A treatment method

Abstract

The present disclosure generally relates to methods of treating mammalian subjects by enhanced cell-based therapeutic methods to promote tissue and neuronal repair, regeneration and / or repair. The present disclosure also encompasses medicaments for treating mammalian subjects and methods of producing the medicaments, wherein the composition comprises exosomes derived from allogeneic mammalian amniotic epithelial cells, wherein the allogeneic mammalian amniotic epithelial cells are from a donor mammal of the same species.

Description

[0001] This application is a divisional application of an invention patent application whose international application date is June 10, 2016, Chinese national application number is 201680047646.2, and the invention name is “A Treatment Method”.

[0002] Application Date

[0003] This application is related to and claims priority from Australian Provisional Patent Application No. 2015902214 filed on June 12, 2015, entitled “A method of treatment”, and Australian Provisional Patent Application No. 2016901349 filed on April 12, 2016, entitled “A method of treatment”, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present disclosure generally relates to methods of treating mammalian subjects by enhanced cell-based therapeutic methods to promote tissue and neuronal repair, regeneration and / or repair. The present disclosure also encompasses medicaments for treating mammalian subjects and methods of producing the medicaments. Background Art

[0005] Bibliographic details of publications cited by the author in this specification are collected alphabetically at the end of the specification.

[0006] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge in any country.

[0007] Modern medicine has been greatly advanced by the identification of chemotherapeutic agents and biologics such as antibiotics. However, many drugs have multiple functions, some affecting off-target physiological effects. Cell-based therapeutics have been proposed as the next pillar of modern medicine (Fishback et al. (2013) Sci Transl Med 5: 179ps7).

[0008] One rate-limiting factor for cell-based therapies is that the products can be inconsistent. This is highlighted in trials involving stem cells. For example, while mesenchymal stem cells (MSCs) are well characterized in the literature and have reached clinical trial levels, they generally require serial passage for use. This can and often does have an adverse impact on when and how the cells can be used.

[0009] One way to address this problem is to use banked mesenchymal progenitor cells. This at least avoids the delay between harvest and treatment. However, this introduces variability in cell potency between donors and does not address the negative effects of serial passaging.

[0010] An interim solution to this problem is to have a "master" cell bank. Again, this does not overcome the inevitable problem that the cells can undergo a limited number of passages before aging and epigenetic / karyotypic changes occur (Schellenberg et al. (2011) Aging (Albany NY) 3:873-888). There is also the risk of immune rejection after repeated administration.

[0011] For example, with the significant improvement of obstetric monitoring and neonatal care, more and more premature infants survive, resulting in an increase in the prevalence of "premature diseases". A particularly debilitating condition is bronchopulmonary dysplasia (BPD), which is an incurable chronic lung disease of very premature infants. It is characterized by alveolar dysplasia and stagnation and destruction of pulmonary capillary structure. BPD is a major condition of neonatal morbidity and mortality. BPD survivors are also at serious risk of obstructive respiratory disease (Doyle et al., (2006) Paediatrics 118: 108-113) and general chronic health impairment and cognitive decline (Lodha et al. (2014) PLoS ONE: e90843) in early adulthood. Although mesenchymal stem cells have been proposed as a possible cell-based therapy for BPD, due to the reasons outlined above, there may be batches of cells with widely different functional effectiveness, which, in addition to causing emotional stress, may also delay other treatment options.

[0012] The problem of damaged pulmonary capillary structure is not limited to premature human infants. The animal racing industry, and in particular the horse racing industry, faces the problem of exercise-induced pulmonary hemorrhage (EIPH). For example, in some jurisdictions, horses that suffer more than two nosebleeds after racing are banned from further competition for life. This can result in catastrophic financial losses. Chemical treatments to prevent or treat EIPH are likely to raise ethical issues in terms of performance enhancement, and in any case such methods are unlikely to regenerate burst capillaries.

[0013] Beneficial effects of human amniotic epithelial cells (hAECs) have been demonstrated (e.g., Hodges et al. (2012) Am J Obstet Gynerol 206:448e8-448e15; Murphy et al. (2012) Cell Transplant 1:1477-1492; Vosdoganes et al. (2013) Cytotherapy 15:1021-1029; Yawno et al. (2013) Dev Neurosci 35:272-282). However, it is necessary to determine the mechanism of action.

[0014] It is therefore clear that the problems with cell-based therapies need to be addressed and an alternative strategy is needed. Summary of the invention

[0015] According to the present invention, vesicle mediators for cellular communication are identified as released from mammalian amniotic epithelial cells (AECs). Vesicles, referred to herein as "amniotic exosomes," are nanometer-sized extracellular vesicles (50-100 nm) that originate from late endosomes and are released from the cell surface.

[0016] This article teaches an improved form of therapy based on mammalian amniotic epithelial cells. The improvement includes the use of nano-sized amniotic exosomes released by epithelial cells, and the repair effect is exerted by activating endogenous repair mechanisms. It is shown herein that amniotic exosomes act directly on immune cells to especially reduce T cell proliferation, increase macrophage phagocytosis, activate stem cells, and inhibit collagen production in activated fibroblasts. It is proposed herein that amniotic exosomes release a group of exosome cargo in the form of proteins (e.g., cytokines) and genetic molecules (e.g., miRNA, mRNA, and non-coding RNA).

[0017] The biogenesis of exosomes involves the formation of intraluminal vesicles by inward budding of the limiting membrane of late endosomes. Late endosomes then fuse with the plasma membrane to release the exosomes. Once secreted, exosomes can be taken up by target cells located in the vicinity of the parent cell or travel to distal sites through the circulation. Mechanistically, exosomes function as complex carriers containing parent cell material. They can contain proteins and genetic material, which will then be transferred to their target cells.

[0018] Therefore, the present invention is based on the development of enhanced methods for cell-based therapies. The present disclosure teaches the use of amniotic exosomes, which are released from mammalian amniotic epithelial cells and have immunomodulatory, pro-regenerative and repair effects. Amniotic exosomes act on immune cells by releasing beneficial proteomics and genetic molecules such as miRNA, mRNA and non-coding RNA to reduce T cell proliferation, increase macrophage phagocytosis and activate endogenous stem cells. It is proposed herein that amniotic exosomes promote tissue repair, regeneration and repair including wound healing, promote cell maintenance, induce neuronal protection including improving neurodegeneration and damage and promote repair and neural regeneration. Amniotic exosomes also inhibit collagen production in activated fibroblasts. It is also proposed that exosomes promote repair and regeneration after systemic vascular diseases or adverse events such as ischemia-reperfusion injury or organ damage, including improving kidney, liver, pancreas, heart and lung damage, and treating fibrotic conditions of these organs (such as liver or lung fibrosis). Exosomes may also be used to promote myelination and therefore have been proposed to be useful in treating demyelinating diseases or conditions such as multiple sclerosis.

