Fibroblast-based therapy for amyotrophic lateral sclerosis

Abstract

The present disclosure relates to methods and compositions for treating or preventing or reducing the risk of having amyotrophic lateral sclerosis (ALS) in an individual. In certain embodiments, the methods and compositions are related to administering to an individual a therapeutically effective amount of a population of fibroblasts, fibroblast exosomes, IL-2, or combinations thereof.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 211,989, filed June 17, 2021, which is incorporated by reference in its entirety.

[0002] Embodiments of the present disclosure encompass at least the fields of cell biology, molecular biology, physiology, and medicine. [Background technology]

[0003] Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative condition that causes muscle atrophy and death within 3–5 years after onset [1]. In the majority of patients (90%), the cause of ALS is idiopathic; however, in about 10% of patients, a familial form of the disease is presented [2]. Specific muscle degeneration is exclusive to motor neurons, begins locally and spreads, resulting in weakness of the limbs, respiratory muscles, and medulla oblongata. Shortly before death, there is an almost complete loss of limb and respiratory function, as well as loss of the ability to chew, swallow, and speak.

[0004] In the United States, ALS is defined as an "orphan disease" with a prevalence of approximately 2 new cases per 100,000 people annually and a total of approximately 5 cases per 100,000 people annually [3]. In the United States [4] and Europe [5], ALS is diagnosed in 1 in 500 to approximately 1 in 1,000 adult deaths, meaning that 500,000 people in the United States will develop the disease in their lifetime. Approximately 10% of ALS cases are inherited, usually as a dominant trait [6]. Both familial ALS (fALS) and sporadic ALS (sALS) can occur simultaneously with frontotemporal lobe dementia (FTLD). In contrast to dementia in Alzheimer's disease (AD), whose cardinal finding is memory loss, FTLD is characterized by behavioral changes and progressive aphasia, occasionally accompanied by movement disorders. While AD is associated with prominent pathology in the hippocampus, the cardinal finding in FTLD, as the name suggests, is early atrophy of the frontal and temporal lobes. From the pathological analysis of autopsy cases with sALS, fALS or ALS-FTLD with diverse genetic causes, four recurring themes have emerged. First, motor neuron death is usually accompanied by deposition of mostly cytoplasmic aggregated proteins, often ubiquitinated. Second, in ALS, the levels and function of RNA and RNA-binding proteins are abnormal. Protein and RNA aggregates are detected in both motor neurons and non-neuronal cells, e.g., astrocytes and microglia. Third, in most cases, it is accompanied by some disturbance of neuronal cytoskeleton assembly and function. Moreover, in almost all cases, motor neuron death is influenced by non-neuronal cells, including oligodendrocytes and cells involved in neuroinflammation (e.g., astroglia and microglia).

[0005] The gene most commonly associated with ALS is the C9ORF72 gene, which harbors a repeat expansion of noncoding GGGGCC hexanucleotide repeats [7], which affects approximately 40% of familial ALS cases [8] and, in some cases, is associated with frontotemporal dementia [9]. The abnormal repeats in the C9ORF72 gene contribute mechanistically to the biology of disease progression. An intriguing study has demonstrated several key possible mechanisms using elegant in vitro models. Specifically, C9ORF72 ALS patient-derived induced pluripotent stem cell (iPSC)-differentiated neurons revealed disease-specific a) nuclear GGGGCCexp RNA foci, (b) dysregulated gene expression, (c) sequestration of the GGGGCCexp RNA-binding protein ADARB2, and (d) susceptibility to excitotoxicity. These pathological and pathogenic features were identified in the brains of ALS patients and were abolished by antisense oligonucleotide-mediated suppression of C9ORF72 transcripts or repeat expansions, despite the presence of repeat-associated non-ATG translation (RAN) products. According to the authors, their data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide potential antisense therapeutics and human pharmacodynamic markers for therapy

[10] . The importance of C9ORF72 repeats in neurodegeneration is supported by studies demonstrating that these repeats are found not only in ALS patients, but also in patients with Alzheimer's disease [11, 12], Parkinson's disease

[13] and other dementias

[14] . Cu / Zn-superoxide dismutase 1 (SOD1) also has an important genetic association with ALS pathogenesis. Additional less common genes associated with ALS include: TAR DNA-binding protein 43 fused in sarcoma (FUS) (TARDBP) and other less frequent mutations.

[0006] Despite significant advances in knowledge of ALS pathology, the only currently available treatment is riluzole, which extends survival by only 3 months and does not improve quality of life. Therefore, it is essential to search for new alternatives to treat ALS, and the present disclosure provides such a solution. Summary of the Invention

[0007] The present invention relates to systems, methods and compositions aimed at reducing and / or ameliorating one or more symptoms of amyotrophic lateral sclerosis (ALS) in mammals. In one embodiment, administration of fibroblasts and / or modified fibroblasts and / or fibroblast exosomes is performed to induce immunological and / or regenerative changes that result in, for example, slowing and / or ameliorating motor neuron degeneration associated with ALS. In one embodiment, fibroblasts and / or modified fibroblasts and / or fibroblast exosomes are utilized to generate immunomodulatory cells that inhibit neuroinflammation and allow stimulation of regenerative processes. In some embodiments, fibroblasts and / or modified fibroblasts and / or fibroblast exosomes are utilized as therapeutic adjuvants.

[0008] In certain embodiments, there is a method for treating or preventing or reducing the risk of having amyotrophic lateral sclerosis (ALS) in an individual, comprising administering to the individual a therapeutically effective amount of a population of fibroblasts, fibroblast exosomes, modified fibroblasts, IL-2, or a combination thereof.In some embodiments, the method further comprises administering to the individual an effective amount of rapamycin, N-acetylcysteine, anti-CD3 antibody, or a combination thereof.The fibroblasts may be allogeneic to the individual, or autologous or xenogeneic to the individual.In some cases, the fibroblasts are mitotically active before administration to the recipient in need of treatment.

[0009] Fibroblasts may be derived from any source and may be isolated from a tissue selected from the group consisting of: a) skin; b) bone marrow; c) blood; d) mobilized peripheral blood; e) gums; f) tonsils; g) placenta; h) Wharton's jelly; i) hair follicles; j) fallopian tubes; k) liver; l) milk teeth; m) vas deferens; n) endometrium; o) menstrual blood; p) omentum; and q) combinations thereof.

[0010] In particular cases, ALS in an individual is associated with elevated inflammatory cytokines compared to age-matched healthy controls, which may be IL-1, IL-2, IL-6, IL-9, IL-11, IL-12, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-27, IL-33, HMGB-1, TNF-α, TNF-β, IFN-α, IFN-β, IFN-γ. In certain embodiments, the fibroblasts are selected for expression of CD73, CD70, CD105, CD16, CD55, CD37, interleukin-10 receptor, and / or interferon gamma receptor.

[0011] Fibroblasts can be selected for expression of CD73, then treated with interferon gamma and expanded for at least one cell division before administration. Fibroblasts and / or modified fibroblasts can be administered in a manner that can stimulate the generation of regulatory T cells. Regulatory T cells can express FoxP3, contain membrane-bound TGF-β, suppress the ability of T cells to proliferate in response to mitogens, and / or suppress the ability of immature dendritic cells to mature into differentiated dendritic cells. In a specific case, maturation of dendritic cells is associated with upregulation of the expression of one or more markers selected from the group consisting of a) HLA-II; b) CD40; c) CD80; d) CD86; and e) combinations thereof. Maturation of dendritic cells can be associated with enhanced ability to activate proliferation of allogeneic T cells. Maturation of dendritic cells can be associated with enhanced ability to induce production of interferon gamma from allogeneic T cells. In certain embodiments, regulatory T cells are activated by exposure to CD3, CD28, interleukin-10, and / or by administration of immature dendritic cells that can express PD-1L. Immature dendritic cells can be maintained in an immature state by culturing under low dose GM-CSF, human chorionic gonadotropin, hypoxia, and / or inhibition of NF-κB activity. Inhibition of NF-κB activity can be achieved by administration of antisense molecules that target NF-κB or molecules in the NF-κB pathway, administration of molecules that can induce RNA interference that target NF-κB or molecules in the NF-κB pathway, gene editing means that target NF-κB or molecules in the NF-κB pathway, administration of decoy oligonucleotides that can block NF-κB or molecules in the NF-κB pathway, and / or administration of small molecule blockers of NF-κB activity. Small molecule blockers of NF-κB activity include calagualin (a fern derivative), conophylline (Ervatamia microphylla), evodiamine (Evodia fructose), and erythropoietin (Evodia fructus).fructus extract), geldanamycin, perillyl alcohol, protein-bound polysaccharides from basidiomycetes, rocaglamide (Aglaia derivative), 15-deoxy-prostaglandin J(2), lead, anandamide, Artemisia vestita, cobrotoxin, dehydroascorbic acid (vitamin C), herbimycin A, isorhapontigenin, manumycin A, pomegranate fruit extract, tetrandrine (plant alkaloid), thienopyridine, acetyl-boswellic acid, 1'-acetoxychavicol acetate (Languas galanga) galanga), Apigenin (plant flavonoid), Cardamomine, Diosgenin, Furonaphthoquinone, Guggulsterone, Falcarindol, Honokiol, Hypestoxide, Garcinone B, Kahweol, Kava (Piper methysticum) derivatives, Mangosteen (Garcinia mangostana), N-Acetylcysteine, Nitrosylcobalamin (vitamin B12 analogue), Picetannol, Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), Quercetin, Rosmarinic acid, Semecarpus anacardiianacardiu) extract, staurosporine, sulforaphane and phenyl isothiocyanate, theaflavin (black tea component), tilianin, tocotrienols, wedelolactone, withanolides, zerumbone, silibinin, betulinic acid, ursolic acid, monochloramine and glycine chloramine (NH2Cl), anethole, Baoganing, black raspberry extract (cyanidin 3-O-glucoside, cyanidin 3-O-(2(G)-xylosylrutinoside), cyanidin 3-O-rutinoside ), Buddlejasaponin IV, Chacospongionolide B, Caragualin, Carbon monoxide, Cardamonin, Cycloepoxydone; 1-Hydroxy-2-hydroxymethyl-3-pent-1-enylbenzene, Decursin, Dexanabinol, Digitoxin, Diterpenes, Docosahexaenoic acid, Extensively oxidized low density lipoprotein (ox-LDL), 4-Hydroxynonenal (HNE), Flavopiridol, [6]-Gingerol; Casparol, Glossogyne tenuifolia, Phytic acid (inositol hexakisphosphate), Pomegranate fruit extract, Prostaglandin A1, 20(S)-Protopanaxatriol (ginsenoside metabolite), Lengiolon, Rottlerin, Saikosaponin-d and / or Normal saline (low Na+ isotonic). In one embodiment, regulatory T cells can be generated in vivo by incubation with mesenchymal stem cell exosomes and exposure of T cells to activators of the interleukin-2 receptor, and can induce proliferation and / or activation of CD4 CD25 T cells.

