao W, Robbins ME. Curr Med Chem. 2009; 16: 130-143).
  • iNOS inducible nitric oxide synthase
  • L-NAME L- nitroarginine methyl ester
  • Injured cells release chemoattractant molecules that trigger nonspecific inflammation (Id., citing Denham JW, Hauer- Jensen M. Radiother Oncol J Eur Soc Ther Radiol Oncol. 2002; 63: 129- 145; Travis EL. Semin Radiat Oncol. 2001 Jul; 11(3): 184-96; Williams JP, et al. Curr Drug Targets. 2010; 11: 1386-1394).
  • thrombosis and ischemia exacerbate local injury leading to further release of inflammatory chemokines and cytokines (Id., citing Boerma M, Hauer-Jensen M. Curr Drug Targets. 2010; 11: 1405-1412; Lefaix JL, Daburon F. Health Phys. 1998; 75: 375-384).
  • Neutrophils are the first inflammatory cells to arrive at the site of injury (Id., citing Abreu MT, et al. J Immunol. 2005; 174: 4453-4460). Increased expression of intercellular adhesion molecule 1 (ICAM-1) (Id., citing Hallahan DE, et al. J Natl Cancer Inst. 2002; 94: 733-741) and platelet endothelial cell adhesion molecule 1 (PECAM-1) (Id., citing Quarmby S, et al. Arterioscler Thromb Vase Biol.
  • ICM-1 intercellular adhesion molecule 1
  • PECAM-1 platelet endothelial cell adhesion molecule 1
  • Platelet-derived growth factor (PDGF) secreted from the M2 subset promotes neoangiogenesis and stimulates the migration of fibroblasts into the injured tissue from either the surrounding stroma or from circulating mesenchymal stem cells (Id., citing Li M, Jendrossek V, Belka C. Radiat Oncol. 2007; 2: 5; Mathew M, Thomas SM. In: Li X, editor. Squamous cell carcinoma. InTech; 2012. pp. 163-174). They also secrete TGF-b, which is heavily implicated in RIF (Id., citing Li MO, et al. Annu Rev Immunol. 2006; 24: 99-146).
  • PDGF Platelet-derived growth factor
  • TGF-b is responsible for a number of functions that contribute to the pathogenesis of this condition, including the production of fibroblasts from bone marrow progenitors (Id., citing Campana F, et al. J Cell Mol Med. 2004; 8: 109-116; Rodemann HP, Bamberg M. Radiother Oncol J Eur Soc Ther Radiol Oncol. 1995; 35: 83-90) and the differentiation of fibroblasts into myofibroblasts (Id., citing Yamold J, Brotons MC. Radiother Oncol J Eur Soc Ther Radiol Oncol.
  • a-SMA alpha- smooth muscle actin
  • myofibroblasts may also derive from circulating bone marrow-derived progenitor cells known as fibrocytes or from epithelial cells undergoing epithelial-mesenchymal transition (EMT) (Id., citing Darby IA, Hewitson TD. Int Rev Cytol. 2007; 257: 143-179).
  • EMT epithelial-mesenchymal transition
  • myofibroblasts secrete excess collagen, fibronectin, and proteoglycans (Id., citing Chithra P, et al. J Ethnopharmacol. 1998; 59: 179-186), and in doing so they are responsible for the increased stiffness and thickening of the tissue (Id., citing Lefaix JL, Daburon F. Health Phys. 1998; 75: 375-384; Martin M, et al. Int J Radiat Oncol Biol Phys. 2000; 47: 277-290).
  • TGF-b promotes decreased matrix metalloproteinase (MMP) activity (especially MMP-2 and MMP-9) and increased activity of tissue inhibitors of metalloproteinases (TIMPs), compounding the already excessive ECM deposition (Id., citing Pardo A, Selman M. Proc Am Thorac Soc. 2006 Jun; 3(4): 383-8).
  • MMP matrix metalloproteinase
  • TGF tissue inhibitors of metalloproteinases
  • myofibroblasts promote endothelial cell proliferation and angiogenesis through the secretion of basic fibroblast growth factor (bFGF) (Id., citing Finlay GA, et al. J Biol Chem.
  • Retroperitoneal fibrosis is a rare condition characterized by inflammation and fibrosis in the retroperitoneal space; most cases are idiopathic, but secondary causes include drugs, infections, autoimmune and inflammatory stimuli, and radiation. Patients may present with pain, and the major clinical sequelae of this condition are related to its involvement with structures in the retroperitoneum, including arteries (leading to acute and chronic renal failure) and ureters (leading to hydronephrosis, the swelling of a kidney due to a build-up of urine).
  • TGF-P-integrin signaling (Rockey DC et al., N Engl J Med. 2015 Mar 19; 372(12): 1138-49, citing Margadant C, Sonnenberg A. EMBO Rep 2010; 11: 97-105).
  • TGF-b affects integrin- mediated cell adhesion and migration by regulating the expression of integrins, their ligands and integrin-associated proteins. Conversely, several integrins directly control TGF-b activation.
  • integrins can interfere with both S mad-dependent and S mad-independent TGF-b signaling in different ways, including the regulation of the expression of TGF-b signaling pathway components, the physical association of integrins with TGF-b receptors and the modulation of downstream effectors.
  • Reciprocal TGF-b- integrin signaling is implicated in normal physiology, as well as in a variety of pathological processes including systemic sclerosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and cancer (Margadant C, Sonnenberg A. EMBO Rep. 2010 Feb; 11(2): 97-105).
  • fibrosing skin disease skin fibroblasts and myofibroblasts are activated through the TGF ⁇ -SMAD signaling pathway (Rockey DC et al., N Engl J Med. 2015 Mar 19; 372(12): 1138-49, citing Jinnin M. J Dermatol 2010; 37: 11-25).
  • Nephrogenic systemic fibrosis a debilitating condition that is marked by widespread organ fibrosis, occurs in patients with renal insufficiency who have been exposed to gadolinium- based contrast material.
  • IgG4-related disease appears to involve autoimmune-driven inflammation that provokes fibrosis in multiple organs, including the pancreas, retroperitoneum, lung, kidney, liver, and aorta (Id., citing Umehara H, et al. Mod Rheumatol 2012; 22: 1-14).
  • 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.
  • a wound-healing response often is described as having three distinct phases- injury, inflammation and repair.
  • 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.
  • 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.
  • thrombin a serine protease required to convert fibrinogen into fibrin
  • Thrombin also can directly activate fibroblasts, increasing proliferation and promoting fibroblast differentiation into collagen- producing myofibroblasts.
  • Damage to the airway epithelium 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.
  • 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.
  • 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-b, may prevent excessive fibroblast activation.
  • PAMPs pathogen-associated molecular patterns
  • NOD-like receptors cytoplasmic proteins that have a variety of functions in regulation of inflammatory and apoptotic responses
  • Endogenous danger signals also can influence local innate cells and orchestrate the inflammatory cascade.
  • Fibrotic lung disease such as idiopathic pulmonary fibrosis
  • IL-la interleukin- 1 alpha
  • IL-Ib interleukin- 1 beta
  • IL-6 interleukin-6
  • TGF-a tumor necrosis factor alpha
  • TGF-b transforming growth factor beta
  • PDGFs platelet-derived growth factors
  • 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.
  • fibrin a fibrous protein that is polymerized to form a“mesh” that forms a clot over a wound site
  • Myofibroblast-derived collagens and smooth muscle actin 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 a-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.
  • 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.
  • EDA extra type III domain of fibronectin
  • the provisional extracellular matrix consists of glycoproteins (such as PDGF), glycosaminoglycans (such as hyaluronic acid), proteoglycans and elastin.
  • glycoproteins such as PDGF
  • glycosaminoglycans such as hyaluronic acid
  • proteoglycans and elastin.
  • Growth factor and TGF-P-activated fibroblasts migrate along the extracellular matrix network and repair the wound. Within skin wounds, TGF-b 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 a- SMA, both of which confer the high contractile activity within myofibroblasts.
  • MMPs matrix metalloproteinases
  • TIMPs tissue inhibitor of metalloproteinases
  • TGF-a serum-derived transforming growth factor alpha
  • MMP-7 matrix metalloproteinase-7
  • cytokines polypeptide mediators known as cytokines, including various lymphokines, interleukins, and chemokines, are important stimuli to collagen deposition in fibrosis. Released by resident tissue cells and recruited inflammatory cells, cytokines are thought to stimulate fibroblast proliferation and increased synthesis of extracellular matrix proteins, including collagen. For example, an early feature in the pathogenesis of idiopathic pulmonary fibrosis is alveolar epithelial and/or capillary cell injury. This promotes recruitment into the lung of circulating immune cells, such as monocytes, neutrophils, lymphocytes and eosinophils.
  • effector cells together with resident lung cells, such as macrophages, alveolar epithelial and endothelial cells, then release cytokines, which stimulate target cells, typically fibroblasts, to replicate and synthesize increased amounts of collagen. Breakdown of extracellular matrix protein also may be inhibited, thereby contributing to the fibrotic process.
  • TGF-b tumor necrosis factor-a
  • PDGF platelet-derived growth factor
  • IGF-1 insulin-like growth factor- 1
  • ET-1 endothelin-1
  • IL-1 interleukin- 1
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • IL-17 interleukin- 17
  • Chemokine leukocyte chemoattractants including the factor Regulated upon Activation in Normal T-cells, Expressed and Secreted (RANTES), are also thought to play an important role.
  • Elevated levels of pro-inflammatory cytokines such as Interleukin 8 (IL-8), as well as related downstream cell adhesion molecules (CAMs) such as intercellular adhesion molecule- 1 (ICAM-1) and vascular cell adhesion molecule- 1 (VCAM-1), matrix metalloproteinases such as matrix metalloproteinase-7 (MMP-7), and signaling molecules such as S100 calcium-binding protein A12 (S100A12, also known as calgranulin C), in the peripheral blood have been found to be associated with mortality, lung transplant-free survival, and disease progression in patients with IPF (Richards et al, Am J Respir Crit Care Med, 2012, 185: 67-76).
  • IL-8 Interleukin 8
  • CAMs cell adhesion molecules
  • IAM-1 intercellular adhesion molecule- 1
  • VCAM-1 vascular cell adhesion molecule- 1
  • MMP-7 matrix metalloproteinases
  • signaling molecules such as S100 calcium-binding protein A
  • TGF-b family of proteins has a potent stimulatory effect on extracellular matrix deposition, and in fact has been used in constructing induced animal models of fibrosis through gene transfer.
  • TGF-bI secreted as a latent precursor, promotes fibroblast procollagen gene expression and protein synthesis.
  • the data suggest that the other mammalian isoforms, TGF ⁇ 2 and TGF-bB, also stimulate human lung fibroblast collagen synthesis and reduce breakdown in vitro.
  • enhanced TGF-bI gene expression is temporally and spatially related to increased collagen gene expression and protein deposition.
  • TGF-bI antibodies reduce collagen deposition in murine bleomycin-induced lung fibrosis, and human fibrotic lung tissue shows enhanced TGF-bI gene and protein expression.
  • TNF-a can stimulate fibroblast replication and collagen synthesis in vitro, and pulmonary TNF-a gene expression rises after administration of bleomycin in mice. Soluble TNF-a receptors reduce lung fibrosis in murine models, and pulmonary overexpression of TNF-a in transgenic mice is characterized by lung fibrosis. In patients with IPF or asbestosis (a chronic inflammatory and fibrotic medical condition affecting the parenchymal tissue of the lungs caused by the inhalation and retention of asbestos fibers), bronchoalveolar lavage fluid-derived macrophages release increased amounts of TNF-a compared with controls.
  • IPF or asbestosis a chronic inflammatory and fibrotic medical condition affecting the parenchymal tissue of the lungs caused by the inhalation and retention of asbestos fibers
  • Endothelin also fulfills the criteria for a profibrotic cytokine. This molecule promotes fibroblast proliferation and chemotaxis and stimulates procollagen production. It is present in the lungs of patients with pulmonary fibrosis, and a recent report suggests that bosentan, an ET-1 receptor antagonist, ameliorates lung fibrosis when administered to experimental animals.
  • the lung is a highly quiescent tissue, previously thought to have limited reparative capacity and a susceptibility to scarring. It is now known that the lung has a remarkable reparative capacity, when needed, and scarring or fibrosis after lung injury may occur infrequently in scenarios where this regenerative potential is disrupted or limited (Kotten, D.N. and Morrisey, E.E., “Lung regeneration: mechanisms, applications and emerging stem cell populations,” Nat. Med. (2014) 20(8): 822-32, citing Beers, MF and Morrisey, EE,“The 3 R’s of lung health and disease - repair, remodeling and regeneration,” J. Clin. Invest. (2011) 121: 2065-73; and Wansleeben, C.
  • the tissues of the lung may be categorized as having facultative progenitor cell populations that can be induced to proliferate in response to injury as well as to differentiate into one or more cell types.
  • the adult lung comprises at least 40-60 different cell types of endodermal, mesodermal, and ectodermal origin, which are precisely organized in an elaborate 3D structure with regional diversity along the proximal-distal axis.
  • epithelial cells include cartilaginous cells of the upper airways, airway smooth muscle cells, interstitial fibroblasts, myofibroblasts, lipofibroblasts, and pericytes as well as vascular, microvascular, and lymphatic endothelial cells, and innervating neural cells.
  • lung epithelial stem/progenitor cells in the different regions of the lung are thought to be determined not only by their intrinsic developmental potential but also by the complex interplay of permissive or restrictive cues provided by these intimately associated cell lineages as well as the circulating cells, soluble and insoluble factors and cytokines within their niche microenvironment (McQualter & Bertoncello., Stem Cells. 2012 May; 30(5); 811- 16).
  • Pulmonary endothelial cell interactions with the extracellular matrix In: Voelkel NF, Rounds S, eds. The Pulmonary Endothelium: Function in Health and Disease. Chichester, West Wales: Wiley-Blackwell, 2009: 51-72). Chemotactic factors elaborated by these cell lineages also orchestrate the recruitment of inflammatory cells, which participate in the remodeling of the niche and the regulation of the proliferation and differentiation of its cellular constituents (McQualter & Bertoncello. Stem Cells. 2012 May; 30(5); 811-16).
  • HSCs bone marrow-derived adult hematopoietic stem cells
  • NSCs brain-derived neural stem cells
  • EpSCs gut- and epidermis- derived adult epithelial stem cells
  • ASCs adipose-derived stem cells
  • Endogenous adult stem cells are embedded within the ECM component of a given tissue compartment, which, along with support cells, form the cellular niche.
  • Such cellular niches within the ECM scaffold together with the surrounding microenvironment contribute important biochemical and physical signals, including growth factors and transcription factors required to initiate stem cell differentiation into committed precursors cells and subsequent precursor cell maturation to form adult tissue cells with specialized phenotypic and functional characteristics.
  • FGF-10 is a component of a multifaceted epithelial-mesenchymal cell signaling network involving BMP, Wnt, and Shh pathways which coordinate the proliferation and differentiation of progenitor cells in the developing lung (reviewed in Morrisey & Hogan. Dev Cell. 2010 Jan 19; 18(1): 8-23).
  • Endothelial-epithelial cell interactions and angiogenic and angiocrine factors elaborated in the lung epithelial stem/progenitor cell microenvironment also play a role in the regulation of endogenous lung epithelial stem/progenitor cell regeneration and repair (Yamamoto et al., Dev Biol. 2007 Aug 1; 308(1): 44-53; Ding et al., Cell. 2011 Oct 28; 147(3): 539-53; Crivellato. Int J Dev Biol. 2011; 55(4-5): 365-75; Grinnell & Harrington. Pulmonary endothelial cell interactions with the extracellular matrix. In: Voelkel NF, Rounds S, eds.
  • EPC derived from human umbilical cord blood, bone marrow, and mobilized peripheral blood
  • the rarity of EPC in the lung have been major impediments in assessing the contribution of endogenous lung EPC in lung vascular repair, and lung regeneration and remodeling (Thebaud & Yoder. Pulmonary endothelial progenitor cells. In: Voelkel NF, Rounds S, eds. The Pulmonary Endothelium: Function in Health and Disease. Chichester, West Wales: Wiley, 2009: 203-16; Yoder. Proc Am Thorac Soc. 2011; 8: 466-70).
  • Lung macrovascular and microvascular endothelial cells can be resolved on the basis of their preferential binding to the lectins Helix pomatia and Griffonia simplicifolica, respectively (King et al., Microvasc Res. 2004; 67: 139-51), but there are no other cell surface markers that can discriminate mature lung endothelial cells and EPC (Yoder. Proc Am Thorac Soc. 2011; 8: 466-70).
  • EPC Yoder. Proc Am Thorac Soc. 2011; 8: 466-70
  • the rarity of EPC has necessitated the ex vivo expansion and passaging of adherent heterogeneous rat (Alvarez et al, Am J Physiol Lung Cell Mol Physiol.
  • lung endothelial cells in liquid culture prior to quantitation and flow cytometric and functional analysis of lung-derived EPC in in vitro assays.
  • lung microvasculature is a rich source of EPC.
  • immunophenotypic and functional properties of EPC in the primary explanted endothelial cells compared with their ex vivo manipulated, selected, and expanded counterparts remains indeterminate.
  • the ability of these endogenous lung EPCs to contribute to vascular repair and remodeling in vivo is also unproven (Yoder. Proc Am Thorac Soc.
  • MSCs Mesenchymal Stem Cells
  • MSCs Mesenchymal stem cells
  • stromal 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 differentiate along a minimum of three lineages (osteogenic, chondrogenic, and adipogenic).
  • 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 CD 14.
  • proliferation potential of MSCs studies have reported that populations of bone marrow- derived MSCs have the capacity to develop into terminally differentiated mesenchymal phenotypes both in vitro and in vivo, including bone, cartilage, tendon, muscle, adipose tissue, and hematopoietic- supporting stroma.
  • Studies using transgenic and knockout mice and human musculoskeletal disorders have reported that MSC differentiate into multiple lineages during embryonic development and adult homeostasis.
  • MSCs are known to undergo phenotypic rearrangements during ex vivo manipulations, losing expression of some markers while also acquiring new ones. (Augello, A. et al. Eur. Cells and Materials (2010) (20): 121-33, citing Jones, et al. 2002 Arthritis Rheum. 46: 3349-60).
  • MSCs are thought to orchestrate wound repair by: (1) structural repair via cellular differentiation; (2) immune-modulation; (3) secretion of growth factors that drive neovascularization and re-epithelialization; and (4) mobilization of resident stem cells.
  • Results indicate that MSCs play several simultaneous roles: limiting inflammation through releasing cytokines; aiding healing by expressing growth factors; altering host immune responses by secreting immuno-modulatory proteins; enhancing responses from endogenous repair cells; and serving as mature functional cells in some tissues such as bone (Phinney, DG and Pittenger, MF. Stem Cells (2017) 35: 851-58).
  • MSCs When labeled and delivered in vivo, MSCs will migrate to sites of tissue injury (Id.). CD44-HA interaction is involved in MSC migratory activities (Zhu, H. et al. Stem Cells (2006) 24: 928- 35).
  • MSCs also can respond to chemotactic signaling molecules acting on pathways other than the SDF- l/CXCR-4 axis, including monocyte chemotactic protein-3 (MCP-3) (Id.). MSCs for cell-based therapy
  • MSCs reduce T-cell proliferation, suppress the inflammatory infiltrates and cytokines and express anti-inflammatory cytokines (Id.).
  • MSCs for animal immune disorder models of arthritis (Id., citing Zhou B, et al. Clin Immunol 2011; 141: 328-337; Gonzalez MA, et al. Arthritis Rheum 2009; 60: 1006-1019; Liu Y, et al. Arthritis Res Ther 2010; 12: R210), SLE (Id., citing Sun L, et al. Stem Cells 2009; 27: 1421-1432; Chang JW, et al.
  • MSCs have been used in clinical trials as an immunomodulator in the treatment of diseases such as GvHD, organ transplantation, diabetes, multiple sclerosis and Crohn’s disease.
  • diseases such as GvHD, organ transplantation, diabetes, multiple sclerosis and Crohn’s disease.
  • Several clinical trials on GvHD and multiple sclerosis have shown complete or partial responses in some patients, with no serious adverse effects (Id., citing Le Blanc K, et al. Lancet 2008; 371: 1579-1586; Perez-Simon JA, et al. Haematologica 2011; 96: 1072- 1076; Muller I, et al. Blood Cells Mol Dis 2008; 40: 25-32; Baron F, et al. Biol Blood Marrow Transplant 2010; 16: 838-847; Arima N, et al.
  • MSCs can even be immuno stimulatory.
  • the mechanisms involved in this process are largely unknown.
  • Zhou et al. (2013) showed that when mouse spleen T cells were stimulated with allogeneic mixed lymphocyte reaction (MLR) or anti-CD3/CD28 beads and treated with autologous bone marrow MSC or MS C -conditioned medium, MSCs had both suppressive and stimulatory functions toward T cells (Zhou Y, et al. Cytotherapy. 2013 Oct; 15(10): 1195-207). This depended on the ratio of MSC to responder T cells, with low numbers of MSC increasing and higher numbers inhibiting T-cell proliferation. Immuno stimulatory function was mediated, in part, by soluble factors.
  • MSC immunosuppression of the MLR was indirect and related to inhibition of antigen-presenting cell maturation. Direct effects of MSC-conditioned medium during anti-CD3/CD28 stimulated proliferation were entirely stimulatory and required the presence of the T-cell receptor. MSC supernatant contained both CCL2 and CCL5 at high levels, but only CCL2 level correlated with the ability to augment proliferation. An anti- CCL2 antibody blocked this proliferative activity. It was therefore determined that CCL2 plays an important role in the immuno stimulatory function of MSC, and that the immunomodulatory role of MSC is determined by a balance between inhibitory and stimulatory factors, suggesting the need for caution when these cells are investigated in clinical protocols.
  • MSCs can acquire immuno stimulatory properties in certain contexts.
  • MSCs cultured with natural killer (NK) cells primed the NK cells for increased release of IFN-g (a cytokine critical for innate and adaptive immunity) in response to IL-12 and IL-18 (interleukins produced by activated antigen-presenting cells).
  • NK natural killer
  • MSC administration has been proposed as an effective therapy to alleviate bleomycin-induced lung injury and fibrosis.
  • a large proportion of these studies have explored the early inflammatory stage rather than the late fibrotic stage (Li, Xiaohong, et al. (2017) Oncotarget 8(60): 102600-102616).
  • MSCs have been used as a cell therapy to treat pulmonary fibrosis (Id., citing Tzouvelekis, A. et al., Curr. Opinion. Pulm. Med. (2011) 17: 368-73).
  • BM-MSC injection (5xl0 5 /mouse in 200 ml of PBS) through the jugular vein immediately after challenge with BLM can significantly reduce pulmonary fibrosis (Id., citing Ortiz, LA, et al. Proc. Natl Acad. Sci. USA (2003) 100: 8407-8411).
  • hMSC human mesenchymal stem cell transplantation directly replaced fibrosis with normal lung cells and reduced IPF symptoms, such as collagen deposition and inflammation (Id., citing Choi, M. et al. Mol. Cells (2014) 37: 133-39).
  • Bleomycin-induced lung injury and fibrosis were significantly reduced by injection of BM-MSCs by downregulating proinflammatory and angiogenic cytokines and nitric oxide metabolites after 4 days of BLM inhalation (Id., citing Lee, SH et al. Respir. Res. (2010) 11: 16). Zhao et al. also showed the therapeutic effects of BM-MSC engraftment in bleomycin-induced lung damage in rats (Id., citing Zhao, F. et al. Transplant Proc (2008) 40: 1700-1705).
  • BM-MSCs home to the lungs after damage, exhibiting epithelioid phenotypes and reducing inflammation and collagen deposition in BLM-induced animal models (Id., citing Ortiz LA, et al. Proc. Natl Acad. Sci. USA (2003) 100: 8407-8411; Ricciardi M. et al. Blood (2013) 122: 5414).
  • Akram et al. found that hMSCs showed a strong migratory response to alveolar epithelial cell injury in a 3D direct-contact wound repair model (Id., citing Akram KM, et al. Respir. Res. (2013) 14: 9).
  • BM-MSCs The migration of BM-MSCs is mediated by some chemotactic factors and their receptors.
  • the chemokine SDF-1 is crucial for migration to injured tissues via interacting with its cognate receptor CXCR4 on the cellular surface (Id., citing Marquez- Curtis, LA and Janowska-Wieczork, A. BioMed. Res. Int. (2013): 2013; 561098).
  • Xu et al. found that SDF-1 significantly promoted the chemotactic migration of BM-MSCs, but this effect was mimicked by lung extracts from mice after bleomycin treatment and was completely inhibited by TN14003, a synthetic specific CXCR4 antagonist (Id., citing Xu, J.
  • MSCs After homing to injured lungs, MSCs can differentiate into type II alveolar epithelial cells (AECs) and be involved in the renewal of the alveolar epithelium in vitro and in vivo (Id., citing Liu, AR. J. Cell Physiol. (2013) 228: 1270-83; Cai, SX et al. Stem Cell Res. Ther. (2015) 6: 65; Liu, A. et al. PLoS One (2014) 9: e90229). MSC differentiation into type II AECs is mainly mediated by the Wnt pathway (Id., citing Ling, L. et al. Gene (2009) 433: 1-7). Liu et al.
  • AECs alveolar epithelial cells
  • b-catenin and glycogen synthase kinase-3P (GSK-3P) in the canonical Wnt pathway were activated during the differentiation of mouse MSCs into type II AECs (Id., citing Liu, AR. J. Cell Physiol. (2013) 228: 1270-83).
  • GSK-3P glycogen synthase kinase-3P
  • Overexpression of b- catenin in mouse MSCs to activate the canonical Wnt ⁇ -catenin pathway further improved their protective effect against epithelial impairment and therapeutic effects for ARDS in mice (Id., citing Cai, SX et al. Stem Cell Res. Ther. (2015) 6: 65).
  • Wnt5a contributes to MSC differentiation into type II AECs through noncanonical c-Jun N- terminal kinase (JNK) or protein kinase C (PKC) signaling in vitro (Id., citing Liu, A. et al. PLoS One (2014) 9: e90229).
  • JNK noncanonical c-Jun N- terminal kinase
  • PLC protein kinase C
  • MSCs administered to mice during the fibrotic stage of a radiation- induced PF model likewise differentiated into fibroblast-like phenotype and aggravated the fibrotic lesion (Id., citing Mora, AL, Rojas, M. J. Cell Biochem. (2008) 105: 641-47).
  • MSCs isolated from bleomycin-injured mice lungs also were more likely to differentiate into fibroblasts in vitro (Id., citing Skurikhin, E.G. et al. Bull Exp. Biol. Med. (2013) 154: 537-43).
  • MSC-derived conditioned medium can also exert a protective effect against BLM-induced lung injury and fibrosis (Id., citing Shen, Q. et al. Mol. Med. Re. (2015) 11: 2831-37).
