Exosome composition containing ganoderma immunomodulatory protein and uses thereof

Abstract

To provide an engineered exosome composition, and uses thereof.SOLUTION: The present disclosure provides an exosome composition comprising a population of enriched exosomes of a fungus containing Ganoderma immunomodulatory protein, a recombinant thereof or a fragment thereof, or a mixture derived from the population of enriched exosomes. The present disclosure also provides a method for treating and / or preventing an oxidative stress related disease in a subject in need thereof, a method for treating and / or preventing inflammation in a subject, and a method for treating and / or preventing a neurological disorder in a subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] Priority information This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 517,206, filed August 2, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in xml format and is hereby incorporated by reference in its entirety.

[0003] FIELD OF THEINVENTION The present disclosure relates to engineered exosome compositions and uses thereof. [Background technology]

[0004] Exosomes are membrane-bound extracellular vesicles (EVs) produced within the endosomal compartment of most eukaryotic cells. Exosomes generally have a size ranging from about 40 nm to 160 nm in diameter (average about 100 nm) of endosomal origin. Their surface consists of a lipid bilayer derived from the plasma membrane of the donor cell, contains cytoplasm from the cell that produced the exosome, and displays membrane proteins on their surface derived from the parent cell. Exosomes were initially thought to be a mechanism for removing unwanted proteins.

[0005] There remains a need to expand the application of exosomes. Summary of the Invention

[0006] The present disclosure provides novel exosome compositions containing enriched populations of engineered fungal, particularly yeast, exosomes.

[0007] Thus, the present disclosure provides an exosome composition comprising a population of enriched fungal exosomes comprising a Ganoderma lucidum immunomodulatory protein, a recombinant form thereof, or a fragment thereof, or a mixture derived from the enriched exosome population.

[0008] In some embodiments, the Ganoderma immunomodulatory protein is from Ganoderma lucidum, Ganoderma tsugae, Ganoderma microsporum, or Ganoderma sinensis. In some further embodiments, the Ganoderma immunomodulatory protein is LZ-8 from Ganoderma lucidum, FIP-gts from Ganoderma tsugae, GMI from Ganoderma microsporum, or FIP-gja from Ganoderma sinensis.

[0009] In some embodiments, the Ganoderma lucidum immunomodulatory protein comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the recombinant Ganoderma lucidum immunomodulatory protein comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the fragment of the Ganoderma lucidum immunomodulatory protein comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2.

[0010] In some embodiments, the fungus belongs to the Saccharomycetaceae family or the Schizosaccharomycetaceae family. Examples of Saccharomycetaceae include, but are not limited to, the genera Kluyveromyces, Sibelindonella, Debaryomyces, Pichia, Saccharomyces, and Casatutania. In another aspect, examples of Schizosaccharomycetaceae include, but are not limited to, the genus Schizosaccharomyces. Examples of yeast include, but are not limited to, Kluyveromyces marxianus (also known as Saccharomyces fragilis, Kluyveromyces fragilis), Kluyveromyces lactis (also known as Saccharomyces lactis), Cyberlindnera jadinii (also known as Candida utilis, Pichia jadinii), Debaryomyces hansenii, Pichia pastoris, Pichia fermentans, and the like. fermentans, Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces florentinus, Kazachstania unispora (also known as Saccharomyces unisporus), Kazachstania turicensis (also known as Saccharomyces florentinus), or Schizosaccharomyces pombe.In particular, in some embodiments of the present disclosure, the yeast belongs to Kluyveromyces lactis (also known as Saccharomyces lactis), Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, or Aspergillus niger.

[0011] In some embodiments of the present disclosure, the concentration of concentrated exosomes in the exosome composition is about 1×10 10 exosomes / mL, approximately 5 x 10 10 exosomes / mL, approximately 1 x 10 11 exosomes / mL, approximately 2 x 10 11 exosomes / mL, approximately 4 x 10 11 exosomes / mL, approximately 6 x 10 11 exosomes / mL, approximately 8 x 10 11 exosomes / mL, approximately 1 x 10 12 exosomes / mL, approximately 2 x 10 12 exosomes / mL, approximately 3 x 10 12 exosomes / mL, approximately 4 x 10 12 exosomes / mL, approximately 5 x 10 12 exosomes / mL, approximately 6 x 10 12 exosomes / mL, approximately 7 x 10 12 exosomes / mL, approximately 8 x 10 12 exosomes / mL, approximately 9 x 10 12 exosomes / mL, or approximately 1 x 10 13 In some further embodiments, the concentration of concentrated exosomes in the exosome composition is greater than about 1×10 12 exosomes / mL ~ 2 x 10 12 exosomes / mL.