[0012] In some embodiments of the method, the interleukin-2 receptor is activated by administration of IL-2. IL-2 is administered at a dose of 0.3×10 per square meter of body surface area. 6 ~3.0×10 6 IU of IL-2 may be administered daily for 1-16 weeks, in some cases.

[0013] In certain embodiments, any method may further comprise administering one or more immunomodulatory compounds, such as oxytocin, prolactin, IL-10, IL-35, CD3 inhibitor, or combinations thereof. The CD3 inhibitor may be an anti-CD3 antibody, such as teplizumab. In certain embodiments, the individual has familial ALS or idiopathic ALS. The individual may have one or more mutations in the C9ORF72 gene. Any method may further comprise administering riluzole to the individual.

[0014] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure so that the detailed description that follows may be better understood. Additional features and advantages will be described below which form the subject of the claims herein. It should be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present design. Those skilled in the art will also appreciate that such equivalent constructions do not depart from the spirit and scope as set forth in the appended claims. The novel features believed to be characteristic of the design disclosed herein, both as to its construction and method of operation, together with further objects and advantages, will be better understood from the following description when considered in conjunction with the accompanying drawings. It is to be expressly understood, however, that each of the drawings is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.

[0015] Detailed Description of the Invention In keeping with long-standing patent law conventions, the words "a" and "an" when used in conjunction with the word "comprising" in this specification, including the claims, indicate "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. Any method or composition described herein may be implemented with respect to any other method or composition described herein, and it is contemplated that different embodiments may be combined.

[0016] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are to be understood to imply the inclusion of the stated step or element or group of steps or elements, but not the exclusion of other steps or elements or group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and no other elements may be present. "Consisting essentially of" means including any elements recited after this phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, but that other elements are not optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0017] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0018] As used herein, the terms "or" and "and / or" are utilized to describe multiple components in combination with or exclusive of each other. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0019] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning within the field of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0020] The term "isolated" as used herein refers to a molecule or biological or cellular material that is substantially free of other materials. In one embodiment, the term "isolated" refers to a nucleic acid, e.g., DNA or RNA, or a protein or polypeptide, or a cell or a cellular organelle, or a tissue or organ, respectively, separated from other DNA or RNA, or a protein or polypeptide, or a cell or a cellular organelle, or a tissue or organ, e.g., present in a natural source. The term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA technology, or chemical precursors or other chemicals if chemically synthesized. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to a polypeptide that is isolated from other cellular proteins, and is meant to encompass both purified and recombinant polypeptides. The term "isolated" is also used herein to refer to a cell or tissue that is isolated from other cells or tissues, and is meant to encompass both cultured or engineered cells or tissues.

[0021] As used herein, "prevent" and similar terms such as "prevented", "preventing" refer to an approach to prevent, inhibit or reduce the likelihood of the occurrence or recurrence of a disease or condition, such as ALS. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, effect, symptoms and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0022] The term "subject" as used herein generally refers to an individual with ALS, or an individual suspected of having ALS across the general population, or an individual at risk of having ALS. A subject can be any living organism or animal subject that is the object of a method or material, including mammals, such as humans, experimental animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject can be a patient, such as a patient who has ALS, is suspected of having ALS, or is known to have ALS. A subject is undergoing treatment or has been treated. A subject can be asymptomatic. A subject can be a healthy individual, but is an individual who wants to prevent ALS. The term "individual" can be used interchangeably in at least some cases. As used herein, a "subject" or "individual" may or may not be housed in a medical facility, or may be treated as an outpatient in a medical facility. An individual may receive one or more pharmaceutical compositions via the Internet. An individual may include any age of human or non-human animal, and thus includes both adults and juveniles. The term is not intended to imply a need for medical treatment, and thus an individual may be a part of an experiment, whether clinical or in support of basic science research, either voluntarily or involuntarily.

[0023] As used herein, "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or condition, and may also include a minimal reduction in one or more measurable markers of the disease or condition (e.g., ALS) being treated. Treatment may optionally include either a reduction or amelioration of symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate a complete eradication or cure of the disease or condition, or its associated symptoms.

[0024] As used herein, unless expressly stated otherwise or clearly indicated otherwise, terms such as "therapeutically effective dose", "therapeutically effective amount", "effective amount" refer to the portion of a compound that has a net positive effect on the health and well-being of a human or other animal. Therapeutic effects may include improving lifespan, quality of life, reducing the number and / or severity of one or more symptoms, and the like. These effects may also include reducing susceptibility to the development of disease or deterioration of health or well-being. Effects may be realized immediately after a single dose and / or treatment, or cumulatively after a series of doses and / or treatments.

[0025] As used herein, unless expressly stated otherwise or clearly implied otherwise, the term "about" refers to a value in the range of plus or minus 10%, e.g., about 1.0 encompasses values ​​from 0.9 to 1.1.

[0026] The embodiments of the present disclosure provide a means of treating or preventing ALS or reducing the risk of having ALS by administration of fibroblasts and / or modified fibroblasts and / or fibroblast exosomes and / or IL-2. In certain embodiments, the present disclosure encompasses the use of fibroblasts and / or modified fibroblasts to induce regulatory T cells (Tregs) that reduce neuroinflammation and allow regenerative processes to occur.

[0027] In certain embodiments, the present disclosure provides the use of fibroblasts and / or modified fibroblasts and / or fibroblast exosomes and / or IL-2 to prevent, inhibit, delay the onset, slow the progression, or ameliorate ALS. In some embodiments of the present disclosure, stimulation of regulatory T cells by fibroblasts in vivo is achieved to reduce inflammation and stimulate regeneration in ALS patients. In some embodiments, the method includes administration of aldesleukin (Proleukin, Novartis), a commercially available IL-2 approved for the treatment of metastatic renal cell carcinoma in the UK. It is produced by recombinant DNA technology using Escherichia coli strains containing a genetically engineered modified human IL-2 gene, and is administered either intravenously or subcutaneously (SC) at a dose that can selectively expand regulatory T cells. This can be performed with and / or without fibroblasts and / or modified fibroblasts and / or fibroblast exosomes.

[0028] Any form of IL-2 may be administered at any suitable dose and by any suitable administration. IL-2 may be administered daily, in at least some cases for 1-16 weeks, at a concentration of 0.3×106 to 3.0×106 IU of IL-2 per square meter of body surface area, and any range of concentrations derivable therein. In certain cases, after a short intravenous infusion, its pharmacokinetic profile is typified by high plasma concentrations, rapid distribution to the extravascular space, and rapid renal clearance. Recommended doses for continuous infusion and subcutaneous injection may be repeated cycles of 18×106 IU / m2 per 24 hours for 5 days and repeated doses of 18×106 IU, respectively. Bioavailability of IL-2 (including aldesleukin) ranges from 31% to 47%, with peak plasma levels reaching 2-6 hours after SC administration. The process of absorption and excretion of subcutaneous IL-2 (including aldesleukin) has been described by a one-compartment model, with an absorption half-life of 45 min and an excretion half-life of 3-5 h

[15] . Natural IL-2 was first identified in 1976 as a growth factor for T lymphocytes. It is produced by human cluster type (CD)4+ T cells and some CD8+ T cells, and is synthesized mainly by activated T cells, especially CD4+ helper T cells. It stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes the expression of cytokines and cytolytic molecules by T cells, promotes the proliferation and differentiation of B cells and the synthesis of immunoglobulins by B cells, and stimulates the generation, proliferation and activation of natural killer (NK) cells. IL-2 is known to play a central role in the generation of immune responses. In cancer clinical trials, high-dose recombinant IL-2 (e.g., IV bolus doses of 600,000 international units (IU) / kg every 8 hours, up to 14 doses) demonstrated antitumor activity in metastatic renal cell carcinoma (RCC) and metastatic melanoma. Thus, such high-dose IL-2 was approved for the treatment of metastatic RCC in Europe in 1989 and in the United States in 1992. Approval was obtained in 1998 for treating patients with metastatic melanoma.Recombinant human IL-2 (aldesleukin) (Proleukin®-Novartis Inc. and Prometheus Labs Inc.) is currently approved by the United States Food and Drug Administration (US FDA). However, IL-2 has a dual function in the immune response in that it not only mediates the proliferation and activity of effector cells, but also plays a crucial role in the maintenance of peripheral immune tolerance. The major mechanism underlying peripheral self-tolerance is IL-2-induced activation-induced cell death (AICD) in T cells. AICD is a process in which fully activated T cells undergo programmed cell death through the engagement of cell surface-expressed death receptors such as CD95 (also known as Fas) or TNF receptors. Re-stimulation of antigen-activated T cells (after previous exposure to IL-2), which express high-affinity IL-2 receptors during proliferation, with antigen via the T cell receptor (TCR) / CD3 complex induces the expression of Fas ligand (FasL) and / or tumor necrosis factor (TNF), rendering the cells susceptible to Fas-mediated apoptosis. This process is IL-2 dependent and mediated through STAT5. The process of AICD in T lymphocytes can establish tolerance not only to self antigens but also to persistent antigens that are apparently not part of the host's makeup, such as tumor antigens.

[0029] For administration of fibroblasts, various protocols and procedures may be utilized. Guidance for administration of cell therapy in ALS may be derived from studies using various mesenchymal stem cell (MSC) approaches for this condition. For example, one of the first clinical interventions using mesenchymal stem cells in ALS was reported by Mazzini et al.