[0019] Amniotic exosomes have beneficial effects not only in humans, but also in non-human mammals. Therefore, the present invention extends to human and veterinary applications. AECs from human subjects are referred to herein as "hAECs".

[0020] An example of a veterinary application is the treatment of exercise-induced pulmonary hemorrhage (EIPH) in racing animals including horses, racing dogs and camels.

[0021] Amniotic exosomes can be produced in large quantities by culturing mammalian amniotic epithelial cells in a bioreactor and isolating amniotic exosomes from the conditioned medium. The epithelial cells can be maintained as an immortalized cell line. Amniotic exosomes can be isolated when needed or stored in a lyophilized state.

[0022] An innovative feature of the present invention is that it is not necessary to determine a compatible donor of mammalian amniotic epithelial cells in order to use amniotic exosomes. Exosomes do not induce adverse immune responses. Instead, donors are selected based on the stage of pregnancy and / or other characteristics (e.g., the health of newborns or full-term infants). However, in one embodiment, amniotic exosomes are derived from hAECs of patients in late pregnancy. Amniotic epithelial cells produce amniotic exosomes, which are at least as good as amniotic epithelial cells in promoting tissue or neuronal repair, regeneration and / or repair of different physiological conditions. However, cell-based therapies do not have any disadvantages. Therefore, one aspect of the present invention is to select donors to determine amniotic epithelial cells that produce amniotic exosomes that can be used to treat the desired condition. This may result in the generation of an amniotic epithelial cell bank. Then, a specific batch of cells is selected based on the disease or condition to be treated.

[0023] The teachings of the present disclosure also encompass pharmaceutical compositions comprising amniotic exosomes, therapeutic kits comprising amniotic exosomes, and / or reagents and bioreactor kits for screening suitable donors or amniotic epithelial cell lines.

[0024] Enhanced or modified forms of cell-based therapies are taught herein. Thus, by using amniotic epithelial cells, improved cell-based therapeutic regimens for treating mammalian (including human) subjects can be achieved herein, the improvements comprising isolating amniotic exosomes from immortalized amniotic epithelial cell lines, and administering systemically or locally to subjects in need of tissue or neuronal repair, regeneration and / or repair (including promoting remyelination). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Some of the drawings contain color representations or entities. Color photographs may be obtained upon request from the patent owner or from the appropriate patent office. If obtained from the patent office, a fee may be charged.

[0026] Figure 1(A) is a photographic representation of electron micrographs of amniotic exosomes, which show a typical cup-shaped morphology and a diameter of approximately 100 nm; (B) is a photographic representation of the expression of exosome biogenesis markers Alix and TSG101. Alix and TSG101 are exosome biomarkers.

[0027] Figure 2 are graphical representations showing that (A) amnion exosomes inhibit T cell proliferation, similar to hAEC conditioned medium; (B) amnion exosomes increase macrophage phagocytosis n=3.

[0028] Figure 3 is a graphical representation showing that amniotic exosomes increase the tissue:air ratio in a mouse model of bronchopulmonary dysplasia (BPD).

[0029] Figure 4 is a graphical representation of the experimental timing used in Example 3, depicting intra-amniotic LPS injection at E16, injection of exosomes / cells at postnatal day 4, and cull points (crosses).

[0030] Figure 5 are graphical representations showing that (A) term exosomes are more immunosuppressive than preterm exosomes; (B) term exosomes are better at increasing phagocytosis by macrophages as shown by pHRodo labeling, n=3 donors per group.

[0031] Figure 6 Figure 2 is a graphical representation showing that amniotic exosomes reverse established lung inflammation and fibrosis in a mouse model of bleomycin-induced pulmonary fibrosis. (A); (B) Female C57BI6 mice aged 6-8 months. 10 μg or 50 μg of exosomes from term hAECs were administered intranasally 7 days after bleomycin challenge.

[0032] Figure 7 is a graphical representation showing that amniotic exosomes reverse activation of primary human lung fibroblasts in vitro. When cultured in the presence of 5 mg / mL transforming growth factor β, the exosomes reduced the protein level of α-smooth muscle actin within 24 hours.

[0033] Figures 8A-8C is a diagrammatic representation showing that amniotic exosomes contain miRNAs that target the cytokine-cytokine receptor signaling pathway. Yellow boxes indicate targets of one or more miRNAs.

[0034] Figures 9A-9B is a graphic representation showing that amniotic exosomes contain miRNAs that target the Wnt signaling pathway. Yellow boxes indicate targets of one or more miRNAs.

[0035] Fig.10is a graphic representation showing that amniotic exosomes contain miRNAs that target the PI3K-Akt signaling pathway. Yellow boxes indicate targets of one or more miRNAs.

[0036] Fig.11 is a graphic representation showing that amniotic exosomes contain miRNAs that target the TGFβ signaling pathway. Yellow boxes indicate targets of one or more miRNAs.

[0037] Fig.12 A is a graphical representation showing the lung regenerative effects of amniotic exosomes, Fig.12 B, C, and D are photographic representations showing the ratio (%) of tissue voids between healthy control hAEC, term exosomes, and preterm exosomes. "Term" exosomes are exosomes isolated from hAEC at the end of pregnancy. "Preterm" exosomes are isolated before term pregnancy.

[0038] Fig.13 A to C are photographic representations showing that amniotic exosomes, like hAECs, trigger regeneration in the lung. Dark staining is evidence of elastin-positive tips.

[0039] Fig.14 is a graphical representation showing that amnion exosomes, but not fibroblast exosomes, trigger an endogenous stem cell response in the lung. The response is significantly greater than that induced by hAECs.

[0040] Fig.15 A and B show Sirius red staining (A) and α-smooth muscle actin (α-SMA) immunohistochemical analysis of liver tissue sections using CCL4+saline and CCL4+exosomes confirmed that amnion exosomes are anti-fibrotic in the liver.

[0041] Fig.16 is a graphical representation showing the differences in proteomic levels between exosomes from term and preterm hAECs.

[0042] Fig.17 is a graphical representation of the comparison of cellular composition between hAECs and total mesenchymal stem cells (MSCs).

[0043] hAEC total exosomes (exo)

[0044] Total MSC exosomes (Anderson et al. (2016) Stem cells http: / / doi.ory / 10.1002 / stem.2298).