[0012] In some embodiments of the present disclosure, the exosome composition contains a population of enriched exosomes having an average particle size of about 150 nm to about 250 nm, about 155 nm to about 245 nm, about 160 nm to about 240 nm, about 165 nm to about 235 nm, about 170 nm to about 230 nm, about 175 nm to about 225 nm, about 180 nm to about 220 nm, about 185 nm to about 215 nm, about 190 nm to about 210 nm, about 195 nm to about 205 nm, or about 200 nm.

[0013] In some embodiments of the present disclosure, the fungus is engineered with a nucleotide sequence encoding a Ganoderma lucidum immunomodulatory protein, a recombinant thereof, or a fragment thereof.

[0014] In some embodiments of the present disclosure, the fungus is cultured in a conditioned medium, an example of which includes, but is not limited to, BMGY medium.

[0015] In some embodiments of the present disclosure, the exosome composition is obtained by filtration. Examples of filtration include, but are not limited to, microfiltration, ultrafiltration, and nanofiltration. In particular, the exosome composition is obtained by performing at least one tangential flow filtration (TFF) step. Examples of TFF include, but are not limited to, performing at least one TFF step using a TFF membrane having a pore size of about 0.8 μm to about 8 μm, or performing at least one TFF step using a TFF membrane having a molecular weight cutoff of about 10 kDa to about 100 kDa. In some embodiments of the present disclosure, the exosome composition is obtained by performing deep filtration using a membrane having a pore size of about 0.22 μm to about 0.45 μm. In some further embodiments, the exosome composition is obtained by performing TFF using a TFF membrane having a pore size of about 0.8 μm to about 8 μm, performing TFF using a TFF membrane having a molecular weight cut off of about 10 kDa to about 100 kDa, and performing depth filtration using a membrane having a pore size of about 0.22 μm to about 0.45 μm.

[0016] In another aspect, the present disclosure provides a method for producing a population of exosomes disclosed herein, comprising culturing a fungus and obtaining a population of enriched exosomes.

[0017] In another aspect, the present disclosure provides a method of treating and / or preventing an oxidative stress associated disease in a subject in need thereof, comprising administering to the subject an exosome composition disclosed herein.

[0018] In another aspect, the present disclosure provides a method of treating and / or preventing inflammation in a subject in need thereof comprising administering to the subject an exosome composition disclosed herein.

[0019] In another aspect, the present disclosure provides a method of treating and / or preventing a neurological disease in a subject in need thereof comprising administering to the subject an exosome composition disclosed herein.

[0020] Examples of neurological diseases include, but are not limited to, neurodegenerative diseases, diseases that have a primary effect outside the nervous system, conditions of the nervous system that arise secondary to a condition or treatment, damage to the nervous system caused by trauma, memory loss, or psychiatric disorders. In particular, examples of neurological diseases include, but are not limited to, peripheral nerve damage caused by physical injury, ischemia, prolonged exposure to cold, damage to the central nervous system, dementia, Alzheimer's disease, Huntington's disease, or Parkinson's disease.

[0021] The present disclosure is described in detail in the following sections. Other features, objects, and advantages of the present disclosure can be found in the detailed description and claims. [Brief description of the drawings]

[0022] [Figure 1]Figure 1 shows that PCR analysis confirms the presence of the gmi gene in the genome of strain mm106. A 339 bp fragment amplified by PCR using GMI-f / GMI-r primers was detected in lanes V and E. [Diagram 2] Figure 2 shows PCR analysis for yeast species identification. The amplicon is an identical approximately 300 bp fragment from both mm166 and the host strain. [Diagram 3] FIG. 3 shows representative histograms demonstrating particle concentration and size distribution of isolated engineered exosomes as measured by nanoparticle tracking analysis (NTA). [Figure 4] Figure 4 shows representative cryo-EM images of engineered exosomes. Scale bar: 100 nm. [Diagram 5] Figure 5 shows SDS-PAGE analysis of engineered exosomes subjected to different filtration processes visualized by Coomassie Brilliant Blue (CBR) staining. Lane M: protein marker with molecular weights indicated in kDa. Lanes 1-5: standard GMI protein at various concentrations (0.2, 0.3, 0.5, 0.6, and 0.8 mg / mL) used as protein concentration controls. Lanes 6-8: supernatant from engineered Pichia pastoris mm106 fermentations after removal of cells via centrifugation loaded at different dilution factors (3x, 4x, and 5x). Lanes 9-11: fermentations after microfiltration (MF) for cell clarification loaded at different dilution factors (2x, 3x, and 4x). Lanes 12-14: Fermentations loaded at different dilution factors (35×, 40×, and 45×) that were further processed by ultrafiltration / nanofiltration (UF / NF) for engineered exome isolation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] It is understood that this invention is not limited to the particular materials and methods described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to limit the scope of the invention, which is limited only by the claims.