[16] who treated ALS patients with ex vivo expanded MSCs from bone marrow. Specifically, bone marrow harvest was performed according to standard procedures by aspiration from the posterior iliac crest. Ex vivo expansion of mesenchymal stem cells was induced according to the Pittenger protocol

[17] . Cells were suspended in 2 ml of autologous cerebrospinal fluid and implanted into the spinal cord by a micrometric pump injector. No patients showed major adverse events such as respiratory failure or death. Minor adverse events were reversible intercostal pain radiation after a mean postoperative period of 3 days (four patients) and reversible leg paresthesia after a mean postoperative period of 6 weeks (five patients). No changes in spinal cord volume or other signs of abnormal cell proliferation were observed. The authors concluded by stating that the procedure of ex vivo expansion and transplantation of autologous mesenchymal stem cells into the human spinal cord appears to be safe and well tolerated by ALS patients. The same group reported a 3-year follow-up of the first patient treated. Seven patients affected by definite ALS were enrolled in the study, and two patients were treated as compassionate use. No patient showed major adverse events such as respiratory failure or death. Minor adverse events were intercostal pain radiation and leg paresthesia, both of which were reversible after a mean of 6 weeks. No changes in spinal cord volume or other signs of abnormal cell proliferation were observed. A significant slowdown in the linear decline of forced vital capacity was evident in four patients 36 months after MSC transplantation

[18] . Two additional studies were performed by the same group on 10 and 19 patients. The longest observation of treated patients was performed 9 years after treatment. No long-term adverse effects were detected and marginal therapeutic effects were seen [19, 20]. In another example, a study by an independent group evaluated the safety of two repeated intrathecal injections of autologous bone marrow (BM)-derived mesenchymal stromal cells (MSCs) in patients with ALS. Eight patients with definite or probable ALS were enrolled.After a 3-month lead-in period, autologous MSCs were isolated twice from BM with an interval of 26 days, then expanded in vitro for 28 days and suspended in autologous cerebrospinal fluid. Of the 8 patients, 7 received 2 intrathecal injections of autologous MSCs (1x10(6) cells / kg), 26 days apart. Clinical or laboratory measurements were recorded to assess safety 12 months after the first MSC injection. The ALS Functional Rating Scale-Revised (ALSFRS-R), Appel ALS score and forced vital capacity were used to assess the disease status of the patients. One patient died before treatment and withdrew from the study. The death was not related to the study and was due to the natural progression of the disease. Except for that patient, no serious adverse events were observed during the 12-month follow-up period. Most adverse events were self-limited or subsided after supportive care within 4 days. The decline in ALSFRS-R scores did not accelerate during the 6-month follow-up period. Two repeated intrathecal injections of autologous MSCs were safe and feasible throughout the 12-month follow-up period

[21] . Subsequent studies by Belarus utilized autologous mesenchymal stem cells (intact cells) injected intravenously or via lumbar puncture (cells involved in neural differentiation). Evaluation of cell-T outcomes after 12 months of follow-up revealed that a slowing of disease progression, as assessed by the ALSFRS-R score, was observed in 10 patients treated with cells. In comparison, no slowing of progression was observed in a control group matched for age and disease status. The control group consisted of 15 patients. The study reported no adverse effects associated with intravenous or intrathecal administration of mesenchymal stem cells

[22] .

[0030] Other injection means for administration are possible, such as intraventricular injection. A study by Baek et al.

[23] evaluated the ability to utilize intraventricular injection directly into the brain by administering cells using an Ommaya reservoir. The Ommaya reservoir is a catheter system typically used to deliver drugs directly into the brain ventricles. It consists of one intraventricular catheter attached to a reservoir implanted under the scalp. It is typically used to treat brain tumors, leukemia / lymphoma or leptomeningeal disease, as well as for intraventricular (ICV) injections of morphine

[24] . Other investigators have previously used the Ommaya reservoir to deliver cell therapy to the brain. To demonstrate the relative safety of this approach, one study in glioma patients administered ex vivo expanded autologous tumor-infiltrating lymphocytes to six patients through the use of the Ommaya reservoir. One patient had a complete response, two had partial responses, and three succumbed to the disease. Most interestingly, no severe adverse effects were observed, despite the fact that activated lymphocytes were injected directly into the brain, an area typically classified as highly susceptible to inflammation

[25] . Along with this rational study, other studies have successfully administered cells to the brain [26-28], and because mesenchymal stem cells are generally considered to be anti-inflammatory, Baek et al. sought to adapt this procedure for use in ALS patients. Bone marrow mesenchymal stem cells were isolated from the bone marrow of a male patient with ALS who had undergone insertion of an Ommaya reservoir. Expanded MSCs (hBM-MSCs: a dose of 1 × 106 cells / kg) were suspended in autologous CSF and transplanted directly into the lateral ventricle of ALS patients via the Ommaya reservoir. Clinical, laboratory and radiological evaluation of the patient revealed no severe adverse effects associated with stem cell therapy. The authors concluded that intraventricular injection of an optimized number of cells was safe and a potential route for stem cell therapy in ALS patients. Intraventricular injection via the Ommaya reservoir makes repeated injections of stem cells easy and reliable, even in patients with much more advanced ALS. Unfortunately, the publication did not provide any insight into the impact on disease progression.

[0031] In another attempt to increase the therapeutic efficacy of mesenchymal stem cells in ALS, researchers have explored in vitro means to increase neurotrophic factor production by manipulation of culture conditions, and these methods may be applied here to any type of fibroblast. A series of studies conducted at Hadassah Medical Center in Jerusalem, Israel, attempted to treat ALS with in vitro engineered MSCs that have been verified to produce higher amounts of neurotrophic factors. In this study, all patients were followed for 3 months before transplantation and 6 months after transplantation. In phase 1 / 2 of the trial, 6 patients with early stage ALS received intramuscular (IM) injections and 6 patients with more advanced disease received intrathecal (IT) transplants. In the second, phase 2a, dose escalation study, 14 patients with early stage ALS received a combination of IM and IT transplants of autologous MSC-NTF cells. Among 12 patients in the Phase 1 / 2 study and 14 patients in the Phase 2a study, ages 20 and 75 years, it was reported that administration of mesenchymal stem cells was found to be safe and well tolerated over the study follow-up period. Most adverse effects were mild and transient, and did not include treatment-related serious adverse events. The rate of progression of forced vital capacity and ALSFRS-R scores in IT (or IT+IM)-treated patients decreased versus the pretreatment period in the 6 months following MSC-NTF cell transplantation (-5.1% to -1.2% / month percentage predicted forced vital capacity, P<.04 and -1.2 to 0.6 ALSFRS-R points / month, P=.052). Of these patients, 13 (87%) were defined as responders for either ALSFRS-R or forced vital capacity, with at least a 25% improvement in the slope of progression 6 months after treatment.

[0032] In some embodiments of the present disclosure, well-known examples of approved drugs that enhance endogenous neural stem cell activity include lithium [29, 30], valproic acid

[31] and human chorionic gonadotropin

[32] , which are utilized with fibroblasts to inhibit and / or treat ALS. Interestingly, the stem cell modifier combination of lithium and valproic acid had already been evaluated in itself in a small study suggesting some possible efficacy. The study recruited 18 patients treated with the combination and compared them to 31 controls carefully paired by age, sex, disease incidence and time who had never been treated with lithium and / or valproate. Assessment of disease with the ALSFRS-R was performed before treatment (baseline), one month after treatment, and every four months until outcome (death or adverse events). The researchers reported that co-treatment with lithium and valproate significantly increased survival, and this treatment also exerted neuroprotection in the patients, as all biochemical markers reached normal levels in treated ALS patients. Biochemical markers were Cu / Zn superoxide dismutase and glutathione peroxidase activity, and reduced glutathione

[33] .

[0033] In one embodiment of the present disclosure, a patient suffering from or at risk for ALS is administered 10 billion to 4 trillion fibroblasts per kilogram of body weight followed by 0.3×106 IU of IL-2 (e.g., aldesleukin) daily. Concentrations for clinical use of IL-2 (including aldesleukin) can be used from literature described for other indications including heart failure

[15] , Wiskott-Aldrich syndrome

[34] , graft-versus-host disease [35, 36], lupus

[37] , and type 1 diabetes [38-40], which are incorporated by reference. In some embodiments of the present disclosure, low doses of IL-2, such as in the form of aldesleukin, are administered daily for 8 weeks at a concentration of 0.3×106 to 3.0×106 IU of IL-2 per square meter of body surface area, or in other embodiments, repeated administration of 1.0×106 to 3.0×106 IU of IL-2 for 5 days. Various types of IL-2 can be utilized. Examples of IL-2 variants, recombinant IL-2, methods of IL-2 production, methods of IL-2 purification, methods of formulation, and the like are well known in the art and can be found, for example, at least, in U.S. Patent Nos. 4,530,787, 4,569,790, 4,572,798, 4,604,377, 4,748,234, 4,853,332, 4,959,314, 5,464,939, 5,229,109, 7,514,073, and 7,569,215, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, low-dose interleukin-2 is provided with one or more activators of co-inhibitory molecules, otherwise known as checkpoints.Such co-inhibitory molecules include CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilin, A2aR, and combinations thereof. In some embodiments of the present disclosure, mesenchymal stem cells are co-administered. Protocols for using MSCs have been previously published and are incorporated by reference [41, 42]. For example, adipose-derived mesenchymal stem cells [43-46], bone marrow-derived mesenchymal stem cells [47-66], placenta-derived mesenchymal stem cells

[67] , amniotic membrane-derived mesenchymal stem cells [68, 69], umbilical cord-derived mesenchymal stem cells [70-76], menstrual blood-derived mesenchymal stem cells

[77] and lung-derived mesenchymal stem cells [78, 79], as well as conditioned medium-derived mesenchymal stem cells [80-87]. Furthermore, the generation of Tregs by mesenchymal stem cells has also been described in the art, and the inventors provide the following references to assist in the practice of the present invention [88-116].

[0034] In one embodiment, ALS patients are administered human IL-2 muteins that preferentially stimulate regulatory T (Treg) cells. As used herein, "preferentially stimulate regulatory T cells" means that the muteins promote the proliferation, survival, activation and / or function of CD3+FoxP3+ T cells over CD3+FoxP3- T cells. The method of measuring the ability to preferentially stimulate Tregs can be measured by flow cytometry of peripheral blood leukocytes, and an increase in the percentage of FOXP3+CD4+T cells among total CD4+T cells, an increase in the percentage of FOXP3+CD8+T cells among total CD8+T cells, an increase in the percentage of FOXP3+T cells compared to NK cells, and / or a greater increase in the expression level of CD25 on the surface of FOXP3+T cells compared to the increase in CD25 expression on other T cells is observed. Preferential growth of Treg cells can also be detected as increased presentation of demethylated FOXP3 promoter DNA (i.e., Treg-specific demethylated region, or TSDR) relative to demethylated CD3 gene in DNA extracted from whole blood, as detected by sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA. IL-2 muteins that preferentially stimulate Treg cells increase the ratio of CD3+FoxP3+ T cells to CD3+FoxP3- T cells in a subject or peripheral blood sample by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%.