[0045] Fig.18is a graphical representation of the comparison of biological processes between hAECs and total MSCs (Anderson et al. (2016) supra).

[0046] hAEC total exosomes

[0047] Total MSC exosomes (Anderson et al. (2016) supra). DETAILED DESCRIPTION

[0048] Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as comprises or comprising, will be understood to imply the inclusion of stated elements or integers or method steps, or groups of elements or integers or method steps, but not the exclusion of any elements or integers or method steps, or groups of elements or integers or method steps.

[0049] As used in this specification, the singular forms "a", "an" and "the" include plural forms unless the context clearly indicates otherwise. Thus, for example, reference to "a disease or condition" includes a single disease or condition as well as two or more diseases or conditions; reference to "an exosome" includes a single exosome as well as two or more exosomes; reference to "the present disclosure" includes single and multiple aspects taught by the present disclosure; and so on. The term "invention" covers multiple aspects taught and enabled herein. "Disease" or "condition" also includes "disorders". All of these aspects are enabled within the scope of the present invention. "Forms" of the present invention cover any variants and derivatives contemplated herein.

[0050] The present disclosure teaches an enhanced cell-based therapy to promote the treatment of a range of diseases or conditions in mammalian subjects, which generally belong to the case of repairing, regenerating and repairing cells, tissues, neural pathways and endocrine pathways. The present disclosure teaches that amniotic exosomes isolated from conditioned medium used to culture mammalian amniotic epithelial cells (AEC) have beneficial immunomodulatory physiological and biochemical properties. In essence, mammalian amniotic exosomes exert effects on immune cells to reduce T cell proliferation, increase macrophage phagocytosis and activate endogenous stem cells by releasing proteomic and genetic molecules such as miRNA, mRNA and non-coding RNA. They also inhibit collagen production in activated fibroblasts. Importantly, amniotic exosomes are not immunogenic, so allogeneic amniotic exosomes can be used.

[0051] Amniotic exosomes also reverse established lung inflammation and pulmonary fibrosis and reversely activate primary lung fibroblasts. This also applies to fibrosis in other organs such as liver, pancreas, heart and kidney. In addition, they contain miRNAs, mRNAs and non-coding RNAs that target cytokine-cytokine receptor signaling pathways, Wnt signaling pathways, PI3K-Akt signaling pathways and TGFβ signaling pathways, as well as signaling pathways involved in a variety of physiological and neurological processes.

[0052] This specification teaches that mammalian amniotic exosomes induce repair, regeneration and repair of cells, tissues including organs, neurological pathways, components of the systemic vasculature, and promote wound healing. It is proposed herein that amniotic exosomes promote the repair, regeneration and repair of the brain and spinal cord after trauma, disease or drug abuse, promote the repair of neuroregenerative conditions, induce the repair of organ damage, and promote repair after stroke or other damage to the brain such as traumatic brain injury. It is proposed that exosomes promote myelin regeneration in the treatment of demyelinating diseases, conditions or disorders such as multiple sclerosis, optic neuritis, Devic's disease, transverse myelitis, acute disseminated encephalomyelitis and adrenoleukodystrophy and adrenomyeloneuropathy. In one embodiment, amniotic exosomes promote the repair of lung damage. This is important in the treatment of bronchopulmonary dysplasia (BPD) in human infants. It also has veterinary applications in the treatment of exercise-induced pulmonary hemorrhage (EIPH) in racing animals such as horses, racing dogs (eg, greyhounds), and camels.

[0053] Thus, the present invention as enabled herein is a method of treating a mammalian subject comprising systemically or locally administering mammalian amniotic exosomes derived from allogeneic mammalian amniotic epithelial cells from a donor mammal of the same species.

[0054] Reference to a "mammalian subject" includes any mammal in need of treatment. In one embodiment, the mammalian subject is a human. The term "AEC" refers to "amniotic epithelial cells." When derived from humans, AECs are referred to as "hAECs."

[0055] Thus, the present specification teaches methods of treating a human subject comprising systemically or locally administering human amniotic exosomes derived from allogeneic human amniotic epithelial cells from a human donor.

[0056] In another embodiment, the mammalian subject is a non-human mammal, such as, but not limited to, a horse, cow, sheep, goat, pig, alpaca, llama, dog, cat, or camel.

[0057] In one embodiment, the mammalian subject is in need of treatment. The term "treatment" encompasses the repair, regeneration or promotion of regeneration and / or repair of cells, tissues and physiological pathways (including neurons and endocrine pathways). Examples include, but the invention is not limited to, repair, regeneration and / or repair of organs after ischemia-reperfusion injury or stroke, internal and surface wounds, ulcers and scars, neurodegenerative conditions, and brain and spinal cord injuries (including traumatic brain injury and spinal cord injury), including circulatory vessels, such as capillaries, arteries and veins including such vessels. It is also proposed that exosomes are used to treat organ fibrosis, such as fibrotic diseases, conditions or disorders of the lungs, liver, heart, kidneys and pancreas. It is also expected that exosomes are used to treat demyelinating diseases, conditions or disorders, or diseases such as multiple sclerosis, optic neuritis, Devic's disease, transverse myelitis, acute disseminated encephalomyelitis and adrenoleukodystrophy and adrenal myeloneuropathy. Amniotic exosomes may be used in clinical applications to treat diseases or conditions as well as as cosmetic agents to promote skin regeneration or scar or wound healing.

[0058] Although it is not intended to limit the present invention to any one theory or mode of action, it is proposed that mammalian amniotic exosomes represent vesicle media for communication from amniotic epithelial cells and release proteomic and genetic molecules that provide a beneficial mixture of molecules to promote repair, regeneration and repair. It is also proposed that the profile of proteomic and genetic molecules will be different depending on the gestational stage of the donor from which the amniotic epithelial cells are obtained. Therefore, the present specification teaches the generation of immortalized mammalian amniotic epithelial cell libraries from different donors at different gestational stages. Epithelial cells are then selected from the library based on the disease or condition of the subject to be treated and based on the profile of proteomic and genetic molecules produced by amniotic exosomes. The present specification teaches that amniotic exosomes with specific proteomic and / or genetic profiles may be preferred depending on the disease or condition to be treated.

[0059] Thus, another aspect of the present teachings is a method of treating a mammalian subject, the method comprising:

[0060] i. optionally identifying the donor;

[0061] ii. selecting immortalized amniotic epithelial cells from the or a donor based on the proteomic and / or genetic profile of amniotic exosomes produced by cultured epithelial cells;

[0062] iii. producing conditioned medium from the selected immortalized amniotic epithelial cells;

[0063] iv. isolating amniotic exosomes from conditioned medium; and

[0064] v. administering the amniotic exosomes systemically or locally to a mammalian subject.