[0024] It must be noted that as used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0025] As used herein, "about" is understood by those of ordinary skill in the art and can vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which they are used, "about" will mean ±10% of the particular term.

[0026] As used herein, the term "exosome" refers to small membrane extracellular vesicles with a diameter of about 30-300 nm that are secreted from producing cells into the extracellular environment. The surface of the exosome comprises a lipid bilayer derived from the membrane of the donor cell, and the lumen of the exosome is topologically identical to the cytoplasm derived from the cell that produces the exosome. Exosomes contain proteins, RNA, lipids, and carbohydrates of the producing cell, although some may be modified or added to the exosome after release from the cell, either through natural processes or by experimental manipulation.

[0027] The term "genetically engineered" or "genetic engineering" with respect to cells refers to the manipulation of genes using genetic material to alter gene copies and / or gene expression levels in the cell. The genetic material can be in the form of DNA or RNA. The genetic material can be delivered into the cell by a variety of means including viral transduction and non-viral transfection. After being genetically engineered, the expression levels of specific genes in the cell can be altered permanently or temporarily.

[0028] As used herein, the terms "treatment", "treating" and the like encompass any treatment of a disease in a mammal, particularly a human, and include (a) preventing the onset of the disease in a subject prone to, but not yet diagnosed as, the disease, (b) inhibiting the disease, i.e., arresting its progression, and (c) relieving the disease, i.e., causing regression of the disease.

[0029] The terms "prevent" or "prophylaxis" are art-recognized and when used in reference to a condition include administering an agent prior to the onset of the condition, which reduces the frequency or severity, or delays the onset of symptoms of a medical condition in a subject, as compared to a subject who has not been administered the agent.

[0030] As used interchangeably herein, the terms "individual," "subject," "host," and "patient" refer to mammals, including but not limited to murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), etc. In particular, the subject is to be vaccinated.

[0031] As used herein, the term "in need of treatment" refers to a judgment made by a medical professional (e.g., in the case of a human, a physician, nurse, medical nurse practitioner, or individual; in the case of an animal, including a non-human animal, a veterinarian) that a subject is in need of treatment or would benefit from treatment. This judgment is within the medical professional's area of ​​expertise, but is made based on a variety of factors, including knowledge that the subject is ill or will become ill as a result of a disease treatable by the compounds of the present disclosure.

[0032] As used herein, the term "neuron" includes neurons and parts thereof, such as the neuronal cell body, axon, or dendrites. The term "neuron" refers to a cell of the nervous system that includes a central cell body or soma and two types of extensions or projections: dendrites, by which the majority of neuronal signals are generally carried to the cell body, and axons, by which the majority of neuronal signals are generally carried from the cell body to effector cells, such as target neurons or muscles.

[0033] As used herein, "neurological disorder" refers to physiological dysfunction or cell death of neurons present in the central or peripheral nervous system or caused by glial cell dysfunction. A non-limiting list of such disorders includes multiple sclerosis, sciatic nerve deficit, brain or spinal cord injury, dementia, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, prion disease, neuropathy, and motor neuron disorder. "Neuropathy" is characterized by neuronal cell death of motor or sensory neurons, and thus neuropathies can be subdivided into motor and sensory neuron disorders.

[0034] The present disclosure provides novel exome compositions containing a population of enriched exomes from Ganoderma lucidum fungi in which immunomodulatory proteins have been engineered.

[0035] Thus, the present disclosure provides exosome compositions containing a population of enriched fungal exosomes including Ganoderma lucidum immunomodulatory proteins, recombinant forms thereof or fragments thereof, or mixtures derived from enriched exosome populations.

[0036] In one embodiment, the immunomodulatory protein or a recombinant thereof is derived from Ganoderma lucidum, Ganoderma tsugae, Ganoderma microsporum, or Ganoderma sinensis. Preferably, the immunomodulatory protein is LZ-8 from Ganoderma lucidum, FIP-gts from Ganoderma tsugae, GMI from Ganoderma microsporum, or FIP-gja from Ganoderma sinensis, or a recombinant thereof.