[0035] In some embodiments of the present disclosure, a patient suffering from ALS is administered an agent having properties that can enhance regulatory T cells by stimulating mesenchymal stem cells administered in an allogeneic host. Proteins, such as antibodies, fusion proteins, and soluble ligands, all of which may be identical to the wild-type protein or contain mutations (i.e., deletion, addition, or substitution of one or more amino acid residues), and the nucleic acid molecules that encode them (or are "antisense" to them; e.g., oligonucleotides that are antisense to nucleic acids that encode target polypeptides, or components (e.g., subunits) of their receptors) are all "agents". Agents of the present invention can be administered systemically, locally, or using cell-based therapy (i.e., agents of the present invention can be administered to a patient by administering to the patient cells that express the agent). The tolerance-restoring agent can be alpha 1-antitrypsin (AAT; sometimes abbreviated as A1AT), also called alpha 1-proteinase inhibitor. AAT is a major serum serine protease inhibitor that inhibits the enzymatic activity of many serine proteases, including neutrophil elastase, cathepsin G, proteinase 3, thrombin, trypsin and chymotrypsin. For example, AAT polypeptide (e.g., purified or recombinant AAT, such as human AAT) or its homologues, biologically active fragments, or other active variants thereof can be administered. Alpha 1 proteinase inhibitors are commercially available for the treatment of AAT deficiency, including ARALAST™, PROLASTIN™, and ZEMAIRA™. AAT polypeptide or its biologically active fragments or variants can be of human origin and can be purified from human tissue or plasma. Alternatively, it can be recombinantly produced. For ease of reading, the phrase "or its biologically active fragments or variants" is not repeated after each reference to AAT. It should be understood that whenever full-length naturally occurring AAT can be used, its biologically active fragments or other biologically active variants (e.g., variants with one or more amino acid residues substituted) can also be used.Similarly, the inventors do not repeat each time that naturally occurring polypeptides (e.g., AAT) can be purified from natural sources or can be recombinantly produced. It should be understood that both forms can be useful. Similarly, the inventors do not repeat each time that polypeptides can be of human or non-human origin. Although there may be advantages in administering human proteins, the present invention is not so limited.

[0036] The method of the present disclosure (e.g., multiple-variable doses of IL-2 alone or in combination with one or more other anti-immunopathic therapies) can be administered to the desired subject, or can be administered if the subject is shown to be a responder to such therapy. In another embodiment, the therapeutic method of the present invention can be avoided if the subject is shown to be a non-responder to this therapy, and an alternative therapeutic regimen, such as targeted and / or non-targeted anti-immunotherapy, can be administered.

[0037] In one embodiment, a multiple variable IL-2 dose method of treating a subject suffering from ALS is provided, comprising: a) administering to the subject an induction regimen comprising continuously administering to the subject interleukin-2 (IL-2) at a dose that increases the subject's plasma IL-2 level and increases the subject's ratio of immunosuppressive T cells to conventional T lymphocytes (Tcons); and b) subsequently administering to the subject at least one maintenance regimen comprising continuously administering to the subject an IL-2 maintenance dose that is higher than the induction regimen dose and that i) further increases the subject's plasma IL-2 level and ii) further increases the ratio of immunosuppressive T cells to Tcons, thereby treating the subject. In one embodiment, the plasma IL-2 level provided by the induction regimen is depleted to a level lower than the previous peak plasma IL-2 level before the induction regimen. The IL-2 maintenance regimen, in some embodiments, can increase the subject's plasma IL-2 level above the peak plasma IL-2 level induced by the induction regimen. The term "multiple-variable IL-2 dose method" refers to a therapeutic intervention that includes two or more IL-2 administrations, where two or more IL-2 administrations use two or more IL-2 doses. Such methods are in contrast to "fixed" administration methods in which a fixed amount of IL-2 is administered in a scheduled manner, for example, daily. The term "induction regimen" refers to continuous administration of IL-2 at a dose that increases the subject's plasma IL-2 levels and increases the subject's immunosuppressive T cell:Tcons ratio. In some embodiments, the regimen is administered until a peak level of plasma IL-2 is achieved. The subject's plasma IL-2 levels and / or immunosuppressive T cell:Tcons ratio can be increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more compared to the baseline ratio before therapy was started.

[0038] Low-dose IL-2 may be utilized, with the term "low-dose IL-2" referring to a dosage range in which immunosuppressive T cells are preferentially enhanced relative to Tcons. In one embodiment, low-dose IL-2 refers to an IL-2 dose that is 50% or less of the "high-dose IL-2" dose used in anti-cancer immunotherapy (e.g., 18 million IU / m2 / day to 20 million IU / m2 / day or more). The upper limit of "low-dose IL-2" may be further limited by treatment adverse events such as fever, chills, asthenia, and fatigue. IL-2 is generally administered according to an amount measured in international units (IU), which is administered relative to body surface area (BSA) per given unit of time. BSA can be calculated by direct measurement or by any number of well-known methods (e.g., Dubois & Dubois formula) as described in the Examples. Generally, IL-2 is administered in terms of IU per m2 of BSA per day. Exemplary low dose IL-2 doses according to the methods of the present invention include any one of 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0×10 IU / m / day, including any value and / or range therebetween. For example, induction regimen doses can range between 0.3×10 IU / m / day and 3.0×10 IU / m / day, and any value or range therebetween.

[0039] Continuous administration may be utilized, the term "continuous administration" referring to administration of IL-2 at regular intervals without any intermittent breaks in between. Thus, no interruption of IL-2 occurs. For example, the induction dose can be administered daily (e.g., once or more per day) for at least 1-14 consecutive days or any range therebetween (e.g., at least 4-7 consecutive days). As described herein, longer acting IL-2 agents and / or IL-2 agents administered by routes other than subcutaneous administration are contemplated. Intermittent intravenous administration of IL-2 as described in the art results in a short IL-2 half-life that is incompatible with increased plasma IL-2 levels and increased immunosuppressive T cell:Tcons ratios according to the present invention. However, once daily subcutaneous IL-2 administration, continuous IV infusion, long acting subcutaneous IL-2 formulations, and the like are contemplated to achieve sustained steady state IL-2 levels.

[0040] As mentioned above, IL-2 can be administered in a pharma- ceutically acceptable formulation and by any suitable route of administration, e.g., subcutaneous, intravenous, intraperitoneal, oral, nasal, transdermal, or intramuscular. In one embodiment, the present invention provides a pharma- ceutically acceptable composition comprising IL-2 in a therapeutically effective amount, formulated with one or more pharma- ceutically acceptable carriers (additives) and / or diluents. The pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, e.g., by subcutaneous, intramuscular or intravenous injection, e.g., as a sterile solution or suspension; (3) topical application, e.g., as a cream, ointment or spray applied to the skin; (4) intravaginally or rectally, e.g., as a pessary, cream or foam; or (5) aerosol, e.g., as an aqueous aerosol, liposomal preparation, or solid particles containing the compound.

[0041] In some embodiments of the present disclosure, monoclonal antibodies (mAbs) against CD3 molecules are utilized for immunomodulation of ALS patients, for example, with or without IL-2 and / or with fibroblasts. This approach has been used previously to induce tolerance to autoimmunity in mouse models of type 1 diabetes. Treatment with anti-CD3 mAbs improved diabetes in NOD mice and prevented recurrent immune responses to transplanted syngeneic islets. This was achieved without the need for continuous immunosuppression and was sustained at a time when T cell numbers were not exhausted and were quantitatively normal. Another approach is to induce specific immunological unresponsiveness by administering autoantigens.

[0042] An example of how different CD3-targeting antibodies can induce different effects is seen in another study by Davis et al., who examined an IgM monoclonal antibody called 38.1, which differs from other anti-CD3 mAbs in that it is rapidly modulated from the cell surface in the absence of a secondary antibody. 38.1 induced an immediate increase in intracellular free calcium [Ca2+]i by highly purified T cells, but did not induce cell entry into the cell cycle in the absence of accessory cells (AC) or protein kinase C-activating phorbol ester. Treated T cells were severely inhibited in their ability to respond to the T cell-stimulating mitogen phytohemagglutanin. The inhibition of responsiveness could be overcome by culturing the cells with supplemental antigen-presenting cells or the cytokine IL-2. These studies demonstrate that a state of T cell unresponsiveness can be induced by modulating CD3 with anti-CD3 mAb in the absence of costimulatory signals. Brief increases in [Ca2+]i due to mobilization of internal calcium stores appear to be sufficient to induce this state of T cell unresponsiveness

[0117] .

[0043] In some circumstances, anti-CD3 antibodies have been shown to program T cells toward antigen-specific tolerance. This is shown in one example from the study by Anasetti et al., who exposed PBMCs to alloantigens for 3–8 days in the presence of anti-CD3 antibodies. They showed no response after restimulation with cells from the original donor, but the PBMCs were still able to respond to a third-party donor. Antigen-specific unresponsiveness was induced by both nonmitogenic and mitogenic anti-CD3 antibodies, but not by antibodies against CD2, CD4, CD5, CD8, CD18 or CD28. This suggested a unique ability of this protein to regulate a program in T cells that is antigen-specific. Unresponsiveness induced by anti-CD3 antibodies in mixed leukocyte cultures persisted for at least 34 days from the initiation of culture and for 26 days after removal of the antibodies. Anti-CD3 antibodies also induced antigen-specific unresponsiveness in a cytotoxic T cell generation assay. Anti-CD3 antibodies did not induce unresponsiveness in previously primed cells.

[0044] The use of anti-CD3 antibodies to practice embodiments of the present disclosure encompasses not only that the antibodies do not result in activation of T cell proliferation and inflammatory cytokine secretion, but that the T cells actually inhibit inflammation and promote regeneration.

[0045] In one embodiment of the present disclosure, the anti-CD3 antibody is given 14 days prior to administration of the mesenchymal stem cells. In one particular embodiment, the 14-day course of anti-CD3 monoclonal antibody utilizes the antibody hOKT3γ1 (Ala-Ala) administered intravenously (1.42 μg / kilogram of body weight on day 1; 5.67 μg / kilogram on day 2; 11.3 μg / kilogram on day 3; 22.6 μg / kilogram on day 4; and 45.4 μg / kilogram on days 5-14). These doses are based on doses previously used for the treatment of transplant rejection

[0119] , which are incorporated by reference. Other types of anti-CD3 molecules and dosing regimens may be used in the context of ALS treatment, and the doses may be selected from examples of the utility of anti-CD3 from the literature described in the following papers and incorporated by reference: prevention of kidney [120-128], liver [129-131], pancreas [132-134], lung

[0135] and heart [136-140] transplant rejection; prevention of graft-versus-host disease

[0141] , multiple sclerosis

[0142] , type 1 diabetes

[0143] ,

[0046] The use of monoclonal antibodies for the implementation of the present invention must be considered with the caveat that in some cases, a cytokine storm may be initiated by antibody administration [144, 145]. In some cases, this is concentration-dependent

[0146] . Treatment for this can be achieved by steroid administration or anti-IL6 antibodies [147-151].