[0065] In one embodiment, the mammalian subject is a human subject. Thus, another aspect of the teachings herein is a method of treating a human subject, the method comprising:

[0066] i. optionally identifying the donor;

[0067] ii. selecting immortalized amniotic epithelial cells from the or a donor based on the proteomic and / or genetic profile of amniotic exosomes produced by cultured epithelial cells;

[0068] iii. producing conditioned medium from the selected immortalized amniotic epithelial cells;

[0069] iv. isolating amniotic exosomes from conditioned medium; and

[0070] v. administering the amniotic exosomes systemically or locally to a human subject.

[0071] In another embodiment, mammalian amniotic exosomes are isolated and their proteomic and genetic profiles are predetermined, and a selected mammalian amniotic exosome library is generated based on the profiles. Specific amniotic exosomes are then selected for treatment.

[0072] Thus, the present specification is a teaching of a method of treating a mammalian subject, the method comprising:

[0073] i. optionally identifying the donor;

[0074] ii. selecting amniotic exosomes from the or a donor based on the proteomic and / or genetic profile of agents released by the exosomes;

[0075] iii. administering the amniotic exosomes systemically or locally to a mammalian subject.

[0076] In one embodiment, the mammalian subject is a human.

[0077] Thus, taught herein is a method of treating a human subject, the method comprising:

[0078] i. optionally identifying the donor;

[0079] ii. selecting amniotic exosomes from the or a donor based on the proteomic and / or genetic profile of agents released by the exosomes;

[0080] iii. administering the amniotic exosomes systemically or locally to a human subject.

[0081] When used in therapy, amniotic exosomes may also be referred to as drugs, agents, therapeutic agents, cell therapy-derived agents, active ingredients, etc. References to "therapy" include both clinical and cosmetic therapies.

[0082] Also taught herein is a method of inducing cell or neuronal repair, regeneration and / or repair in a mammalian subject, the method comprising systemically or locally administering allogeneic amniotic exosomes to the mammalian subject for a time or under conditions sufficient to induce cell or neuronal repair.

[0083] In one embodiment, the present invention enables a method of inducing cell or neuronal repair, regeneration and / or repair in a human subject, the method comprising systemically or locally administering allogeneic amniotic exosomes to the human subject for a time or under conditions sufficient to induce cell or neuronal repair.

[0084] In another embodiment, contemplated herein is the use of mammalian amniotic exosomes in the manufacture of a medicament for cell or neuronal repair, regeneration and / or repair in a mammalian subject.

[0085] In one embodiment, the mammal is a human.

[0086] Therefore, the present specification also teaches the use of human amniotic exosomes in the manufacture of a medicament for cell or neuronal repair in a human subject. The present specification also teaches the use of human amniotic exosomes in the manufacture of a medicament for treating demyelinating diseases, conditions or disorders (such as but not limited to multiple sclerosis).

[0087] The present invention teaches an isolated sample of amniotic exosomes derived from amniotic epithelial cells. This includes an isolated sample of human amniotic exosomes from human amniotic epithelial cells. It is proposed to use these amniotic exosomes in improved cell-based therapeutic regimens. Therefore, the present invention extends to a pharmaceutical composition comprising allogeneic mammalian amniotic exosomes selected for use in treating a mammalian subject, the pharmaceutical composition also comprising one or more pharmaceutically acceptable carriers, excipients and / or diluents.

[0088] In one embodiment, the mammalian subject is a human subject.

[0089] Therefore, the present invention teaches a pharmaceutical composition comprising human allogeneic amniotic exosomes for treating a human subject, the pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers, excipients and / or diluents.

[0090] Additionally, the composition may be a cosmetic composition comprising human allogeneic amniotic exosomes for use in treating a human subject, the cosmetic composition further comprising one or more cosmetically acceptable carriers, excipients and / or diluents.

[0091] The pharmaceutically acceptable carrier may contain a physiologically acceptable compound, which, for example, acts to stabilize the amniotic exosomes. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or excipients including water or saline or other stabilizers and / or buffers. Detergents may also be used to stabilize or increase or decrease the absorption of amniotic exosomes (including liposome carriers). Pharmaceutically acceptable carriers and formulations are known to those skilled in the art and are described in detail in the scientific and patent literature, for example, see Remington's Pharmaceutical Sciences (1990), 18th edition, Mack Publishing Company, Easton ("Remington's").

[0092] Other physiologically acceptable compounds include preservatives that can be used to prevent the growth or action of microorganisms in amniotic exosome preparations. Various preservatives are well known, including, for example, ascorbic acid. Those skilled in the art will appreciate that the choice of pharmaceutically acceptable carriers (including physiologically acceptable compounds) depends, for example, on the route of administration of the amniotic exosomes of the present invention and the specific physiological or biochemical characteristics of the proteins and nucleic acids produced by the exosomes.

[0093] Administration of amniotic exosomes in the form of a pharmaceutical composition can be performed by any convenient means known to those skilled in the art and according to the disease or condition or site of injury. Routes of administration include, but are not limited to, respiratory, intratracheal, nasopharyngeal, intravenous, intraperitoneal, intrathoracic, subcutaneous, intracranial, intradermal, intramuscular, intraoccularly, intrathecal, intracerebral, intranasal, rectal, topical, patch, bandage, and implant. In one embodiment, amniotic exosomes can be sprayed onto, for example, a subject with severe burns.

[0094] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions.

[0095] Sterile injectable solutions in the form of dispersions are generally prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the amniotic exosomes.

[0096] For parenteral administration, amniotic exosomes can be formulated with a pharmaceutical carrier and administered in the form of a suspension. Illustrative suitable carriers are water, saline, dextrose solution, fructose solution, ethanol or oils of animal, vegetable or synthetic origin. The carrier may also contain other ingredients, such as preservatives, buffers, etc. When amniotic exosomes are administered intrathecally, they may also be formulated in the cerebrospinal fluid.

[0097] For transmucosal or transdermal administration, penetrants suitable for permeation barriers can be used to deliver the medicament. Such penetrants are generally known in the art, such as bile salts and fusidic acid derivatives for transmucosal administration. In addition, detergents can be used to promote penetration. Transmucosal administration can be by nasal sprays or use suppositories, such as Sayani and Chien (1996) Crit Rev Ther Drug Carrier Syst 13: 85-184.

[0098] The amniotic exosomes of the present invention can also be administered in a sustained delivery or sustained release mechanism, which can deliver exosomes internally over a period of time. For example, the formulations of the present invention can include biodegradable microspheres or capsules or other biodegradable polymer configurations that can sustain delivery of amniotic exosomes (e.g., Putney and Burke (1998) Nat Biotech 16: 153-157).