[0037] In one embodiment, the immunomodulatory protein is derived from Ganoderma microsporum (GMI) or a recombinant form thereof (reGMI). More preferably, the immunomodulatory protein (GMI or reGMI) has the amino acid sequence: (1)-Leu-Ala-Trp-Asn-Val-Lys-(LAWNVK; SEQ ID NO: 1) and (2)-Asp-Leu-Gly-Val-Arg-Pro-Ser-Tyr-Ala-Val-(DLGVRPSYAV; SEQ ID NO: 2), the amino acid sequence: MSDTALIFTLAWNVKQLAFDYTPNWGRGRPSSFIDTVTFPTVLTDKAYTYRVVVSGKDLGVRP SYAVESDGSQKINFLEYNSGYGIADTNTIQVYVIDPDTGNNFIVAQWN (SEQ ID NO: 3) or the amino acid sequence: EAEAEFMSDTALIFTLAWNVKQLAFDYTPNWGRGRPSSFIDTVTFPTVLTDKAYTYRVVVSGKDLGVRPSYAVESDGSQKINFLEYNSGYGIADTNTIQVYVIDPDTGNNFIVAQWNYLEQKLISEEDLNSAVDHHHHHH (SEQ ID NO: 4).

[0038] The choice of host cell is an important consideration in exosome production, as different cell types have different exosome production capacities. In some embodiments, the fungus is listed in Table 1.

[0039] [Table 1]

[0040] In particular, in some embodiments of the present disclosure, the fungus belongs to the genus Kluyveromyces lactis (also known as Saccharomyces lactis), Pichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe.

[0041] P. pastoris has a well-documented safety profile across the pharmaceutical, food, and cosmetic industries and has been widely used for recombinant protein production since the mid-1980s. Ciofalo et al. (Ciofalo et al., 2006. Safety evaluation of lipase enzyme preparation, expressed in Pichia pastoris, intended for use in the degumming of edible vegetable oil. Regul Toxicol Pharmacol. 45(1):1-8) conducted a comprehensive safety evaluation of lipase enzyme preparations expressed in P. pastoris, further validating the reliability of the strain for industrial applications. Furthermore, in 1993, the U.S. Food and Drug Administration (FDA) approved the use of P. pastoris dried yeast as a food additive in animal feed and drinking water, strengthening the regulatory safety profile.

[0042] In some embodiments, engineered fungal cells are adapted for exosome production. These eukaryotic species are selected for their high yield and stability of protein production. The safety of using these eukaryotic species is well established. Without wishing to be limited by theory, the use of eukaryotic cells provides a potentially safer alternative for exosome production, as opposed to mammalian cells, which share the potential for zoonotic infection when considering the use of exosomes for human therapy. In some embodiments, appropriate safety procedures are taken in the manipulation and manufacture of these cells to minimize potential risks.

[0043] Host yeast and fungal cell lines are screened for endogenous exosome production using standard protocols for exosome enrichment and characterization. In some embodiments, briefly, at scales of less than 1 mL, exosomes are isolated using centrifugation and size exclusion filtration, while for scales of less than 100 liters, filtration methods are applied. Genetic (target-specific) engineering of the host cell line is achieved by modified homologous recombination using linear DNA fragments containing homologous regions, desired modifications, and selection markers. In engineering host cells for exosome production, all of the parameters are considered, including transformation strategy / efficiency, genetic engineering target, and selection or screening strategy.

[0044] In some embodiments, the fungus is genetically engineered with nucleotides encoding Ganoderma lucidum immunomodulatory proteins, recombinants thereof, or fragments thereof. The transformation strategy depends on the number of samples to be manipulated in a single process and the type of host cell. Both physical and chemical methods for the genetic transformation of fungal cells have been established. In some embodiments, transformation involves physical methods, electroporation, instead of chemical methods, due to its transformation efficiency and ability to optimize conditions.

[0045] The engineered target (cargo load) depends on the desired application of the exosomes. For example, exosomes can be engineered to load bioactive proteins such as antibodies, cytokines, or growth factors - any desired coding sequence. Target proteins are selected based on their function and application.

[0046] A selection or screening strategy is used to select cells with the highest levels of exosome expression. In some embodiments, antibiotic / auxotrophic selection of cells with stable integration of the genetic material is applied. Antibiotic / auxotrophic selection is used to identify successfully engineered cells. In some embodiments, engineered hosts and corresponding exosomes are specifically characterized for expression of the target molecule using quantitative polymerase chain reaction (qPCR) or Western blotting. Purity, size and yield of engineered exosomes are assessed using NTA. Assays to assess cargo loading of engineered exosomes, such as assessing protein content, are also used.