  • MSC-CM prevented PF by reducing pulmonary inflammation, fibrosis score, collagen deposition, and cell apoptosis.
  • MSC-CM also protected human non- small-cell lung cancer epithelial cells (A549) from bleomycin-induced apoptosis.
  • bleomycin injury does resolve in some cases, and also responds to use of anti-inflammatory and antifibrotic agents (Borzone G, et al. Am J Respir Crit Care Med. 2001 Jun; 163(7): 1648-53).
  • MSCs have been proposed to possess the capacity to secrete a broad range of bioactive molecules, such as growth factors, cytokines, and chemokines (Li, Xiaohong, et al. (2017) Oncotarget 8(60): 102600-102616, citing Monsel, A. et al. Expert Opin. Biol. Ther. (2016) 16: 859-71; Caplan, A. and Correa, D. Cell Stem Cell (2011) 9: 11-15; Kosuma, GD, et al. Stem Cells Dev. (2017) 26: 617-31). These bioactive molecules regulate local immune response to establish a regenerative microenvironment and subsequently inhibit inflammation and repair the injured tissues (Id.).
  • bioactive molecules such as growth factors, cytokines, and chemokines
  • MSCs have been intensively studied in basic cardiovascular research (Chou SH, et al. Cell Transplant. 2014; 23(4-5): 513-29). Since Wakitani et al. reported that MSCs can differentiate in vitro into a myogenic phenotype, there has been a growing body of evidence that MSCs are effective in improving the cardiac performance of the ischemia/reperfusion (IR) heart (Id., citing Wakitani, S, et al. Muscle Nerve 18: 1417- 1426; 1995). In vitro differentiation of MSCs into cells resembling cardiomyocytes prompted early expectation of their capacity to regenerate these cells in vivo.
  • IR ischemia/reperfusion
  • stromal cell lines, primary stromal cells and MSCs from different species and different tissue sources exhibited a modified phenotype with the adoption of myotube morphology, expression of immature action potentials, and a variety of cardiac- specific genes (e.g., MEF-2A/MEF-2D) and peptides (e.g., myosin, desmin, actinin, atrial natriuretic peptides) (Id., citing Chung, YS, et al. Chin. J. Physiol. 54: 205-218; 2011; Moscoso, I, et al. Transplant. Proc. 37: 481-482; 2005; Rangappa, S, et al. Ann.
  • MEF-2A/MEF-2D cardiac- specific genes
  • peptides e.g., myosin, desmin, actinin, atrial natriuretic peptides
  • Engraftment and differentiation rates of MSCs are relatively low compared with other cellular effects they render, including their paracrine actions (Golpanian S, et al. Physiol Rev. 2016 Jul; 96(3): 1127-1168, citing Leri A, et al. Stem Cell Res: 631-646, 2014). Still, numerous reports regarding these mechanisms of action for both autologous and allogeneic MSCs exist in the literature, albeit conflicting evidence (Id., citing Kim PJ, et al. Circ Res: e40-e50, 2015; Makkar RR, et al. J Cardiovasc Pharmacol Ther: 225-233, 2005; Price MJ, et al.
  • LAD left anterior descending
  • the engrafted cells however began to appear morphologically indistinguishable from the host cardiac myocytes and, starting as early as 2 wk post-injection, revealed de novo expression of desmin, b-myosin heavy chain, a-actinin, cardiac troponin T, and phospholamban with sarcomeric organization of contractile proteins (Id., citing Toma C, et al. Circulation: 93-98, 2002).
  • mice that were administered human placenta-derived amniotic MSCs (AMCs) following left anterior descending (LAD) artery ligation did not demonstrate any immunohistological evidence of engraftment, while those that were treated with c-kit + AMCs showed engraftment yet no cardiac differentiation of cells (Id., citing Kim PJ, et al. Circ Res: e40-e50, 2015).
  • AMCs left anterior descending (LAD) artery ligation
  • Type I collagen is the most frequently found collagen in fibrotic tissues, and the presence of tissue fibrosis has been associated with dysregulation of myocyte regeneration and repair (Id., citing Alexakis C, et al. Am J Physiol Cell Physiol: C661-C669, 2007; Brack AS, et al. Science: 807-810, 2007). Indeed, exposure of MSCs to type I collagen leads to a downregulation of growth and inflammatory gene factors with a resultant decrease in MSC-induced myoblast proliferation potential (Id., citing De Lisio M, et al.
  • MMPs Matrix metalloproteinases
  • ECM extracellular matrix
  • MSCs release a combination of various MMPs and tissue inhibitors that are involved in extracellular remodeling (Id., citing Molina EJ, et al. J Tissue Engineering Regenerative Med: 85-91, 2009).
  • the ratio of MMPs to tissue inhibitors can be modulated by overexpression of certain factors, which can reverse the process of cardiac remodeling (Id., citing Shu T, et al. Tissue Cell: 217-222, 2010; Tang J, et al. Molecules Cells: 9-19, 2010).
  • MSCs are capable of regulating the ECM degradative potential of cardiac fibroblasts, thereby supporting an indirect antifibrotic mechanism (Id., citing Mias C, et al. Stem Cells: 2734-2743, 2009).
  • a decreased incidence of acute rejection has also been demonstrated in patients receiving MSCs at the time of kidney transplantation (Id., citing Tan J, et al. JAMA 307: 1169-1177, 2012).
  • MSC therapy may be an attractive strategy for renal repair, most clinical trials involve only early phases of kidney disease (Id., citing Squillaro T, et al. Cell Transplant 25: 829-848, 2016), and the potential of MSC-based therapy to prevent or ameliorate chronic kidney disease (CKD) is only beginning to be elucidated. Unlike acute alterations elicited in AKI, regression of longstanding structural remodeling like fibrosis is difficult to attain with any therapeutic intervention (Zhu XY, et al. Stem Cells. 2013 Sep; 31(9): 1731-1736).
  • the route of MSC delivery may affect their efficiency for kidney repair.
  • MSC intravenously infused into baboons were observed for 9-21 months, estimated levels of engraftment in the kidney, lung, liver, thymus, and skin ranged from 0.1-2.7% (Zhu XY, et al. Stem Cells. 2013 Sep; 31(9): 1731-1736, citing Devine SM, et al. Blood. 2003; 101: 2999-3001).
  • the intravenous route lags in delivery efficiency, because MSC may initially be trapped in the lungs (Id., citing Fischer UM, et al. Stem Cells Dev.
  • Intra-arterial infusion of MSC was the most effective route to achieve immunomodulation in rat kidney transplantation, possibly by avoiding lodging in the pulmonary circulation, allowing MSC to home to the injured kidney (Id., citing Zonta S, et al. Transplant Proc. 2010;42:1336-1340). Indeed, a retention of 12-14% of intra-arterially injected MSC was observed in one study of experimental ischemic CKD (Id., citing Eirin A, et al. Stem Cells. 2012; 30: 1030-1041).
  • the decrease over time in the paracrine/endocrine effects of MSC may be more important for CKD than AKI, in which the injurious trigger might have been removed.
  • Repeated weekly administration of MSC improves their protective effects in the rat remnant kidney, primarily via paracrine effects (Id., citing Lee SR, et al. Ren Fail. 2010; 32: 840-848). Whether CKD would benefit from multiple MSC administration awaits further testing in CKD models.
  • hypoxic preconditioning enhances MSC recruitment and functional recovery from IRI (Id., citing Liu H, et al. PLoS One. 2012; 7: e34608), but remains to be tested in CKD.
  • liver fibrosis was induced by intraperitoneal or subcutaneous injection of CCL (carbon tetrachloride, a potent hepatotoxin).
  • CCL carbon tetrachloride
  • liver fibrosis such as biliary fibrosis.
  • MSCs may exert a beneficial effect. Indeed, some studies have mentioned the differentiation of MSCs into hepatocyte-like cells (Id., citing Jung KH, et al. Liver Int. 2009; 29: 898-909; Li Q, et al. PLoS One. 2013; 8: e62363) and/or the expression of metalloproteinases by MSCs (Id., citing Chang YJ, et al. Life Sci. 2009; 85: 517-525; Rabani V, et al. Cell Biol Int. 2010; 34: 601-605; Tanimoto H, et al. Cell Tissue Res. 2013; 354: 717-728). The promotion of hepatocyte proliferation and modulation of inflammation have also been proposed (Id., citing Li Q, et al. PLoS One. 2013; 8: e62363).
  • IFN-g interferon-gamma
  • MSCs can be genetically modified using adeno-associated vims vectors to express interferon- alpha (IFN-a) as a therapeutic strategy tested in a mouse model for lung melanoma metastasis [Id., citing Ren, et al., Stem Cells (2008) 26: 2332-38].
  • IFN-a interferon- alpha
  • MSCs have been proposed as viral packaging cell lines for transport of viruses and transduction into target tissues (Id., citing Silva, F., Nardi, N., Med. Hypoth. (2006) 67:922-925; Hakkarainen, T. et al., , Human Gene Therapy (2007) 18:627-641, Ricks, D.M., et al. Stem Cells Dev. (2008) 17:441-450).
  • W02010/053350 disclosed that one or more factors excreted from MSCs is able to inhibit viral replication of hepatitis C virus (HCV) and hepatitis B virus (HBV). At least one factor is larger than 50 kD, because filtration of the exudate through a 3 kD, 10 kD and/or through a 50 kD filter, in which all compounds less than the indicated size were filtered out of the exudate, resulted in a concentrated MSC preparation with an enrichment of antiviral activity. These results were interpreted as indicating that one or more large (>50 kD) compounds are responsible for this effect.
  • HCV hepatitis C virus
  • HBV hepatitis B virus
  • At least one antiviral compound(s) seemed to have a molecular mass between 10 and 50 kD (approx. 80-400 aa), equivalent to the mass of all interferons (180-200 aa) and most chemokines, cytokines and growth factors.
  • the antiviral activity of this latter compound is less than of the >50kD compound(s).
  • Heat treatment did not decrease the antiviral activity, indicating that, if the compound is of a proteinaceous structure, the activity is mainly residing in the primary structure of the protein(s) and is independent of a specific folding of the protein.
  • One embodiment disclosed is a method for treating viral infections in a mammal by exposing the cells in which the virus is replicating to MSCs or to the exudate of MSCs.
  • the MSCs are obtained from human bone marrow, liver or spleen, although other sources for MSC could be considered.
  • MSCs from bone marrow are commercially available, MSCs from liver and or spleen can be prepared as indicated in the examples.
  • the exudate of the MSCs can, if necessary, be enriched by passing it through a 10 kD cut-off filter, discarding the filtrate and resuspending the retentate.
  • the remaining exudate can be enriched in antiviral activity, and any toxic or pathogenic low molecular substance will be deleted from it by this filtration step. Further, resuspending the retentate enables using medium or solvent that is acceptable in pharmaceutical formulations.
  • MSC-CM induces angiogenesis in infarcted myocardium (Id., citing Chimenti et al. Circ. Res., 106 (2010): 971-980; Deuse et al. Circulation, 120 (2009): S247-S254; Li et al. Am. J. Physiol. Heart Circ. Physiol., 299 (2010): H1772-H1781).
  • MSCs secrete cardioprotective microparticles with a hydrodynamic radius ranging from 50 to 65 nm (Id., citing Chen et al., 2011; Lai et al. J. Mol. Cell. Cardiol. (2010), 48: 1215-1224).
  • the therapeutic efficacy of such MSC-derived extracellular vesicles (EVs) was independent of the tissue source of the MSCs. For example, exosomes from human embryonic stem cell-derived MSCs were similar to those derived from other fetal tissue sources (e.g. limb, kidney). This suggested that secretion of therapeutic EVs may be a general property of all MSCs (Id., citing Lai et al. Stem Cell Res., 4 (2010): 214-222).
  • Exosomes are released by most if not all cell types, including platelets, blood cells, dendritic cells, mast cells, T cells, B cells, epithelial cells, endothelial cells, mesenchymal stem cells, smooth muscle cells, neuronal cells and many tumor cells.
  • cell types including platelets, blood cells, dendritic cells, mast cells, T cells, B cells, epithelial cells, endothelial cells, mesenchymal stem cells, smooth muscle cells, neuronal cells and many tumor cells.
  • Exosomes are further enriched in tetraspanins, like CD9, CD63, CD81 and CD82, which are important molecules for protein-protein interactions in cellular membranes. Tetraspanins bind many proteins, including integrins and MHC molecules [Id., citing Thery, C. , et al. Nat. Rev. Immunol. (2009) 9: 581-93; Escola, JM. J. Biol. Chem. (1998) 273: 20121-27; Keller, S. et al. Immunol. Lett (2006) 107: 102-108; Stoorvogel, W. et al. Traffic (2002) 3: 321-330].
  • Rab proteins a highly conserved family of small GTPases that functional as molecular switches and coordinate membrane traffic [Id., citing Stenmark, H. Nat. Rev. Mol. Cell Biol. (2009) 10: 513-25] are often observed in exosomes by mass- spectrometry. Exosomes are also rich in annexins, membrane trafficking proteins that are involved in fusion events. Furthermore cytoskeletal proteins like myosine, actin and tubulin are present in exosomes. Finally, metabolic enzymes, antigen presentation molecules, ribosomal proteins and signal transduction molecules are shown to be present in exosomes [Id., citing Mathivanan, S., et al. Proteomics (2008 ) 8: 4083-99].
  • exosomes also incorporate (functional) nucleic acids, most notably small RNA molecules [Id., citing Zomer, A. et al. Commun. Integr. Biol. (2010) 3: 447-50; Gibbings, D, Voinnet, O. Trends Cell Biol. (2010) 20: 491- 501].
  • RNA molecules detected in exosomes the class of 22nt long, non-coding miRNAs has received attention since the discovery that miRNAs can be functionally transferred to recipient cells [Id., citing Pegtel, DM et al. Proc. Nat. Acad. Sci. USA (2010) 107: 6328-33, Valadi, H. et al. Nat.
  • MiRNAs regulate gene expression by binding imperfectly to the 3' untranslated region of the target mRNA that results in translational repression of the mRNA into protein [Id., citing Bartel, DP. Cell (2004) 116: 281-97; Brennecke, J. et al. PLoS Biol. (2005) 3: e85].
  • Exosomes also contain specific proteins depending on the cell of origin. As examples, exosomes from tumor cells contain tumor antigens, platelet-derived exosomes contain coagulation factors, and exosomes from dendritic cells express toll-like receptor ligands [Zhu, L. et al., Artificial Cells, nanomedicine and biotechnology (2016) 46 (53): S166-S179, citing Jong, AY et al. J. Extracell. Vesicles (2017) 6: 1294368; Sobo-Vujanovic, A. et al., Cell Immunol. (2014) 289: 119-127]. Exosome-mediated functions vary depending on the condition or the origin of the cells [Id., citing Kim, OY, et al. Semin. Cell Dev. Biol. (2017) 67: 74-82]
  • Exosome function It is believed that exosomes can regulate the bioactivities of recipient cells by the transportation of lipids, proteins, and nucleic acids while circulating in the extracellular space. [Zhang, J. et al. Genomic sProteomics Bioinformatics (2015) 13: 17-24, citing Liao J., et al. Int J Mol Sci. 2014;15:15530-15551]. Several reports have shown that exosomes play important roles in immune response, tumor progression, and neurodegenerative disorders. Esther et al.
  • Exosomes derived from SGC7901 promoted the proliferation of SGC7901 and another gastric cancer cell line, BGC823 [Id., citing Qu J.L., et al. Dig Liver Dis. 2009;41:875-8].
  • BGC823 another gastric cancer cell line
  • CD147-positive exosomes derived from epithelial ovarian cancer cells promoted angiogenesis in endothelial cells in vitro [Id., citing Millimaggi D., et al. Neoplasia. 2007;9:349-357]. Webber et al.
  • miRNAs are not randomly incorporated into exosomes. Guduric-Fuchs et al. analyzed miRNA expression levels in a variety of cell lines and their respective derived exosomes, and found that a subset of miRNAs (e.g., miR-150, miR-142-3p, and miR-451) preferentially enter exosomes [Id., citing Guduric-Fuchs J., et al. BMC Genomics. 2012;13:357]. Similarly, Ohshima et al.
  • let-7 miRNA family members in exosomes derived from the gastric cancer cell line AZ-P7a with those from other cancer cell lines, including the lung cancer cell line SBC-3/DMS-35/NCI-H69, the colorectal cancer cell line SW480/SW620, and the stomach cancer cell line AZ-521.
  • members of the let-7 miRNA family are abundant in exosomes derived from AZ-P7a, but are less abundant in exosomes derived from other cancer cells [Id., citing Ohshima K., et al. PLoS One. 2010;5:el3247].
  • exosomal miRNA expression levels are altered under different physiological conditions.
  • the level of miR-21 was lower in exosomes from the serum of healthy donors than those glioblastoma patients [Id., citing Skog J., et al. Nat Cell Biol. 2008;10:1470-1476]. Levels of let-7f, miR-20b, and miR-30e-3p were lower in vesicles from the plasma of non- small-cell lung carcinoma patients than normal controls [Id., citing Silva J., et al. Eur Respir J. 2011;37:617-623]. Different levels of eight exosomal miRNAs, including miR-21 and miR141, were also found between benign tumors and ovarian cancers [Id., citing Taylor D.D., et al. Gynecol Oncol. 2008;110:13-21].
  • nSMase2 The neural sphingomyelinase 2 (nSMase2)-dependent pathway.
  • nSMase2 is the first molecule reported to be related to miRNA secretion into exosomes.
  • Kosaka et al. found that overexpression of nSMase2 increased the number of exosomal miRNAs, and conversely inhibition of nSMase2 expression reduced the number of exosomal miRNAs [Id., citing Kosaka N., et al. J Biol Chem. 2013;288:10849-10859].
  • hnRNPs nuclear ribonucleoproteins
  • miRNA induced silencing complex (miRISC)-related pathway. It is well known that mature miRNAs can interact with assembly proteins to form a complex called miRISC.
  • the main components of miRISC include miRNA, miRNA- repressible mRNA, GW 182, and AG02.
  • the AG02 protein in humans which prefers to bind to U or A at the 5' end of miRNAs, plays an important role in mediating mRNA:miRNA formation and the consequent translational repression or degradation of the mRNA molecule [Id., citing Frank F., et al. Nature. 2010;465:818-822]. Recent studies recognized a possible correlation between AG02 and exosomal miRNA sorting.
  • exosomal miRNAs in cell-released exosomes can circulate with the associated vehicles to reach neighboring cells and distant cells. After being delivered into acceptor cells, exosomal miRNAs play functional roles. Although it is difficult to completely exclude the effects of other exosomal cargos on recipient cells, miRNAs are considered the key functional elements.
  • the functions of exosomal miRNAs can be generally classified into two types. One is the conventional function, i.e., miRNAs perform negative regulation and confer characteristic changes in the expression levels of target genes.
  • Exosomal miR-92a derived from K562 cells, significantly reduced the expression of integrin a5 in the human umbilical vein endothelial (HUVEC) cells and enhanced endothelial cell migration and tube formation [Id., citing Umezu T., et al. Oncogene. 2013;32:2747-2755].
  • the other one is a novel function that has been identified in some miRNAs when they are studied as exosomal miRNAs rather than intracellular miRNAs.
  • Exosomal miR-21 and miR- 29a in addition to the classic role of targeting mRNA, were first discovered to have the capacity to act as ligands that bind to toll-like receptors (TLRs) and activate immune cells [Id., citing Fabbri M., et al. Proc Natl Acad Sci U S A. 2012;109:E2110-E2116].
  • Exosomal miRNAs can stably exist in the blood, urine, and other body fluids of patients, and exosomes can reflect their tissue or cell of origin by the presence of specific surface proteins [Id., citing Simons M., et al. Curr Opin Cell Biol. 2009;21:575-581, Mathivanan S., et al.
  • exosomal miRNAs show potential for use as noninvasive biomarkers to indicate disease states.
  • exosomal miRNAs can be used to aid in clinical diagnosis [Id., citing Skog J., et al. Nat Cell Biol. 2008;10:1470-1476; Silva J., et al. Eur Respir J. 2011;37:617-623; Taylor D.D., et al.
  • a set of exosomal miRNAs including let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a, can be used as the diagnostic biomarker of colorectal cancer [Id., citing Ogata-Kawata H., et al. PLoS One. 2014;9:e92921].
  • Another set, miR-1290 and miR-375 can be used as the prognostic marker in castration-resistant prostate cancer [Id., citing Huang X., et al. BMC Genomics. 2013; 14:319].
  • Exosomal miRNAs can stably exist in the blood, urine, and other body fluids of patients, and exosomes can reflect their tissue or cell of origin by the presence of specific surface proteins [Id., citing Simons M., et al. Curr Opin Cell Biol. 2009;21:575-581, Mathivanan S., et al. J Proteomics. 2010;73:1907-1920, Gross J.C., et al. Nat Cell Biol. 2012;14:1036-1045]. Furthermore, the amount and composition of exosomal miRNAs differ between patients with disease and healthy individuals. Thus, exosomal miRNAs show potential for use as noninvasive biomarkers to indicate disease states.
  • exosomal miRNAs can be used to aid in clinical diagnosis [Id., citing Skog J., et al. Nat Cell Biol. 2008; 10: 1470- 1476; Silva J., et al. Eur Respir J. 2011;37:617-623; Taylor D.D., et al. Gynecol Oncol. 2008;110:13-21; Rabinowits G., et al. Clin Lung Cancer. 2009;10:42-46].
  • a set of exosomal miRNAs including let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a, can be used as the diagnostic biomarker of colorectal cancer [Id., citing Ogata- Kawata H., et al. PLoS One. 2014;9:e92921].
  • Another set, miR-1290 and miR-375 can be used as the prognostic marker in castration-resistant prostate cancer [Id., citing Huang X., et al. BMC Genomics. 2013; 14:319] .
  • exogenous miRNAs can also be sorted into exosomes, which has been experimentally confirmed by Pegtel et al. [Id., citing Pegtel D.M., et al. Proc Natl Acad Sci U S A. 2010;107:6328-6333] and Meckes et al. [Id., citing 79], who observed that human tumor viruses can exploit exosomes as delivery vectors to transfer their exogenous miRNAs to other non-infected cells [Id., citing Pegtel D.M., et al. Proc Natl Acad Sci U S A.
  • exogenous small RNAs have also been transferred by exosomes by mimicking the molecular mechanism of endogenous miRNAs transportation.
  • MSC-derived EVs comprising exosomes and microvesicles
  • MSC-derived EVs which include exosomes and microvesicles (MV), are involved in cell-to-cell communication, cell signaling, and altering cell or tissue metabolism at short or long distances in the body, and can influence tissue responses to injury, infection, and disease (Phinney, DG and Pittenger, MF. Stem Cells (2017) 35: 851-58). Their content includes cytokines and growth factors, signaling lipids, mRNAs, and regulatory miRNAs (Id.). The content of MSC EVs is not static; they are a product of the MSC tissue origin, its activities, and the immediate intercellular neighbors of the MSCs (Id.).
  • MSCs secrete a plethora of biologically active proteins (Id., citing Tremain N, et al. Stem Cells 2001; 19: 408-418; Phinney DG, et al. Stem Cells 2006; 24: 186-198; Ren J, et al. Cytotherapy 2011; 13: 661-674).
  • MSC-derived EVs recapitulate to a large extent the enormous broad therapeutic effects previously attributed to MSCs, most studies fall short of rigorously validating this hypothesis (Id.)
  • various groups have compared the potency of MSCs versus MSC-derived EVs, and in some cases MSC-conditioned media, in animal models of myocardial infarction (Id., citing Bian S, et al. J Mol Med (Berlin) 2014; 92: 387- 397), focal cerebral ischemia (Doeppner TR, et al. Stem Cells Transl Med 2015; 4: 1131— 1143), gentamicin-induced kidney injury (Reis LA, et al.
  • MSC-derived EVs may function largely via horizontal transfer of mRNAs, miRNAs, and proteins, which then function by a variety of mechanisms to alter the activity of target cells. For example, it has been reported that transfer of IGF-1R mRNA from MSC-derived exosomes to cisplatin-damaged proximal tubular epithelial cells sensitized the epithelial cells to the renal-protective effects of locally produced IGF-1 (Id., citing Tomasoni S, et al. Stem Cells Dev 2013; 22: 772-780). With respect to miRNAs, those contained within MSC-derived EVs have been shown to inhibit tumor growth (Id., citing Katakowski M, et al.
  • CNS cystinosin
  • exosomes produced from adipose-derived MSCs contain neprilysin, an enzyme that degrades the amyloid beta (Ab) peptide, and that coculture of N2a cells engineered to overexpress human Ab with ASCs significantly reduced the levels of secreted Ab40 and Ab42 by exo some -mediated transfer of neprilysin (Id., citing Katsuda T, et al. Sci Rep (2013); 3: 1197).
  • EVs from marrow and umbilical cord-derived MSCs were shown to inhibit the growth and to induce apoptosis of U87MG glioblastoma cells in vitro whereas those from adipose -derived MSCs promoted cell growth but had no effect on U87MG survival (Id., citing Del Fattore, A. et al. Expert Opin. Biol. Ther. (2015) 15: 495-504).
  • exosomes prepared from different tissue-specific MSCs have measurably different effects on neurite outgrowth in primary cortical neurons and dorsal root ganglia explant cultures (Id., citing Lopez- Verrilli et al. Neuroscience 2016; 320: 129-139).
  • RNAs as critical regulators in the expression and function of eukaryotic genes and genomes
  • RNA silencing (Id.).
  • the central theme that runs throughout is that the small RNAs serve as specificity factors that direct bound effector proteins to target nucleic acid molecules via base-pairing interactions (Id.).
  • the core component of the effector machinery is a member of the Argonaute protein superfamily (Id.).
  • siRNAs and miRNAs are the most broadly distributed in both phylogenetic and physiological terms and are characterized by the double- stranded nature of their precursors (Id.).
  • piRNAs are primarily found in animals, exert their functions most clearly in the germline, and derive from precursors that are poorly understood, but appear to be single stranded (Id.).
  • siRNAs and miRNAs bind to members of the Ago clade of Argonaute proteins
  • piRNAs bind to members of the Piwi clade (Id.).
  • RNA silencing The signature components of RNA silencing are Dicers, Agos, and -21-23 nt duplex-derived RNAs (Id.). Both siRNA and miRNA small RNAs depend on Dicer enzymes to excise them from their precursors, and Ago proteins to support their silencing effector functions (Id.).
  • RNase III enzymes which are dsRNA-specific nucleases, are the source of miRNA/siRNA biogenesis (Id.).
  • One class of large RNase III enzymes has several domains in a specific order from the amino to carboxy terminus: a DEXD/H ATPase domain, a DUF283 domain, a PAZ domain, two tandem RNase III domains, and a dsRNA-binding domain (Id.). Some members of this family differ slightly from this arrangement (Id.).
  • PAZ and RNase III domains play central roles in excising siRNAs preferentially from ends of dsRNA molecules.