[0099] In preparing the pharmaceutical compositions of the present invention, various formulation techniques can be used and manipulated to alter biodistribution. Many methods for altering biodistribution are known to one of ordinary skill in the art. Examples of such methods include protecting exosomes in vesicles composed of substances such as proteins, lipids (e.g., liposomes), carbohydrates, or synthetic polymers. For a general discussion of pharmacokinetics, see, e.g., Remington's.

[0100] Depending on the method of administration, the pharmaceutical composition of the present invention can be administered in various unit dosage forms. Such dosages are generally of an advisory nature and are adjusted according to the specific treatment situation. The amount of amniotic exosomes sufficient to achieve this is defined as an "effective amount". The dosage regimen and effective amount, i.e., the "dosing regimen", used for this purpose will depend on various factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical condition, age, drug formulation, and the concentration or selection of amniotic exosomes. When calculating the patient's dosing regimen, the mode of administration is also taken into account. The dosing regimen must also take into account the clearance rate of the pharmaceutical composition, etc. See, for example, Remington's; Egleton and Davis (1997), Peptides 18: 1431-1439; Langer (1990), Science 249: 1527-1533. In one embodiment, from 0.05 μg to 100 μg of amniotic exosomes are administered. Included herewith are from 0.1 μg to 50 μg and 0.1 μg to 20 μg and any amount therebetween.

[0101] According to these methods, amniotic exosomes or pharmaceutical compositions comprising the same can be co-administered in combination with one or more other agents. "Co-administration" mentioned herein refers to simultaneous administration with the same formulation or two different formulations by the same or different routes, or sequential administration by the same or different routes. "Sequential" administration mentioned herein refers to the time difference of seconds, minutes, hours or days between the administration of amniotic exosomes and another agent. Co-administration may occur in any order. Examples of agents that can be co-administered include cytokines. Typically, the selection of another agent is based on the disease or condition to be treated.

[0102] Alternatively, targeted therapy can be used to deliver amniotic exosomes to certain cell types or locations in the body by using targeting systems, such as antibodies or cell-specific ligands. Targeting may be desirable for a variety of reasons, such as to facilitate localized treatment of the area in need of treatment.

[0103] The production of amniotic exosomes is also taught herein. Conveniently, this can be accomplished in a bioreactor, which can be in the form of a batch culture reactor or a continuous flow culture reactor. Typically, amniotic epithelial cells are immortalized and used to inoculate growth medium in a bioreactor. The resulting conditioned medium is then collected, the amniotic exosomes are isolated, and formulated for immediate use or storage, for example by freeze drying for later use.

[0104] Kits are also contemplated herein. The kits may be therapeutic or diagnostic. Therapeutic kits may include a selected batch of freeze-dried amniotic exosomes and one or more other pharmaceutically acceptable carriers, excipients and / or diluents and / or another active agent. Diagnostic kits may include reagents for determining the proteomic or genetic profile of a batch of amniotic exosomes.

[0105] Example

[0106]

[0066] Various aspects taught herein will now be further described by the following non-limiting examples.

[0107] Example 1

[0108] Production of amniotic exosomes

[0109] A protocol for isolating amniotic exosomes was developed ( Figure 1). This is the first description of amniotic exosomes and the validation of their biological activity. Primary isolates of hAEC were cultured in serum-free medium (Ultraculture media, Lonza) for 96 hours, followed by continuous ultracentrifugation at 110,000g to remove cells and process the conditioned medium for exosome isolation. Approximately 1.5-2 μg of purified exosomes were consistently purified per million hAEC, regardless of gestational age. This can be scaled up in bioreactor cultures without contamination by apoptotic bodies.

[0110] The ability of amnion exosomes to exert a similar effect was tested. Amnion exosomes inhibited T cell proliferation to a similar extent as hAEC conditioned medium, with a clear dose effect (0.1 μg vs. 1 μg). Depletion of exosomes from hAEC conditioned medium (ExD CM) abolished this effect ( Figure 2 A), indicating that amniotic exosomes are the main mediator of T cell inhibition. Amniotic exosomes can directly increase the phagocytic activity of macrophages ( Figure 2 B) These findings suggest that the immunomodulatory effects of hAEC-conditioned medium are primarily attributable to exosomes.

[0111] Example 2

[0112] Activity of amniotic exosomes

[0113] To determine whether amniotic exosomes are functional in vivo, 1 μg aliquots of amniotic exosomes were injected intravenously into BPD mice on postnatal day 4, and the tissue:air ratio was assessed on postnatal day 14. Amniotic exosomes effectively reversed alveolar simplification ( Figure 3 ). Amniotic exosomes played a major role, preventing or reversing the deleterious changes in lung architecture by reducing the simplicity of alveolar development and recruiting endogenous stem cells, while resolving inflammation in BPD mice.

[0114] In summary, the data suggest that amniotic exosomes modulate host immune events and lung repair in a manner similar to their parental cells. It is proposed that amniotic exosomes can recapitulate the regenerative capacity of hAECs in vivo. By revealing the nature of amniotic exosome cargo, they may be used to exert profound immunomodulatory and pro-repair effects.

[0115] A BPD mouse model was used to determine that neonatal administration of amniotic exosomes could restore lung structure, activate the lung stem cell niche, and modulate inflammation in BPD mice to levels comparable to hAEC-treated animals. It was further determined that this would lead to improvements in long-term physiological outcomes such as pulmonary hypertension and lung function. The proteomic and mRNA / miRNA content of amniotic exosomes was analyzed to identify specific pathways associated with hAEC-mediated repair.

[0116] Example 3

[0117] Repair effect of amniotic exosomes in BPD mice

[0118] The data suggest that amniotic exosomes exert immunomodulatory and pro-regenerative effects in vitro and in vivo. To understand how amniotic exosomes affect cellular crosstalk during repair and to determine whether amniotic exosomes alone are sufficient to recapitulate the repair effects of hAECs in animal models of BPD, two doses of amniotic exosomes (1 μg and 10 μg) were compared with the effects of an optimized dose of hAECs. Fibroblasts and fibroblast exosomes were used as controls.