[0047] Overall, the transfection strategy, genetic engineering target, and selection or screening strategy are optimized depending on the particular application of the engineered exosomes. By carefully selecting the target molecule and optimizing the transformation and selection strategy, exosomes with high expression and activity of the desired protein molecule are engineered.

[0048] In some embodiments, the culture conditions for fungal cells are important to produce a high yield of recombinant protein and corresponding exosomes. Culture media typically contain a carbon source, a nitrogen source, trace elements, vitamins, and other components. The selection of these components affects the yield and quality of the final product.

[0049] A carbon source is an essential component of the culture, with glucose being the most commonly used, although other carbon sources such as maltose, galactose, fructose, and glycerol are also applicable.

[0050] Cells require nitrogen for the synthesis of amino acids, which are the building blocks of proteins. In some embodiments, ammonium sulfate is the most commonly used nitrogen source in the culture. Other sources, such as yeast extract and peptones, are also applicable.

[0051] In some embodiments, the culture medium contains trace elements. Examples of trace elements include, but are not limited to, iron, zinc, magnesium, and other elements essential for cell growth. In some embodiments, the trace elements are in the form of inorganic salts. In some embodiments, the concentrations and ratios of these trace elements and vitamins such as biotin and pantothenic acid are optimized.

[0052] In some embodiments, the culture medium further comprises other additives such as buffers, pH adjusters (acid / base), and antibiotics to prevent contamination. In some embodiments, the selection and concentrations of these components are optimized.

[0053] In some embodiments, other environmental factors are also optimized, including temperature, aeration, and dissolved oxygen levels. The optimal pH for cell growth and end product quality vary depending on the strain and genetic engineering target. In some embodiments, temperature is maintained at a level that promotes cell growth while allowing optimal protein folding and stability along with exosome integrity. In some embodiments, aeration is adjusted to affect oxygen availability and cell growth rate.

[0054] In some embodiments, chemically defined or animal-origin free (AOF) media containing well-defined amounts of each component were used to ensure reproducibility and consistency of product quality. Fed-batch cultivation strategies, in which nutrients are added continuously or intermittently, were also applied to further improve yields.

[0055] Without wishing to be bound by theory, it is believed that optimization of fungal cell culture conditions involves careful adjustment of components in the medium, pH, temperature, aeration, and other factors that may affect the yield, quality, and stability of recombinant proteins and exosomes.

[0056] In some embodiments, a physical-based method, specifically tangential flow filtration (TFF), is used for exosome isolation to ensure scale-up capability. In some embodiments, engineered exosomes in engineered cell culture medium are isolated using a three-stage TFF process followed by nanofiltration. First, cell clarification is performed by subjecting the fermentation broth to microfiltration using a 0.45 μm membrane with 0.22 μm filtered reverse osmosis (RO) water as the dialysis buffer. Then, further engineered exosome isolation is achieved by ultrafiltration using a 10 kDa membrane with 0.22 μm filtered phosphate buffered saline (PBS, pH 7.4) as the dialysis buffer. Finally, the isolated engineered exosomes are subjected to nanofiltration using a 0.22 μm membrane.

[0057] Without wishing to be bound by theory, it is believed that these physically based methods offer several advantages compared to ultracentrifugation for exosome enrichment in both scale-down and scale-up models, particularly for subsequent industrial applications. These methods have been optimized to produce high yields of pure engineered exosomes and are easily scaled up for large-scale production.

[0058] In some embodiments of the present disclosure, the exosome composition acts as an antioxidant. In another aspect, the present disclosure provides a method for treating and / or preventing an oxidative stress associated disease in a subject in need thereof, comprising administering to the subject an exosome composition disclosed herein.

[0059] Examples of oxidative stress-related diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), ataxia, Friedreich's ataxia, autism, obsessive-compulsive disorder, attention deficit hyperactivity disorder, migraine, stroke, traumatic brain injury, cancer, inflammation, autoimmune diseases, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, stenosis, restenosis, atherosclerosis, metabolic syndrome, endothelial dysfunction, vasospasm, diabetes, among others. These conditions include, but are not limited to, chronic heart disease, aging, chronic fatigue, coronary heart disease, cardiac fibrosis, myocardial infarction, hypertension, angina, Prinzmetal angina, ischemia, angioplasty, hypoxia, Keshan disease, glucose-6-phosphate dehydrogenase deficiency, faba toxicosis, ischemia-reperfusion injury, rheumatoid and osteoarthritis, asthma, chronic obstructive pulmonary disease (e.g., emphysema and bronchitis), allergies, acute respiratory distress syndrome, chronic kidney disease, kidney transplantation, nephritis, ionizing radiation damage, sunburn, dermatitis, melanoma, psoriasis, macular degeneration, retinal degeneration, and cataract formation.