  • PAZ domains are shared with Argonaute proteins and are specialized to bind RNA ends, especially duplex ends with short ( ⁇ 2 nt) 3’ overhangs.
  • An end engages the Dicer PAZ domain, and the substrate dsRNA then extends approximately two helical turns along the surface of the protein before it reaches a single processing center that resides in a cleft of an intramolecular dimer involving the RNase III domains.
  • Each of the two RNase IIII active sites cleaves one of the two strands, leading to staggered duplex scission to generate new ends with ⁇ 2-3’ nt overhangs.
  • This general model pertains equally to pre-miRNA stem-loop substrates and to long, perfectly base-paired dsRNAs. In some species, different functional categories of small RNAs exhibit slightly different lengths; this appears to be dictated by the distance between the PAZ domain and the processing center in the relevant Dicer enzyme (Id.).
  • ATPase domain probably vary among different forms of Dicer (Id.). ATP promotes dsRNA processing by Drosophila Dicer 2 and C. elegans Dcr-1, and mutations predicted to cripple ATPase activity in Drosophila Dicer-2 specifically abolish dsRNA processing. In contrast, ATP is dispensable for dsRNA processing by human Dcr (hDcr), and an ATPase defective mutant exhibits no processing defect (Id.).
  • Dicers isolated from their natural sources generally are found in a heterodimeric complex with a protein that contains two or three double stranded Ras binding domains (dsRBDs); the Ras-binding domain (RBD) is an independent domain of about 75 residues, which is sufficient for GTP-dependent binding of Ras and other G alpha GTPases.
  • dsRBDs Ras binding domains
  • Both hDcr and Drosophila Dcr-2 process dsRNAs effectively in the absence of the heterodimeric partner (TRBP and R2D2, respectively).
  • TRBP and R2D2 heterodimeric partner
  • Dicer in silencing extends beyond dsRNA processing and into the pathway of RISC assembly; this activity is much more dependent on the dsRBD partner protein (Id.).
  • the Argonaute superfamily can be divided into three separate subgroups: the Piwi clade that binds piRNAs, the Ago clade that associates with miRNAs and siRNAs, and a third clade described in nematodes. All gene regulatory phenomena involving ⁇ 20-30 nt RNAs are thought to require one or more Argonaute proteins, which are the central, defining components of an RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the double- stranded products of Dicer enter into a RISC assembly pathway that involves duplex unwinding, culminating in the stable association of only one of the two strands with the Ago effector protein. This guide strand directs target recognition by Watson-Crick base pairing; the other strand of the original small RNA duplex (the passenger strand) is discarded (Id.).
  • Argonaute proteins are defined by the presence of four domains: the PAZ domain (shared with Dicer enzymes), the PIWI domain that is unique to the Argonaute superfamily, and the N and Mid domains.
  • the overall protein structure is bi-lobed, with one lobe consisting of the PAZ domain and the other lobe consisting of the PIWI domain flanked by N-terminal (N) and middle (Mid) domains.
  • the Argonaute PAZ domain has RNA 3’ terminus binding activity, and the co-crystal structures reveal that this function is used in guide strand binding.
  • the other end of the guide strand engages a 5’ phosphate binding pocket in the Mid domain, and the remainder of the guide tracks along a positively charged surface to which each of the domains contributes.
  • the protein-DNA contacts are dominated by sugar-phosphate backbone interactions.
  • Guide strand nucleotides 2-6 which are especially important for target recognition, are stacked with their Watson-Crick faces exposed and available for base pairing (Id.).
  • the PIWI domain adopts an RNase H-like fold that in some cases can catalyze guide strand-dependent endonucleolytic cleavage of a base pair target.
  • This initial cut represents the critical first step in a subset of small RNA silencing events that proceed through RNA destabilization.
  • Not all Argonaute proteins have endonucleolytic activity, and those that lack it usually also lack critical active-site residues that coordinate a presumptive catalytic metal ion (Id.).
  • MicroRNAs are found in plant and animal branches of Eukaryotes and are encoded by a bewildering array of genes. Transcription of miRNAs is typically performed by RNA polymerase II, and transcripts are capped and polyadenylated. Although some animal miRNAs are individually produced from separate transcription units, many more are produced from transcription units that make more than one product. A transcript may encode clusters of distinct miRNAs, or it may encode miRNA and protein. The latter type of transcript is organized such that the miRNA sequence is located within an intron. Many new animal miRNAs are thought to arise from accumulation of nucleotide sequence changes and not from gene duplication (Id.).
  • the resulting primary or pri-miRNA transcript extends both 5’ and 3’ from the miRNA sequence, and two sequential processing reactions trim the transcript into the mature miRNA. Processing depends on the miRNA sequence folding into a step-loop structure.
  • a typical animal pri-miRNA consists of an imperfectly paired stem of ⁇ 33 bp, with a terminal loop and flanking segments.
  • the first processing step which occurs in the nucleus, excises the stem-loop from the remainder of the transcript to create a pre-miRNA product.
  • a nuclear member of the RNase III family carries out this cleavage reaction.
  • Drosha catalyzes pri-miRNA processing, it depends on a protein cofactor, which contains two dsRBD domains and stably associates with the ribonuclease to form the microprocessor complex (Id.).
  • the second processing step excises the terminal loop from the pre-miRNA stem to create a mature miRNA duplex of approximately 22 bp length.
  • the pre- miRNA is exported from the nucleus, and the canonical Dicer enzyme carries out the cleavage reaction in the cytoplasm (Id.).
  • MicroRNAs behave like traditional polymeric products of gene activity, such that most species of a miRNA have highly exact ends, although there is a little variation. This feature of miRNAs has probably allowed them to interact with greater specificity on substrate mRNAs without a need for stringent complementarity or large overlap (Id.).
  • DGCR8 dsRBD domain binding partner protein
  • Drosha carries out the cleavage reaction, it relies upon DGCR8 to serve as a molecular anchor that properly positions Drosha’ s catalytic site the correct distance from the stem- flank junction.
  • the endpoint of the stem is a critical determinant for one end of the mature miRNA (Id.).
  • Dicer The second cut performed by Dicer defines the other end of the mature miRNA.
  • Dicer will cleave anywhere along a dsRNA molecule but has a strong preference for the terminus.
  • the PAZ domain of Dicer interacts with the 3’ overhang at the terminus and determines the cleavage site in a ruler-like fashion.
  • the RNase III catalytic sites are positioned two helical turns or 22 bp away from the terminus/P AZ portion of the Dicer-RNA complex (Id.).
  • Id. Dicer-RNA complex
  • the mature miRNA duplex is a short-lived entity; it is rapidly unwound when it associates with an Ago protein. Unwinding occurs so rapidly after duplex formation, because the two processes are physically coupled due to Ago2’s presence in a complex with Dicer and TRBP, the double- stranded RNA binding protein that loads siRNA into the RISC (Id.).
  • miRNA unwinding is accompanied by differential strand retention, i.e., one strand is retained while the other strand is lost. Strand retention is based on the relative thermodynamic stability of the duplex’s ends. Although the rule is that the 5’ terminus of the retained strand is at the less stably base-paired end of the duplex, this rule is not absolute. The other strand is appreciably detected in Ago complexes, lending ambiguity to the notion of strand asymmetry. Although either strand can become stably associated with Ago proteins, the more commonly associate strand is termed the miRNA strand; the other strand is called the miRNA* strand. miRNA unwinding is not accompanied by cleavage of the ejected strand by the associated Ago (Id.).
  • the mammalian Dicer/Ag/miRNA complex is associated with other proteins, e.g., Gemin3, Gemin4, MovlO, and Imp8, as well as the mammalian protein GW182, associate with Ago2.
  • GW182 is both necessary and sufficient for miRNA-bound Ago to silence gene expression.
  • miRNA-bound Ago in association with GW 182 can be thought of as the miRISC complex (Id.).
  • An miRNA acts as an adaptor for miRISC to specifically recognize and regulate particular mRNAs. If miRISC is tethered to a heterologous RNA recognition factor, the factor enables miRISC to recognize and repress mRNAs that lack miRNA-binding sites. With few exceptions, miRNA-binding sites in animal mRNAs lie in the 3’ untranslated region (UTR) and are usually present in multiple copies. Most animal miRNAs bind with mismatches and bulges, although a key feature of recognition involves Watson-Crick base pairing of miRNA nucleotides 2-8, representing the seed region (Id.).
  • miRISC regulates translation have been subject to ongoing debate. The fundamental issue of whether repression occurs at translation initiation or post-initiation has not yet been resolved. There are three competing models for how miRISC represses initiation. One proposes that there is competition between miRISC and elF4E for binding to the mRNA 5’ cap structure. A second model has proposed that miRISC stimulates de-adenylation of the mRNA tail; translation is repressed because the cap and PABPl-free tail of the deadenylated mRNA are unable to circularize.
  • a third model has proposed that miRISC blocks association of the 60S ribosomal subunit with the 40S preinitiation complex, i.e., the recruitment of eIF6 by miRISC may repress translation by preventing the assembly of translationally competent ribosomes at the start codon (Id.).
  • the cell’s position in the cell cycle is one such context.
  • miRNA let-7 and an artificial miRNA CXCR-4 repress translation in proliferating human cells, but change into translational activators when the cell cycle is arrested at the G1 checkpoint by serum starvation.
  • Aphidicollin-induced arrest at G1 also generates translational activation, whereas nocodazole-induced arrest at G2/M generates translational repression.
  • Lymphocyte growth arrest induces TNFa expression that is required for macrophage maturation; miR-369- 3p switches from a repressor to an activator of TNFa translation when cells in culture are growth arrested (Id., citing Vasudevan, S.et al. Science (2007) 318: 1931-34).
  • Binding site position is another context. Interaction of miR-lOa with the 5’UTR of certain ribosomal subunit mRNAs leads to their activated translation, whereas interaction with the 3’UTR leads to repression (Id., citing Orom, UA et al. (2008) Mol. Cell 30: 460-71). [00209] Another context is how small RNA regulation is organized and modulated within the cell. Ago proteins are frequently associated with membrane trafficking compartments, such as the Golgi and ER (Id., citing Cikaluk, D.E. et al. Mol. Biol. Cell (1999) 10: 3357-72).
  • miRISC factors might become anchored in certain subcellular compartments, e.g., P bodies or GW bodies, two separate pools of sequestered non-translating RNAs (Patel, PH, et al. PLos One (2016) 11(3): e015029).
  • Subunits of miRISC miRNAs, Ago and GW1821
  • GW bodies are not essential for miRNA repression, GW body formation requires an intact miRNA pathway (Carthew, RW and Sontheimer, EJ. Cell (2009) 136: 642-55).
  • HIV for instance exploits the Endosomal sorting complex Required for Transport (ESCRT) machinery for the formation of a particular exosome-like vesicle that is closely involved with viral budding from the plasma membrane [Id., citing Meckes, DG et al. J. Virol. (2011) 85: 12844-54, Benaroch, P. et al. Retrovirol. (2010) 7: 29; Penchen-Matthews, A., et al. Trends Microbiol. (2004) 12: 310-316] that allows a stealthy infection of neighboring cells i.e. without the use of envelope glycoproteins (Env) and/or retroviral receptors [Id., citing gould, SJ et al. Proc. Nat.
  • ESCRT Endosomal sorting complex Required for Transport
  • exosomes released from HIV infected cells may contain co-receptors (CCR5) that when transferred to neighboring/recipient cells may enhance their susceptibility to infection by HIV promoting viral spread [Id., citing Mack, M. et al. Nat. Med. (2000) 6: 769-75].
  • CCR5 co-receptors
  • Another interesting observation is the specific release and transport of the HIV Nef protein to neighboring cells via exosomes or‘nanotubes’ [Id., citing daSilva, LL. et al. J. Virol. (2009) 83: 6578-90].
  • Nef is able to alter the endosomal system altogether by increasing the number of endosomes, lysosomes and MVBs [Id., citing Mack, M. et al. Nat. Med. (2000) 6: 769-75; Madrid, R. et al. J. Biol. Chem. (2005) 280: 5032-44].
  • Nef is widely considered as an HIV virulence factor and one mechanism maybe the secretion via exosomes that is associated with the induction of apoptosis of responding CD4 + T cells [Campbell, TD et al. ethn. Dis. (2008) 18: S2-S9].
  • Virus-infected cells also package virus-encoded RNAs into exosomes that are delivered into non-infected recipient cells. [Id.].
  • exosomes in body fluids may change significantly with disease. For example, there are increased numbers of BAL exosomes in sarcoidosis patients compared with healthy volunteers [Alipoor, SD et al. Mediators of Inflammation (2016) 5628404., citing Qazi, KR, et al. Thorax, (2010) 65 (11): 1016-1024] BAL exosomes from sarcoidosis patients induce the production of inflammatory cytokines by PBMCs and promote the release of CXCL-8 by airway epithelial cells through delivery of pathogen-associated proinflammatory mediators [Qazi, KR, et al. Thorax (2010) 65 (11): 1016-1024].
  • exosomal content may also provide valuable information about disease status [Id., citing Tesselaar, MET et al. Journal of Thrombosis and Haemostasis (2007) 5 (3): 520-527].
  • MicroRNAs are implicated in modulating gene expression by interfering with mRNA translation most commonly by destabilizing mRNA thereby facilitating degradation.
  • One miRNA may target a large number of genes, and the targets of an miRNA may belong to a variety of functional groups.
  • the 3’-UTR of a single mRNA transcript may be the target for several different miRNAs, miRNA-mediated regulation of gene expression has been found to affect many cellular functions, including innate and antiviral responses. [Brogaard, L. et al., Sci. Reports (2016) 6:21812].
  • miRNAs are predicted to function as inhibitors of numerous mRNAs involved in ECM production and fibrosis [van Rooij, E. et al. Proc. Natl. Acad. Sci. USA (2008) 105: 13027-32]. These investigators showed that cardiac hypertrophy and heart failure are accompanied by characteristic changes in the expression of a collection of specific microRNAs (miRNAs), which act as negative regulators of gene expression, and that myocardial infarction (MI) in mice and humans also results in the dysregulation of specific miRNAs, which are similar to but distinct from those involved in hypertrophy and heart failure.
  • miRNAs specific microRNAs
  • MI myocardial infarction
  • the MI-regulated miRNAs are members of the miR-29 family, which are down-regulated in the region of the heart adjacent to the infarct.
  • the miR- 29 family targets a cadre of mRNAs that encode proteins involved in fibrosis, including multiple collagens, fibrillins, and elastin.
  • down-regulation of miR-29 would be predicted to derepress the expression of these mRNAs and enhance the fibrotic response.
  • down-regulation of all three members of the miR-29 family with anti-miRs in vitro and in vivo induced the expression of collagens, and enhanced the fibrotic response. This down-regulation remained present even after initial infarct healing had taken place.
  • miR-29 is an important regulator of the INF-g pathway in helper T cells and that this regulation is mediated in part through the Thl cell transcription factor T-bet. They showed that miR-29 regulates helper T cell differentiation by repressing multiple target genes, including at least two that are independently capable of inducing the Thl cell gene expression program. miRNA-deficient helper T cells exhibit abnormal INF-g production and decreased proliferation. Multiple members of the miR-17 and miR-92 families enhanced miRNA- deficient T cell proliferation, whereas miR-29 largely corrected their aberrant INF-g expression.
  • Eomesodermin Eomes
  • T-bet CD8+ T cells Eomesodermin
  • INF-g production Although not usually expressed at functionally relevant amounts in helper T cells, Eomes was abundant in miRNA-deficient cells and was upregulated after miR-29 inhibition in wild type cells.
  • T bet and Eomes facilitates the cooperative maintenance of the pool of antiviral CD8+ T cells during chronic viral infection.
  • T-bet is reduced in virus- specific CD8+ T cells; this reduction correlates with T cell dysfunction.
  • Eomes mRNA expression is up-regulated in exhausted CD8+ T cells during chronic infection.
  • miR-122 is an indispensable factor in supporting hepatitis C virus (HCV) replication [Li, Y., et al. J. Virol.
  • miR-125b and miR-223 directly target human immunodeficiency virus type 1 (HIV-1) mRNA, thereby attenuating viral gene expression in resting CD4+ T cells [Id., citing Huang, J, et al.,. Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes. Nat. Med. (2007( 13:1241-1247), and miR- 198 modulates HIV-1 replication indirectly by repressing the expression of ccntl [Id., citing Sung, T. L., and A. P. Rice. 2009. miR-198 inhibits HIV-1 gene expression and replication in monocytes and its mechanism of action appears to involve repression of cyclin Tl. PLoS Pathog. 5:el000263], a cellular factor necessary for HIV-1 replication.
  • HIV-1 human immunodeficiency virus type 1
  • viruses may promote their life cycles by modulating the intracellular environment through actively regulating the expression of multiple cellular microRNAs.
  • human T-cell lymphotropic virus type 1 (HTLV-1) modulates the expression of a number of cellular microRNAs in order to control T-cell differentiation [Li, Y., et al., J. Virol. (2010) 84(6): 3023-32, citing Bellon, M. et al. Deregulation of microRNA involved in hematopoiesis and the immune response in HTLV-I adult T-cell leukemia. Blood (2009) 113:4914-4917].
  • hCMV human cytomegalovirus
  • HSV-1 human cytomegalovirus
  • hCMV human cytomegalovirus
  • HIV-1 Human immunodeficiency vims type 1 down-regulate s the expression of many cellular miRNAs [Id., citing Yeung ML, et al. Retrovirology (2005) 2: 81], and for miR- 17/92, miRNA suppression is required for efficient virus replication [Id., citing Triboulet R, et al. Science (2007)315: 1579-1582]
  • Influenza viruses are ubiquitous, causing acute respiratory disease and substantial morbidity and mortality each year [Bakre, A. et al., PLoS One (2013) 6: e66796., citing Thompson WW, et al. (2004) Influenza- associated hospitalizations in the United States. JAMA 292: 1333-1340; Thompson WW, et al. (2003) Mortality associated with influenza and respiratory syncytial vims in the United States. JAMA 289: 179-186; MMWR (2010) Estimates of Deaths Associated with Seasonal Influenza - United States, 1976-2007. Atlanta, GA: Centers for Disease Control and Prevention pp. 1057-1062].
  • Influenza viruses belong to the family Orthomyxoviridae, are enveloped, and have an eight segmented, negative-sense, single-stranded RNA genome that encodes up to 11 proteins [Id., citing Palese P SM (2007) Fields Virology; Knipe DM HP, editor. Philadelphia: Raven].
  • the viral envelope contains the surface glycoproteins and antigenic determinants, hemagglutinin (HA) and neuraminidase (NA), as well as the membrane ion channel protein, M2.
  • the matrix protein (Ml) provides structure and secures the viral ribonucleoprotein (vRNP) complexes consisting of viral RNA coupled to nucleoprotein (NP) and the three polymerase proteins (PB1, PB2 and PA).
  • the remaining viral proteins include the nonstmctural proteins, NS1 and NS2, and the recently identified PB1-F2 protein found in some vims species.
  • the vims must infect a host cell to co-opt host proteins and pathways for the successful generation of progeny vims host cell pathways affected. Overlap was identified in pathways used for vims entry [Id., citing Shapira SD, et al. (2009) Cell 139: 1255-1267; Karlas, A.
  • a number of miRNAs have been demonstrated to bind to influenza PB1 mRNA and inhibit viral replication in vitro [Brogaard, et al., Sci. Repts. (2016) 6: 21812, citing Song, L. et al., J. Virol. (2010) 84: 8849-60].
  • Three studies have investigated the role of circulating miRNA during influenza A infection in human patients. Each study employed a different type of sample material, namely whole blood [Id., citing Tambyah, P. et al., PLoS One (2013) 8: e76811], PBMCs [Id., citing Song, H., et al., BMC Infect. Dis.
  • a porcine model used for demonstration of the temporal dynamics of miRNA expression after influenza A vims challenge from the first days of infection to after the infection had cleared showed that there is a time factor to consider when assessing the relation and involvement of cell-associated circulating miRNAs in response to influenza A vims infection.
  • ssc-miR29a was initially down-regulated (24 h p.L), but later up-regulated (72h and 14d pi) in the porcine model, whereas human studies have reported only down-regulation of its homolog has-miR29a-3p.
  • phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual- specificity protein phosphatase PTEN
  • PTEN dual-specificity protein phosphatase
  • Akt protein kinase B
  • FOX03A a pro-apoptotic transcription factor downstream of PTEN and Akt
  • miR-150p was reported by one study to be up-regulated [Id., citing Tambyah, P. et al., PLoS One (2013) 8: e76811], while it was found to downregulated in another. [Id., citing Song, H., et al., BMC Infect. Dis. (2013) 13: 257].
  • hsa-miR-150p was found to be downregulated in porcine leukocytes 72 h and 114 d after H1N2 challenge in pigs. [Id.]
  • Viral infection triggers host responses that engage signaling networks which have a fundamental role in the anti-viral response.
  • HPKs human protein kinases
  • HPKs human protein kinases
  • PLC protein kinase C
  • ERK extracellular signal-regulated kinase ERK
  • NF- kb a key mediator, is induced by accumulation of viral HA, NP and Ml proteins [Id., citing Ludwig S, et al. (2004) FEBS Letters 561: 37-43; Wei L, et al. (2006) J Biol Chem 281: 11678-11684; Wang X, et al. (2000) J Virol 74: 11566-11573; Pinto R, et al. (2011) I Antiviral Res 92: 45-56; Pauli EK, et al. (2008) PLoS Pathog 4: el000196; Pahl HL, Baeuerle PA (1995) J Virol. 69: 1480-1484; Nimmerjahn F, et al.
  • Influenza virus infection modulates multiple cellular miRNAs, and miR-323, miR-491, and miR-654 have been shown to inhibit viral replication by binding to the viral PB1 gene [Id., citing Liu H, et al. (2010) MiR26a and miR939 regulate the replication of H1N1 influenza virus in MDCK cell. Wei Sheng Wu Xue Bao 50: 1399-1405]. miR-507 and miR-136 have potential binding sites within the viral PB2 and HA genes [Id., citing 49]. miR-26a and miR-939 regulate the replication of H1N1 influenza virus in MDCK cells [Id., citing Liu H, et al. (2010) Wei Sheng Wu Xue Bao 50: 1399-1405].
  • Influenza virus A/WSN/33 was chosen for primary and validation screens; because it is a lab-adapted strain, another influenza strain (influenza A/New Caledonia/20/ 199 was tested to validate the HPK hits.
  • Human kinase genes identified as important for influenza virus replication include: NPR2 (natriuretic peptide receptor B/guanylate cyclase B; MAP3K1 (mitogen-activated protein kinase kinase kinase 1), DYRK3 (dual specificity tyrosine (Y) -phosphorylation regulated kinase 3); EPPHA6 (EPH receptor A6; TPK1 (thiamin pyrophosphokinase 1); PDK2 (pyruvate dehydrogenase kinase, isozyme 2) C90RF96 (chromosome 9 open reading frame 96); EXOSC10 (exosome component 10); NEKS (never in mitosis gene
  • the validated HPKs affect critical pathways during influenza infection and replication, Four of the 6 validated HPKs, i.e. CDK13, NEK8, PLK4 and SGK3, have roles in cell cycle regulation. Similarly, two of 3 anti- viral HPKs (MAP3K1, DYRK3) are also implicated in regulation of cell cycle. SGK3 belongs to the three member family of serum glucocorticoid kinases (SGK1, 2 and 3), and has been shown to regulate influenza vRNP nuclear export into the cytoplasm [Id., citing Alamares-Sapuay, JG et al. J. Virol. (2013) 87: 6020-26].
  • MAP3K1 is a multifunctional protein and important for induction of IFN-b induction in response to poly I: C challenge via IRF-3 activation [Yoshida R, et al. (2008) TRAF6 and MEKK1 play a pivotal role in the RIG-Tlike helicase antiviral pathway. J Biol Chem 283: 36211-36220].
  • MAP3K1 also inhibits expansion of virus specific CD8+ T cells [Id., citing Labuda T, et al. (2006) MEK kinase 1 is a negative regulator of virus-specific CD8(+) T cells. Eur J Immunol 36: 2076-2084].
  • DYRK3 belongs to a family of dual specificity tyrosine kinases that activate by auto phosphorylation and catalyze phosphorylation of histone H3 and H2B. DYRK3 phosphorylates and activates sirtuin 1 (SIRT1) turnover, causes deacetylation of p53 and increased apoptosis [Id., citing Guo X, et al.
  • Influenza virus infection upregulates mTORCl signaling pathway [Id., citing Mata MA, et al. (2011) Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor. Nat Chem Biol 7: 712-719] and inhibition of mTORCl can significantly delay mortality due lethal challenge of influenza virus in mice [Id., citing Murray JL, et al. (2012) Inhibition of influenza A vims replication by antagonism of a PI3K-AKT-mTOR pathway member identified by gene-trap insertional mutagenesis.
  • DYRK3 has been shown to stabilize P-granule like structures and the mTORCl pathway during cellular stress. Inactivation of DYRK3 traps mTORCl inside cytosolic stress granules while activation of DYRK3 promotes dissolution of stress granules and release of mTORCl [Wippich F, et al. (2013) Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORCl signaling. Cell 152: 791-805].
  • CDK13, HK2, NEK8, PANK4, PLK4, SGK3 increased or decreased viral replication of A/New Caladonia/20/99 infection as measured by influenza NP localization and influenza M gene levels.
  • HK2, NEK8, PANK4, PLK4 have been identified as important for influenza vims replication in other influenza genome screens.
  • miRNAs regulate multiple aspects of the host response to infection.
  • the number of miRNAs that were validated to be affected was limited.
  • miRNAs of HPKs important for influenza replication include the following. Targeting DYRK3, CDK13 and SGK3 did not alter HPK expression or viral replication and were not discussed further. No effect of miR-149* modulation on influenza NP staining was evident suggesting that under the conditions of assay, NEK 8 transcript modulation by miR-149* did not have any effect on viral replication. Indeed, miR-149* induction during influenza infection has been reported to occur only post 72 hrs [Id., citing Loveday, E Ket al. J. Virol (2012) 86: 6109-6122].
  • miR- 149 is known to induce apoptosis by repressing Aktl and E2F1.
  • MAP3K1 transcript expression was significantly up-regulated by miR-548d inhibitor treatment; the mimic down- regulated MAP3K1 transcript expression.
  • miR-29a or miR-138* treatments did not have any appreciable effect on MAP3K1 expression; miR29a has been implicated in regulation of MAPK1, and miR-29b has been implicated in regulation of DUYRK3 and CDK13.
  • miR- 548d inhibitor/mimic treatments did not alter MAP3K1 protein expression; similar to the NEK8 findings, MAP3K1 transcript modulation by miR-548d did not alter viral replication.