[0119] The effects of term and preterm amniotic exosomes on lung repair were evaluated using a BPD mouse model that combines two major contributors to human BPD, perinatal inflammation and postnatal hyperoxia. Despite the limitations of using rodents to model complex diseases such as BPD, this model lends itself to detailed molecular analyses. Rodent studies are relatively affordable for the evaluation of dose effects, with long-term studies investigating outcomes in adolescence and adulthood. Briefly, 0.2 μg of lipopolysaccharide (LPS) in 5 μL of saline was injected into each amniotic sac of E16 mouse embryos using a microglass needle (inner diameter: 70-80 μm) and a microinjector (IM-300, Narashige). Once born, newborn mouse pups and their nursing dams were placed in a hyperoxic chamber (65% oxygen) or room air. Nursing dams were rotated every 48 hours to prevent oxygen toxicity. This combination of prenatal inflammation and postnatal hyperoxia results in lung injury similar to human BPD (Vosdoganes et al. (2013) Cytotherapy 15:1021-1029; Nold et al. Proc. Natl Acad. Sci USA 110:14384-14389). The therapy was administered on postnatal day 4. The experimental timeline depicting intra-amniotic LPS injection at E16, exosome / cell injection on postnatal day 4, and collection points (crossover) is shown in Figure 4 middle.

[0120] Exosomes or cells were administered intravenously via the superficial temporal vein using the same equipment as described for intra-amniotic injection and a wider glass needle (100-120 μm inner diameter). The final injection volume was 10 μL, which was well tolerated by 4-day-old mice. On days 7 and 14 after birth, pups were captured for assessment of immunological changes as well as lung stem cell recruitment and lung repair. Both groups of animals were then transferred to room air after weaning and tested at 4 and 10 weeks of age to assess the effects of neonatal therapy on long-term outcomes (e.g., pulmonary hypertension, cardiovascular and respiratory function in adolescence and early adulthood).

[0121] hAEC was isolated from full-term (37-40 weeks) human pregnancy. Primary isolates were used for experiments. hAEC from 6 donors were mixed equally to provide a unified population for all animal experiments. Animals receiving hAEC received a single injection of 100,000 hAEC on the 4th day after birth. For amniotic exosomes, a portion of mixed hAEC was placed in culture medium (10 million per 25mL Ultraculture culture medium, Lonza) for 96 hours. Exosomes were then isolated from conditioned culture medium. The exosome properties of the separated precipitates were determined by Western blotting of exosome markers (TSG101 and Alix) and the size and identification were observed by electron microscopy. Exosomes were resuspended in saline and administered at a dose of 1 μg or 10 μg on the 4th day after birth.

[0122] Human lung fibroblasts do not support lung repair and are suitable as a control cell type (Moodley et al. (2010) Am J Respir Crit Care Med i: 643-651). Human lung fibroblasts or fibroblast exosomes obtained using the same culture protocol as above were administered. Fibroblasts were administered at the same dose as hAECs, and fibroblast exosomes were administered at a higher dose (10 μg). The experimental groups are described in Table 1.

[0123] Table 1 Experimental groups

[0124]

[0125] Immunological changes

[0126] Lungs were collected and processed for flow cytometry as described above (Nold et al. (2013) supra; Tan et al. (2015) Stem Cell Res. Ther. 6: 8). CD45+ fractions were sorted, and a combination of surface markers and intracellular cytokine staining was used to assess changes in the number, phenotype, and activation state of T cells (CD3, CD4, CD25, IFNγ, IL-4, IL17A, FoxP3), macrophages (CD11b, F4 / 80, CD86, MHCII), neutrophils (CD11c, Ly6G), B cells (B220), and NK cells (NK1.1). Bronchoalveolar lavage fluid was collected to measure changes in cytokines using Proteome Profiler (R&D Systems) as described above (Nold et al., (2013) supra).

[0127] Lung stem / progenitor cell recruitment

[0128] Based on the standard CD45- / CD31- / Sca-1+ / EpCam+, the changes in the BASC population were determined by flow sorting (Lee et al. (2014) Cell 156:440-455). This uses the CD45+ fraction of cells from the above immune cell study. AT2 was sorted based on flow sorting with high CD31- / Sca-1- / autofluorescence. The differences in transcriptional profiles were determined using single-cell digital PCR (Fluidigm, qdPCR37K). The flow-sorted single cells were captured on a 96-well microfluidic plate (C1 Single Cell Autoprep System, Fluidigm), where cell lysis, RNA isolation, preamplification, and cDNA conversion will occur. The samples were then loaded onto a microfluidic chip card for digital PCR. The data were analyzed using the SINGULAR v2.0 analysis tool set. Since niche activation pathways for BASC and AT2 are poorly described, a custom 48:48deltaGene assay was used that covers stem cell pluripotency, activation, recruitment, and differentiation, including the recently described BMP1 / NFATc1 / Thrombospondin-1 axis (Lee et al. (2014) supra).

[0129] Simplified alveolar development

[0130] Quantitative image analysis measurements of tissue:air ratios were performed to determine the extent of simplification of alveolar development in all experimental groups.

[0131] Activation of the host stem cell niche

[0132] Immunohistochemical staining of BASCs at the terminal bronchioles (SPC+CC10+) was performed to determine the activation status of the lung stem cell niche (Lee et al. (2014) supra).

[0133] The goal is to know whether the changes in lung architecture and recruitment of endogenous lung stem cells extend to long-term improvement in lung function and reduction of secondary complications.

[0134] Physiological research

[0135] Recovered adolescent (4 weeks old) and young adult (10 weeks old) mice were subjected to pulmonary function tests and echocardiography.

[0136] Echocardiography

[0137] Mice were anesthetized with 3% isoflurane and continued at 1-2% to reach a heart rate of 350-450bpm. Vevo2100 ultrasound (Monash Bioimaging) and 40MHz linear transducer (transducer) were used to perform PW Doppler measurement of pulmonary artery acceleration time along the left parasternal long axis section angled forward. Right ventricular wall thickness was measured by applying M mode along the right parasternal long axis section. Invasive pulmonary function tests were performed using the same group of mice. Tracheostomy was performed on them with an 18G cannula, which was connected to an online ultrasonic nebulizer, a ventilator and an additional pressure sensor (FlexiVent, SCIREQ, Montreal, Canada). Airway resistance and compliance were assessed by exposing mice to increasing concentrations of methacholine (1-30mg / mL, 3 minutes per cycle). Forced expiratory volume, vital capacity and deep inspiration capacity (inspiratory capacity) were obtained. Unlike unrestrained whole-body plethysmography, this does not require animal training and allows for short pauses in mechanical ventilation to perform the measurement maneuver, during which a predetermined pressure or volume waveform is measured. This overcomes traditional challenges with plethysmography, such as excessive dead space and inaccurate measurements.