[0060] In another aspect, the present disclosure provides a method for treating and / or preventing inflammation in a subject in need thereof comprising administering to the subject an exosome composition disclosed herein.

[0061] Many other diseases involve oxidative stress and inflammation in affected tissues, including inflammatory bowel disease; inflammatory skin diseases; mucositis associated with radiation and chemotherapy; ophthalmic diseases such as uveitis, glaucoma, macular degeneration, and various forms of retinopathy; transplant failure and rejection; ischemia-reperfusion injury; chronic pain; degenerative diseases of the bones and joints, including osteoarthritis and osteoporosis; asthma and cystic fibrosis; seizure disorders; and neuropsychiatric disorders, including schizophrenia, depression, bipolar disorder, post-traumatic stress disorder, attention deficit disorder, autism spectrum disorders, and eating disorders such as anorexia nervosa.

[0062] In another aspect, the present disclosure provides a method for treating and / or preventing a neurological disease in a subject in need thereof comprising administering to the subject an exosome composition disclosed herein.

[0063] Examples of neurological disorders include, but are not limited to, neurodegenerative disorders, diseases of the nervous system that are secondary to a disease, condition, or treatment that has a temporary effect on the rest of the nervous system, damage to the nervous system caused by trauma, memory loss, or psychiatric disorders. In particular, examples of neurological disorders include, but are not limited to, damage to peripheral nerves caused by physical injury, ischemia, prolonged exposure to cold, damage to the central nervous system, dementia, Alzheimer's disease, Huntington's disease, or Parkinson's disease.

[0064] The following examples are provided to aid those of ordinary skill in the art in practicing the present disclosure. EXAMPLES

[0065] Analytical methods used for characterization and quality control Nanoparticle Tracking Analysis (NTA) NTA was performed using a NANOSIGHT™ NS300 (MALVERN PANALYTICAL®, UK). Samples were diluted in particle-free phosphate buffered saline (PBS) to a concentration of 1 × 10 8 ~1×10 9 Concentrations between 100 and 150 particles / mL were achieved. Five 60 second videos were recorded for each sample and analyzed using NTA 3.0 software. Measurements were performed at room temperature.

[0066] Cryo-Electron Microscopy (cryo-EM) Exosome samples (1-3 μL) were applied to glow discharge holed carbon grids and vitrified using a VITROBOT™ Mark IV (FEI®, USA) at 98% humidity and 4° C. The grids were blotted for 4.5 s before being immersed in liquid ethane.

[0067] SDS-PAGE analysis Protein samples from different stages of the filtration process were mixed with 4x LAEMMLI™ sample buffer (BIO-RAD®) and heated at 95°C for 5 min. Samples (20 μg total protein per lane) were loaded onto a 12% SDS-polyacrylamide gel along with protein molecular weight markers. Electrophoresis was performed at 100V for 90 min using the MINI-PROTEAN™ TETRA CELL SYSTEM (BIO-RAD®). Gels were stained with Coomassie Brilliant Blue R-250™ (Bio-Rad®) for 1 h and destained overnight in a solution of 40% methanol and 10% acetic acid. Images of the gels were captured using the IMAGESCANNER™ III IMAGING SYSTEM (GE®).

[0068] Protein quantification Protein concentrations were determined using BCA (PIERCE™) according to the manufacturer's instructions, with bovine serum albumin (BSA) as the standard.

[0069] Example 1 Host cell selection, engineering, and screening of engineered strains The selected engineered Pichia pastoris strain, designated mm106, was obtained from the commercially available P. pastoris strain KM71H(aox1::ARG, arg4) (INVITROGEN®) using recombinant DNA techniques. This strain was engineered to express the proteins set forth in SEQ ID NOs: 1-4, as described in U.S. Patent No. 7,601,808.

[0070] Construction of GMI expression vector The gmi gene was cloned from G. microsporum using primers with EcoRI / Xbal restriction sites as described in U.S. Patent No. 7,601,808. The PCR-amplified DNA fragment was purified by agarose gel electrophoresis, digested with EcoRI and Xbal, and ligated into the corresponding sites of pPICZαA to generate the Gmi expression vector.