  • miR-34c The most significant effects were observed for miR-34c and the PLK4 gene. While miR-34b and let-7i inhibitor/mimic treatments had no substantial effects on PLK4 transcript and protein expression, miR-34c mimic considerably up-regulated PLK4 transcript and protein expression, as well as influenza NP levels. It was hypothesized that during influenza vims infection, NS1 mediated p53 up-regulation triggers miR-34c activity to regulate cell cycle through Myc, PLK4 and NEK8. miR-34c alters PLK4 activity by modulating the activity of either p53 or by stabilizing PLK4 translation.
  • a systematic profiling of cellular microRNAs in lung tissues from ice infected with rl918 or a nonlethal seasonal influenza vims was performed to identify miRNAs whose expression patterns differentiated the host response to rl918 and Tx/91 infections, and the potential functions of differentially expressed miRNAs was assessed by analyzing the predicted target genes whose expression was inversely correlated with the expression of these miRNAs.
  • miRNAs Because of their high abundance in lung tissue, 18 miRNAs were the focus of the analysis. These miRNAs demonstrated distinct expression patterns between the infections of r 1918 and Tx/91 in mouse lungs. For example, miR-193 was strongly downregulated during rl 918 infection, while it was upregulated during Tx/91 infection. In contrast, miR-709 was strongly upregulated during r 1918 infection, while it was strongly downregulated during Tx/91 infection. miR-223 and miR-21, which were strongly upregulated in r 1918 infection, were moderately upregulated only upon Tx/91 infection. On the other hand, while strongly downregulated in r 1918 infection, miR-29a and miR-29b were moderately downregulated only upon Tx/91 infection. Finally, the expression levels of miR- 200a, miT-34a, and miR-30a were downregulated in rl918 infection but were below the cutoff in Tx/91 infection. [Id.]
  • MiR-223 and Let-7 have been shown to be involved in immune responses, and miR-223 is a negative modulator of neutrophil activation and neutrophil-mediated killing [Id., citing Jopling, CLM, et al. Science (2006) 309: 1577-81].
  • a decreased expression level of Let-7 is associated with the activation of NF-KB in response to microbial challenge [Id., citing Hu, G., et al. J. Immunol. (2009) 183z: 1617-24].
  • Upregulation of miR-21 is closely related to airway inflammation [Id., citing Lu, TX, et al. J. Immunol.
  • a symptom of lethal rl918 infection, and miR-34a is associated with tumorigenesis, as the mutual activation of MiR-34a and p53 has been shown both in a human cell line [Id., citing Uamakuchi, M. et al. Proc. Natl Acad. Sci. USA (2008) 105: 13421-26] and in patients [Id., citing Mraz, M. e al. Leukemia (2009) 23: 1159-63].
  • a stable expression of miR-200a is critical in maintaining the phenotype of epithelial cells [Id., citing Gregory, PA, et al. 2008.
  • the miR- 200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB 1 and SIP1. Nat. Cell Biol. 2008) 10:593-6011]
  • microRNAs predominately function as repressors of target gene expression, these investigators therefore focused only on the targets whose expression was inversely correlated with the expression of their corresponding microRNAs.
  • microRNA and cellular gene expression was directly compared between r 1918 and Tx/91 infections to assess expression changes.
  • an upregulation means that a microRNA or a cellular gene was expressed more abundantly during r 1918 infection relative to Tx/91 infection, while a downregulation means that a microRNA or a cellular gene was expressed less abundantly during r 1918 infection relative to Tx/91 infection.
  • miR-29a was significantly downregulated in r 1918 infection at all three time points, the HG test results from days 3 and 5, but not day 1, had P values of ⁇ 0.05; by inference, miR-29a-mediated regulation on target gene expression occurred predominately at a later stage of infection. Furthermore, although miR-34a was downregulated in r 1918 infection at all three time points, the HG test results from only day 3 had a P of ⁇ 0.05, suggesting that miR-34a-mediated regulation on gene expression may be transient. [Id.]
  • the inversely correlated targets of four microRNAs are associated with the immune response.
  • the targets of Fet-7f are associated with lymphocyte-mediated immune response, while the targets of miR-200a are associated with viral gene replication and the JAK-STAT signaling pathway, which is closely related to the type I IFN-mediated innate immune response.
  • the inversely correlated miR-34a targets are associated with calcium ion homeostasis, which is critical for immune cell activation.
  • the targets of miR-27a are associated with regulation of the immune response.
  • the inversely correlated targets of three microRNAs, including miR-652, miR-27a, and miR-27b, are associated with apoptosis and cell death.
  • Type 1 interferon pathway The analysis of microRNA targets indicated that the type I IFN pathway was subject to microRNA-mediated regulation, since key genes in this pathway, such as IFNAR1 and STAT2, are direct targets of miR-200a, and their expression was inversely correlated with the expression of this microRNA).
  • many IFN-stimulated genes demonstrated increased expression levels in rl918-inf ected lungs compared with those in the Tx/91 -infected lungs [Id., citing Kash, JC, et al. Nature (2006) 443:578-581; Kobasa, D., et al. Nature (2007) 445:319-323].
  • CREB pathway The cyclic AMP (cAMP) responsive element binding protein, CREB, is a transcription factor that regulates the expression of hundreds of genes.
  • cAMP cyclic AMP
  • CREB-null mice die immediately after birth from respiratory distress [Id., citing Rudolph, DA, et al. Proc. Natl. Acad. Sci. U. S. A. (1998) 95:4481-4486], as CREB is involved in critical functions, including T-cell development [Id., citing Rudolph, DA, et al. Proc. Natl. Acad. Sci. U. S. A.
  • CREB CREB is involved in critical functions, including T-cell development) and cell survival [Id., citing Bonni, A, et al. Science (1999) 286:1358-1362].
  • the activity of CREB is regulated by multiple upstream pathways, including insulin-like growth factor, Ca2+, and G protein-coupled receptor signaling pathways.
  • Many key genes in the CREB upstream pathways are miR-233 targets, and concomitant with the strongly increased expression of miR-233 in rl918-inf ected lungs, the miR-223 targets in the CREB upstream pathways were significantly downregulated. The data therefore suggested that upregulation of miR-223 may repress the activity of CREB.
  • influenza virus infection induces changes in the cellular microRNAome and that unique patterns of differential expression of microRNAs may contribute to the extreme virulence of rl918 influenza virus infection by regulating the expression of cellular targets involving immune response and other critical cellular functions.
  • Regulating cellular microRNA expression may be a common activity among different influenza viruses; however, the pathogenic capacity of the viruses may eventually be determined by the expression pattern of a specific microRNA or a group of microRNAs.
  • miR-200a was known only for regulating the epithelial- to-mesenchymal transition by targeting zebl and sipl [Id., citing Park, SM, et al. Genes Dev. (2008) .22:894-907].
  • zebl and sipl the investigators not only found inversely correlated expression of miR-200a and sipl but also found evidence implicating miR-200a in the type I IFN response.
  • the aberrant activation of the type I IFN pathway during r 1918 infection may contribute to an unconstrained inflammatory response [Id., citing Kash, JC, et al. Nature (2006) 443:578-581; Kobasa, D., et al. Nature (2007) 445:319-323].
  • Increased expression of miR-200a target genes in the type I IFN pathway, including IFN-a receptors and STAT2/4 was found; the upregulation of these cellular genes, owing to the downregulation of miR-200a, may induce profound effects during infection. As these genes are located at the upstream end of the IFN signaling cascade, a moderate expression change could be exponentially amplified at the downstream end of the cascade.
  • ISGs IFN-stimulated genes
  • An increase in the expression of STAT2/4 caused by the downregulation of miR-200a may induce an overwhelming increase of ISG expression.
  • Massive induction of ISG expression has been observed in mice and macaques infected with the rl918 virus [Id., citing Kash, JC, et al. Nature (2006) 443:578-581; Kobasa, D., et al. Nature (2007) 445:319- 323]).
  • miR-223 may indirectly repress the activity of the transcription factor CREB by regulating its upstream pathways.
  • miR-223 may downregulate CREB activity by repressing three such upstream pathways, namely the IGF-1 receptor, Ca2+ channel, and GPCR pathways.
  • miR-223 also repressed the expression of crucial intermediate molecules in these pathways, such as PI3K, PP2A, and PKA.
  • crucial intermediate molecules in these pathways such as PI3K, PP2A, and PKA.
  • One critical function of CREB is maintaining cell survival and growth in vivo (20).
  • HIV Human immunodeficiency virus
  • miRNA-29a which targets the nef-3’UTR, is a potent inhibitor of HIV-1 replication [Id., citing Sunm, G. et al., Nucleic Acids Res. (2012) 40: 2181-96); Ahluwalia, JK, et al., Retrovirology (2008) 5: 117; Nathans, R. et al., Mol.
  • Nef may inhibit Kaposi’s sarcoma-associated herpesvirus (KSHV) replication to promote viral latency and contribute to the pathogenesis of AIDS-related malignancies.
  • KSHV Kaposi’s sacoma-associated herpesvirus
  • KS Kaposi’s sarcoma
  • PEL primary effusion hymphoma
  • Castleman multicentric Castleman’s disease.
  • hsa- miR-1258 a Nef-upregulated miRNA
  • RTA major lytic switch protein
  • Ectopic expression of hsa-miR-1258 impaired RTA synthesis and enhanced Nef-mediated inhibition of KSHV replication, whereas repression of hsa-miR-1258 has the opposite effect. Mutation of the seed sequence in the RTA 3’ UTR was found to abolish downregulation of RTA by hsa-miR-1258.
  • miRNAs affect HIV-1 replication indirectly by affecting host cell HIV-1 dependency factors that regulate HIV-1 integration and transcription, and directly by binding to viral transcripts and inhibiting translation.
  • miR-29a The role of miR-29a in HIV- 1 replication and latency. J. Virus Eradication (2017) 3: 185-191, citing Swaminathan, G. et al. J. Mol. Biol. (2014) 426: 1178-97].
  • miR-144 targets cellular LEDGF/p75, which plays a critical role in guiding reverse-transcribed genomes to the intronic regions of highly expressed genes. [Id., citing Ruelas, DS, Green, WC. Cell (2013) 155: 519-29].
  • miRNAs can reinforce HIV-1 latency through their regulation of constitutively expressed factors that control cellular proliferation regardless of the cell’s infection status. It has been hypothesized that miRNAs known to target cyclin Tl, whose association with CDK9 in the POTEFb complex is essential for hIV-1 transcription (miR-27b, miR-29b, miR-150, miR-198, and miR-223) might play a key role in HIV-1 latency regulation. [Id. citing Chiang, K. et al. J. Virol (2012) 86: 3244-52]. miR-17-5p and miR- 20a are involved in the epigenetic control of HIV-1 replication.
  • PCAF CBP-associated factor
  • a histone acetyltransferase that enhances HIV-1 transcription by acetylating both the histone proteins and the p65 component of NF- k B
  • miR-155 another regulator of NF- KB activity, inhibits the ubiquitinating effect of TRIM-32 on IkBa, this enhancing the availability of IKB and the following sequestration of NFKB in the cytoplasm [Id. citing Ruelas, DS, et al. J. Biol. Chem. (2015) 290: 13736-48]
  • miRNAs can enhance HIV-1 infection by inhibiting cellular repressors of viral replication.
  • miR-34a and miR-217 downregulate SIRT-1, a p65 and Tat deacetylase, thus enhancing their efficiency under HIV-1 transcription [Id., citing Zhang, HS et al., FEBS Lett. (2012) 586: 4203-7; Zhang, HS, et al., Biochim. Biophys. Acta (2012) 1823: 1017-23]
  • Host miRNAs also can bind directly to viral transcripts.
  • miR-28, miR-125b, miR-150, miR-223, and miR-382 were shown to downregulate transcripts containing a 1.2 kb fragment from HIV-1 3’UTR harboring target sequences for these miRNAs; mutations in these putative miRNA targets relieved the inhibition.
  • Virus transcript downregulation was reported to take place in resting, but not stimulated CD4+ T cells. Inhibition of the five miRNAs simultaneously was reported to cause reactivation of HIV-1 infection in CD4 T cells from HIV-1 on combined retroviral therapy [Id. citing Huang, J. et al. Nat. Med. (2007) 13: 1241-47].
  • miR-29a allows the RISC to bind HIV-1 mRNA, and that the miR-29a-HIV-l- mRNA-RISC complex then associates with P-bodies, the cytoplasmic substructures where Ago-proteins, miRNAs and untranslated mRNAs accumulate, together with other enzymes involved in mRNA turnover and translational repression [Id. citing Winter, J. et al. Nat. Cell Biol. (2008) 11: 228-34], where mRNA translational repression takes place.
  • Retrovirology (2014) 11: 108] indicates that miR-29a interacts with HIV-1 transcripts, silencing viral production and infectivity, but results from two other studies have questioned whether this interaction may exist in vitro [Id., citing Sun, G. et al. Nucleic Acid Res. (2012) 40: 2181-96, Whisnant, AW et al. MBio (2013) 4: e000193], and suggested that the virus can escape miR-29a-mediated restriction when cellular miR-29a expression is at physiological concentrations. [00251]
  • Evidence suggests that miR-29a downregulates Nef protein levels in infected cells [Id., citing Ahluwalia, J. et al. Retrovirology (2008) 5: 117; Patel, P. et al.,
  • exosomal membrane- sorting pathway can be co-opted by retroviruses for the generation of Trojan virions.
  • Trojan virions [Willis, GR et al., Front. Cardiovasc. Med. (2017) 4: article 63].
  • miRNAs are selectively retained in cells, while others are preferentially secreted.
  • the human immunodeficiency virus encodes prototypic retroviral proteins (Gag, Pol, Env) as regulatory (Rev, Tat) and accessory (Nef, Vif, Vpr, Vpu/Vpx) proteins, the last group being dispensable for virus replication in vitro, but important for persistence and disease in an immunocompetent host [Aqil, M. et al., J. Extracell. Vesicles (2014) 3. doi: 10342/jev.v3.23129, citing Das SR, Jameel S. Indian J Med Res. 2005;121:315-32].
  • Nef is the largest of the four HIV accessory proteins, is expressed early in infection and at far higher levels than the other early proteins, Tat and Rev [Id., citing Landi A, et al Curr HIV Res. 2011;9:496-504]. It is primarily localized at cellular membranes, which include endosomal membranes, the perinuclear region and the inner surface of plasma membrane. Nef is also released from cells, either in microvesicles (MVs) [Id., citing Campbell TD, et al. Ethn Dis.
  • MVs microvesicles
  • Nef-GFP Nef-green fluorescent protein
  • vesicles were shown to be taken up by Jurkat CD4+ T cells, in which the Nef-GFP fusion protein localized mainly to the cytoplasm as punctate structures [Id., citing Campbell TD, et al. Ethn Dis. 2008;18:S2-14-9].
  • the vesicles are likely to enter recipient cells via endocytosis, which had been reported in other systems [Id., citing Svensson KJ, et al. J Biol Chem. (2013) 288:17713-24].
  • Nef exosomes could also fuse with Nef-deficient HIV-1 virions and restore the infectivity of mature particles [Id., citing Campbell, TD et al. Ethn. Dis.
  • Nef-expressing U937 cells secreted on an average about 60% more exosomes than control cells. Nef exosomes are enriched in miRNAs that can target proinflammatory cytokines and other genes involved in key pathways like JAK-STAT signaling, MAPK signaling and apoptosis. Further, an overwhelming percentage of miRNAs that can potentially target HIV-1 are secreted out of Nef-expressing cells into exosomes. As reported earlier for other cell types, U937 cells expressing Nef also showed increased exosome secretion. There was differential expression of about 50% of detected miRNAs under the influence of Nef. We observed significant changes in the levels of several miRNAs that regulate innate immune responses, especially the proinflammatory cytokines. These include miR-16, miR-125b, miR-146a, miR-146b-3p and miR-181a, which are reduced in Nef- expressing U937 cells.
  • Host miRNAs also target viral transcripts and limit replication.
  • miR-18, miR- 19a, miR-20a, miR-21 and miR-29b were found to be downregulated in Nef expressing monocytes.
  • a comparison of miRNA expression patterns in resting and activated CD4+ T cells and use of specific antagomirs concluded that miR-28, miR-125b, miR-150, miR-223 and miR-382 target the nef/3'LTR region and contribute to HIV latency in resting CD4+ T cells; similar data were also reported for monocytes and macrophages [Id., citing Wang X, et al. Blood. 2009;113:671-4; Sun G, et al. Nucleic Acids Res. 2012;40:2181-96].
  • Profiling demonstrated reduced levels of miR-125b and miR-223 in Nef-expressing monocytes, whereas miR-382 was not detected.
  • miR-29a, miR-29b, miR-149, miR-324-5p and miR-378 have been reported to target conserved regions of the HIV-1 genome, including the nef gene [Id., citing Sun G, et al. Nucleic Acids Res. 2012;40:2181-966]. Of these, miR-29b was found to be downregulated in Nef-expressing cells.
  • a majority of miRNAs that inhibit HIV replication including miR-17, miR-19a, miR-19b, miR-20a, miR-26a, miR-28, miR-29a, miR-29b, miR-29c, miR-92a, miR-125b, miR-149, miR-150, miR-223, miR-324-5p, miR- 378 and miR-382 were present at 1.5-folds or higher levels in Nef exosomes. Further, when correlated with the reported in silico analysis of miRNA target sites in the HIV genome, an overwhelming majority of miRNAs that can potentially target HIV-1 genomes were present at increased levels in exosomes secreted by Nef-expressing cells.
  • Nef expression reduced cellular levels of several host miRNAs that target innate immune responses and viral transcripts by exosome-mediated export, which likely modifies the host cell environment to favor virus replication.
  • TGFb a key growth factor elevated in the wound site, stimulated upregulation of miR-21 in MSCs as well as in multipotential C3H10T1/2 cells, and promoted proliferation and differentiation of these cells in vitro. Consistently, knockdown of miR-21 in the wound bed delayed the healing process.
  • MSCs secrete microvesicles (MVs) (Id., citing Bruno S, et al. J. Am. Soc. Neprhol. (2009) 20: 1053-67; Collino, F. et al. PLoS One (2010) 5: el 1803).
  • An miRNA profile on MVs from MSCs and HLSCs showed that MVs contained a pattern of miRNAs shared with their cells of origin (Collino F, et al. PLoS One (2010) 5: el 1803).
  • MVs are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the surface membranes of most cell types (Guo, L. et al. Exptl Hematol. (2011) 39: 608-616, citing Lotvall, J., Valadi, H. Cell Adh. Mgr. (2007) 1: 156-58). Accumulating data suggest that MVs may serve as a means of cell-to-cell communication through which genetic information or gene products are transferred and cell activities are regulated (Id., citing Carmussi, et al. Kidney Int. (2010) 78: 838-848; Lotvall, J., Valadi, H. Cell Adh. Mgr.
  • MSC EVs have been shown to harbor a variety of mRNAs and miRNAs (Carmussi, et al. Kidney Int. (2010) 78: 838-848; Lotvall, J., Valadi, H. Cell Adh. Mgr. (2007) 1: 156-58; Chen, TS et al. Nucleic Acids Res. (2010) 38: 215-224).
  • Differential miRNA expression profiles in MSCs and MVs derived from MSCs have been observed (Carmussi, et al. Kidney Int. (2010) 78: 838-848; Chen, TS et al. Nucleic Acids Res. (2010) 38: 215-224).
  • Microarray analysis for the presence of miRNAs revealed that the secreted RNA contained many miRNAs that were essentially a subset of those in MSCs. 9 of the 13 members in one of the most highly conserved and developmentally important human let-7 family were expressed in MSCs (citing Jerome T, et al. Curr. Genomics. 2007; 8: 229- 233; Roush S, Slack FJ. Trends Cell Biol. 2008; 18: 505-516.).
  • hsa-let-7a hsa- let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f, hsa-let-7g, hsa-let-7i, and hsa-miR-98.
  • CM conditioned medium
  • the passenger miRNA sequences of -7b and -7d also were detected in the CM and not detectable in MSCs (Id.).
  • MSCs secretion of miRNAs including passenger miRNA sequences is a selective, and not a random process, by MSCs.
  • Microarray analysis also revealed the presence of miRNA-923, a degradative product of ribosomal RNA (Id.). Therefore, while the secretion did not contain intact rRNA, it contained degraded ribosomal RNA and possibly degraded mRNA (Id.).
  • MSCs were found to preferentially secrete miRNA in the precursor instead of the mature form; these pre-miRNAs were enriched in MVs, which were readily taken up by neighbor cells, suggesting a potential mechanism in regulation of activities of other cells (Id.).
  • MSC EVs in treatment of organ fibrosis have been descried from MSC-exosomes in several disease models, e.g., promoting functional recovery and nruovascular plasticity following traumatic brain injury [Willis, GR et al. Front. Cardiovasc. Med. (2017) 4: article 63, citing Zhang, Y et al. J. Nerurosthetic. (2015) ' l l: 856-67]; reducing myocardial infarction size [Id., citing Lai, RC, et al. Stem Cell Res. (2010) 4: 214-22; Arslan, F. et al. Stem Cell Res.
  • MSC-derived EVs have shown protective effects in several models of organ injury and fibrosis.
  • MEx-preconditioned BM-derived monocytes alleviated core features of pulmonary fibrosis and lung inflammation.
  • Proteomic analysis revealed that MEx therapy reprogrammed bone marrow derived monocytes to a nonclassical anti-inflammatory phenotype.
  • a bolus dose of purified MSC- exosomes significantly improved lung morphology and pulmonary development, decreased lung fibrosis, and ameliorated pulmonary vascular remodeling in a neonatal hyperoxia- induced murine model of bronchopulmonary dysplasia (BPD).
  • mouse lungs in this model present a development stage resembling that of a human preterm neonate between 14 and 28 weeks gestation, these lungs, albeit in the saccular stage, are competent for proper gas exchange, whereas human preterm neonates often require supplemental oxygen and surfactant administration.
  • MSC-exosomes can modulate proinflammatory signaling and immune responses in the hyperoxic lung via modulation of lung macrophage phenotype.
  • MSC-derived EVs protected against renal injury by reducing levels of creatinine, uric acid, lymphocyte response and fibrosis through shuttling miR-let7c to induce renal tubular cell proliferation (Kusuma GD, et al. Front Pharmacol. 2018; 9: 1199, citing Wang B, et al. Mol Ther. 2016 Aug; 24(7): 1290-301).
  • a murine model of carbon tetrachloride-induced hepatic injury concurrent treatments of MSC- EVs attenuated the injury by increasing the proliferation, survival and prevented the apoptosis of hepatocytes (Id., citing Tan CY, et al.
  • MSC-EVs delivery of miR-133b directly to neurite cells reportedly enhanced the outgrowth of neurites resulting in increased proliferation of neuroblasts and endothelial cells (Id., citing Xin H, et al. Stem Cells. 2013 Dec; 31(12): 2737-46). Additionally, Anderson et al. showed through a comprehensive proteomic analysis that MSC-derived EVs mediated angiogenesis via NF-KB signaling (Anderson JD, et al. Stem Cells. 2016 Mar; 34(3): 601-13), while Zhang et al. (Stem Cells Transl Med. 2015 May; 4(5): 513-22) showed that UC MSC-EVs mediated angiogenesis via the Wnt4/b-catenin pathway.
  • Cocks akievirus B (CVB), a member of the Picornaviridae family, is a common enterovirus that can cause various human systemic inflammatory disease, such as myocarditis, meningitis, and pancreatitis; the six CVB serotypes are each responsible for different diseases and symptoms.
  • CVB Cocks akievirus B
  • Viral myocarditis is a common cause of dilated cardiomyopathy and sudden cardiac death. .
  • Several preclinical stem cell therapies have made some progress in reducing inflammation and improving myocardial function, but they still have limitations. [Id., citing Wemer, L. et al. J. Mol. Cell Cardio. (2005) 39: 691-97; Miteva, K. et al. Stem Cells Trans. Med. (2017) 6: 1249-61; Van Linthout, S. et al. Eur. Heart J. (2011) 32: 2168-78].
  • Cardiac progenitor cells are a group of heterogeneous cells distributed throughout the heart and able to differentiate into several cell types, including cardiomyocytes, vascular smooth muscle cells, and endothelial cells. Direct transdifferentiating into cardiac tissue is considered unlikely.
  • the mechanism of adult stem cell therapy has been tested to be mediated through paracrine release of EVs containing growth factors and cytokines to exert anti-apoptosis effects, suppress immunity, and promote angiogenesis. [Id., citing Le, T., Chang, J. Cell Death Discov. (2016) 2: 16052; Chimenti, I. et al. Circulation Res. (2010) 106: 971-80].
  • exosomes could avoid many of the limitations of cell therapy.
  • exosomes isolated from expanded rat CPCs isolated and cultured from rat heart tissue were determined. Exosomes were isolated and purified using ExoQuick- TCTM Exosome Isolation Reagent (System Biosciences, USA). The presence and size of exosomes was determined using transmission electron microscopy. H9C2 cells also were used as an in vitro model; H9C2 myoblasts are a cell model used as an alternative for cardiomyocytes. To assess in vitro uptake of the CPC exosomes by H9C2 cells, purified exosomes were labeled with green fluorescent labeling (DiO perchlorate, Dio).
  • TCID50 medium tissue culture infectious dose signifies the concentration at which 50% of the cells are infected when a test tube or well plate upon which cells have been cultured is inoculated with a diluted solution of viral fluid.
  • mice were injected with CVB3 containing 10 4 TCID50 through the tail vein to establish the viral myocarditis model.
  • Rats infected with CVB3 Nancy strain were injected with CPC-Ex i.v. at 24 h p.i. All rats were sacrificed by cervical dislocation at 7 days p.i. and heart tissue acquired. Tissue structure was observed using H & E staining and TEM. Apoptosis of myocardial cells was detected by TdT-mediated dUTP nick-end labeling (TUNEL) and immunofluorescence.
  • TUNEL TdT-mediated dUTP nick-end labeling
  • Apoptosis was determined by FACS analysis of cells stained with Annexin-V FITC and propidium iodide by flow cytometry. CVB3-induced apoptosis was inhibited by CPC-Ex at 12, 24 and 48 h. p.i.
  • Akt/mTOR signaling pathway plays an important role in anti-apoptosis and regulation of cellular transcription by activating p70S6K and 4EBP1.
  • p70S6K is a serine/threonine-protein kinase that acts downstream of mTOR signaling in response to growth factors and nutrients to promote cell proliferation, cell growth and cell cycle progression.
  • Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) is a member of a family of translation repressor proteins, and a well- known substrate of mechanistic target of rapamycin (mTOR) signaling pathway. [Qin, X et al. Cell Cycle (2016) 15(6): 781-86].
  • CPC-exosomes could inhibit CVB3 replication, thereby reducing the CVB3-induced apoptosis by inhibiting phosphorylation of 4EBP1.
  • TOP mRNAs are vertebrate transcripts which contain a 5'terminal oligopyrimidine tract (5'TOP), encode for ribosomal proteins and elongation factors 1 alpha and 2, and are candidates for growth-dependent translational control mediated through their 5'TOP. (Avni, D. et al. Nucleic Acid Res. (1997) 25(5): 995- 1001).