[0138] It is proposed that amniotic exosomes have beneficial effects in their ability to trigger macrophage polarization, induce Treg expansion, and reduce neutrophil and dendritic cell activation in BPD mice. It is proposed that the immunological changes were greater with a 10μg dose of amniotic exosomes compared to 1μg of control hAEC. Therefore, the reversal of simplification of alveolar development was greater in animals receiving higher doses of amniotic exosomes. This implies that long-term physiological outcomes are improved, such that there will be a dose-dependent reduction in right ventricular wall thickening, reduction in pulmonary hypertension, and restoration of normal lung function. No changes were expected when hAECs or amniotic exosomes were administered to healthy mice. Fibroblasts or fibroblast exosomes have not been proposed to have an effect on immune cells, lung repair, or long-term physiological outcomes.

[0139] Example 4

[0140] Unique mediators in amniotic exosomes

[0141] The gestational age of hAEC donors has a significant effect on their repair ability (Lim et al. (2013) Placenta 34: 486-492). A comparison was made between exosome cargoes collected from full-term and premature hAECs. In preparation, amniotic exosomes from full-term and premature donors were administered, and it was shown that alveolar development simplification was reversed only in animals that received amniotic exosomes from full-term donors, indicating that the ability to activate immunomodulatory and regenerative pathways in premature amniotic exosomes was significantly impaired. When exosome cargoes were initially analyzed for presence / absence of proteomics, 242 and 21 unique proteins were identified in full-term and premature donors, respectively. Using gene ontology analysis, it was determined that full-term amniotic exosomes contain mediators of cell signaling associated with wound healing, apoptosis, vascular development, acute inflammation, and epithelial cell development.

[0142] For proteomic analysis, in-solution trypsin digestion of amniotic exosomes (term and preterm, n = 10 per group) was performed, followed by liquid chromatography and mass spectrometry for absolute quantification (WEHI Proteomics Laboratory, Melbourne, Australia). Data were acquired using a Q-Exactive combined quadrupole-orbitrap mass spectrometer adapted to a Nano-ESI source (Proxeon) coupled to a nanoACQUITY UPLC system (Waters). Peak lists for each LC-MS / MS run were merged into a single MASCOT file and searched against the human Ref-Seq protein database (1% false discovery rate). Quantitative proteomic data were analyzed using Pipeline Pilot (Accelrys) and Spotfire (TIBCO). Abundance differences were assessed using the Wilcoxon signed-rank test. The UniProt database classifies proteins based on function, subcellular localization, and designates genes involved in wound healing, cell survival, and immune regulation.

[0143] For nucleic acid analysis, digital gene expression profiling was performed using Massive Analysis (MACE, GenXpro GmbH) at the end of cDNA. This enables to capture and quantify a small amount of transcripts, such as receptors and transcription factors that are usually lost in microarrays, with a depth of about 20 times greater than RNASeq (1-20 copies per million transcripts). MACE is optimized to sequence small RNAs and miRNAs from exosomes, and combines the advantages of qPCR arrays and RNASeq by marking each cDNA molecule. It determines the variable polyadenylation (alternative polyadenylation) that affects the stability and biological relevance of mRNA-miRNA interactions and thereby determines transcripts. Gene ontology enrichment and gene set enrichment analysis for paired comparisons were performed.

[0144] There will be unique molecular signatures between term and preterm amniotic exosomes, which are implicated in their pro-repair and regenerative effects.

[0145] Example 5

[0146] Pro-regenerative effect

[0147] The pro-regenerative effects of amniotic exosomes in a neonatal mouse model of bronchopulmonary dysplasia were demonstrated. Alveolar pruning ( Fig.12A to D). The term "BPD" refers to the bronchopulmonary dysplasia mouse model animal.

[0148] Example 6

[0149] Mechanism of action of exosomes

[0150] The ability of human exosomes derived from term fetuses to induce lung regeneration was tested with human amniotic epithelial cells (hAEC). Fig.13 A to Fig.13 C. Full-term exosomes restored the secondary septal crest, such as Fig.13 Medium to dark staining (elastin positive) is shown in the distal end.

[0151] in addition, Fig.14 The researchers showed that amniotic exosomes triggered an endogenous stem cell response in the lung. In fact, amniotic exosomes were more than two times more effective than hAECs.

[0152] It was also observed that amniotic exosomes could directly stimulate enhanced growth of exogenous lung stem cells in alveolar, bronchiolar, and mixed lung tissues exposed to exosomes relative to controls.

[0153] Example 7

[0154] Exosomes fight fibrosis in the liver

[0155] Liver fibrosis was induced in adult mice aged 8-12 weeks by intraperitoneal injection of carbon tetrachloride three times a week for 12 weeks. Exosomes (1 μg) were injected twice a week in week 8. Fig.15 In A and B. Fibroblastic cells were determined using Sirius Red assay and α-smooth muscle actin (SMA) expression assay. α-SMA plays a role in fibroblast contraction. α-SMA expression was determined using standard assays and the Sirius Red or α-SMA positive area per region was determined. The inflammatory macrophage protein CCL14 was used. CCL14+exosomes resulted in significantly fewer fibrotic cells per region compared to CCL14+saline controls ( Fig.15 A and B).

[0156] Example 8

[0157] Proteomic cargo

[0158] Fig.16 The proteomic cargo between term exosomes and preterm hAECs was shown to be largely the same. There were more differences between the proteomic cargo of term and preterm hAECs. The proteins tested are listed in Tables 2a and 2b. Fig.17A useful comparison of cellular components between hAECs and total MSCs is shown. Fig.18 The biological processes between hAECs and total MSCs were also compared.

[0159] Table 2a Proteomic cargo in amniotic exosomes: proteins common to MSCs

[0160]

[0161]

[0162] Table 2b Proteomic cargo in amniotic exosomes: hACE-unique proteins

[0163]

[0164]

[0165] Example 9

[0166] Exosomes promote myelination

[0167] Amniotic exosomes were tested in an animal model of multiple sclerosis. It is expected that the exosomes will promote remyelination and can be used to treat multiple sclerosis and other conditions such as optic neuritis, Devic's disease, transverse myelitis, acute disseminated encephalomyelitis, and adrenoleukodystrophy and adrenomyeloneuropathy.

[0168] Example 10

[0169] exosome activity

[0170] Exosomes isolated from the conditioned medium of human amniotic epithelial cells have immunomodulatory and pro-regenerative effects.Amniotic exosomes contain (among other factors) high levels of HLA-G.

[0171] The immunosuppressive effect of amniotic exosomes corresponds to the gestational age of the donor. This corresponds to the gestational age-related donor efficacy, which we have previously published in (Lim et al. Humans (2013) supra).