[0071] Construction and selection of mm106 strain Competent P. pastoris KM71H cells were prepared according to the Pichia Expression Kit (INVITROGEN®) manual. The GMI expression vector was linearized with Pmel and transformed into the competent cells. Candidate strains were selected based on antibiotic resistance, with higher resistance corresponding to higher recombinant GMI expression levels. The strain that showed the highest production level, designated mm106, was selected as the producer strain.

[0072] Confirmation of the identity of the gmi gene in the production strain Ey166 PCR analysis confirmed the presence of the gmi gene in the genome of strain mm106. A 339 bp fragment was amplified from mm106 genomic DNA using primers GMI-f (SEQ ID NO: 5, GAATTCATGTCCGACACTGCCTT) and GMI-r (SEQ ID NO: 6, GTTCCACTGGGCAACAATGAAA), designed from the 5' and 3' ends of the gmi gene, respectively (Figure 1). This fragment was visualized by agarose gel electrophoresis, but no signal was detected from the host strain.

[0073] Furthermore, the internal transcribed spacer primers ITS1 (SEQ ID NO: 7, TCCGTAGGTGAACCTGCGG) and ITS4 (SEQ ID NO: 8, TCCTCCGCTTATTGATATGC) used for yeast species identification amplified identical approximately 300 bp fragments from both mm166 and the host strain (Figure 2). Sequencing of these fragments (data not shown) confirmed that both strains belonged to the species P. pastoris.

[0074] [Example 2 Manufacturing Process and Process Control] fermentation: A frozen culture (stored at -80°C in 15% glycerol) was prepared from one batch culture of clone mm106 and inoculated into 400 mL of YPD medium to obtain a preculture. This preculture (OD600>0.4) was inoculated into 3 L of YPD medium (initial OD600=0.05-0.1) to obtain a seed culture. This seed culture (OD600=12-15) was used for batch fermentation (agitation: 200-500 rpm; aeration: air+O2, 0.5-0.7 vvm; pressure: 0.1-0.3 kg / cm2). 2 The culture was inoculated into 40 L of BMGY medium (initial OD600 = 0.6-0.9) for either fed-batch fermentation (DO spike; feeding 5 kg 50% glycerol in 2 h) or fed-batch fermentation (DO spike; feeding methanol at a flow rate of 60-80 g / h for 44 h).

[0075] Post-fermentation process: The cells were filtered using 0.22 μm filtered RO water as the dialysis buffer, followed by 0.45 μm, 0.6-0.8 μm 2 The vesicles were isolated by microfiltration using a 10 kDa, 0.5 m 2 The ultrafiltration was carried out using a membrane and a dialysis buffer of 0.22 μm filtered phosphate buffered saline, PBS (pH 7.4). The product was filtered using 0.22 μm, 0.6-0.8 μm filters. 2 The mixture was subjected to final filtration by nanofiltration using a membrane.

[0076] Isolation and characterization of engineered exosomes The fermentation filtrate of engineered Pichia pastoris mm106 was subjected to exosome isolation using tangential flow filtration (TFF). The supernatant was concentrated approximately 10-fold during this process. Nanoparticle tracking analysis (NTA) of the concentrated sample revealed the presence of particles with diameters ranging from approximately 50 to 250 nm. This size distribution is consistent with that predicted for exosomes and small extracellular vesicles (Figure 3 and Table 2).

[0077] [Table 2]

[0078] To further characterize these particles, cryo-electron microscopy (cryo-EM) was used. Cryo-EM images confirmed the presence of circular structures with diameters (50-250 nm) consistent with those observed by NTA. Importantly, these particles exhibited characteristic membrane structures including single and double lipid bilayers, providing strong evidence that they are extracellular vesicles / exosomes. Grey regions were observed within these vesicles representing encapsulated proteins that are presumed to be GMI cargo.

[0079] To analyze the protein content of engineered exosomes, SDS-PAGE was performed on samples from different stages of the filtration process. Coomassie Brilliant Blue (CBR) staining was used to visualize the gels. The SDS-PAGE results demonstrated that the major cargo in engineered exosomes was indeed the GMI protein.

[0080] Notably, the TFF ultrafiltration process not only achieved a 10-fold enrichment of exosomes but also maintained a high recovery (>85%) of GMI proteins. This high recovery highlights the efficiency of the TFF method in enriching engineered exosomes while preserving their protein cargo.

[0081] Taken together, these findings provide strong evidence for the successful generation and isolation of engineered exosomes containing GMI as the primary cargo. Consistency across multiple analytical techniques (NTA, cryo-EM, and SDS-PAGE) strengthens the identification of these particles as exosomes and confirms their intended protein content.