  • Akt2 p70S6K and 4EBP1 overexpression groups, CPC-exosomes promoted CVB3-induced apoptosis, viral capsid protein VP1 expression and cleavage of caspase 3.
  • EV composition is determined not only by the cell type but also by the physiological state of the producer cells. This diversity of mechanisms by which EVs are generated and confer effects provides both opportunities and challenges for developing EV- based therapeutics (Gyorgy B, et al. Annu Rev Pharmacol Toxicol. (2015) 55: 439-464). As described above, MSCs have been shown to be both immunosuppressive and immunostimulatory, depending on the context, and many questions about EVs remain. For example, many methods are used to isolate EVs, and EV contents and properties overlap with those of the cells of origin and other EV types. Formalizing EV nomenclature and defining attributes is a work in progress.
  • EV-mediated transfer between proximal cells, for example, when diffusional barriers lead to local accumulation of secreted EVs rather than transfer of EVs via the circulation, where EV concentrations may be lower.
  • EV-mediated signaling is dose-dependent (Id., citing Yu S, et al. J. Immunol. 2007; 178: 6867-75), so the tuning of EV dose may enable the balancing of potential deleterious and therapeutic effects of EV administration. Understanding the role of EV dose is also important for achieving therapeutic efficacy.
  • EV binding is mediated by receptors that interact with either universal EV molecules, such as lipids and carbohydrates, or specific peptides present on subsets of EVs.
  • cells internalize EVs by processes that include receptor-mediated phagocytosis or endocytosis via receptors that include T cell immunoglobulin- and mucin- domain-containing molecule-4 (TIM4), which binds to phosphatidylserine (PS) on EVs; scavenger receptors; integrins; and complement receptors (Id., citing Record M, et al. Biochem. Pharmacol. 2011; 81: 1171-82). How EV cargo is released into the cytoplasm after entry into recipient cells is unclear.
  • TIM4 T cell immunoglobulin- and mucin- domain-containing molecule-4
  • EVs may potentially pass through cells within the multivesicular body compartment, which could explain how EVs cross the blood-brain barrier (BBB) (i.e., via a transendothelial route).
  • BBB blood-brain barrier
  • Endocytotic mechanisms must circumvent the lysosomal degradative pathway, and direct fusion between the EV and target cell plasma membrane or endocytotic membrane does not always ensure functionality of the contents.
  • EV cargo can be degraded by recipient cells, thereby inhibiting therapeutic delivery but limiting the impact of off-target delivery.
  • the fate of EVs within the body and cells remains poorly understood and requires additional investigation to elucidate how these processes impact functional EV-mediated delivery (Id.).
  • the WNT protein WNT-5A is largely known to exert its effects b-catenin independent and has been found upregulated in IPF fibroblasts (Id., citing Vuga LJ, et al. Am J Respir Cell Mol Biol. 2009 Nov; 41(5): 583-9).
  • lung fibroblasts are a source of EVs and demonstrate autocrine effects of EVs on fibroblast proliferation, which was enhanced by TGF-b (Id.).
  • MSC-derived exosomes were found to induce dermal fibroblast proliferation (Id., citing McBride JD, et al. Stem Cells Dev. 2017 Oct 1;26(19) : 1384-1398).
  • Fibroblast-derived EVs did not promote myofibroblast differentiation, but rather decreased mRNA levels of myofibroblast markers.
  • MSC-EVs have also been reported to suppress myofibroblast differentiation (Id., citing Fang S, et al. Stem Cells Transl Med.
  • WNT transport on EVs has important implications with respect to the signaling range of WNT proteins, which is generally thought to be rather short and limited to close neighboring cells. EV-mediated transport can contribute to a larger signaling range of WNT proteins and thus determine the signaling outcome on other cells.
  • WNT-5A has also been reported to promote processes as fibroblast adhesion (Id., citing Kawasaki A, et al. Cell Signal. 2007 Dec; 19(12): 2498-506) or invasion (Id., citing Waster P, et al. Int J Oncol. 2011 Jul; 39(1): 193-202), as well as epithelial-mesenchymal transition (Id., citing Gujral TS, et al. Cell. 2014 Nov 6; 159(4): 844- 56). WNT-5A bound EVs in IPF broncho alveolar lavage fluid (BALF) were shown to contribute to the functional effects, thus suggesting that fibroblast derived EVs can be found in IPF BALF. This work further raises the more general question whether EVs promote lung fibrosis development or might have a protective role in vivo (Id.).
  • IPF broncho alveolar lavage fluid BALF
  • IPF treatments have included corticosteroids, azathioprine, cyclophosphamide, anticoagulants, and N-acetylcysteine (Raghu G. et al, Am J Respir Crit Care Med., 183(6): 788-824, 2011).
  • supportive therapies such as oxygen therapy and pulmonary rehabilitation are employed routinely.
  • none of these have definitely impacted the long term survival of IPF patients, which further highlights the unmet medical need for treatment options in IPF.
  • InterMune's oral small-molecule Esbriet® pirfenidone
  • Esbriet® thus became the first medication specifically indicated for the treatment of IPF; due to equivocal trial outcomes and drug side effects, the drug's utility is viewed with skepticism in the United States, and did not receive an FDA approval based on the data submitted at that time.
  • Example 1 The AETHER trial described in Example 1 was a single single-center, nonrandomized, non-placebo-controlled phase I study of 9 patients with mild to moderate IPF to assess the effect of infusion of hMSCs on clinical outcome. Patients with other infiltrative diseases, connective tissue disease, pulmonary hypertension, peripheral capillary oxygen saturation ⁇ 93% at rest at sea level, life expectancy shorter than 1 year, and those actively listed for any organ transplant were excluded.
  • Donor eligibility included testing for antibodies against HIV-1/2, human T-lymphocyte virus PII, hepatitis C vims, hepatitis B core (IgG and IgM), and cytomegalovirus; nucleic acid testing for HIV-1, hepatitis C virus, and West Nile vims; and testing for the surface antigen of the hepatitis B vims, Trypanosoma cruzi enzyme-linked immunosorbent assay, and rapid plasma reagin. This raised the possibility of treating viral infection in potential donors.
  • corticosteroids are used for the treatment of acute respiratory distress syndrome (ARDS) and pulmonary fibrosis.
  • ARDS acute respiratory distress syndrome
  • pulmonary fibrosis a chronic respiratory disease fibrosis fibrosis fibrosis fibrosis fibrosis fibrosis fibrosis fibrosis fibrosis fibrosis fibrosis fibrosis fibrosis fibrosis fibrosis fibrosis.
  • ARDS acute respiratory distress syndrome
  • pulmonary fibrosis pulmonary fibrosis
  • Exosomes act as intercellular communicators to facilitate direct transfer of proteins, lipids and nucleic acids between cells in vitro and in vivo. Pioneering work has demonstrated that exosomal cargos changed markedly during viral infection: (1) exosomes can participate directly in viral replication; (2) exosomes modulate immune response during viral infections; (3) exosomal RNAs and proteins might be selected as novel biomarkers for the diagnosis of viral infections; and (4) exosomes can also be designed as therapeutics to attenuate viral replication.
  • Exosomes are released by a wide range of cell types present within the lung including endothelial cells, stem cells, epithelial cells, alveolar macrophage, and tumor cells, although epithelial cells are reported to be the main source of lung-derived exosomes [Id., citing Y. Fujita, N. Kosaka, J. Araya, K. Kuwano, and T. Ochiya,“Extracellular vesicles in lung microenvironment and pathogenesis,” Trends in Molecular Medicine, vol. 21, no. 9, pp. 533-542, 2015].
  • Exosomes released by airway epithelial cells contain mucins and alpha 2,6- linked sialic acid which have a neutralizing effect on human influenza vims infection [Id., citing N. T. Eissa,“The exosome in lung diseases: message in a bottle,” Journal of Allergy and Clinical Immunology, vol. 131, no. 3, pp. 904-905, 2013].
  • Membrane-tethered mucins within epithelial cell-derived exosomes affect the structural properties, conformation, and surface charge of exosomes. The properties of exosomes contribute to mucociliary defense by the lung’s innate immune system [Id., citing M. C. Rose and J. A.
  • Exosomes can modulate the immune response during viral infection by increasing the function of macrophages and NK cells, and delivering antiviral molecules among cells, as well as by inhibiting immune responses directly or indirectly and influencing cytokine-mediated signaling pathways and cytokine production.
  • exosomes released from DCs laden with pathogen-derived antigens can protect against infection [Id., citing A. I. Masyuk, T. V. Masyuk, and N. F. LaRusso,“Exosomes in the pathogenesis, diagnostics and therapeutics of liver diseases,” Journal of Hepatology, vol. 59, no. 3, pp. 621-625, 2013].
  • exosomes can also contain exogenous viral RNAs and be involved in the spreading of infection [Id., citing H. S. Chahar, X. Bao, and A. Casola, “Exosomes and their role in the life cycle and pathogenesis of RNA viruses,” Viruses, vol. 7, no. 6, pp. 3204-3225, 2015].
  • exosomes due to their biological properties, exosomes have also been proposed as possible delivery vectors for therapeutic purposes [Id., citing E. van der Pol, A. N. Boing, P. Harrison, A. Sturk, and R. Nieuwland,“Classification, functions, and clinical relevance of extracellular vesicles,” Pharmacological Reviews, vol. 64, no.
  • NK cells Natural killer (NK) cells are innate immune lymphocytes that destroy infecting or transformed cells without the need for activation, in contrast to T and B cells.
  • NK cells Human natural killer (NK) cells have been reported to release exosomes in both resting and activated condition. [Fais, S. Oncoimmunology (2013) 2 : 1, e :2237, citing Lugini, L. et al. J. Immunol. (2012) 189: 2833-42].
  • the NK cell-derived exosomes not only express both typical NK markers (i.e., CD56) and killer proteins (i.e., FASL and perforin) but also exert antitumor and immune homeostatic activities.
  • NK cells secrete exosomes in a constitutive way and independently from their activation status.
  • NK cell-derived express detectable amounts of the activating receptor NKG2D, whereas natural cytotoxicity receptors (NCRs), the other NK-cell receptors that mediate cytotoxic functions (i.e., NKp30, NKp46 and NKp44), are less expressed.
  • NCRs natural cytotoxicity receptors
  • Perforin was detected in exosomes purified from both NK-cell culture supernatants and the plasma of healthy individuals, whereas FASL was undetectable in plasmatic exosomes.
  • perforin-containing plasmatic exosomes were exclusively associated with NK-cell but not CD8+ T-cell markers; this is in line with a previous report showing that perforin is highly expressed by resting NK cells, but not by resting CD8+ T lymphocytes.
  • exosomes can serve as vectors, e.g., for gene delivery. While adeno-associated virus (AAV)-containing exosomes can be used for delivering genes to the cardiomyocytes and to the heart, they are susceptible to blocking by AAV neutralizing antibodies. AAV exosomes purified so as to minimize contamination with free AAVs were used to deliver the sarcoplasmic reticulum calcium ATPase gene (SERCA2a) in a mouse model with myocardial infarction. AAVExo vectors showed a significant enhancement in gene transduction compared to free AAVs and were more resistant to neutralizing antibodies than AAV both in vitro and in vivo. AAVExo-SERCA2a outperformed conventional AAV vectors in preserving cardiac function in presence and absence of neutralizing antibodies. [Liang, Y. et al. Circulation (2017) 136: A15439].
  • AAV sarcoplasmic reticulum calcium ATPase gene
  • Exosomes may mitigate apoptosis and block viral replication by inhibiting phosphorylation and suppressing pro-apoptosis factors. Moreover, the synergistic anti- apoptosis effect of exosomes relies on the Akt and caspase-3 dependent pathways.
  • the described invention has utility as both a diagnostic and therapeutic candidate utilized in a precision medicine method for diagnosing and optimizing long term health and therapeutic benefit for a susceptible subject with an acute lung injury progressive to pulmonary fibrosis caused by viral infection by, without limitation, SARS-CoV, CoVID- 19, MERS and other emerging respiratory viruses.
  • the described invention provides a precision medicine method for optimizing therapeutic benefit for a susceptible subject at risk of pulmonary complications of an acute lung injury caused by a severe infection with a respiratory virus, comprising:
  • EVs from the subject is dysregulated compared to the healthy control
  • the medical treatment comprises: administering a therapeutic amount of a pharmaceutical composition comprising either:
  • the therapeutic amount upregulates expression of miR-29a, downregulates expression of miR-199-3p, or both, and effectively delays development of the pulmonary complications.
  • the administering occurs by inhalation.
  • the population of EVs is derived from peripheral blood mononuclear cells (PBMCs) of the recovered patient.
  • the PBMCs comprise T lymphocytes, B lymphocytes and NK cells.
  • the population of EVs is derived from T lymphocytes of the recovered patient.
  • the population of EVs is derived from B lymphocytes of the recovered patient.
  • the population of EVs is derived from NK cells of the recovered patient.
  • the population of EVs is derived from urine of the recovered patient.
  • the population of EVs is modified by a viral vector.
  • the pulmonary complications comprise a fibrotic lung disease.
  • the fibrotic disease is a fibrotic lung disease.
  • the fibrotic lung disease is pulmonary fibrosis.
  • the isolating step (b) comprises purifying the urine-derived exosomes by one or more of: a) ultracentrifugation; b) sucrose density gradient centrifugation; c) column chromatography; d) size exclusion; or e) filtration through a device containing an affinity matrix selective towards the EVs.
  • the urine-derived EVs are characterized by sedimentation at about 100,000 x g, a buoyant density in sucrose of about 1.10-1.21 g/ml, and an average diameter of from about 30 nm to about 200 nm.
  • the average diameter of the EVs ranges from about 140 nm to about 150 nm.
  • the EVs comprise microvesicles whose diameter is >200 nm.
  • the predisposition to develop a fibrotic disease comprises onset of pulmonary fibrosis at an age earlier than the healthy control; typical predispositions based on genetic, environmental and lifestyle factors if not for the respiratory virus infection, or both.
  • the susceptible subject includes a very young individual, an elderly individual, an individual who is suffering from a respiratory disease; an individual who is receiving immunosuppressant therapy; an individual with a long term health condition; and an individual who is physically weak due to malnutrition or dehydration.
  • the severe infection with the respiratory virus comprises an acute lung injury, acute respiratory distress syndrome, or both.
  • the severe infection is caused by a respiratory virus selected from the group consisting of human coronavirus; human influenza virus, human herpesvirus, hantavirus; human cytomegalovirus; or human immunodeficiency virus.
  • the dysregulated miRNAs comprise one or more of miR-134-5p, miR-196b-5p, miR-629-5p, miR-206, miR-192-5p, miR-320c, miR- 125a-3p, miR-215-5p, miR-642a-3p, miR-576-3p, miR-3679-5p, miR-134-5p, miR-196b-5p, miR-629-5p, or miR-206. miR-let7d; miR-29a, miR 20-b,miR-29c, or miR-199.
  • the one or more miRNAs is downregulated compared to the healthy control.
  • the one or more miRNAs is upregulated compared to the healthy control.
  • the method further comprises steps of detecting a level of expression of one or more WNT proteins in the EVs from the urine samples; and determining that expression of one or more of the WNT proteins in the urine sample from the subject is dysregulated compared to the healthy control.
  • the one or more WNT proteins comprise WNT-5A.
  • the method further comprises
  • the method further comprises obtaining a bronchoalveolar lavage fluid (BALF) sample from the subject and from the healthy control; isolating EVs from the BALF samples; detecting a level of expression of one or more WNT proteins in the EVs; and comparing the level of expression of the one or more WNT proteins in the EVs from the subject and from the healthy control; wherein an increase in the levels of the one or more WNT proteins in the EVs from the subject indicates the subject has a fibrotic disease.
  • the one or more WNT proteins comprise WNT- 5A.
  • the fibrotic disease is selected from one or more of a fibrotic lung disease, a fibrotic cardiac disease, a fibrotic renal disease, a fibrotic hepatic disease, a fibrotic skin disease, a fibrotic pancreatic disease, a fibrotic eye disease, a fibrotic joint disease, a fibrotic bone marrow disease, a fibrotic brain disease, a fibrotic intestinal disease, a fibrotic peritoneum disease, or a fibrotic retroperitoneum disease.
  • the fibrotic disease is a fibrotic
  • the miR-29a mimic has at least about 70% sequence homology with SEQ ID NO: 1.
  • the miR-199-3p inhibitor has at least about 70% sequence homology with SEQ ID NO: 2.
  • the described invention provides a method of diagnosing and treating a lung injury caused by a severe infection by a respiratory vims fibrotic disease in a susceptible subject comprising
  • the therapeutic amount upregulates expression of miR-29a, downregulates expression of miR-199-3p, and treats the fibrotic disease.
  • the administering occurs by inhalation.
  • the sequence of the miR-29a mimic is at least about 70% homologous with SEQ ID NO: 1.
  • the sequence of the miR- 199-3p inhibitor is at least about 70% homologous with SEQ ID NO: 2.
  • the upregulated expression of miR-29a is effective to decreases expression of MMP-2.
  • the downregulated expression of miR-199-3p is effective to upregulates CAV-1 expression.
  • the lung injury includes acute respiratory failure, acute respiratory distress syndrome, or both.
  • the acute lung injury is progressive to a fibrotic lung disease.
  • the fibrotic lung disease is pulmonary fibrosis.
  • the described invention provides a method of attenuating a severe infection with a respiratory vims comprising a lung injury treating a fibrotic disease in a subject in need thereof comprising administering to the subject a therapeutic amount of a pharmaceutical composition comprising a population of EVs comprising one or more of an miRNA derived from normal, healthy MSCs, an miR-29a mimic, an miR-199-3p inhibitor, or both, and a pharmaceutically acceptable carrier, and wherein the pharmaceutical composition is effective to upregulates expression of miR-29a, downregulates miR-199-3p, or both, wherein the method attenuates the severe virus infection and the lung injury.
  • the respiratory virus is selected from the group consisting of human coronavirus; human influenza, human herpesvirus, hantavirus; human cytomegalovirus; and human immunodeficiency vims.
  • the susceptible subject includes a very young individual, an elderly individual, an individual who is suffering from a respiratory disease; an individual who is receiving immunosuppressant therapy; an individual with a long term health condition; and an individual who is physically weak due to malnutrition or dehydration.
  • the population of EVs is derived from peripheral blood mononuclear cells (PBMCs) of the recovered patient.
  • the PBMCs comprise T lymphocytes, B lymphocytes and NK cells.
  • the population of EVs is derived from T lymphocytes of the recovered patient. According to some embodiments, the population of EVs is derived from B lymphocytes of the recovered patient. According to some embodiments, the population of EVs is derived from NK cells of the recovered patient. According to some embodiments, the population of EVs is derived from urine of the recovered patient. According to some embodiments, the population of EVs is modified by a viral vector. According to some embodiments, the exosomes are synthetic.
  • Attenuating the lung injury accomplishes one or more of decreasing one or more symptoms of a fibrotic lung disease, increasing repair of a lung injury, restoring lung function, reducing or eliminating a need for other active agents or therapeutics; and slowing progression of fibrotic lung disease.
  • the exosomes are derived from a patient who has recovered from a viral infection. According to some embodiments, the exosomes are synthetic.
  • the lung injury is an acute lung injury.
  • the acute lung injury comprises inflammation, acute respiratory failure, or both.
  • lung injury includes acute respiratory distress syndrome.
  • the method treats progression to a fibrotic lung disease.
  • the fibrotic lung disease is pulmonary fibrosis.
  • the upregulated expression of miR-29a is effective to decreases expression of MMP-2.
  • the downregulated expression of miR-199-3p is effective to upregulates CAV-1 expression.
  • the miR-29a mimic has at least about 70% sequence homology with SEQ ID NO: 1.
  • the miR-199-3p inhibitor has at least about 70% sequence homology with SEQ ID NO: 2.
  • the normal healthy MSCs are obtained from a human subject.
  • the MSCs are derived from a tissue selected from the group consisting of amniotic membrane, chorionic membrane, umbilical cord tissue, bone marrow, and adipose tissue.
  • the EVs are characterized by sedimentation at about 100,000 x g, a buoyant density in sucrose of about 1.10-1.21 g/ml, and an average diameter of from about 30 nm to about 200 nm.
  • the average diameter of the EVs ranges from about 140 nm to about 150 nm.
  • the step of administering occurs nasally, intratracheally, orally, parenterally, topically, or by inhalation. According to some embodiments, the administering occurs by inhalation.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • the additional therapeutic agent is selected from one or more of an immunomodulatory agent, an analgesic agent, an anti-inflammatory agent, an anti-infective agent, an anti-malarial agent, an anti-viral agent, an anti-fibrotic compound, an anti-fibrotic agent, or a proton pump inhibitor.
  • the immunomodulatory agent is a corticosteroid.
  • the corticosteroid is selected from prednisone, azathioprine, mycophenolate, mycophenolate mofetil, colchicine, interferon-gamma lb, and combinations thereof.
  • the analgesic agent is selected from codeine, hydrocodone, oxycodone, methadone, hydromorphone, morphine, fentanyl, and combinations thereof.
  • the anti-inflammatory agent is selected from aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac nabumetone, naproxen, nintedanib, oxaprozin, pirfenidone, piroxicam, salsalate, sulindac, tolmetin, and combinations thereof.
  • the anti-viral agent is selected from acyclovir, gancidovir, foscamet; ribavirin; amantadine, azidodeoxythymidine /zidovudine), nevirapine, a tetrahydroimidazobenzodiazepinone (TIBO) compound; efavirenz; remdecivir, and delavirdine.
  • acyclovir gancidovir, foscamet
  • ribavirin amantadine, azidodeoxythymidine /zidovudine
  • nevirapine a tetrahydroimidazobenzodiazepinone (TIBO) compound
  • TIBO tetrahydroimidazobenzodiazepinone
  • the anti-malarial agent is selected from an aryl aminoalcohol compound selected from quinine, quinidine, chloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, and tafenoquine; an antifolate compound, selected from pyrimethamine, proguanil, chlorproguanil, trimethoprim; an artemisinin compound selected from artemisinin, dihydroartemisinin, artemether, artesunate; and atovaquone,
  • the anti-infective agent is selected from amoxicillin, doxycycline, demeclocycline; eravacycline, minocycline, ormadacycline, tetracycline, cephalexin, defotaxime, cetazidime, cefuroxime, ceftaroline; ciprofloxacin, levofloxacin, moxifloxacin
  • the anti- fibrotic agent is selected from nintedanib, pirfenidone, and combinations thereof.
  • the proton pump inhibitor is selected from omeprazole, lansoprazole, dexlansoprazole, esomeprazole, pantoprazole, rabeprazole, ilaprazole, and combinations thereof.
  • the method of attenuating a severe virus infection comprising a lung injury in a subject in need thereof further comprises:
  • Fibrosis markers selected from the group consisting of integrin mRNA, collagen type lal mRNA, miR29, c-jun protein; estrogen receptor alpha (ERa), androgen receptor (AR), caveollin-1 protein; pAKT/AKT protein in the EVs obtained from the subject and from the healthy control prior to treatment; and
  • the described invention has utility as a diagnostic and therapeutic candidate utilized to assess the health of donor lungs and to clinically aid in their resuscitation, since mR-29a is specifically involved in the downregulation of MMP2; MMP-2 levels correlate with poor organ function in donor lungs undergoing ex vivo lung perfusion; bronchiolocentric fibrosis is essentially represented by the pathological pattern of constrictive fibrotic bronchiolitis obliterans; and bronchiolitis obliterans may result from alloimmune chronic processes after lung transplantation.
  • a method for resuscitating donor lungs comprises administering ex vivo a therapeutic amount of a pharmaceutical composition comprising a population of EVs comprising one or more of an miRNA derived from normal, healthy MSCs, an miR-29a mimic, an miR-199-3p inhibitor, and a pharmaceutically acceptable carrier, and wherein the pharmaceutical composition upregulates expression of miR-29a, downregulates miR-199-3p, or both, wherein the method restores lung function.
  • the exosomes are derived from a patient who has recovered from an infection with a respiratory virus or has been exposed to anti- viral antibodies through treatment.
  • the respiratory virus is selected from the group consisting of human coronavirus; human influenza, human herpesvirus, hantavirus; human cytomegalovirus; and human immunodeficiency virus.
  • the population of EVs is derived from peripheral blood mononuclear cells (PBMCs) of the recovered patient.
  • the PBMCs comprise T lymphocytes, B lymphocytes and NK cells.
  • the population of EVs is derived from T lymphocytes of the recovered patient.
  • the population of EVs is derived from B lymphocytes of the recovered patient.
  • the population of EVs is derived from NK cells of the recovered patient.
  • the population of EVs is derived from urine of the recovered patient.
  • the population of EVs is modified by a viral vector.
  • the exosomes are synthetic.
  • FIG. 1 is a flow chart of clinical trial participants in the Allogeneic Human Cells (hMSC) in patients with Idiopathic Pulmonary Fibrosis via Intravenous Delivery (AETHER) trial.
  • hMSC Allogeneic Human Cells
  • AETHER Intravenous Delivery
  • FIG. 2A-FIG. 2D are a series of graphs showing secondary efficacy outcomes during the 60-week study period.
  • FIG. 2A Changes in mean % predicted FVC for overall study period.
  • FIG. 2B Changes in % predicted diffusing capacity of the lungs for carbon monoxide (Dlco).
  • FIG. 2D A representative HRCT scan at baseline, week 24, and week 48, showing decreasing fibrotic reticulation in all lobes at 100 x 10 6 dose.
  • FIG. 3A-FIG. 3B show bleomycin-induced lung injury evidenced by micro computed tomography (mCT) 7 days post-instillation.
  • FIG. 3A Representative mCT transverse and coronal lung sections acquired from aged (22 month-old) male C57BF/6 mice at baseline (left) and 7 days following intratracheal bleomycin (BFM, 2.0 units/kg) administration (right) demonstrating increased lung density and loss of airspaces.
  • FIG. 3B Saline treatment did not result in evidence of lung injury on mCT scan at baseline (left) or 7 days post-instillation (right).
  • FIG. 4A-FIG. 4E show that allogeneic adipose-derived mesenchymal stem cells (ASCs) administered 12 days after bleomycin (BFM)-instillation reduce severity of pulmonary fibrosis and collagen content in aged mice. Histological sections of lung tissue were stained with Masson’ s-Trichrome.
  • FIG. 4A and 4B show representative photomicrographs (20x and 40x magnification) of lung sections from saline-treated control mice (FIG. 4A) and BFM-treated mice (FIG. 4B).
  • FIG. 4C Administration of ASCs 12 days after BFM-instillation resulted in reduced severity of pulmonary fibrosis.