[0047] In some embodiments of the present disclosure, administration of PGE1 and / or various natural anti-inflammatory compounds is provided to reduce TNF-α production as a result of anti-CD3 administration, as described in this article

[0152] , which is incorporated by reference. In further embodiments of the present invention, administration of anti-CD3 may be performed with an endothelial protective agent and / or an anticoagulant to reduce clotting associated with CD3 modulators

[0153] . In some embodiments, anti-CD3 antibodies may be used in combination with tolerogenic cytokines, such as interleukin-10, to enhance the number of angiogenesis-supporting T cells. The safety of anti-CD3 and IL-10 administration has previously been demonstrated in clinical trials

[0154] .

[0048] The phrase "pharmacologically acceptable" is used herein to refer to agents, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. The phrase "pharmacologically acceptable carrier" as used herein means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting the subject chemical from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that can function as pharma- ceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch, potato starch; (3) cellulose and its derivatives, e.g. sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatine; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; (10) glycols, e.g. (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide, aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) butyl phosphate solution; and (21) other non-toxic compatible substances used in pharmaceutical formulations. Formulations useful in the methods of the invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and / or parenteral administration. The formulations may be conveniently provided in unit dosage form and may be prepared by any method well known in the art of pharmacy.The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the host being treated, the particular method of administration. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form is generally the amount of compound that produces a therapeutic effect. Generally, out of 100%, this amount ranges from about 1% to about 99%, preferably about 5% to about 70%, most preferably about 10% to about 30% of active ingredient.

[0049] In one embodiment, the Treg cell surface protein is selected from the group consisting of CD25, GITR, TIGIT, CTLA-4, neuropilin, OX40, LAG3, and combinations thereof, and said Tregs are isolated from a tissue source bearing said surface protein and optionally expanded ex vivo before administration to a patient suffering from ALS.

[0050] In one embodiment of the present disclosure, the use of ex vivo manipulations is used to generate an environment suitable for the survival of regulatory T cells following administration from an exogenous source or to enhance the survival of endogenous regulatory T cells. Since the development of renal dialysis in the late 1940s by William Kolff, the extracorporeal removal of various physiological or pathological agents has been part of medical practice. Advanced means of extracorporeal removal of various substances have been demonstrated in the case of immune complex removal [156-159], antibodies [160-165], viruses [166-168], soluble receptors

[0169] , and even cells [170, 171]. These methodologies may be used to optimize the efficacy of the present invention to remove regulatory T cell inhibitory compounds such as TNF-α, interferon-γ, or interleukin-33, among others.

[0051] In some embodiments, HGF stimulatory factors are added to enhance the proliferation of regulatory T cells [172-175].

[0052] In one embodiment, the disclosure teaches the use of activation of fibroblasts prior to therapeutic use and / or administration of agents that act as "regenerative adjuvants" for said fibroblasts. The cells in the formulation exhibit typical fibroblast morphology when grown in culture monolayer. Specifically, the cells may assume an elongated spindle or spindle appearance with elongated extensions, or the cells may appear as larger, flattened stellate cells that may have a cytoplasmic leading edge. A mixture of these morphologies may also be observed. The cells express proteins characteristic of normal fibroblasts, including the fibroblast-specific marker CD90 (Thy-1), a 35 kDa cell surface glycoprotein, and collagen, an extracellular matrix protein. The fibroblast administration formulation is an autologous cell therapy product composed of a suspension of autologous fibroblasts grown from a biopsy of each individual's own skin using standard tissue culture procedures. In one embodiment, the fibroblasts of the invention can also be used to generate other cell types for tissue repair or regeneration.

[0053] The fibroblasts utilized in this disclosure are, in one embodiment, produced by explantation from a biopsy of the recipient's own skin (for autologous preparations) or a healthy donor's skin (for allogeneic preparations). In some embodiments, fibroblasts from a young donor are used. In another embodiment, the fibroblasts are transfected with genes that allow for enhanced growth and overcoming the Hayflick limit. Following induction of cell proliferation in culture using standard cell culture techniques. Skin tissue (dermal and epidermal layers) may be biopsied from the subject's post-auricular area. In one embodiment, the starting material is comprised of three 3 mm punch skin biopsies collected using standard aseptic practice. The biopsies are collected by the investigator and placed in vials containing sterile phosphate buffered saline (PBS). The biopsies are returned to the manufacturing facility in a refrigerated transporter at 2-8°C. In one embodiment, after arrival at the manufacturing facility, the biopsies are inspected and, if accepted, are transported directly to the manufacturing area. At the start of the process, the biopsy tissue is washed prior to enzymatic digestion. After washing, Liberase digestion enzyme solution is added without mincing and the biopsy tissue is incubated for 1 hour at 37.0±2°C. The time of biopsy tissue digestion is a critical process parameter that can affect the viability and growth rate of cells in culture. Liberase is a collagenase / neutral protease enzyme cocktail available formulated from Lonza Walkersville, Inc. (Walkersville, Md.) and unformulated from Roche Diagnostics Corp. (Indianapolis, Ind.). Alternatively, other commercially available collagenases such as Serva Collagenase NB6 (Helidelburg, Germany) may be used. After digestion, the enzyme is neutralized by the addition of Initiation Growth Media (IMDM, GA, 10% fetal bovine serum (FBS)), and the cells are pelleted by centrifugation and resuspended in 5.0 ml of Initiation Growth Media. Alternatively, no centrifugation is performed and complete inactivation of the enzyme occurs by the addition of Initiation Growth Media alone. For the initiation of cell growth and proliferation, add initial growth medium prior to seeding the cell suspension into a T-175 cell culture flask.T-75, T-150, T-185 or T-225 flasks can be used in place of the T-75 flasks. The cells are incubated at 37 ± 2.0 °C, 5.0 ± 1.0% CO2 and fed with fresh complete growth medium every 3-5 days. All feedings in this process are done by removing half of the complete growth medium and replacing the same volume with fresh medium. Alternatively, full feedings can be performed. Cells should not remain in the T-175 flask for more than 30 days before passaging. Confluence is monitored throughout the process to ensure proper seeding density during culture splitting. When cell confluence is 40% or greater in the T-175 flask, they are passaged by removing the spent medium, washing the cells and treating them with trypsin-EDTA to release the adherent cells in the flask into solution. The cells are then trypsinized and seeded into T-500 flasks for continued cell growth. Alternatively, one or two T-300 flasks, One Layer Cell Stacks (1 CS), One Layer Cell Factory (1 CF) or Two Layer Cell Stacks (2 CS) can be used instead of the T-500 flasks. Morphology is assessed before each passage and harvest to monitor culture purity throughout the entire process. Morphology is assessed by comparing observed samples to visual standards for morphological examination of cell cultures. Cells exhibit typical fibroblast morphology when grown in culture monolayers. Cells may appear as either elongated spindles or spindles with elongated extensions, or larger flattened stellate cells that may have a leading edge of cytoplasm. A mixture of these morphologies may also be observed. Fibroblasts in less confluent areas may be of similar shape but randomly oriented. The presence of keratinocytes in the cell cultures is also assessed. The keratinocytes appear rounded and irregularly shaped; at higher confluence, they appear organized like cobblestones. At lower confluence, the keratinocytes are observable in small colonies.Cells are incubated at 37 ± 2.0 °C, 5.0 ± 1.0% CO2 and passaged in T-500 flasks every 3-5 days and in 10-layer cell stacks (10CS) every 5-7 days. Cells should not remain in the T-500 flasks for more than 10 days before passaging. Quality control (QC) release tests for the safety of the Bulk Drug Substance include sterility and endotoxin tests. When cell confluence in the T-500 flask is 95% or higher, cells are passaged into 10CS culture vessels. Alternatively, two 5-layer cell stacks (5CS) or 10-layer cell factories (10CF) can be used instead of the 10CS. 10CS. Passaging into 10CS is performed by removing the spent medium, washing the cells, and treating with trypsin-EDTA to release the adherent cells in the flask into solution. The cells are then transferred to the 10CS. Additional complete growth medium is added to neutralize the trypsin, and the cells from the T-500 flask are pipetted into a 2L bottle containing fresh complete growth medium. The contents of the 2L bottle are transferred to a 10CS and seeded across all layers. The cells are then incubated at 37±2.0°C, 5.0±1.0% CO2, and fed fresh complete growth medium every 5-7 days. The cells should not remain in the 10CS for more than 20 days before passaging. In one embodiment, the passaged dermal fibroblasts are rendered substantially free of immunogenic proteins present in the medium by incubating the expanded fibroblasts in protein-free medium for a period of time, and the first harvest is when the cells are at 95% or greater cell confluence in the 10CS. Harvesting is performed by removing the spent medium, washing the cells, treating with trypsin-EDTA to release adherent cells into solution, and adding additional complete growth medium to neutralize the trypsin. Cells are harvested by centrifugation, resuspended, and in-process QC tests are performed to determine total viable cell count and cell viability.

[0054] Therapies provided herein may include administering therapeutic agents (e.g., fibroblasts, fibroblast-derived exosomes, etc.) alone or in combination. Therapeutic agents may be administered in any suitable manner known in the art. For example, the first and second treatments may be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second treatments are administered in separate compositions. In some embodiments, the first and second treatments are administered in the same composition. Embodiments of the present disclosure relate to compositions and methods that include therapeutic compositions. Different therapies may be administered in one composition or in multiple compositions, for example, two compositions, three compositions, or four compositions. Various combinations of agents may be used. The therapeutic agents of the present disclosure (e.g., fibroblasts) may be administered by the same or different routes of administration. In some embodiments, the therapeutic agents are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally. The appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0055] The treatment may include various "unit doses." A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered, as well as the particular route and formulation, are within the skill of those of ordinary skill in the clinical arts to determine. A unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. In some embodiments, a unit dose includes a single administrable dose.

[0056] The amount to be administered depends on the desired therapeutic effect, both depending on the number of treatments and the unit dose. It is understood that an effective dose refers to the amount required to achieve a particular effect. In practice, it is contemplated that in some embodiments, a dose ranging from 10 mg / kg to 200 mg / kg can affect the protective capacity of these agents. Thus, doses are contemplated to include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such doses can be administered multiple times over the course of a single day and / or multiple days, weeks, or months.