[0082] While the present disclosure has been described in conjunction with specific embodiments shown, many alternatives thereto, as well as modifications and variations thereon, will be apparent to those skilled in the art, and such alternatives, modifications and variations are deemed to be within the scope of the present disclosure.

Claims

1. An exosome composition comprising a population of enriched fungal exosomes containing a Ganoderma immunomodulatory protein, a recombinant thereof, or a fragment thereof, or a mixture derived from the enriched exosome population.

2. 2. The exosome composition of claim 1, wherein the Ganoderma immunomodulatory protein is LZ-8 from Ganoderma lucidum, FIP-gts from Ganoderma tsugae, GMI from Ganoderma microsporum, or FIP-gja from Ganoderma sinensis.

3. 2. The exosome composition of claim 1, wherein the Ganoderma lucidum immunomodulatory protein comprises the amino acid sequence of SEQ ID NO:

3.

4. 2. The exosome composition of claim 1, wherein the recombinant Ganoderma lucidum immunomodulatory protein comprises the amino acid sequence of SEQ ID NO:

4.

5. 2. The exosome composition of claim 1, wherein the fragment of a Ganoderma lucidum immunomodulatory protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2.

6. The fungi include Kluyveromyces marxianus (also known as Saccharomyces fragilis, Kluyveromyces fragilis), Kluyveromyces lactis (also known as Saccharomyces lactis), Cyberlindnera jadinii (also known as Candida utilis, Pichia jadinii), Debaryomyces hansenii, Pichia pastoris, Pichia fermentans, Saccharomyces bayanus, and the like. bayanus, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces florentinus, Kazachstania unispora (also known as Saccharomyces unisporus, Kazachstania turicensis (also known as Saccharomyces florentinus), Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, or Aspergillus niger. niger.

7. The concentration of concentrated exosomes in the exosome composition is about 1×10 10 2. The exosome composition of claim 1, wherein the exosome composition comprises more than exosomes / mL.

8. 2. The exosome composition of claim 1, wherein the exosome composition contains a population of enriched exosomes having an average particle size of about 150 nm to about 250 nm.

9. 2. The exosome composition of claim 1, wherein the fungus is engineered with a nucleotide encoding a Ganoderma lucidum immunomodulatory protein, a recombinant thereof, or a fragment thereof.

10. 2. The exosome composition of claim 1, wherein the exosome composition is obtained by microfiltration, ultrafiltration, or nanofiltration.

11. 2. The exosome composition of claim 1, wherein the exosome composition is obtained by performing at least one step of tangential flow filtration (TFF).

12. 2. The exosome composition of claim 1, wherein the exosome composition is obtained by performing at least one TFF step using a TFF membrane having a pore size of about 0.8 μm to about 8 μm.

13. 2. The exosome composition of claim 1, wherein the exosome composition is obtained by performing at least one TFF step using a TFF membrane with a molecular weight cutoff of about 10 kDa to about 100 kDa.

14. 2. The exosome composition of claim 1, wherein the exosome composition is obtained by performing depth filtration using a membrane having a pore size of about 0.22 μm to about 0.45 μm.

15. 2. The exosome composition of claim 1, wherein the exosome composition is obtained by performing TFF using a TFF membrane having a pore size of about 0.8 μm to about 8 μm, performing TFF using a TFF membrane having a molecular weight cut off of about 10 kDa to about 100 kDa, and performing depth filtration using a membrane having a pore size of about 0.22 μm to about 0.45 μm.

16. 13. A method for treating and / or preventing an oxidative stress-related disease in a subject in need thereof, comprising administering to the subject the exosome composition of claim 1.

17. 13. A method for treating and / or preventing inflammation in a subject in need thereof, comprising administering to the subject the exosome composition of claim 1.

18. 13. A method for treating and / or preventing a neurological disease in a subject in need thereof, comprising administering to the subject the exosome composition of claim 1.

19. 19. The method of claim 18, wherein the neurological disorder is a neurodegenerative disease, a disease that has a primary effect outside the nervous system, a nervous system condition secondary to a condition or treatment, nervous system damage caused by trauma, memory loss, or a psychiatric disorder.

20. 19. The method of claim 18, wherein the neurological disease is peripheral nerve damage caused by physical injury, ischemia, prolonged exposure to cold, damage to the central nervous system, dementia, Alzheimer's disease, Huntington's disease, or Parkinson's disease.

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