  • FIG. 4A-FIG. 4E show that allogeneic adipose-derived mesenchymal stem cells (ASCs) administered 12 days after bleomycin (BFM)-instillation reduce severity of pulmonary fibrosis and collagen content in aged mice. Histological sections of lung
  • FIG. 4D Degree of pulmonary fibrosis on histological sections was measured by semi-quantitative Ashcroft score. BFM instillation resulted in increased Ashcroft score compared to saline controls. Treatment with ASCs 12 days following BFM administration resulted in decreased Ashcroft score.
  • FIG. 5 shows that bleomycin (BLM)-induced AKT activation is inhibited by treatment with allogeneic adipose-derived mesenchymal stem cells (ASCs).
  • BLM bleomycin
  • ASCs allogeneic adipose-derived mesenchymal stem cells
  • FIG. 6A-FIG. 6D show that lung miR-29a expression is increased and miR- 199-3p expression is decreased by infusion of allogeneic adipose-derived mesenchymal stem cells (ASCs) following bleomycin (BLM)-induced downregulation.
  • ASCs allogeneic adipose-derived mesenchymal stem cells
  • BBM bleomycin
  • Aged C57B1/6 mouse lung expression of miR-29a is decreased by 21 -day sacrifice in response to bleomycin (BLM) lung injury compared to saline controls. Treatment with allogeneic ASCs on day 12 post-BLM infusion resulted in higher expression of miR-29a versus BLM-only controls.
  • FIG. 7 shows that in vitro transfection of miR-29a mimic (SEQ ID NO: 1) and miR-199-3p inhibitor (SEQ ID NO: 2) regulates downstream targets MMP-2 and CAV-1 in human myofibroblasts.
  • miR-29a mimic SEQ ID NO: 1
  • miR-199-3p inhibitor SEQ ID NO: 2
  • Myofibroblasts were transfected with scrambled controls (Control) or miR-29a and -199-3p (29/199).
  • FIG. 8A and FIG. 8B show a schematic (FIG. 8A) of the strategy to determine if E2 is protective and androgens detrimental to fibrosis development, and if E2 is protective and androgens detrimental to fibrosis development in a mouse model, and to determine the contribution of AR and ER in the development of lung fibrosis using myofibroblasts isolated from lungs of male and female patients with IPF (FIG. 8B).
  • FIG. 9 shows that ASCs isolated from post-menopausal (old) adipose have decreased estrogen receptor expression and response to estrogen compared to ASCs isolated from pre-menopausal (young) adipose.
  • FIG. 9A compares ERa protein expression for pre menopausal and post-menopausal tissue.
  • FIG. 9B compares ERP protein expression for pre menopausal and post-menopausal tissue.
  • FIG. 9C premenopausal
  • FIG. 9D premenopausal
  • FIG. 10 shows that ASCs isolated from post-menopausal (old) adipose have decreased catalase expression compared to ASCs isolated from pre-menopausal (young) adipose.
  • FIG. 11 shows lung histology 21 days post-BLM treatment and infusion of pre-menopausal ASCs (“pre- ASCs”) or post-menopausal ASCs (“post-ASCs””).
  • FIG. 11 panels 4, 8, 12 post- hASCs transfected with catalase activator.
  • Infusion of pre-hASC transfected with inhibitor did not reduce severity of fibrosis in the lung compared to post-hASCs transfected with catalase activator (hASCs+activator, FIG. 11, panels 10 and 12).
  • FIG. 12 shows that pre-menstrual hASCs transfected with catalase inhibitor increased lung hydroxyproline (collagen accumulation, FIG. 12A), and TNFa mRNA expression (FIG. 12B, relevant endpoints in the lung fibrosis model.
  • FIG. 13A and FIG. 13B show that wound healing is enhanced in post- menopausal ASCs after transfection with catalase activator.
  • FIG. 13A is a plot showing % wound healing on the Y-axis; the x axis identifies the samples tested: control media, pre- menopausal human ASC (control); inhibitor at 48 hours, inhibitor for 72 hours, control post- human-ASCs; activator at 48 hours, and activator at 72 hours.
  • FIG. 13B shows tissue treated (from top to bottom) with Control media (a). pre-hASC control (b); pre-hASC + inhibitor (c), post -hASC control (d); and post-hASC + activator.
  • the white arrows indicate wound edges after initial wounding.
  • FIG. 14 shows graphs showing that IPF myofibroblast derived EVs increase pro-fibrotic markers in 3D lung punches, either by increasing integrin (FIG. 14A), c-Jun (Fig. 14B), or by decreasing the expression of caveolin-l(FIG. 14C), microRNA 29a (miR-29) (FIG. 14D), and miR-let 7D (FIG. 14E).
  • FIG. 15 panels A, B, C, D, E, and F are pictures showing the effects of IPF and non-IPF lung fibroblast-derived EVs on representative ex vivo lung punches from an aging male mouse at 20x (panels A, B, C) and 40x (panels D, E, F).
  • Panels A and D media control
  • Panels B and E IPF lung fibroblast-derived exosomes
  • Panels C and F non-IPF lung fibroblast derived exosomes.
  • FIG. 16 shows graphs showing that mesenchymal stem cell-derived EVs reduced fibrosis in an established model of pulmonary fibrosis.
  • FIG. 16A Ashcroft score;
  • FIG. 16B hydroxyproline (collagen content) P ⁇ 0.05.
  • FIG. 17 is a picture showing that EVs derived from IPF lung fibroblasts prevent ex vivo wound healing.
  • FIG. 18 is a representative picture showing that EVs performed equally to whole cell MSCs in an ex vivo wound healing assay.
  • FIG. 18A whole cell MSC;
  • FIG. 18B exosomes;
  • FIG. 18C control.
  • FIG. 19A, FIG. 19B, FIG. 19C show that mesenchymal stem cell-derived EVs are equally efficacious as whole cells in preventing pulmonary fibrosis.
  • FIG. 19A shows representative pictures from BLM control (panel 1), BLM + hASC whole cell (panel 2), BLM + hASC exosomes (panel 3); BLM + mASC (panel 4); and BLM + mASC exosomes (panel 5).
  • FIG. 19B is a graph comparing Ashcroft scores.
  • FIG. 19C is a graph comparing collagen content.
  • FIG. 20 shows two graphs depicting changes in expression of anti-fibrotic marker caveolin-1 (FIG. 20A) and profibrotic marker c-jun (FIG. 20B) after injection of ASC-derived EVs.
  • EVs or media were injected into a lung punch from an aging mouse treated for 10 days with bleomycin. Punches were collected after 4 days and analyzed.
  • FIG. 21 shows urine-derived exosome expression of t integrin mRNA n (FIG. 21A), collagen type lal mRNA (FIG. 21B), profibrotic c-Jun protein (FIG. 21 C); pAK/pAKT ratio (FIG. 21D); and MMP-9 activity (FIG. 21E) for media control, urine exosomes from control subjects; and urine exosomes from IPF subjects.
  • Expression of integrin mRNA, collagen type lal mRNA, c-June protein; and pAkt/AKT ratio all of which are markers for IPF, were altered in the urine derived exosomes from patients with IPF compared to the urine derived exosomes derived from controls.
  • FIG. 22 shows photographs of ex vivo lung punches (4 mm) from the agarose- infused young and old mouse lungs that were injected with ASC derived exosomes and collected after 4 days.
  • FIG. 23 shows results obtained when exosomes derived from young ASCs were injected into lung punches isolated from day 10 post-BLM treated lung (right panels). Media control (left panels) received treatment with media only. Results show that punches treated with ASC exosomes (FIG. 23B) display reduced a smooth muscle actin expression compared to the control (FIG. 23A). The ASC exosomes treated punches also show an increase in anti-fibrotic CAV-1 (FIG. 23D) compared to the control (FIG. 23C); and a decrease in pro-fibrotic c-Jun (FIG. 23F) compared to the control (FIG. 23E). No modification in b actin was detected (FIG. 23H, FIG. 23G) This demonstrates that treatment of ex vivo lung punches with ASC exosomes modifies ex vivo lung punch tissue.
  • FIG. 24 left panels show ex vivo mouse punches injected with a media control; right panels show punches injected with ASC exosomes.
  • FIG. 24A and FIG. 24B show trichrome histology of ex vivo mouse punches following contact with a media control (FIG. 24A) and ASC exosomes (FIG. 24B).
  • FIG. 24C, 24D show results of immunofluorescent staining for surfactant protein C (SPC).
  • SPC surfactant protein C
  • Surfactant proteins are mainly expressed by distal lung epithelial cells. SPC is a pulmonary surfactant protein. SPC therefore was used as a marker for alveolar cell regeneration.
  • FIG. 24E control
  • FIG. 24E control
  • FIGS. 24F show results of immunofluorescent staining for aquaporin 5 (AQP5).
  • Aquaporin is a water channel protein, which plays a role in the generation of pulmonary secretions.
  • Punches treated with ASC exosomes express increased Surfactant Protein C (SPC) and Aquaporin 5 (AQP5) compared to the control.
  • SPC Surfactant Protein C
  • AQP5 Aquaporin 5
  • the left panels show a few alveolar type 2 cells.
  • the right panels show an increase in both alveolar type 1 and type 2 cells, mostly type 2, with a little of type 1. This demonstrates that punch treatment with ASC exosomes increases expression of alveolar cell type 2 and type 1.
  • FIG. 25 shows results of experiments in which exosomes derived from either fibroblasts isolated from young male control lungs or myofibroblasts isolated from IPF lungs were injected into a naive aging mouse lung punch and parameters associated with pulmonary fibrosis, namely integrin, miR-29, c-jun protein, ERa, and CAV-1 protein levels measured.
  • FIG. 25 shows that expression of integrin mRNA, MIR-29a, Caveolin-1 protein; c-June protein; and estrogen receptor alpha, all of which are markers for IPF, were altered in the exosomes from patients with IPF compared to the normal control. More specifically, the level of integrin mRNA was increased (FIG.
  • FIG. 25A miR-29 decreased [FIG. 25B] shows, antifibrotic CAV-1 protein decreased (FIG. 25C), profibrotic c-jun protein increased (FIG. 25D), ERa protein increased (FIG. 25E) compared to a media control and exosomes prepared from control lung fibroblasts. EVs from control lungs increased CAV-1 protein, an anti-fibrotic marker.
  • FIG. 26 shows results of experiments in which exosomes derived from the urine of subjects without lung disease (control) or from the urine of patients with IPF were injected into naive aging mouse punches.
  • the result show that EVs derived from the urine of patients with IPF likewise display an increase in integrin mRNA expression (FIG. 26A), an increase in collagen lal mRNA expression (FIG. 26 B); an increase in profibrotic c-Jun protein expression (FIG. 26C) and an increase in the ratio of pAKT/AKT (FIG. 26D) compared to a urine control and a media control.
  • FIG. 27A shows results obtained when human lung punches were injected with exosomes derived from normal fibroblasts (control fib) and exosomes derived from IPF lung myofibroblasts (IPF fib) and collected 4 days later. Punches were processed for mRNA and protein expression.
  • FIG. 27A shows that expression of aV integrin and of type I collagen in lungs injected with IPF fibroblast-derived exosomes is increased compared to the control.
  • FIG. 27B shows that ERa protein expression in punches contacted with IPF urine was increased compared to controls.
  • Fig. 27C shows that anti-fibrotic caveolin-1 protein expression was decreased in IPF urine and IPF fib samples compared to the controls.
  • FIG. 28 A, 26B-28B, 28C, 28D, and 28E show time course of distribution of ExoGlowTM labeled exosomes injected via tail vein in a mouse 8 days after treatment with BLM.
  • FIG. 28A distribution after 5 minutes.
  • FIG. 28B after 30 minutes, the distribution of ExoGlowTM indicates migration of exosomes to the lungs.
  • FIG. 28C shows the distribution at 2 hours.
  • FIG. 28D shows distribution at 8 hours.
  • FIG. 28E shows that at 20 hours, the distribution of ExoGlow indicates migration of exosomes to the kidneys.
  • FIG. 30 shows the vivid Exo-GlowTM fluorescence 24 hours post-injection of Exo-GlowTM exosomes in kidney (1) and liver (2) at the 90 mg dosing. At 24 hour, exosomes cannot be detected in the lungs, spleen and heart ((3, 4, 5) at the 90 mg dosing.
  • FIG. 31A and FIG. 31B show the exosomes membrane opposed to the membrane of the type I alveolar epithelial cell, and the nanoparticles are seen within the alveolar epithelial cell.
  • the arrows in FIG. 31A, FIG. 31B (higher magnification) show that the exosomes cell membrane is still intact.
  • FIG. 32A and FIG. 32B show exosomes containing nanoparticles being engulfed.
  • FIG. 33A, 33B, 33C, and 33D show 15 month old mouse lung punch injected with urine derived exosomes from control subjects without lung disease.
  • FIG. 34A, 34B, 34C, 34D, and 34E show 15 month old mouse lung punch injected with urine derived exosomes from IPF subjects.
  • the lung punch shows evidence of collagen bands not seen when urine-derived exosomes are injected (See Fig 33 punches).
  • Urine-derived exosomes labeled with nanoparticles (gold arrow) are evident in AEC I cells in panels D and E
  • FIG. 35 shows Ex-Glow mice following tail vein injection of urine-derived exosomes imaged with IVIS (in vivo imaging system) at 5 minutes (I), 24 hours (II) and 19 days (III).
  • A corresponds to PBS injection (negative control).
  • Panels B and D urine derived exosomes from control subjects;
  • Panels C and E urine derived exosomes from IPF subjects. Regardless of source (control or IPF subject), exosomes were located in the lung 5 mins post tail vein injection and continued to be present at 24 hours. The signal was no longer evident at 19 days.
  • FIG. 36A, 36B and 36C show urine-derived exosomes expression of selected microRNAs.
  • FIG. 36A miR-let7d/U6; FIG. 36B MiR-29a/U6; FIG. 36C MiR-199/U6.
  • the relative expression of mirR-let7d (A) and mir-29A (B) in urine derived exosomes from IPF subjects was decreased compared to exosomes from control subjects.
  • the relative expression of miR-199 (C) in urine derived exosomes from IPF subjects was increased compared to urine-derived exosomes from control subjects.
  • FIG. 37 compares miRNA expression in urine exosome injected explants (FIG 37A, miR-199a; FIG. 37B, miR-29a; FIG. 37C let7d); to miRNA expression in fibroblast exosome injected explants (FIG. 37D, miR0199-3p; FIG. 37E, miR-29a; FIG. 37F, miR-let7d.
  • the results show that microRNA expression of the punches changes after injection with exosomes. IPF exosomes confer disease to the punch by changing the miR to mimic what is seen in IPF lungs.
  • 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%.
  • adipocyte refers to the functional cell type of fat, or adipose tissue that is found throughout the body, particularly under the skin. Adipocytes store and synthesize fat for energy, thermal regulation and cushioning against mechanical shock. Although the lineage of adipocytes is still unclear, it appears that mesenchymal stem cells can differentiate into two types of lipoblasts, one that give rise to white adipocytes and the other to brown adipocytes. Both types of adipocytes store fat. Adipose tissue may be brown or white adipose tissue, derived from, for example, subcutaneous, omental/visceral, mammary, gonadal, periorgan or other adipose tissue site.
  • adipose stem cell refers to pluripotent stem cells, mesenchymal stem cells, and more committed adipose progenitors and stroma obtained from adipose tissue.
  • 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.
  • 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.
  • allogeneic refers to being genetically different although belonging to or obtained from the same species.
  • the term“allograft” as used herein refers to a transplant of tissue from an allogeneic donor of the same species.
  • the term“allograft immunity” as used herein refers to any immune response by the host against a transplanted tissue.
  • alveolus or“alveoli” as used herein refers to an anatomical structure that has the form of a hollow cavity.
  • 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.
  • amino acid is used to refer to an organic molecule containing both an amino group and a carboxyl group; those that serve as the building blocks of naturally occurring proteins are alpha amino acids, in which both the amino and carboxyl groups are linked to the same carbon atom.
  • amino acid residue or“residue” are used interchangeably to refer to an amino acid that is incorporated into a protein, a polypeptide, or a peptide, including, but not limited to, a naturally occurring amino acid and known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
  • the amino acids may be L- or D-amino acids.
  • An amino acid may be replaced by a synthetic amino acid which is altered so as to increase the half-life of the peptide or to increase the potency of the peptide, or to increase the bioavailability of the peptide.
  • Isoleucine Isoleucine
  • Leucine L
  • Methionine M
  • Valine V
  • Amnionic membranes develop from extra-embryonic tissue and consist of a fetal component (the chorionic plate) and a maternal component (the decidua, meaning the lining of the pregnant uterus), which are held together by the chorionic villi and connect the cytotrophoblastic shell of the chorionic sac to the decidua basalis.
  • the fetal component which includes the amniotic and chorionic fetal membranes, separates the fetus from the endometrium.
  • the amniochorionic membrane forms the outer limits of the sac that encloses the fetus, while the innermost layer of the sac is the amniotic membrane.
  • the amniotic membrane consists of (A) an epithelial monolayer, (B) a thick basement membrane, (C) a compact layer, (D) a fibroblast layer, and (E) a spongy layer.
  • the amniotic epithelium the innermost layer nearest to the fetus, and in contact with the amniotic fluid, consists of a single layer of cells uniformly arranged on the basement membrane.
  • the epithelial layer can be removed while the basement membrane and stromal surfaces remain morphologically intact.
  • the basement membrane is composed of a network of reticular fibers.
  • the compact layer of stromal matrix adjacent to the basement membrane forms the main fibrous skeleton of the AM.
  • the collagens of the compact layer are secreted by mesenchymal cells situated in the fibroblast layer.
  • Interstitial collagens (types I and III) predominate and form parallel bundles that maintain the mechanical integrity of the AM.
  • Collagens type V and VI form filamentous connections between interstitial collagens and the epithelial basement membrane.
  • the fibroblast layer is composed of a loose fibroblast network embedded in a mass of reticulum.
  • the spongy layer of the stromal matrix sits adjacent to the chorionic membrane, and represents the tissue of the extraembryonic coelom, which is compressed between the amnion and the chorion. It contains a nonfibrillar meshwork of mostly type III collagen.
  • the spongy layer is loosely connected to the chorionic membrane; hence the AM is easily separated from the chorion by means of blunt dissection (Niknejad, H. et al, Eur. Cells and Materials (2008) 15: 88-99).
  • amniotic stem cells 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.
  • angiogenic factor refers to any of a group of substances present in the circulation (most of which are polypeptides— e.g., angiogenin, fibroblast growth factor, transforming growth factors and some lipids), which play a role in blood vessel formation (angiogenesis).
  • polypeptides e.g., angiogenin, fibroblast growth factor, transforming growth factors and some lipids
  • angiogenic factors such as VEGF, IGF, PDGF, HGF, FGF, TGF, Angiopoeitin-1, and stem cell factor (SCF) have been found to differ amongst bone-derived-, cartilage-derived-, and adipose-derived MSCs (Peng et al., 2008, Stems Cells and Development, 17: 761-774).
  • 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.
  • 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.
  • 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.
  • antibody refers to a polypeptide or group of polypeptides comprised of at least one binding domain that is formed from the folding of polypeptide chains having three-dimensional binding spaces with internal surface shapes and charge distributions complementary to the features of an antigenic determinant of an antigen.
  • An antibody typically has a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light” and one "heavy” chain. The variable regions of each light/heavy chain pair form an antibody binding site.
  • a “targeted binding agent” is an antibody, or binding fragment thereof, that preferentially binds to a target site. According to some embodiments, the targeted binding agent is specific for only one target site.
  • the targeted binding agent is specific for more than one target site.
  • the targeted binding agent may be a monoclonal antibody and the target site may be an epitope.
  • epitope refers to that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding pocket of an antibody.
  • Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies.
  • An antibody other than a "bispecific” or "bifunctional” antibody is understood to have each of its binding sites identical.
  • An antibody substantially inhibits adhesion of a receptor to a counter-receptor when an excess of antibody reduces the quantity of receptor bound to counter-receptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitro competitive binding assay).
  • An antibody may be an oligoclonal antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti-idiotypic antibody, and an antibody that can be labeled in soluble or bound form, as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques.
  • An antibody may be from any species.
  • antibody also includes binding fragments of the antibodies of the invention; exemplary fragments include Fv, Fab, Fab', single stranded antibody (svFC), dimeric variable region (Diabody) and di-sulphide stabilized variable region (dsFv).
  • exemplary fragments include Fv, Fab, Fab', single stranded antibody (svFC), dimeric variable region (Diabody) and di-sulphide stabilized variable region (dsFv).
  • minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the described invention, providing that the variations in the amino acid sequence maintain at least about 75%, and in some embodiments, at least about 80%, about 90%, about 95%, and about 99% sequence identity to the antibodies or immunoglobulin molecules described herein. Conservative amino acid replacements are contemplated.
  • amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains.
  • Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. For example, computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. See, for example, Bowie et al. Science 253:164 (1991), which is incorporated by reference in its entirety.
  • the term“antigen” refers to a molecule, e.g., a peptide, polypeptide, protein, fragment, or other biological moiety, which elicits an antibody response in a subject, or is recognized and bound by an antibody.
  • autocrine signaling refers to a type of cell signaling in which a cell secretes signal molecules that act on itself or on other adjacent cells of the same type.
  • binding and its other grammatical forms as used herein means a lasting attraction between chemical substances. Binding specificity involves both binding to a specific partner and not binding to other molecules. Functionally important binding may occur at a range of affinities from low to high, and design elements may suppress undesired cross-interactions. Post-translational modifications also can alter the chemistry and structure of interactions.“Promiscuous binding” may involve degrees of structural plasticity, which may result in different subsets of residues being important for binding to different partners. “Relative binding specificity” is a characteristic whereby in a biochemical system a molecule interacts with its targets or partners differentially, thereby impacting them distinctively depending on the identity of individual targets or partners.
  • biomarker refers to a peptide, a protein, a nucleic acid, an antibody, a gene, a metabolite, or any other substance used as an indicator 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.
  • indicator 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.
  • 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.
  • Cavs 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, IR. Adv. Exp. Med. Biol. (2012) 729: 29-50). Cavs are both positive and negative regulators of cell signaling in and/or out of caveolae, invaginated lipid raft domains whose formation is caveolin expression dependent. Caveolins and rafts have been implicated in membrane compartmentalization; proteins and lipids accumulate in these membrane microdomains where they transmit fast, amplified and specific signaling cascades.
  • CAV1 caveolin 1
  • CCL18 CCC motif chemokine ligand 18
  • CCL18 refers to a small protein derived from alveolar macrophages that acts as a chemo-attractant.
  • CCL18 is mainly secreted by antigen-presenting cells such as monocytes, macrophages and dendritic cells (Guiot, J. et al. Lung (2017) 195(3): 273-280, citing Hieshima K, et al. J Immunol. 1997; 159(3): 1140-49).
  • alveolar macrophages are believed to be the main source of CCL18 in the lung and play a role in the pathogenesis of pulmonary fibrosis (Id., citing Prasse A, et al. Am J Respir Crit Care Med. 2006; 173(7): 781- 92). Serum CCL18 is increased in IPF but is not specific of the disease (Id., citing Prasse A, et al. Am J Respir Crit Care Med. 2006; 173(7): 781-92; Prasse A, et al. Arthritis Rheum. 2007; 56(5): 1685-93; Luzina IG, et al. J Cell Physiol. 2006; 206(1): 221-8).
  • pirfenidone one of the specific anti-fibrotic therapies in IPF, significantly suppressed the expression of CCL18 on macrophages (Id., citing Saito Y, et al. Immunopharmacol Immunotoxicol. 2016; 38(6): 46471). Baseline concentration > 150 ng/ml is associated with higher mortality (Id.).
  • chorion 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).
  • a matrix type on the inner side more compact
  • a fibrin type on the outer side more reticulate
  • 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, GL, Am. J. Obstet. & Gynec. (1960) 79 (6): 1070-73
  • “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.
  • the term "comparison window" refers to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • conditioned medium refers to spent culture medium harvested from cultured cells containing metabolites, growth factors, RNA and proteins released into the medium by the cultured cells.
  • contact and its various grammatical forms as used herein refers to a state or condition of touching or of immediate or local proximity.
  • culture medium refers to a substance containing nutrients in which cells or tissues are cultivated for controlled growth.
  • cytokine 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.
  • 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 TNFa and lymphotoxin
  • immunoglobulin super-family members including interleukin 1 (IL- 1);
  • 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.
  • the term“derived from” is meant to encompass any method for receiving, obtaining, or modifying something from a source of origin.
  • 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.
  • the term“differentiation” as used herein refers to a process of development with an increase in the level of organization or complexity of a cell or tissue, accompanied by a more specialized function.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the particle size is 0.5 mm.
  • size of the dispersed particle is in the range of approximately 1 mm to 0.5 mih.
  • 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.
  • 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.
  • down-regulation 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).
  • down-regulation can refer to instances where protein translation from transcripts produced by the polynucleotide sequence is reduced or eliminated.
  • 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.
  • exogenous refers to that which is naturally occurring, incorporated within, housed within, adherent to, attached to, or resident in.
  • exogenous refers to that which is non-naturally occurring, or that is originating or produced outside of a specific EV, cell, organism, or species.
  • 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).
  • FACS fluorescence- activated cell sorting
  • ESCRT machinery refers to an evolutionarily- conserved, multi-subunit membrane remodeling complex originally identified in yeast for its essential role in the biogenesis of intraluminal vesicles (ILVs) upon a class of endosome called the multivesicular body (MVB), whose role in mammalian cells includes a number of topologically equivalent membrane modeling events, see Olmos, Y., Oarlton, JG. Curr. Opin. Cell Biol. (2016) 38: 1-11, citing Katzmann, DJ et al. Cell (2001) 106: 145-55; Babst, M. et al. dev. Cell (2002) 3: 283-89; Babst, M. et al., dev. Cell (2002) 3 : 272-82]
  • exacerbation refers to an increase in the severity of a disease or any of its signs or symptoms.
  • expand and its various grammatical forms as used herein refers to a process by which dispersed living cells propagate in vitro in a culture medium that results in an increase in the number or amount of viable cells.
  • 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.
  • 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.
  • fibroblast refers to a connective tissue cell that makes and secrets collagen protein. Fibroblasts, the most common cell type found in connective tissues, play an important role in healing wounds. Like other cells of connective tissue, fibroblasts are derived from primitive mesenchyme (a type of loose connective tissue derived from all three germ layers and located in the embryo). In certain situations, epithelial cells can give rise to fibroblasts, a process called epithelial-mesenchymal transition.
  • myofibroblasts refers to fibroblasts in wound areas that have some characteristics of smooth muscle, such as contractile properties and fibers, and are believed to produce, temporarily, type III collagen.
  • fragment and its other grammatical forms are meant to refer to portions of a nucleic acid, polynucleotide or oligonucleotide shorter than the full sequence of a reference.
  • sequence of bases in a fragment is unaltered from the sequence of the corresponding portion in the molecule from which it arose; there are no insertions or deletions in a fragment in comparison with the corresponding portion of the molecule from which it arose.