[0057] In some embodiments, between about 105 to about 1013 cells per 100 kg are administered to a human per injection. In some embodiments, between about 1.5x106 to about 1.5x1012 cells per 100 kg are injected. In some embodiments, between about 1x109 to about 5x1011 cells per 100 kg are injected. In some embodiments, between about 4x109 to about 2x1011 cells per 100 kg are injected. In some embodiments, between about 5x108 to about 1x1012 cells per 100 kg are injected. In some embodiments, a single administration of cells is provided. In some embodiments, multiple administrations are provided. In some embodiments, multiple administrations are provided over 3-7 consecutive days. In some embodiments, 3-7 administrations are provided over 3-7 consecutive days. In some embodiments, 5 administrations are provided over 5 consecutive days. In some embodiments, a single administration of between about 105 and about 1013 cells per 100 kg is provided. In some embodiments, a single administration of between about 1.5x108 and about 1.5x1012 cells per 100 kg is provided. In some embodiments, a single administration of between about 1x109 and about 5x1011 cells per 100 kg is provided. In some embodiments, a single administration of about 5x1010 cells per 100 kg is provided. In some embodiments, a single administration of 1x1010 cells per 100 kg is provided. In some embodiments, multiple administrations of between about 105 and about 1013 cells per 100 kg are provided. In some embodiments, multiple administrations of between about 1.5x108 and about 1.5x1012 cells per 100 kg are provided. In some embodiments, multiple administrations of between about 1x109 and about 5x1011 cells per 100 kg are provided over 3 to 7 consecutive days. In some embodiments, multiple doses of about 4×10 9 cells per 100 kg are provided over 3-7 consecutive days. In some embodiments, multiple doses of about 2×10 11 cells per 100 kg are provided over 3-7 consecutive days.In some embodiments, five doses of about 3.5×109 cells are provided over five consecutive days. In some embodiments, five doses of about 4×109 cells are provided over five consecutive days. In some embodiments, five doses of about 1.3×1011 cells are provided over five consecutive days. In some embodiments, five doses of about 2×1011 cells are provided over five consecutive days.

[0058] In one embodiment, fibroblasts are cultured using means known in the art to preserve fibroblast viability and proliferation capacity. The present invention can be applied to both individualized autologous exosome preparations and exosome preparations obtained from established cell lines for experimental or biological use. In one embodiment, the present invention is more specifically based on the use of chromatographic separation methods to prepare membrane vesicles, in particular to separate membrane vesicles from potential biological contaminants, said microvesicles being exosomes and the cells utilized to generate said exosomes being fibroblasts.

[0059] Indeed, the applicant has now demonstrated that membrane vesicles, in particular exosomes, can be purified and have the ability to inhibit pain. In one embodiment, strong or weak, preferably strong, anion exchange can be performed. Moreover, in a particular embodiment, the chromatography is performed under pressure. Thus, more specifically, it can consist of high performance liquid chromatography (HPLC). Different types of supports may be used to perform anion exchange chromatography. More preferably, these may include cellulose, poly(styrene-divinylbenzene), agarose, dextran, acrylamide, silica, ethylene glycol-methacrylate copolymer, or mixtures thereof, such as agarose-dextran mixtures. To illustrate this, different chromatographic devices composed of the above supports, in particular the following gels, can be mentioned: for example, POROS®, SEPHAROSE®, SEPHADEX®, TRISACRYL®, TSK-GEL SW OR PW®, SUPERDEX® and SEPHACRYL®, which are suitable for the application of the present invention. Thus, in certain embodiments, the present disclosure relates to a method for preparing membrane vesicles, in particular exosomes, from a biological sample, such as a tissue culture containing fibroblasts, comprising at least one step in which the biological sample is treated by anion exchange chromatography on a support, alone or in mixtures, selected from cellulose, poly(styrene-divinylbenzene), silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate copolymer, optionally functionalized.

[0060] Moreover, in order to improve the chromatographic resolution, it is preferred within the scope of the present invention to use supports in the form of beads. Ideally, these beads have a homogeneous and calibrated diameter with a porosity high enough to allow the penetration of the objects under chromatography (i.e. exosomes). In this way, taking into account the diameter of exosomes (generally 50 nm to 100 nm), it is preferred to use highly porous gels, in particular of 10 nm to 5 μm, more preferably about 20 nm to about 2 μm, even more preferably about 100 nm to about 1 μm, for the application of the present invention. In the case of anion exchange chromatography, the support used must be functionalized with a group capable of interacting with anionic molecules. Generally, this group is composed of amines, which may be tertiary or quaternary, defining respectively a weak or strong anion exchanger. It is particularly advantageous within the scope of the present invention to use strong anion exchangers. In this way, according to the present invention, the above-mentioned chromatographic supports functionalized with quaternary amines are used. Thus, according to a more specific embodiment of the present invention, the anion exchange chromatography is carried out on a support functionalized with a quaternary amine. Even more preferably, the support should be selected from poly(styrene-divinylbenzene), acrylamide, agarose, dextran and silica, alone or in mixture, and functionalized with a quaternary amine. Examples of supports functionalized with quaternary amines include gels SOURCEQ, MONO Q, Q SEPHAROSE®, POROS® HQ and POROS® QE, FRACTOGEL® TMAE type gels and TOYOPEARL SUPER® Q gels.

[0061] One support for carrying out anion exchange chromatography includes poly(styrene-divinylbenzene). An example of this type of gel that can be used within the scope of the present invention is SOURCE Q gel, in particular SOURCE 15 Q (Pharmacia). This support offers the advantage that the internal pores are very large, thus allowing rapid diffusion of the exosomes to the functional groups while providing a low resistance to the circulation of liquid through the gel, a parameter that is particularly important for exosomes, taking into account their size. The biological compounds retained in the column can be eluted in various ways, in particular using a gradient of increasing concentration, for example the passage of saline from 0 to 2 M. Sodium chloride solutions can in particular be used, varying in concentration, for example from 0 to 2 M. The different fractions thus purified are detected by measuring their optical density (OD) using continuous spectrophotometric readings at the column outlet. As a guide, under the conditions used in the examples, fractions containing membrane vesicles were eluted at an ionic strength comprised between about 350 and 700 mM, depending on the type of vesicle.

[0062] To carry out this chromatography step, different types of columns can be used according to the requirements and the volumes to be processed. For example, depending on the preparation, it is possible to use columns of about 100 μl up to 10 ml or more. In this way, the available supports have a capacity that can reach, for example, 25 mg of protein per ml. For this reason, a 100 μl column has a capacity of about 2.5 mg of protein, allowing the processing of about 2 l of culture supernatant (which represents, for example, a volume of 100-200 ml per preparation after concentration by a factor of 10-20), taking into account the sample in question. It will be understood that larger volumes can also be processed, for example by increasing the volume of the column. Furthermore, in order to carry out the invention, it is also possible to combine an anion exchange chromatography step with a gel permeation chromatography step. Thus, according to a particular embodiment of the invention, a gel permeation chromatography step is added to the anion exchange step, either before or after the anion exchange chromatography step. Preferably, in this embodiment, the permeation chromatography step is carried out after the anion exchange step. Moreover, in a particular variant, the anion exchange chromatography step is replaced by a gel permeation chromatography step. The present application also demonstrates that gel permeation liquid chromatography can be used to purify membrane vesicles, particularly when this step is combined with anion exchange chromatography or other processing steps of the biological sample, as described in detail below.

[0063] To carry out the gel permeation chromatography step, supports are preferably used that are selected from silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate copolymer or mixtures thereof, such as agarose-dextran mixtures. By way of example, supports such as SUPERDEX® 200HR (Pharmacia), TSK G6000 (TosoHaas), SEPHACRYL® S (Pharmacia) are preferably used for gel permeation chromatography. The method according to the invention can be applied to different biological samples. In particular, these can consist of biological fluids (bone marrow, peripheral blood, etc.), culture supernatants, cell lysates, pre-purification solutions or any other compositions including membrane vesicles from a subject.

[0064] In this regard, in a particular embodiment of the invention, the biological sample is a culture supernatant of membrane vesicle-producing fibroblasts.

[0065] Furthermore, according to a preferred embodiment of the present invention, the biological sample is treated prior to the chromatography step in such a way that the membrane vesicles are enriched (enrichment step).Thus, in a particular embodiment, the present invention relates to a method for preparing membrane vesicles from a biological sample, characterized in that it comprises at least b) a concentration step for preparing a sample enriched in membrane vesicles, and c) a step in which the sample is treated by anion exchange chromatography and / or gel permeation chromatography.

[0066] In one embodiment, the biological sample is the culture supernatant that is treated to enrich membrane vesicles.In particular, the biological sample can be composed of a pre-purified solution obtained from the culture supernatant of a population of membrane vesicle-producing cells or from a biological fluid by processing such as centrifugation, clarification, ultrafiltration, nanofiltration and / or affinity chromatography, in particular clarification and / or ultrafiltration and / or affinity chromatography.Thus, the preferred method of preparing membrane vesicles according to the present invention more specifically comprises the following steps: a) culturing a population of membrane vesicle-producing cells (e.g., exosomes) under conditions that allow the release of vesicles, b) concentrating the sample into membrane vesicles, and c) anion exchange chromatography and / or gel permeation chromatography processing of the sample.

[0067] As mentioned above, the sample (e.g., supernatant) concentration step may comprise one or more centrifugation, clarification, ultrafiltration, nanofiltration and / or affinity chromatography steps on the supernatant. In a first particular embodiment, the concentration step comprises (i) removal of cells and / or cell debris (clarification), optionally followed by (ii) concentration and / or affinity chromatography steps. In another particular embodiment, the concentration step comprises an affinity chromatography step, optionally preceded by a cell and / or cell debris removal (clarification) step. A preferred concentration step according to the invention comprises (i) removal of cells and / or cell debris (clarification), (ii) concentration and (iii) affinity chromatography. Cells and / or cell debris may be removed, for example, by centrifugation of the sample at low speed, preferably below 1000 g, for example between 100 and 700 g. Preferred centrifugation conditions during this step are, for example, about 300 g or 600 g for a period of 1 to 15 minutes.

[0068] Cells and / or cell debris can also be removed by filtration of the sample, possibly in combination with the above-mentioned centrifugation. Filtration can in particular be carried out by successive filtration using filters of decreasing porosity. For this purpose, filters with a porosity of more than 0.2 mM, for example between 0.2 and 10 mM, are preferentially used. It is particularly possible to use a series of filters with a porosity of 10 mM, 1 mM, 0.5 mM, followed by 0.22 mM.