[0172] In a neonatal mouse model of bronchopulmonary dysplasia, amniotic exosomes reversed lung injury. Intravenously injected exosomes significantly improved the tissue:air ratio compared with saline and are consistent with our results that term amniotic exosomes outperformed preterm exosomes in their ability to ameliorate BPD-associated lung injury in vitro. This was associated with activation of the lung's endogenous stem cell niche, the bronchoalveolar ductal junction. Figure 5 It is suggested that amniotic exosomes may be used to treat pulmonary fibrosis and fibrosis of other organs.

[0173] In a mouse model of bleomycin-induced pulmonary fibrosis, amniotic exosomes reversed established lung inflammation and fibrosis. Intranasal administration of amniotic exosomes 7 days after bleomycin challenge significantly reduced the percentage of activated myofibroblasts (α-smooth muscle actin positive) in the lungs. This was consistent with a decrease in collagen deposition in the lungs. The results were shown in Figure 6 middle.

[0174] Amniotic exosomes directly reversed the activation of primary human lung fibroblasts in vitro. When cultured in the presence of 5 ng / mL transforming growth factor-β, amniotic exosomes reduced the protein level of α-smooth muscle actin within 24 hours. The results were shown in Figure 7 middle.

[0175] Amniotic exosomes contain miRNAs that target the cytokine-cytokine receptor signaling pathway, as shown in Figure 8, where yellow boxes indicate the targets of one or more miRNAs.

[0176] Amniotic exosomes contain miRNAs that target the Wnt signaling pathway, as shown in FIG9 , wherein yellow boxes indicate targets of one or more miRNAs.

[0177] Amniotic exosomes contain miRNAs that target the PI3K-Akt signaling pathway, such as Fig.10 As shown, yellow boxes indicate targets of one or more miRNAs.

[0178] Amniotic exosomes contain miRNAs that target the TGFβ signaling pathway, such as Fig.11 As shown, yellow boxes indicate targets of one or more miRNAs.

[0179] Clearly, amnion exosomes are as potent, if not more potent, than AECs (e.g., hAECs) and possess a vast capacity to induce cellular and molecular repair mechanisms in a wide range of physiological and neural processes.

[0180] Those skilled in the art will appreciate that, in addition to those specifically described, the disclosure described herein may be varied and modified. It should be understood that the disclosure is intended to protect all such variations and modifications. The disclosure is also capable of individually or collectively realizing all steps, features, compositions and compounds mentioned or indicated in this specification, and capable of realizing any and all combinations of any two or more of the steps or features or compositions or compounds.

[0181] References:

[0182] Anderson et al. (2016) Stem Cells. http: / / doi.org / 10.1002 / stem.2298Doyle et al. (2006) Paediatrics 118:108-113

[0183] Egleton and Davis (1997) Peptides 18:1431-1439

[0184] Fishback et al. (2013) Sci Transl Med 5:179ps7)

[0185] Hodges et al. (2012) Am J Obstet Gynerol 206:448e8-448e15 Langer (1990) Science 249:1527-1533

[0186] Lee et al. (2014) Cell 156:440-455

[0187] Lim et al. (2013) Placenta 34:486-492

[0188] Lodha et al. (2014) PLoS ONE:e90843

[0189] Putney and Burke (1998) Nat Biotech 16:153-157

[0190] Remington's Pharmaceutical Sciences (1990), 18th Edition, Mack Publishing Company, Easton, PA.

[0191] Moodley et al. (2010) Am J Respir Crit Care Med i:643-651 Murphy et al. (2012) Cell Transplant 21:1477-1492

[0192] Nold et al. Proc. Natl Acad. Sci USA 110:14384-14389

[0193] Schellenberg et al. (2011) Aging (Albany NY) 3:873-888

[0194] Sayani and Chien (1996) Crit Rev Ther Drug Carrier Syst 13:85-184 Tan et al (2015) Stem Cell Res Ther 6:8

[0195] Vosdoganes et al. (2013) Cytotherapy 15:1021-1029

[0196] Yawno et al. (2013) Dev Neurosci 35:272-282

Claims

1. Use of mammalian amniotic exosomes in the manufacture of a medicament for reducing or inhibiting T cell proliferation and / or increasing macrophage phagocytosis in a demyelinating disease in a mammalian subject, wherein the mammalian amniotic exosomes are mammalian amniotic exosomes derived from allogeneic mammalian amniotic epithelial cells, and the allogeneic mammalian amniotic epithelial cells are from a donor mammal of the same species.

2. The use of claim 1, wherein the mammalian subject is a human.

3. The use of claim 1, wherein the mammalian subject is a non-human mammal.

4. The use of claim 3, wherein the non-human mammalian subject to be treated is a racing animal selected from the group consisting of a horse, a dog and a camel.

5. The use of claim 1, wherein the mammalian subject is in need of tissue or neuronal repair, regeneration and / or restoration.

6. The use of claim 1, wherein the demyelinating disease is multiple sclerosis, optic neuritis, Devic's disease, transverse myelitis, acute disseminated encephalomyelitis, or adrenoleukodystrophy.

7. The use according to claim 1, wherein the demyelinating disease is adrenomyeloneuropathy.

8. The use of claim 1, wherein the amniotic exosomes contain miRNAs targeting cytokine-cytokine receptor, Wnt, PI3K-Akt and TGFβ signaling pathways.

9. The use of any one of claims 1 to 8, wherein the amniotic exosomes are derived from a pool of immortalized mammalian amniotic epithelial cell lines.

10. The use of any one of claims 1 to 8, wherein the amniotic exosomes are selected from a library of lyophilized amniotic exosomes derived from immortalized mammalian amniotic exosomes.

11. Use of mammalian amniotic exosomes in the manufacture of a medicament for treating bronchopulmonary dysplasia (BPD) in a mammalian subject, wherein the mammalian amniotic exosomes are mammalian amniotic exosomes derived from allogeneic mammalian amniotic epithelial cells, and the allogeneic mammalian amniotic epithelial cells are from a donor mammal of the same species.

12. The use of claim 11, wherein the mammalian subject is a human.

13. The use of claim 11, wherein the mammalian subject is a non-human mammal.

14. The use of claim 11, wherein the mammalian amniotic exosomes are human amniotic exosomes.

15. Use of amniotic epithelial cell exosomes in the manufacture of a medicament for an improved cell-based therapeutic regimen for treating a mammalian subject with a demyelinating disease, the improvement comprising isolating amniotic exosomes from an immortalized amniotic epithelial cell line and administering systemically or locally to a subject in need of reducing or inhibiting T cell proliferation and / or increasing macrophage phagocytosis.

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