  • a fragment of a nucleic acid or polynucleotide is 15 or more bases in length, or 16 or more, 17 or more, 18 or more, or 19 or more, or 20 or more, or 21 or more, or 22 or more, or 23 or more, or 24 or more, or 25 or more, or 26 or more, or 27 or more, or 28 or more, or 29 or more, 30 or more, 50 or more, 75 or more, 100 or more bases in length, up to a length that is one base shorter than the full length sequence.
  • RNA refers to a DNA polynucleotide sequence that expresses an RNA (RNA is transcribed from the DNA polynucleotide sequence) from a coding region, which RNA can be a messenger RNA (encoding a protein) or a non-protein- coding RNA (e.g., a crRNA, tracrRNA, or gRNA herein).
  • A“gene” may refer to the coding region alone, or may include regulatory sequences upstream and/or downstream to the coding region (e.g., promoters, 5 '-untranslated regions, 3 '-transcription terminator regions).
  • a coding region encoding a protein can alternatively be referred to herein as an“open reading frame” (ORF).
  • ORF an“open reading frame”
  • a gene that is“native” or“endogenous” refers to a gene as found in nature with its own regulatory sequences; such a gene is located in its natural location in the genome of a host cell.
  • A“chimeric” gene refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature (i.e., the regulatory and coding regions are heterologous with each other). Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • A“foreign” or“heterologous” gene refers to a gene that is introduced into the host organism by gene transfer.
  • Foreign/heterologous genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes.
  • the polynucleotide sequences in certain embodiments disclosed herein are heterologous.
  • A“transgene” is a gene that has been introduced into the genome by a gene delivery procedure (e.g., transformation).
  • A“codon- optimized” open reading frame has its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.
  • GGO Ground-glass opacity
  • CT computed tomography
  • GGO can be a manifestation of a wide variety of clinical features, including malignancies and benign conditions, such as focal interstitial fibrosis, inflammation, and hemorrhage (Id., citing Park CM, et al. Nodular ground-glass opacity at thin-section CT: histologic correlation and evaluation of change at follow-up. Radiographics (2007) 27:391- 408).
  • growth factor 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. These pathways stimulate the accumulation of proteins and other macromolecules, e.g., by increasing their rate of synthesis, decreasing their rate of degradation, or both.
  • FGF Fibroblast Growth Factor
  • FGF1 is also known as acidic FGF
  • FGF2 is sometimes called basic FGF (bFGF)
  • FGF7 sometimes goes by the name keratinocyte growth factor.
  • FGFs can activate a set of receptor tyrosine kinases called the fibroblast growth factor receptors (FGFRs).
  • FGFRs fibroblast growth factor receptors
  • 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.
  • a dormant tyrosine kinase i.e., a protein that can phosphorylate another protein by splitting ATP
  • 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.
  • 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.
  • IGF-1 Insulin-Like Growth Factor
  • 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.
  • GH growth hormone
  • STAT5B signal transducer and activator of transcription 5B
  • IGF-1 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.
  • GH growth hormone
  • 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-l-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.
  • PI3 kinase inhibitor LY294002 and wortmannin
  • the data indicate that IGF-1 increases MSC migratory responses via CXCR4 chemokine receptor signaling which is PI3/Akt dependent.
  • TGF-b Transforming Growth Factor Beta
  • the TGF-b superfamily includes the TGF-b 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 Mullerian inhibitory factor, which is involved in mammalian sex determination.
  • TGF-b family members TGF-bI, 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-bI increases the amount of extracellular matrix epithelial cells make both by stimulating collagen and fibronectin synthesis and by inhibiting matrix degradation.
  • TGF ⁇ s may be critical in controlling where and when epithelia can branch to form the ducts of kidneys, lungs, and salivary glands.
  • VEGF Vascular Endothelial Growth Factor
  • 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.
  • VEGF's normal function creates new blood vessels during embryonic development, after injury, and to bypass blocked vessels.
  • 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.
  • 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.
  • 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.
  • hybridization refers to the binding of two single stranded nucleic acid molecules to each other through base pairing. Nucleotides will bind to their complement under normal conditions, so two perfectly complementary strands will bind (or‘anneal’) to each other readily. However, due to the different molecular geometries of the nucleotides, a single inconsistency between the two strands will make binding between them more energetically unfavorable. Measuring the effects of base incompatibility by quantifying the rate at which two strands anneal can provide information as to the similarity in base sequence between the two strands being annealed.
  • nucleic acid or amino acid sequences refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., at least about 60% or about 65% identity, or at least about 70-95% identity, or at least 95% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, at least about 60% to 95% or greater sequence identity are considered to be substantially identical.
  • the described identity can exist over a region that is at least about 15 to 25 amino acids or nucleotides in length, or over a region that is about 50 to 100 amino acids or nucleotides in length.
  • Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245), as known in the art.
  • the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity.
  • CFUSTAFW does take sequence gaps into account in its identity calculations.
  • BEAST and BEAST 2.0 algorithms Altschul Nucl. Acids Res. 25 (1977), 3389-3402.
  • the BLASTP program uses as defaults a wordlength (W) of 3, and an expectation (E) of 10.
  • the described invention also relates to nucleic acid molecules, the sequence of which is degenerate in comparison with the sequence of an above-described hybridizing molecule.
  • the term "being degenerate as a result of the genetic code” means that due to the redundancy of the genetic code, different nucleotide sequences code for the same amino acid.
  • the described invention also relates to nucleic acid molecules which comprise one or more mutations or deletions, and to nucleic acid molecules which hybridize to one of the herein described nucleic acid molecules, which show (a) mutation(s) or (a) deletion(s).
  • the terms “immune response” and “immune-mediated” are used interchangeably herein to refer to any functional expression of a subject’s immune system, against either foreign or self-antigens, whether the consequences of these reactions are beneficial or harmful to the subject.
  • immunogen and its various grammatical forms as used herein refers to a substance that elicits an immune response
  • immunomodulatory refers to a substance, agent, or cell that is capable of augmenting or diminishing immune responses directly or indirectly, e.g., by expressing chemokines, cytokines and other mediators of immune responses.
  • infection refers to the initial entry of a pathogen into a host; and the condition in which a pathogen has become established in or on cells or tissues of a host; such a condition does not necessarily constitute or lead to a disease.
  • infectious disease refers to any disease which, under natural conditions, can be transmitted from one individual to another by a causal agent which passes either directly or indirectly from the infected to the non-infected individual.
  • a transmissible disease may be transmitted from the affected individual to another by any means.
  • infectious agent or“causal agent” are used interchangeably to refer to a pathogen that causes an infection.
  • inhalation refers to the act of drawing in a medicated vapor with the breath.
  • nasal insufflation refers to the act of delivering air, a gas, or a powder under pressure to a cavity or chamber of the body.
  • nasal insufflation relates to the act of delivering air, a gas, or a powder under pressure through the nose.
  • inhalation delivery device 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.
  • inhalation delivery device include, but are not limited to, a nebulizer, a metered-dose inhaler, and a dry powder inhaler (DPI).
  • the term“infuse” and its other grammatical forms as used herein refers to introduction of a fluid other than blood into a vein.
  • 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%.
  • inhibitor 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.
  • 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.
  • 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.
  • KL-6 Kelbs von den Lungen-6
  • MUC1 cluster 9
  • Serum levels of KL-6 are elevated in a variety of interstitial lung diseases that are characterized by alveolar epithelial cell damage.
  • Serum KL-6 concentrations are associated with alveolar epithelial barrier dysfunction, as they have been shown to correlate with indices of alveolar capillary permeability. Serum baseline level > 1000 U/ml is associated with worse prognosis and >1300 U/ml with increased risk of acute exacerbation (Guiot, J. et al. Lung (2017) 195(3): 273-280).
  • liposome refers to a synthetic, spherical extracellular vesicle consisting of one or more phospholipid bilayers surrounding a hollow or aqueous core.
  • lung consolidation refers to a chest X-ray finding indicating the presence of a radio-opaque area in the lung.
  • the opacification is caused by fluid or solid material within the airways or lung parenchyma.
  • lung function or“pulmonary function” are used interchangeably to refer to the process of gas exchange called respiration (or breathing).
  • 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.
  • 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.
  • lymphocyte refers to a small white blood cell formed in lymphatic tissue throughout the body and in normal adults making up about 22-28% of the total number of leukocytes in the circulating blood that plays a large role in defending the body against disease.
  • lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens. This commitment, which exists before the first contact of the immune system with a given antigen, is expressed by the presence on the lymphocyte’s surface membrane of receptors specific for determinants (epitopes) on the antigen. Each lymphocyte possesses a population of receptors, all of which have identical combining sites.
  • lymphocytes differs from another clone in the structure of the combining region of its receptors and thus differs in the epitopes that it can recognize. Lymphocytes differ from each other not only in the specificity of their receptors, but also in their functions. Two broad classes of lymphocytes are recognized: the B-lymphocytes (B- cells), which are precursors of antibody-secreting cells, and T-lymphocytes (T-cells),
  • B-lymphocytes are derived from hematopoietic cells of the bone marrow.
  • a mature B-cell can be activated with an antigen that expresses epitopes that are recognized by its cell surface.
  • the activation process may be direct, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B-cell activation), or indirect, via interaction with a helper T-cell, in a process referred to as cognate help.
  • cognate help In many physiological situations, receptor cross-linkage stimuli and cognate help synergize to yield more vigorous B-cell responses.
  • Cross-linkage dependent B-cell activation requires that the antigen express multiple copies of the epitope complementary to the binding site of the cell surface receptors because each B-cell expresses Ig molecules with identical variable regions. Such a requirement is fulfilled by other antigens with repetitive epitopes, such as capsular polysaccharides of microorganisms or viral envelope proteins. Cross-linkage-dependent B- cell activation is a major protective immune response mounted against these microbes. (Paul, W. E.,“Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4 th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
  • Cognate help allows B-cells to mount responses against antigens that cannot cross-link receptors and, at the same time, provides costimulatory signals that rescue B cells from inactivation when they are stimulated by weak cross-linkage events.
  • Cognate help is dependent on the binding of antigen by the B-cell’s membrane immunoglobulin (Ig), the endocytosis of the antigen, and its fragmentation into peptides within the endosomal/lysosomal compartment of the cell. Some of the resultant peptides are loaded into a groove in a specialized set of cell surface proteins known as class II major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the resultant class II/peptide complexes are expressed on the cell surface and act as ligands for the antigen- specific receptors of a set of T-cells designated as CD4+ T-cells.
  • the CD4+ T-cells bear receptors on their surface specific for the B-celTs class II/peptide complex.
  • B-cell activation depends not only on the binding of the T cell through its T cell receptor (TCR), but this interaction also allows an activation ligand on the T-cell (CD40 ligand) to bind to its receptor on the B-cell (CD40) signaling B-cell activation.
  • T helper cells secrete several cytokines that regulate the growth and differentiation of the stimulated B-cell by binding to cytokine receptors on the B cell. (Paul, W. E.,“Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4 th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
  • the CD40 ligand is transiently expressed on activated CD4+ T helper cells, and it binds to CD40 on the antigen- specific B cells, thereby transducing a second costimulatory signal.
  • the latter signal is essential for B cell growth and differentiation and for the generation of memory B cells by preventing apoptosis of germinal center B cells that have encountered antigen.
  • Hyperexpression of the CD40 ligand in both B and T cells is implicated in the pathogenic autoantibody production in human SLE patients. (Desai-Mehta, A. et al,“Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production,” J. Clin. Invest., 97(9): 2063-2073 (1996)).
  • T-lymphocytes derive from precursors in hematopoietic tissue, undergo differentiation in the thymus, and are then seeded to peripheral lymphoid tissue and to the recirculating pool of lymphocytes. T-lymphocytes or T cells mediate a wide range of immunologic functions. These include the capacity to help B cells develop into antibody- producing cells, the capacity to increase the microbicidal action of monocytes/macrophages, the inhibition of certain types of immune responses, direct killing of target cells, and mobilization of the inflammatory response. These effects depend on their expression of specific cell surface molecules and the secretion of cytokines. (Paul, W.
  • T cells differ from B cells in their mechanism of antigen recognition. Immunoglobulin, the B cell’s receptor, binds to individual epitopes on soluble molecules or on particulate surfaces. B-cell receptors see epitopes expressed on the surface of native molecules. Antibody and B-cell receptors evolved to bind to and to protect against microorganisms in extracellular fluids. In contrast, T cells recognize antigens on the surface of other cells and mediate their functions by interacting with, and altering, the behavior of these antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • dendritic cells 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.
  • APC antigen-presenting cell
  • 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.
  • MHC proteins which present foreign antigen to the T cell receptor
  • costimulatory proteins which bind to complementary receptors on the T cell surface
  • cell-cell adhesion molecules which enable a T cell to bind to the antigen-presenting cell (APC) for long enough to become activated.
  • T-cells are subdivided into two distinct classes based on the cell surface receptors they express.
  • the majority of T cells express T cell receptors (TCR) consisting of a and b chains.
  • TCR T cell receptors
  • a small group of T cells express receptors made of g and d chains.
  • oc/b T cells are two important sublineages: those that express the coreceptor molecule CD4 (CD4+ T cells); and those that express CD8 (CD8+ T cells). These cells differ in how they recognize antigen and in their effector and regulatory functions.
  • CD4+ T cells are the major regulatory cells of the immune system. Their regulatory function depends both on the expression of their cell-surface molecules, such as CD40 ligand whose expression is induced when the T cells are activated, and the wide array of cytokines they secrete when activated. [00475] T cells also mediate important effector functions, some of which are determined by the patterns of cytokines they secrete. The cytokines can be directly toxic to target cells and can mobilize potent inflammatory mechanisms.
  • T cells particularly CD8+ T cells, can develop into cytotoxic T- lymphocytes (CTLs) capable of efficiently lysing target cells that express antigens recognized by the CTLs.
  • CTLs cytotoxic T- lymphocytes
  • T cell receptors recognize a complex consisting of a peptide derived by proteolysis of the antigen bound to a specialized groove of a class II or class I MHC protein.
  • the CD4+ T cells recognize only peptide/class II complexes while the CD8+ T cells recognize peptide/class I complexes.
  • the TCR’s ligand i.e., the peptide/MHC protein complex
  • APCs antigen -presenting cells
  • class II MHC molecules bind peptides derived from proteins that have been taken up by the APC through an endocytic process. These peptide-loaded class II molecules are then expressed on the surface of the cell, where they are available to be bound by CD4+ T cells with TCRs capable of recognizing the expressed cell surface complex.
  • CD4+ T cells are specialized to react with antigens derived from extracellular sources.
  • class I MHC molecules are mainly loaded with peptides derived from internally synthesized proteins, such as viral proteins. These peptides are produced from cytosolic proteins by proteolysis by the proteasome and are translocated into the rough endoplasmic reticulum. Such peptides, generally nine amino acids in length, are bound into the class I MHC molecules and are brought to the cell surface, where they can be recognized by CD8+ T cells expressing appropriate receptors.
  • T cell system particularly CD8+ T cells, the ability to detect cells expressing proteins that are different from, or produced in much larger amounts than, those of cells of the remainder of the organism (e.g., vial antigens) or mutant antigens (such as active oncogene products), even if these proteins in their intact form are neither expressed on the cell surface nor secreted.
  • vial antigens e.g., vial antigens
  • mutant antigens such as active oncogene products
  • Helper T cells are T cells that stimulate B cells to make antibody responses to proteins and other T cell-dependent antigens.
  • T cell-dependent antigens are immunogens in which individual epitopes appear only once or a limited number of times such that they are unable to cross-link the membrane immunoglobulin (Ig) of B cells or do so inefficiently.
  • B cells bind the antigen through their membrane Ig, and the complex undergoes endocytosis. Within the endosomal and lysosomal compartments, the antigen is fragmented into peptides by proteolytic enzymes and one or more of the generated peptides are loaded into class II MHC molecules, which traffic through this vesicular compartment.
  • the resulting peptide/class II MHC complex is then exported to the B-cell surface membrane.
  • T cells with receptors specific for the peptide/class II molecular complex recognize this complex on the B-cell surface.
  • B-cell activation depends both on the binding of the T cell through its TCR and on the interaction of the T-cell CD40 ligand (CD40L) with CD40 on the B cell.
  • T cells do not constitutively express CD40L. Rather, CD40L expression is induced as a result of an interaction with an APC that expresses both a cognate antigen recognized by the TCR of the T cell and CD80 or CD86.
  • CD80/CD86 is generally expressed by activated, but not resting, B cells so that the helper interaction involving an activated B cell and a T cell can lead to efficient antibody production.
  • CD40L on T cells is dependent on their recognition of antigen on the surface of APCs that constitutively express CD80/86, such as dendritic cells.
  • Such activated helper T cells can then efficiently interact with and help B cells.
  • Cross-linkage of membrane Ig on the B cell even if inefficient, may synergize with the CD40L/CD40 interaction to yield vigorous B-cell activation.
  • the subsequent events in the B-cell response, including proliferation, Ig secretion, and class switching (of the Ig class being expressed) either depend or are enhanced by the actions of T cell-derived cytokines.
  • CD4+ T cells tend to differentiate into cells that principally secrete the cytokines IL-4, IL-5, IL-6, and IL-10 (T H2 cells) or into cells that mainly produce IL-2, IFN-g, and lymphotoxin (T H1 cells).
  • T H2 cells are very effective in helping B-cells develop into antibody-producing cells
  • T H1 cells are effective inducers of cellular immune responses, involving enhancement of microbicidal activity of monocytes and macrophages, and consequent increased efficiency in lysing microorganisms in intracellular vesicular compartments.
  • T H1 cells Although the CD4+ T cells with the phenotype of T H1 cells (i.e., IL-4, IL-5, IL-6 and IL-10) are efficient helper cells, T H1 cells also have the capacity to be helpers. (Paul, W. E.,“Chapter 1: The immune system: an introduction,” Fundamental Immunology, 4 th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
  • T cells also may act to enhance the capacity of monocytes and macrophages to destroy intracellular microorganisms.
  • interferon-gamma IFN-g
  • helper T cells enhances several mechanisms through which mononuclear phagocytes destroy intracellular bacteria and parasitism including the generation of nitric oxide and induction of tumor necrosis factor (TNF) production.
  • the T H1 cells are effective in enhancing the microbicidal action because they produce IFN-g.
  • two of the major cytokines produced by T H2 cells IL-4 and IL-10, block these activities.
  • a controlled balance between initiation and downregulation of the immune response is important to maintain immune homeostasis.
  • Both apoptosis and T cell anergy are important mechanisms that contribute to the downregulation of the immune response.
  • a third mechanism is provided by active suppression of activated T cells by suppressor or regulatory CD4+ T (Treg) cells. (Reviewed in Kronenberg, M. et al, “Regulation of immunity by self-reactive T cells,” Nature 435: 598-604 (2005)).
  • CD4+ Tregs that constitutively express the IL-2 receptor alpha (IL-2Roc) chain are a naturally occurring T cell subset that are anergic and suppressive.
  • CD4+ CD25+ Tregs are a naturally occurring T cell subset that are anergic and suppressive.
  • Depletion of CD4 + CD25 + Tregs results in systemic autoimmune disease in mice. Furthermore, transfer of these Tregs prevents development of autoimmune disease.
  • Human CD4 + CD25 + Tregs are generated in the thymus and are characterized by the ability to suppress proliferation of responder T cells through a cell-cell contact-dependent mechanism, the inability to produce IL-2, and the anergic phenotype in vitro.
  • Human CD4 + CD25 + T cells can be split into suppressive (CD25 hlgh ) and nonsuppressive (CD25 low ) cells, according to the level of CD25 expression.
  • a member of the forkhead family of transcription factors, FOXP3 has been shown to be expressed in murine and human CD4 + CD25 + Tregs and appears to be a master gene controlling CD4 + CD25 + Treg development. (Battaglia, M. et al.,“Rapamycin promotes expansion of functional CD4+CD25+Foxp3+ regulator T cells of both healthy subjects and type 1 diabetic patients,” J. Immunol., 177: 8338-8347 (200)).
  • CTL Cytotoxic T Lymphocytes
  • the CD8+ T cells that recognize peptides from proteins produced within the target cell have cytotoxic properties in that they lead to lysis of the target cells.
  • the mechanism of CTL-induced lysis involves the production by the CTL of perforin, a molecule that can insert into the membrane of target cells and promote the lysis of that cell.
  • Perforin- mediated lysis is enhanced by a series of enzymes produced by activated CTLs, referred to as granzymes.
  • Many active CTLs also express large amounts of fas ligand on their surface. The interaction of fas ligand on the surface of CTL with fas on the surface of the target cell initiates apoptosis in the target cell, leading to the death of these cells.
  • CTL-mediated lysis appears to be a major mechanism for the destruction of virally infected cells.
  • MHC Major Histocompatibility Complex
  • HLA Human leukocyte antigen
  • Both species have three main MHC class I genes, which are called HLA- A, HLA-B, and HLA-C in humans, and H2-K, H2-D and H2-L in the mouse. These encode the a chain of the respective MHC class I proteins.
  • the other subunit of an MHC class I molecule is p2-microglobulin.
  • the class II region includes the genes for the a and b chains (designated A and B) of the MHC class II molecules HLA-DR, HLA-DP, and HLA- DQ in humans.
  • the genes for the TAP1:TAP2 peptide transporter are also in the MHC class II region.
  • the PSMB (or LMP) genes that encode proteasome subunits the genes encoding the DMa and BMb chains (DMA and DMB), the genes encoding the a and b chains of the DO molecule (DOA and DOB, respectively), and the gene encoding tapasin (TAPBP).
  • the class II genes encode various other proteins with functions in immunity.
  • the DMA and DMB agenes seeking the subunits of the HLA-DM molecule that catalyzes peptide binding to MHC class II molecules are related to the MHC class II genes, as are the DOA and DOB genes that encode the subunits of the regulatory HLA-DO molecule.
  • MHC-like molecules while not encoded by the same gene group as true MHCs, have the same folding and overall structure of MHCs, and specifically MHC class I molecules, and thus possesses similar biological functions such as antigen presentation.
  • MHC Class I-like molecules are nonclassical MHC type molecules, while including Cdld also include CDla, CDlb, CDlc, CDle, and MR1 are also expressed on APCs and can activate various subsets of T cells. Kumar and Delovitch (2014)“Different subsets of natural killer T cells may vary in their roles in health and disease.” Immunology 142: 321-336.
  • Other non-classical histocompatibility molecules include MR1, which activate MAIT cells.
  • MSCs meenchymal stem cells
  • MSCs 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 differentiate along a minimum of three lineages (osteogenic, chondrogenic, and adipogenic).
  • MSCs commonly are known as osteochondrogenic, osteogenic, or chondrogenic, since a single MSC has shown the ability to differentiate into chondrocytes or osteoblasts, depending on the medium.
  • MSCs secrete many biologically important molecules, including interleukins 6, 7, 8, 11, 12, 14, and 15, M-CSF, Flt-3 ligand, SCF, LIF, bFGF, VEGF, P1GF and MCP1 (Majumdar, et al, J. Cell Physiol. 176: 57-66 (1998); Kinnaird et al, Circulation 109: 1543- 49 (2004)).
  • MSCs lack typical hematopoietic antigens, namely CD14, CD34, and CD45 (Pittenger et al, Science 284: 143-47 (1999)).
  • 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 l,2-dichloro-l,l,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),
  • the propellant includes a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or mixtures thereof.
  • a hydrofluorocarbon is used as the propellant.
  • HFC-227 and/or HFC- 134a are used as the propellant.
  • matrix metalloproteinases 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 IO, et al. PLoS Med. 2008; 5(4): e93).
  • 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).
  • MMP-7 MMP-7
  • FVC forced vital capacity
  • DLCO DLCO
  • microRNA refers to a class of small, non-coding RNA molecules, usually from about 18 to about 28 nucleotides in length. MicroRNAs are partially complementary to one or more messenger RNA (mRNA) molecules, and function in posttranscriptional regulation of gene expression and RNA silencing.
  • mRNA messenger RNA
  • the term“mimic” as used herein means a compound or substance that chemically resembles a parent compound or substance and retains at least a degree of the desired function of the parent compound or substance.
  • the term“modulate” as used herein means to regulate, alter, adapt, or adjust to a certain measure or proportion.
  • NK cells naturally killer cells
  • lymphocytes in the same family as T and B cells classified as group I innate lymphocytes.
  • NK cells have an ability to kill invading pathogens cells without any priming or prior activation, in contrast to cytotoxic T cells, which need priming by antigen presenting cells.
  • NK cells secrete cytokines such as IFNy and TNFa, which act on other immune cells, like macrophages and dendritic cells, to enhance the immune response.
  • Activating receptors on the NK cell surface recognize molecules expressed on the surface of cancer cells and infected cells and switch on the NK cell. Inhibitory receptors act as a check on NK cell killing.
  • MHCI receptors Most normal healthy cells express MHCI receptors, which mark them as “self.” Inhibitory receptors on the surface of the NK cell recognize cognate MHCI, which switches off the NK cell, preventing it from killing. Once the decision is made to kill, the NK cell releases cytotoxic granules containing perforin and granzymes, which leads to lysis of the target cell. Natural killer reactivity, including cytokine secretion and cytotoxicity, is controlled by a balance of several germ-line encoded inhibitory and activating receptors such as killer immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs).
  • KIRs killer immunoglobulin-like receptors
  • NCRs natural cytotoxicity receptors
  • the presence of the MHC Class I molecule on target cells serves as one such inhibitory ligand for MHC Class I-specific receptors, the Killer cell Immunoglobulin-like Receptor (KIR), on NK cells.
  • KIR Killer cell Immunoglobulin-like Receptor
  • Engagement of KIR receptors blocks NK activation and, paradoxically, preserves their ability to respond to successive encounters by triggering inactivating signals. Therefore, if a KIR is able to sufficiently bind to MHC Class I, this engagement may override the signal for killing and allows the target cell to live. In contrast, if the NK cell is unable to sufficiently bind to MHC Class I on the target cell, killing of the target cell may proceed.
  • abnormal healthy subject refers to a subject having no symptoms or other evidence of a fibrotic condition.
  • nucleic acid is used herein to refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and, unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single- stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
  • nucleotide is used herein to refer to a chemical compound that consists of a heterocyclic base, a sugar, and one or more phosphate groups.
  • the base is a derivative of purine or pyrimidine
  • the sugar is the pentose deoxyribose or ribose.
  • Nucleotides are the monomers of nucleic acids, with three or more bonding together in order to form a nucleic acid.
  • Nucleotides are the structural units of RNA, DNA, and several cofactors, including, but not limited to, CoA, FAD, DMN, NAD, and NADP.
  • Purines include adenine (A), and guanine (G); pyrimidines include cytosine (C), thymine (T), and uracil (U).
  • organ refers to a differentiated structure consisting of cells and tissues and performing some specific function in an organism.
  • paracrine signaling refers to short range cell-cell communication via secreted signal molecules that act on adjacent cells.