[0069] A concentration step may also be carried out to reduce the volume of the sample processed during the chromatographic stage. In this way, the concentration can be obtained by centrifuging the sample at high speed, for example between 10,000 and 100,000 g, in order to cause the sedimentation of the membrane vesicles. This may consist of a series of fractional centrifugations, the last of which is carried out at about 70,000 g. The membrane vesicles in the resulting pellet may be taken up in a suitable buffer, in a smaller volume, for the subsequent steps of the method. The concentration step may also be carried out by ultrafiltration. In fact, this ultrafiltration allows both the concentration of the supernatant and the initial purification of the vesicles. According to a preferred embodiment, the biological sample (for example, the supernatant) is subjected to ultrafiltration, preferably tangential ultrafiltration. Tangential ultrafiltration consists in concentrating and fractionating the solution between two compartments (filtrate and retentate) separated by a membrane of a determined cut-off threshold. The separation is carried out by applying a flow in the retentate compartment and a transmembrane pressure between this compartment and the filtrate compartment. Ultrafiltration can be carried out using different systems such as spiral membranes (Millipore, Amicon), flat membranes or hollow fibers (Amicon, Millipore, Sartorius, Pall, GF, Sepracor).Within the scope of the present invention, it is advantageous to use membranes with a cut-off threshold of less than 1000 kDa, preferably between 300 kDa and 1000 kDa, or even more preferably between 300 kDa and 500 kDa.

[0070] The affinity chromatography step can be carried out in various ways, using different chromatographic supports and materials. It is advantageously a non-specific affinity chromatography that aims to retain (i.e. bind) certain contaminants present in the solution and not to retain the entities of interest (i.e. exosomes). It is therefore a negative selection. Preferably, dye affinity chromatography is used that allows the removal (i.e. retention) of contaminants such as proteins and enzymes, for example albumins, kinases, dehydrogenases, clotting factors, interferons, lipoproteins, or cofactors. More preferably, the support used in this chromatographic step is a support such as that used for ion exchange chromatography, functionalized with a dye. As a specific example, the dye may be selected from Blue SEPHAROSE® (Pharmacia), YELLOW 86, GREEN 5 and BROWN 10 (Sigma). The support is more preferably agarose. It is understood that any other support and / or dye or reactive group that allows the retention (binding) of contaminants from the treated biological sample can be used in the present disclosure.

[0071] In one embodiment, a membrane vesicle preparation method within the scope of the present disclosure comprises the following steps: a) culturing a population of membrane vesicle (e.g., exosome) producing cells under conditions allowing the release of vesicles, b) treating the culture supernatant with at least one ultrafiltration or affinity chromatography step to produce a biological sample enriched in membrane vesicles (e.g., exosomes), and c) treating said biological sample with anion exchange chromatography and / or gel permeation chromatography. In a preferred embodiment, said step b) comprises filtration of the culture supernatant followed by ultrafiltration, preferably tangential filtration. In another preferred embodiment, said step b) comprises clarification of the culture supernatant followed by dye affinity chromatography, preferably by Blue SEPHAROSE®.

[0072] Furthermore, after step c), the recovered material may be subjected to one or more additional treatment and / or filtration steps d), if applicable, in particular for sterilization purposes. In this filtration treatment step, preferentially filters with a diameter of 0.3 μm or less are used, or even more preferentially filters with a diameter of 0.25 μm or less are used. Such filters have, for example, a diameter of 0.22 μm. After step d), the material obtained is distributed in a suitable storage medium in a suitable device, for example a bottle, a tube, a bag, a syringe, etc. The purified vesicles thus obtained may be refrigerated, frozen or used immediately. Thus, a particular preparation method within the scope of the invention comprises at least the following steps: c) an anion exchange chromatography and / or gel permeation chromatography treatment of the biological sample, and d) a filtration step, in particular a sterile filtration, of the material recovered after step c). In a first variant, the method according to the invention comprises c) an anion exchange chromatography treatment of the biological sample, and d) a filtration step, in particular a sterile filtration, on the material recovered after step c).

[0073] In another embodiment, the method according to the invention comprises c) a gel permeation chromatography treatment of the biological sample, and d) a filtration step, in particular a sterile filtration, on the material recovered after step c).According to a third variant, the method according to the invention comprises c) an anion exchange chromatography treatment of the biological sample, followed or preceded by gel permeation chromatography, and d) a filtration step, in particular a sterile filtration, on the material recovered after step c).

[0074] Embodiments of the present disclosure include a method of inhibiting and / or treating amyotrophic lateral sclerosis (ALS), comprising administering a population of fibroblasts capable of inducing regenerative and / or immunomodulatory effects in a patient suffering from ALS. In some embodiments, the fibroblasts are allogeneic to the recipient, and in some embodiments, the fibroblasts are either autologous or xenogeneic to the recipient. In some cases, the fibroblasts are mitotically active prior to administration to the recipient in need of treatment. The fibroblasts may be isolated from a tissue selected from the group consisting of: a) skin; b) bone marrow; c) blood; d) mobilized peripheral blood; e) gums; f) tonsils; g) placenta; h) Wharton's jelly; i) hair follicles; j) fallopian tubes; k) liver; l) milk teeth; m) vas deferens; n) endometrium; o) menstrual blood; and p) omentum. ALS is associated with an elevation of one or more inflammatory cytokines, such as IL-1, IL-2, IL-6, IL-9, IL-11, IL-12, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-27, IL-33, HMGB-1, TNF-α, TNF-β, IFN-α, IFN-β, IFN-γ, compared to age-matched healthy controls. Fibroblasts may be administered with a concentration of interleukin-2 sufficient to selectively upregulate the activity and / or number of regulatory T cells. Interleukin-2 may be administered in the absence of fibroblasts. Any administration that includes interleukin-2 may also include rapamycin, N-acetylcysteine, and / or antibodies against CD3, including those that can enhance the proliferation and / or activity of regulatory T cells.

[0075] Any fibroblast cell used herein may include expression of CD73, CD70, CD105, CD16, CD55, CD37, interleukin-10 receptor and / or interferon gamma receptor. Fibroblast cells may include expression of CD73, and then be treated with interferon gamma and allowed to grow for at least one cell division, and then be administered. Any fibroblast cell and / or modified fibroblast cell and / or fibroblast exosome may be administered in a manner that can stimulate the generation of regulatory T cells. Regulatory T cells may express FoxP3, contain membrane-bound TGF-β, suppress the ability of T cells to proliferate in response to mitogens, and / or suppress the ability of immature dendritic cells to mature into differentiated dendritic cells. Dendritic cell maturation may be associated with upregulation of the expression of markers selected from the group consisting of a) HLA-II; b) CD40; c) CD80; and / or d) CD86. Maturation of dendritic cells may be associated with enhanced ability to activate proliferation of allogeneic T cells and / or enhanced ability to induce interferon gamma production from allogeneic T cells. Regulatory T cells can be activated by exposure to CD3 and / or CD28 and / or IL-10, and / or regulatory T cells can be activated by administration of immature dendritic cells. Immature dendritic cells can express PD-1L, can be maintained in an immature state by culture in low dose GM-CSF, can be maintained in an immature state by culture in human chorionic gonadotropin, can be maintained in an immature state by culture under hypoxia, and / or can be maintained in an immature state by inhibition of NF-κb activity. Inhibition of NF-κB activity may be achieved by administration of antisense molecules targeting NF-κB or molecules in the NF-κB pathway, administration of molecules capable of inducing RNA interference targeting NF-κB or molecules in the NF-κB pathway, gene editing means targeting NF-κB or molecules in the NF-κB pathway, and / or administration of decoy oligonucleotides capable of blocking NF-κB or molecules in the NF-κB pathway. Small molecule blockers of NF-κB activity include calagualin (a fern derivative),Conophylline (Ervatamia microphylla), Evodiamine (Evodiae fructus), Geldanamycin, Perillyl Alcohol, Protein-bound Polysaccharides from Basidiomycetes, Rocaglamide (Aglaia derivative), 15-Deoxy-Prostaglandin J(2), Lead, Anandamide, Artemisia vestita, Cobrotoxin, Dehydroascorbic Acid (Vitamin C), Herbimycin A, Isorhapontigenin, Manumycin A, Pomegranate Fruit Extract, Tetrandrine (Plant Alkaloid), Thienopyridine, Acetyl-Boswellic Acid, 1'-Acetoxychavicol Acetate (Languagensis galanga) galanga), Apigenin (plant flavonoid), Cardamomine, Diosgenin, Furonaphthoquinone, Guggulsterone, Falcarindol, Honokiol, Hypestoxide, Garcinone B, Kahweol, Kava (Piper methysticum) derivatives, Mangosteen (Garcinia mangostana), N-Acetylcysteine, Nitrosylcobalamin (vitamin B12 analogue), Picetannol, Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), Quercetin, Rosmarinic acid, Semecarpus anacardii anacardiu) extract, staurosporine, sulforaphane and phenyl isothiocyanate, theaflavin (black tea component), tilianin, tocotrienol, wedelolactone, withanolides, zerumbone, silibinin, betulinic acid, ursolic acid, monochloramine and glycine chloramine (NH2Cl), anethole, Baoganing, black raspberry extract (cyanidin 3-O-glucoside, cyanidin 3-O-(2(G)-xylosylrutinoside), cyanidin 3-O-rutinoside), buddleia saponin IV, cacospongionolide B, caragualin, carbon monoxide, cardamonin,Cycloepoxydone; 1-hydroxy-2-hydroxymethyl-3-pent-1-enylbenzene, decursin, dexanabinol, digitoxin, diterpenes, docosahexaenoic acid, extensively oxidized low density lipoprotein (ox-LDL), 4-hydroxynonenal (HNE), flavopiridol, [6]-gingerol; casparol, Glosogyne tenuifolia, phytic acid (inositol hexakisphosphate), pomegranate fruit extract, prostaglandin A1, 20(S)-protopanaxatriol (ginsenoside metabolite), lengiolon, rottlerin, saikosaponin-d, saline (low Na+ isotonic),

[0076] Regulatory T cells can be activated by incubation with mesenchymal stem cell exosomes. Regulatory T cells can be generated in vivo by exposure of T cells to activators of the interleukin-2 receptor and can induce proliferation and / or activation of CD4 CD25 T cells.

[0077] In some embodiments, the interleukin-2 receptor is activated by administration of IL-2, including aldesleukin, in certain embodiments, the IL-2, including aldesleukin, is administered daily for 1 to 16 weeks at a concentration of 0.3 x 106 to 3.0 x 106 IU of IL-2 per square meter of body surface area.