  • nanoparticles 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.
  • pill refers to fine particles of solid or liquid matter suspended in a gas or liquid.
  • peripheral blood mononuclear cell refers to a type of white blood cells that contains one nucleus, such as a lymphocyte or a macrophage.
  • 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.
  • formulation and “composition” are used interchangeably herein to refer to a product of the described invention that comprises all active and inert ingredients.
  • 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.
  • 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.
  • pluripotent stem cell refers to the ability to develop into multiple cells types, including all three embryonic lineages, forming the body organs, nervous system, skin, muscle, and skeleton.
  • A“pluripotent stem cell,”“PSC,” or“pluripotent cell” is a cell that has the ability under appropriate conditions of producing progeny of several different cell types that are derivatives of all of the three germinal layers (endoderm, mesoderm, and ectoderm).
  • pluripotent stem cells are embryonic stem (ES) cells, embryonic germ stem (EG) cells, embryonic carcinoma (EC) cells, induced pluripotent stem (iPS) cells, and adult stem cells.
  • PSCs may be derived from any organism of interest, including, e.g., primate, human, canine, feline, murine, equine, porcine, avian, camel, bovine, ovine, etc.
  • polynucleotide “polynucleotide sequence”, and“nucleic acid sequence” are used interchangeably herein. These terms encompass nucleotide sequences and the like.
  • a polynucleotide may be a polymer of DNA or RNA that is single- or double- stranded, that optionally contains synthetic, non-natural or altered nucleotide bases.
  • a polynucleotide may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
  • Nucleotides can be referred to by a single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively),“C” for cytidylate or deoxycytidylate (for RNA or DNA, respectively), “G” for guanylate or deoxyguanylate (for RNA or DNA, respectively),“U” for uridylate (for RNA),“T” for deoxythymidylate (for DNA),“R” for purines (A or G),“Y” for pyrimidines (C or T),“K” for G or T,“H” for A or C or T,“I” for inosine,“W” for A or T, and“N” for any nucleotide (e.g., N can be A, C, T, or G, if referring to a DNA sequence; N can be A, C, U, or
  • precision medicine 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.
  • primer refers to a nucleic acid which, when hybridized to a strand of DNA, is capable of initiating the synthesis of an extension product in the presence of a suitable polymerization agent.
  • the primer is sufficiently long to uniquely hybridize to a specific region of the DNA strand.
  • a primer also may be used on RNA, for example, to synthesize the first strand of cDNA.
  • progenitor cell refers to an early descendant of a stem cell that can only differentiate, but can no longer renew itself. Progenitor cells mature into precursor cells that mature into mature phenotypes. Hematopoietic progenitor cells are referred to as colony-forming units (CFU) or colony-forming cells (CFC). The specific lineage of a progenitor cell is indicated by a suffix, such as, but not limited to, CFU-E (erythrocytic), CFU-F (fibroblastic), CFU-GM (granulocytic/macrophage), and CFU-GEMM (pluripotent hematopoietic progenitor).
  • CFU-E erythrocytic
  • CFU-F fibroblastic
  • CFU-GM granulocytic/macrophage
  • CFU-GEMM pluripototent hematopoietic progenitor
  • pulmonary compliance 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 DJ, O’ Grady KF. 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:
  • This total compliance may be related to lung compliance and thoracic (chest wall) compliance by the following relation:
  • C T total compliance (e.g., 100 mL/cm 3 ⁇ 40)
  • C L lung compliance (e.g., 200 mL/cm 3 ⁇ 40)
  • C Th thoracic compliance (e.g., 200 mL/cm H20)
  • 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.
  • 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.
  • reference sequence refers to a sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence.
  • repair refers to any correction, reinforcement, reconditioning, remedy, making up for, making sound, renewal, mending, patching, or the like that restores function.
  • 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.
  • soluble and solubility refer to the property of being susceptible to being dissolved in a specified fluid (solvent).
  • insoluble refers to the property of a material that has minimal or limited solubility in a specified solvent.
  • the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • 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.
  • solvate refers to a complex formed by the attachment of solvent molecules to that of a solute.
  • solvent refers to a substance capable of dissolving another substance (termed a“solute”) to form a uniformly dispersed mixture (solution).
  • 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.
  • self renewal 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.
  • adult (somatic) stem cells 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.
  • SP-A surfactant protein A
  • SP-D surfactant protein D
  • type II pneumocytes nonciliated bronchiolar cells
  • submucosal glands submucosal glands
  • 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).
  • 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).
  • the term “susceptible subject” as used herein refers to an individual vulnerable to developing infection when their body is invaded by an infectious agent.
  • individuals vulnerable to developing a serious lung infection include, without limitation, the very young, the elderly, those who are ill; those who are receiving immunosuppressants; those with long term health conditions; and those who are physically weak, e.g., due to malnutrition or dehydration.
  • suspension 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.
  • the most common suspensions are those of solids in liquid.
  • symptom 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.
  • 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.
  • 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.
  • a therapeutically effective dosage i.e., dose and frequency of administration
  • 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.
  • therapeutic effect 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.
  • tissue refers to a collection of similar cells and the intercellular substances surrounding them.
  • 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.
  • transformation refers to the transfer of a nucleic acid molecule into a host organism or host cell by any method.
  • a nucleic acid molecule that has been transformed into an organism/cell may be one that replicates autonomously in the organism/cell, or that integrates into the genome of the organism/cell, or that exists transiently in the cell without replicating or integrating.
  • Non-limiting examples of nucleic acid molecules suitable for transformation are disclosed herein, such as plasmids and linear DNA molecules.
  • treat 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.
  • 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.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and“transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (2001), Ausubel et al. (2002), and other laboratory manuals.
  • viral vector refers to a means of gene transfer for modifying specific cell types or tissues that can be manipulated to express therapeutic genes wherein additional DNA segments can be ligated into a viral genome.
  • exemplary viral vectors include, without limitation, replication defective adenoviruses, adeno-associated viruses, retroviruses (g-retroviruses and lentiviruses), poxviruses, baculoviruses, and herpes simplex viruses.
  • the terms“wild type,”“naturally occurring,” or grammatical equivalents thereof are meant to refer to an amino acid sequence or a nucleotide sequence that is found in nature and includes allelic variations; that is, an amino acid sequence or a nucleotide sequence that usually has not been intentionally modified. Accordingly, the term “non-naturally occurring,”“synthetic,”“recombinant,” or grammatical equivalents thereof, are used interchangeably to refer to an amino acid sequence or a nucleotide sequence that is not found in nature; that is, an amino acid sequence or a nucleotide sequence that usually has been intentionally modified.
  • nucleic acid once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations, however, such nucleic acids, once produced recombinantly, although subsequently replicated non- recombinantly, are still considered recombinant for the purpose of the described invention.
  • the described invention provides a composition comprising a population of isolated EVs.
  • the pharmaceutical composition contains the composition comprising a population of isolated EVs and a pharmaceutically acceptable carrier.
  • the population of EVs is derived from peripheral blood mononuclear cells (PBMCs).
  • the PBMCs comprise T lymphocytes, B lymphocytes and NK cells.
  • the population of EVs is derived from T lymphocytes.
  • the population of EVs is derived from B lymphocytes.
  • the population of EVs is modified by a viral vector.
  • the population of EVs is derived from NK cells.
  • the population of EVs is derived from a patient who has recovered from a viral infection or has been exposed to anti-viral antibodies through treatment.
  • the population of EVs is derived from peripheral blood mononuclear cells (PBMCs) of the recovered patient.
  • PBMCs peripheral blood mononuclear cells
  • the PBMCs comprise T lymphocytes, B lymphocytes and NK cells.
  • the population of EVs is derived from T lymphocytes of the recovered patient.
  • the population of EVs is derived from B lymphocytes of the recovered patient.
  • the population of EVs is derived from NK cells of the recovered patient. According to some embodiments, the population of EVs is derived from urine of the recovered patient. According to some embodiments, the population of EVs is modified by a viral vector.
  • the EVs are membrane (i.e., lipid bilayer) vesicles derived from mesenchymal stem cells (MSCs).
  • MSCs mesenchymal stem cells
  • the MSCs are allogeneic to a subject for whom administration of the pharmaceutical composition is contemplated.
  • the MSCs are autologous to a subject for whom administration of the pharmaceutical composition is contemplated.
  • the source of MSCs is a tissue autologous to the recipient subject.
  • the source of the MSCs is a tissue allogeneic to the recipient subject.
  • the tissue is mammalian.
  • the tissue is human.
  • the source of the MSCs is placental tissue obtained from one or more areas, including both material and fetal tissue, e.g., amniotic membrane, chorionic membrane, or umbilical cord.
  • the source of MSCs is adipose tissue.
  • the adipose tissue is subcutaneous white adipose tissue.
  • the source of MSCs is bone marrow, umbilical cord tissue, dental pulp, lung tissue, or heart tissue.
  • the source of the MSCs is a body fluid.
  • the body fluid is peripheral blood, umbilical cord blood, or amniotic fluid.
  • hAMSC Human amniotic mesenchymal cells
  • hCMSC human chorionic mesenchymal cells
  • isolations are usually performed with term amnion dissected from the deflected part of the fetal membranes to minimize the presence of maternal cells.
  • 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).
  • 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).
  • hCMSCs are isolated from both first- and third-trimaster 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).
  • 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).
  • MSCs from the umbilical cord matrix 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).
  • 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 x 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 x g, 20 min). Cells at the interface are removed, and washed twice in sterile PBS.
  • 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% CO2 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-derived stromal/stem cells 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.
  • 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 x g for 10 min; aspirating floating adipocytes, lipids and liquid, leaving the stromal vascular fraction (SVF) pellet; resuspending the SVF pellet in 160 mM NH4CI and incubating for 10 minutes at room temperature; centrifuging at 400 x g for 10 min at room temperature; layering cells on Percoll or Histopaque gradient; centrifuging at 1000 x g for 30 minutes at room temperature; washing cells twice with PBS and centrifuging at 400 x g for 10
  • SVF
  • An exemplary protocol for expansion and subculture of human MSCs includes the following steps: Preocating a tissue culture vessel with 5 mg/mL of PRIME-XV MatrlS 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 TrupLETM Express to each T-75 flask, and tilting the flask in all directions to disperse the TrypLETM Express evenly over the cells; incubating the cells at 37°C, 5% CO 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;
  • 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 AI, et al, Biomed Res Int. 2017: 2851906).
  • 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.
  • MSCs may be cultured from tissue biopsies or transplanted tissues.
  • tissue-derived MSCs 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 lx PBS. The cell suspension is filtered through a cell strainer (40-pm) and collected in a 50-ml tube. Red blood cells are removed by incubating cells in lx RBC lysis buffer (BioLegend) for 5 min at room temperature. Then, 2 x 10 7 cells are seeded onto a collagen I-coated, 10-cm dish using MesenCult medium containing lx 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.
  • Wako collagenase
  • 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 NJ), 100 U penicillin, 100 U streptomycin and 2mM L-Glutamine (Life Technologies/Gibco). Cells are allowed to adhere overnight and nonadherent cells washed out with medium changes.
  • MSC expansion medium Iscove modified Dulbecco medium (IMDM, Life Technologies) and 20% FBS supplemented with 10 ng/mL recombinant human bFGF (Peprotech, Rocky Hill NJ), 100 U penicillin, 100 U streptomycin and 2mM L-Glutamine (Life Technologies/Gibco). Cells are allowed to adhere overnight and nonad
  • 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 x 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 x g for 10 min at room temperature and the supernatant discarded.
  • the cell pellet is resuspended in 50 mL buffer and centrifuged at 200 x 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% CO2. 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 is formed at 2 weeks after fertilization in the amniotic cavity of early gestation (Kim EY, et al, BMB Rep. (2014) Mar; 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) Apr-Jun; 98(2): 119-26). Many studies have identified amniotic fluid (AF) as a source of MSCs.
  • AF amniotic fluid
  • 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 MS, et al., Hum Reprod. (2004) Jun; 19(6): 1450-6; De Coppi P, et al., Nat. Biotechnol. (2007) Jan; 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 AR, et al., Med Sci Monit.(2002) Nov; 8(11): RA253-7).
  • AF-MSCs are easily isolated and offer advantages of nontumorigenicity and low immunogenic activity. (Id.).
  • 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 MS, et al., Hum Reprod. (2004) Jun; 19(6): 1450-6).
  • Tsai MS et al., Hum Reprod. (2004) Jun; 19(6): 1450-6
  • primary in situ cultures are set up in tissue culture-grade dishes using Chang medium (Irvine Scientific, Santa Ana, CA). Metaphase selection and colony definition is based on the basic requirements for prenatal cytogenetic diagnosis in amniocytes (Moertel CA, et al., (1992); Prenat Diagn 12, 671-683).
  • 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 a-modified minimum essential medium (a-MEM; Gibco-BRL) supplemented with 20% fetal bovine serum (FBS; Hyclone, Logan, UT) and 4 ng/ml basic fibroblast growth factor (bFGF; R&D systems, Minneapolis, MN) in a 25 cm 2 flask and incubated at 37°C with 5% humidified CO2 for MSC culture.
  • a-MEM a-modified minimum essential medium
  • FBS fetal bovine serum
  • bFGF basic fibroblast growth factor
  • surface antigens such as SH3, SH4, CD29, CD44 and HLA-A,B,C (MHC class I) may be found, and CD10, CDl lb, CD14, CD34, CD117, HLA-DR,DP,DQ (MHC class II) and EMA are absent (Tsai MS, et al., Hum Reprod. (2004) Jun; 19(6): 1450-6; Pittenger MF, et al., Science (1999) 284, 143-7; Colter DC, et al., Proc Natl Acad Sci USA 98, 78415; Young HY, et al., Anat Rec (2001) 264, 51-62).
  • 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.
  • confluent MSCs are cultured 1 to 3 weeks in differentiation medium, and lipid droplet staining is carried out by S Red Oil (Sigma).
  • flow cytometry can be used to characterize cell markers expressed on the surface of the isolated MSCs.
  • the phenotype of the adherent MSCs is CD73+, CD90+, CD105+, CD34-, CD45-.
  • the EVs contain microvesicles, exosomes, or both.
  • the EVs have a diameter ranging from about 30 nm to 200 nm, i.e., at least 30 nm, at least 31 nm, at least 32 nm, at least 33 nm, at least 34 nm, at least 35 nm, at least 36 nm, at least 37 nm, at least 38 nm, at least 39 nm, at least 40nm, at least 41 nm, at least 42 nm, at least 43 nm, at least 44 nm, at least 45 nm, at least 46 nm, at least 47 nm, at least 48 nm, at least 49 nm, at least 50 nm, at least 51 nm, at least 52 nm, at least 53 nm, at least 54 nm, at least 55 nm, at least 56 nm, at least
  • the EVs appear to have a cup- shaped morphology. According to some embodiments, they sediment at about 100,000xg and have a buoyant density in sucrose of about 1.10 to about 1.21 g/ml.
  • the EVs comprise proteins, nucleic acids, or both, including RNA species, such as miRNAs.
  • the EVs are produced by transfection (meaning introduction of one or more foreign nucleic acid molecules into a eukaryotic cell usually followed by expression of those nucleic acid molecules in the cell) with an miRNA-29a mimic, an miRNA-199 inhibitor, or both.
  • the extracelluar vesicles are isolated EVs.
  • the term“an isolated population of EVs” as used herein refers to a population of EVs that is physically separated from its natural environment.
  • isolated populations of EVs can be physically separated, in whole or in part, from tissue or cells with which the populations naturally exist.
  • a composition comprising isolated EVs may be substantially free of cells or cell components, or it may be free or substantially free of conditioned media.
  • the concentration of isolated EVs may be higher than the concentration EVs present in unmanipulated conditioned media.
  • the population of EVs comprises an enriched subpopulation of EVs.
  • the EVs can be isolated from conditioned media harvested from cultured MSCs containing metabolites, growth factors, RNA and proteins released into the medium by the cultured MSCs.
  • a method for harvesting EVs from MSCs involves first culturing MSCs under standard conditions until they reach about 70% confluency, and then culturing the cells in a serum-free media for 24 hours. The conditioned media is then collected and subjected to differential centrifugation at 400xg for 10 minutes and 12000xg for 10 minutes in order to remove whole cells and cellular debris, producing a clarified conditioned media. The clarified conditioned media then is concentrated by ultrafiltration using a 100 kDa MWCO filter (Millipore), and then centrifuged again at 12000xg for 10 minutes.
  • EVs then are isolated using size exclusion chromatography by loading the concentrated clarified conditioned media on a PBS -equilibrated Chroma S-200 column (Clontech), eluting with PBS, and collecting fractions of 350-550 microliters. Fractions containing EVs are identified and potentially pooled. Protein concentration is measured using a standard Bradford assay (Bio-Rad). Aliquots of the enriched extracellular vesicle preparations can be stored at -80°C.
  • EVs can be isolated from plasma.
  • plasma is centrifuged at room temperature at 2000 x g for 20 minutes. The supernatant is then transferred to a new microcentrifuge tube and centrifuged at 10,000 x g 20 minutes. The supernatant is then transferred to a new microcentrifuge tube. 100 pL of PBS is added to the sample and then is mixed by vortexing. 60 pL of the Exosome Precipitation Reagent is added and was mixed thoroughly by vortexing. The samples are incubated at room temperature for 10 minutes and then are centrifuged at 10,000 x g for 5 minutes at room temperature.
  • the supernatant is discarded and the samples are centrifuged again at 10,000 x g for 30 seconds.
  • the residual supernatant is discarded and pellets are resuspended in 200 pL of PBS for RNA extraction and miRNA profiling.
  • EVs can be isolated from bronchoalveolar lavage fluid (BALF).
  • BALF bronchoalveolar lavage fluid
  • BALF is diluted with an equal volume of PBS and transferred to 50-ml tubes.
  • the tubes are centrifuged for 30 minutes at 2,000 x g at 4°C.
  • the supernatant is then transferred to ultracentrifuge tubes or bottles without pellet contamination and centrifuged for 45 min at 12,000 x g, 4°C.
  • the supernatant is then transferred to ultracentrifuge tubes or bottles and centrifuged for 2 hours at 110,000 x g, 4°C.
  • the pellets are then resuspended in 1 ml PBS and pooled in one of the tubes.
  • the tube can be filled with PBS to dilute the resuspension in a large volume.
  • the suspension is then filtered through a 0.22-mm filter, collected in a fresh ultracentrifuge tube or bottle, and centrifuged for 70 minutes at 110,000 x g, 4°C.
  • the supernatant is poured off.
  • the pellet is resuspended in 1 ml PBS, and then the tube filled with PBS and centrifuged for 70 min at 110,000 x g, 4°C.
  • the supernatant is then discarded and the pellet resuspended in 30 to 100 ml sterile PBS and used or stored at -80°C.
  • EVs also can be purified by ultracentrifugation of the clarified conditioned media at 100,000xg. According to some embodiments, they also can be purified by ultracentrifugation into a sucrose cushion. GMP methods for extracellular vesicle purification from dendritic cells have been described in J Immunol Methods. 2002; 270: 211-226, which is incorporated by reference herein.
  • EVs can be purified by differential filtration through nylon membrane filters of defined pore size. For example, a first filtration though a large pore size will retain cellular fragments and debris; a subsequent filtration through a smaller pore size will retain EVs and purify them from smaller size contaminants.
  • the EV preparation can comprise synthetically engineered EVs.
  • these synthetic EVs can be synthesized in vitro.
  • the synthetic populations of EVs can be engineered to express an miRNA-29a mimic, an miRNA-199 inhibitor, or both.
  • the miRNA-29a mimic, miRNA-199 inhibitor, or both may or may not comprise nucleic acids that encode the parent miRNA-29a, miRNA-199, or both.
  • the synthetic EVs comprise liposome membranes. Liposome synthesis is known in the art, and liposomes may be purchased from commercial sources.
  • the basic strategies involved in the preparation of liposomes include: steps for separation of lipids from organic solvent; steps for dispersion of lipids in an aqueous medium; purifying the resultant liposomes; and steps for analyzing the manufactured liposomes.
  • Exemplary methods for dispersion of the lipids in the aqueous medium are outlined below.
  • Sonication is likely the most commonly used method for the dispersion of lipids, particularly for the manufacture of small unilamellar vesicles (SUVs).
  • a bath type sonicator or a probe sonicator is used to produce the liposomes passively.
  • the probe sonication method requires a high input of energy to enhance the dispersion; because this creates heat, the vessel must be placed in a water or ice bath to control the temperature.
  • the sonication method is limited by its low internal volume or ability to encapsulate large molecules. Additionally, the phospholipids and internal molecules may be subject to degradation, resulting in an unsuccessful encapsulation.
  • the French pressure cell method uses a process of extrusion and pushes multilamellar vesicles (MLVs) through a small orifice to disperse the lipids.
  • MLVs multilamellar vesicles
  • the resulting liposomes tend to be larger than with the sonication method and it recalls encapsulated solutes longer than SUVs.
  • the manufacturing process requires particularly high temperatures and there is a restricted working volume.
  • the SUVs are frozen for a short period and then allowed to thaw over a long timeframe. This disperses the lipids and leads to the formation of large unilamellar vesicles (LUVs).
  • the freeze-thaw method is limited by the concentration of phospholipids and ionic strength of the medium.
  • lipid film hydration lipid film hydration
  • micro-emulsification membrane extrusion
  • dried reconstituted vesicles Factors to be considered include physicochemical characteristics of the material to be encapsulated, the medium in which lipid vesicle will be dispersed, concentration and potential toxicity of the encapsulated substance, the process of administration of the vesicles, size, polydispersity and shelf-life of vesicles, as well as reproducibility of safe and efficient products.
  • the invention contemplates immediate use of EV preparations or short- and/or long-term storage of EV preparations, for example, in a cryopreserved state prior to use.
  • Proteinase inhibitors are typically included in freezing media as they provide extracellular vesicle integrity during long-term storage. Freezing at -20°C is not preferable since it is associated with increased loss of extracellular vesicle activity. According to some embodiments, the EV preparations are quick frozen at -80°C to preserve activity. (See, for example, Kidney International (2006) 69, 1471-1476, incorporated herein by reference). Additives to the freezing media similar to those used for cryopreservation of intact cells, including, without limitation, DMSO, glycerol and polyethylene glycol, may be used in order to enhance preservation of extracellular vesicle biological activity.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed.
  • “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel (1990) GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors examples include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • Additional vectors include minichromosomes such as bacterial artificial chromosomes, yeast artificial chromosomes, or mammalian artificial chromosomes.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type such as a cell of the respiratory tract.
  • Tissue-specific regulatory elements are known in the art.
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector.
  • the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that includes guide polynucleotide targeting a viral RNA.
  • Regulatory sequences operatively linked to a nucleic acid can be chosen that direct the continuous expression of the nucleic acid molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression.
  • the composition of the described invention may be prepared in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • the compositions of the described invention may be formulated as a dispersible dry powder for delivery by inhalation or insufflation (either through the mouth or through the nose, respectively).
  • Dry powder compositions may be prepared by processes known in the art, such as lyophilization and jet milling, as disclosed in International Patent Publication No. WO 91/16038 and as disclosed in U.S. Pat. No. 6,921,527, the disclosures of which are incorporated by reference.
  • the composition of the described invention may be placed within a suitable dosage receptacle in an amount sufficient to provide a subject with a unit dosage treatment.
  • the dosage receptacle can be one that fits within a suitable inhalation device to allow for the aerosolization of the dry powder composition by dispersion into a gas stream to form an aerosol and then capturing the aerosol so produced in a chamber having a mouthpiece attached for subsequent inhalation by a subject in need of treatment.
  • a dosage receptacle includes any container enclosing the composition known in the art such as gelatin or plastic capsules with a removable portion that allows a stream of gas (e.g., air) to be directed into the container to disperse the dry powder composition.
  • gases e.g., air
  • Further exemplary containers also include those used in conjunction with Glaxo's Ventolin® Rotohaler brand powder inhaler or Fison's Spinhaler® brand powder inhaler.
  • Another suitable unit-dose container which provides a superior moisture barrier is formed from an aluminum foil plastic laminate.
  • the pharmaceutical-based powders is filled by weight or by volume into the depression in the formable foil and hermetically sealed with a covering foil-plastic laminate.
  • Such a container for use with a powder inhalation device is described in U.S. Pat. No. 4,778,054 and is used with Glaxo's Diskhaler® (U.S. Pat. Nos. 4,627,432; 4,811,731; and 5,035,237).
  • Glaxo's Diskhaler® U.S. Pat. Nos. 4,627,432; 4,811,731; and 5,035,237).
  • the dry powder may be produced by a spray drying process.
  • the composition of the invention may be formulated as a solution.
  • An exemplary formulation is sterile, contains a sufficient amount of the active, and is not harmful for the proposed application.
  • the compositions of the described invention may be formulated as aqueous suspensions wherein the MSCs, EVs or both are in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • Such excipients include, without limitation, suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia), dispersing or wetting agents including, a naturally-occurring phosphatide (e.g., lecithin), or condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyl-eneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate).
  • compositions of the described invention also may be formulated in the form of dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water.
  • the active ingredient in such powders and granules is provided in admixture with a dispersing or wetting agent, suspending agent, and one or more preservatives.
  • a dispersing or wetting agent, suspending agent, and one or more preservatives are exemplified by those already mentioned above. Additional excipients also may be present.
  • the pharmaceutical composition is packaged in an inhalation device, including, for example, but not limited to a nebulizer, a metered-dose inhaler (MDI), and a dry powder inhaler (DPI).
  • an inhalation device including, for example, but not limited to a nebulizer, a metered-dose inhaler (MDI), and a dry powder inhaler (DPI).
  • MDI metered-dose inhaler
  • DPI dry powder inhaler
  • the pharmaceutical composition is a liquid for aerosolized delivery using a nebulizer.
  • the flow-rate of the pharmaceutical composition is at least 0.3 ml/min, and the pharmaceutical composition is delivered as 2 mm particles, with distribution into deepest alveoli.

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Abstract

La présente invention concerne des compositions et des procédés pour traiter un sujet sensible au risque de complications pulmonaires d'une lésion pulmonaire aiguë provoquée par une infection grave à un virus respiratoire et pour restaurer la fonction pulmonaire à des poumons donneurs. Les procédés comprennent l'administration d'une quantité thérapeutique d'une composition pharmaceutique comprenant des vésicules extracellulaires (EV) comprenant un ou plusieurs miARN et un excipient pharmaceutiquement acceptable. La population d'EV peut être dérivée d'un patient étant rétabli d'une infection par le virus respiratoire ou ayant été exposé à des anticorps anti-viraux à travers un traitement; ladite population peut également être dérivée de MSC d'un individu sain, peut être modifiée par un vecteur viral, ou peut être synthétique.
PCT/US2020/036182 2019-06-06 2020-06-04 Procédés pour atténuer une infection virale et pour traiter une lésion pulmonaire WO2020247675A1 (fr)

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