[0078] In certain embodiments, one or more immunomodulatory compounds, such as oxytocin, prolactin, IL-10 and / or IL-35, are co-administered to enhance the generation of regulatory T cells in vivo. EXAMPLES

[0079] The following examples are included to demonstrate certain non-limiting aspects of the present disclosure. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in implementing the disclosed subject matter. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the disclosed subject matter. Example 1

[0080] Individuals who are at risk of having ALS or have ALS, which is greater than the average person in the population, can be subjected to the methods and compositions of the present disclosure.Individuals may or may not have a genetic predisposition to ALS.Individuals may have relatives who have or have had ALS.Individuals may be subjected to one or more tests to determine that they have ALS or are at risk of having ALS, including by genetic testing and / or other analysis.

[0081] The individual may be administered a therapeutically effective amount of fibroblasts, modified fibroblasts and / or fibroblast exosomes, and in certain embodiments, also administered a therapeutically effective amount of IL-2. The amount of IL-2 is sufficient to cause the stimulation of regulatory T cells in the individual. The fibroblasts, modified fibroblasts and / or fibroblast exosomes may be administered simultaneously with, before and / or after IL-2 administration. One or more administrations to the individual may be administered over a defined period of time, or one or more administrations to the individual may be administered throughout the lifespan of the individual once initiated.

[0082] In certain embodiments, after administration of the therapy, the regulatory T cells in the individual may be stimulated to express FoxP3 and / or contain membrane-bound TGF-β. The regulatory T cells may suppress the ability of T cells to proliferate in response to mitogens. The regulatory T cells may suppress the ability of immature dendritic cells to mature into differentiated dendritic cells, and the maturation of said dendritic cells may be associated with upregulation of the expression of one or more markers selected from the group consisting of a) HLA-II; b) CD40; c) CD80; d) CD86; and e) combinations thereof. (References) 1. Peters, OM, M. Ghasemi, and RH Brown, Jr., Emerging mechanisms of molecular pathology in ALS. J Clin Invest, 2015. 125(5): p. 1767-79. 2. Ling, SC, M. Polymenidou, and DW Cleveland, Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron, 2013. 79(3): p. 416-38. 3. 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[0083] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described herein. As one skilled in the art will readily appreciate from this disclosure, any currently existing or hereafter developed process, machine, manufacture, composition of matter, means, method or step that performs substantially the same function or achieves substantially the same result as the corresponding embodiment described herein may be utilized in accordance with the present disclosure. Therefore, the appended claims are intended to include within their scope such processes, machines, manufactures, compositions of matter, means, methods or steps.

Claims

1. 1. A method of treating, preventing, or reducing the risk of having amyotrophic lateral sclerosis (ALS) in an individual, comprising administering to the individual a therapeutically effective amount of a population of fibroblasts, fibroblast exosomes, modified fibroblasts, IL-2, or a combination thereof.

2. The method of claim 1, further comprising administering to the individual an effective amount of rapamycin, N-acetylcysteine, an anti-CD3 antibody, or a combination thereof.

3. The method described in claim 1 or 2, wherein the fibroblasts are mitotically active prior to administration to a recipient in need of treatment.

4. A method described in any one of claims 1 to 3, wherein the ALS in the individual is accompanied by elevated inflammatory cytokines compared to age-matched healthy controls.

5. The inflammatory cytokine is IL-1, IL-2, IL-6, IL-9, IL-11, IL-12, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, 5. The method according to claim 4, which is IL-22, IL-23, IL-27, IL-33, HMGB-1, TNF-α, TNF-β, IFN-α, IFN-β and / or IFN-γ.

6. A method according to any one of claims 1 to 5, wherein the fibroblasts are selected for expression of CD73, CD70, CD105, CD16, CD55, CD37, interleukin-10 receptor and / or interferon gamma receptor.

7. A method described in any one of claims 1 to 6, wherein the fibroblasts are selected for expression of CD73, then treated with interferon gamma and allowed to grow for at least one cell division before administration.

8. A method described in any one of claims 1 to 7, wherein the fibroblasts and / or modified fibroblasts are administered in a manner capable of stimulating the production of regulatory T cells.

9. The method described in claim 8, wherein the regulatory T cells express FoxP3.

10. The method described in claim 8 or 9, wherein the regulatory T cells contain membrane-bound TGF-β.

11. A method described in any one of claims 8 to 10, wherein the regulatory T cells suppress the ability of T cells to proliferate in response to a mitogen.

12. A method described in any one of claims 8 to 11, wherein the regulatory T cells suppress the ability of immature dendritic cells to mature into differentiated dendritic cells.

13. The method of claim 12, wherein the maturation of the dendritic cells is associated with upregulation of expression of one or more markers selected from the group consisting of: a) HLA-II; b) CD40; c) CD80; d) CD86; and e) combinations thereof.

14. The method described in claim 12 or 13, wherein maturation of the dendritic cells is associated with an enhanced ability to activate proliferation of allogeneic T cells.

15. A method described in any one of claims 12 to 14, wherein maturation of the dendritic cells is associated with an enhanced ability to induce interferon-γ production from allogeneic T cells.

16. A method described in any one of claims 8 to 15, wherein the regulatory T cells are activated by exposure to CD3 and CD28.

17. A method described in any one of claims 8 to 16, wherein the regulatory T cells are activated by exposure to interleukin-10.

18. A method described in any one of claims 8 to 17, wherein the regulatory T cells are activated by administration of immature dendritic cells.

19. The method described in claim 18, wherein the immature dendritic cells express PD-1L.

20. The method of claim 18 or 19, wherein the immature dendritic cells are maintained in an immature state by culturing in low-dose GM-CSF.

21. A method according to any one of claims 18 to 20, wherein the immature dendritic cells are maintained in an immature state by culturing in human chorionic gonadotropin.

22. The method of claim 21, wherein the immature dendritic cells are maintained in an immature state by culturing under hypoxia.

23. The method of claim 21 or 22, wherein the immature dendritic cells are maintained in an immature state by inhibiting NF-κb activity.

24. The method of claim 23, wherein the inhibition of NF-κB activity is achieved by administration of an antisense molecule that targets NF-κB or a molecule within the NF-κB pathway.

25. The method described in claim 23 or 24, wherein the inhibition of NF-κB activity is achieved by administering a molecule capable of inducing RNA interference targeting NF-κB or a molecule within the NF-κB pathway.

26. A method according to any one of claims 23 to 25, wherein the inhibition of NF-κB activity is achieved by gene editing means targeting NF-κB or a molecule within the NF-κB pathway.

27. ​​A method described in any one of claims 23 to 26, wherein the inhibition of NF-κB activity is achieved by administration of a decoy oligonucleotide capable of blocking NF-κB or a molecule within the NF-κB pathway.

28. The method of any one of claims 23 to 27, wherein the inhibition of NF-κB activity is achieved by administration of a small molecule blocker of NF-κB activity.

29. The method of claim 29, wherein the small molecule blocker of NF-κB activity is selected from the group consisting of caragualin (fern derivative), conophylline (Ervatamia microphylla), evodiamine (Evodiae fructus component), geldanamycin, perillyl alcohol, protein-bound polysaccharides from basidiomycetes, rocaglamide (Aglaia derivative), 15-deoxy-prostaglandin J(2), lead, anandamide, Artemisia vesiculata vestita), Cobrotoxin, Dehydroascorbic Acid (Vitamin C), Herbimycin A, Isorhapontigenin, Manumycin A, Pomegranate Fruit Extract, Tetrandrine (Plant Alkaloid), Thienopyridine, Acetyl-Boswellic Acid, 1'-Acetoxychavicol Acetate (Languas galanga), Apigenin (Plant Flavonoid), Cardamomine, Diosgenin, Furonaphthoquinone, Guggulsterone, Falcarindol, Honokiol, Hypestoxide, Garcinone B, Kahweol, Kava (Piper methysticum) Derivatives, Mangosteen (Garcinia mangostana) mangostana), N-acetylcysteine, nitrosylcobalamin (vitamin B12 analogue), piceatannol, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), quercetin, rosmarinic acid, Semecarpus anacardiu extract, staurosporine, sulforaphane and phenyl isothiocyanate, theaflavin (a component of black tea), tilianin, tocotrienol, wedelolactone, withanolides, zerumbone, silibinin, betulinic acid, ursolic acid, monochloramine and glycine chloramine (NH2Cl), anethole, Baoganing, black raspberry extract (cyanidin 3-O-glucoside, cyanidin 3-O-(2(G)-xylosylrutinoside), cyanidin 3-O-rutinoside ), Buddleia saponin IV, chacospongionolide B, caragualin, carbon monoxide, cardamonin, cycloepoxydone; 1-hydroxy-2-hydroxymethyl-3-pent-1-enylbenzene, decursin, dexanabinol, digitoxin, diterpenes, docosahexaenoic acid, extensively oxidized low-density lipoprotein (ox-LDL), 4-hydroxynonenal (HNE), flavopiridol, [6]-gingerol; casparol, Glossogyn tenuifolia (Glossogyn tenuifolia), phytic acid (inositol hexakisphosphate), pomegranate fruit extract, prostaglandin A1, 20(S)-protopanaxatriol (ginsenoside metabolite), lengiolon, rottlerin, saikosaponin-d, and physiological saline (low Na+ isotonic).

30. A method according to any one of claims 8 to 29, wherein regulatory T cells are activated by incubation with mesenchymal stem cell exosomes.

31. The method of any one of claims 8 to 30, wherein the regulatory T cells are generated in vivo by exposure of T cells to an activator of the interleukin-2 receptor and are capable of inducing proliferation and / or activation of CD4 CD25 T cells.

32. A method according to any one of claims 1 to 31, wherein the interleukin-2 receptor is activated by administration of IL-2.

33. The method of any one of claims 1 to 32, wherein the IL-2 is administered daily for 1 to 16 weeks at a concentration of 0.3 x 10 6 to 3.0 x 10 6 IU of IL-2 per square meter of body surface area.

34. A method described in any one of claims 1 to 33, further comprising administering one or more immunomodulatory compounds.

35. The method of claim 34, wherein the compound is oxytocin, prolactin, IL-10, IL-35, a CD3 inhibitor, or a combination thereof.

36. The method described in claim 35, wherein the CD3 inhibitor is an anti-CD3 antibody.

37. The method of claim 36, wherein the anti-CD3 antibody is teplizumab.

38. A method described in any one of claims 1 to 37, wherein the individual has familial ALS.

39. A method described in any one of claims 1 to 37, wherein the individual has idiopathic ALS.

40. A method described in any one of claims 1 to 39, wherein the individual has one or more mutations in the C9ORF72 gene.

41. A method described in any one of claims 1 to 40, further comprising administering riluzole to the individual.