Exosome composition containing ganoderma lucidum immunomodulatory protein and application thereof

Abstract

The present disclosure pertains to an exosome composition comprising an enriched population of fungal exosomes, or a mixture derived from the enriched population of exosomes, the enriched fungal exosomes containing a Ganoderma lucidum (Ganoderma lucidum) immunomodulatory protein, a recombinant thereof, or a fragment thereof. The disclosure also relates to 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 condition in a subject.

Description

[0001] priority

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 517,206, filed on August 2, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing which is submitted electronically in .xml format and is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates to an engineered exosome composition and uses thereof. Background Art

[0006] Exosomes are membrane-bound extracellular vesicles (EVs) produced in the endosomal compartment of most eukaryotic cells. Exosomes generally have a size range of about 40nm to 160nm (average about 100nm) in diameter and are derived from endosomes. Its surface consists of a lipid bilayer from the cell membrane of the donor cell, and it contains cytosol from the cell that produces the exosomes, and displays membrane proteins from the parent cell on the surface. Exosomes were originally thought to be a mechanism for removing unwanted proteins.

[0007] There is still a need to expand the application of exosomes. Summary of the invention

[0008] The present disclosure provides a novel exosome composition comprising an enriched exosome population of an engineered fungus, particularly yeast.

[0009] Therefore, the present disclosure provides an exosome composition comprising an enriched fungal exosome population or a mixture derived from an enriched exosome population, wherein the enriched fungal exosomes contain a Ganoderma immunomodulatory protein, a recombinant thereof, or a fragment thereof.

[0010] In some embodiments, the Ganoderma lucidum immunomodulatory protein is derived from Ganoderma lucidum, Ganoderma tsugae, Ganoderma microsporum, or Ganoderma sinensis. In some other embodiments, the Ganoderma lucidum immunomodulatory protein is LZ-8 derived from Ganoderma lucidum, FIP-gts derived from Ganoderma tsugae, GMI derived from Ganoderma microsporum, or FIP-gja derived from Ganoderma sinensis.

[0011] 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 Ganoderma lucidum immunomodulatory protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2.

[0012] In some embodiments, the fungus belongs to the family Saccharomycetaceae or Schizosaccharomycetaceae. Examples of the family Saccharomycetaceae include, but are not limited to, Kluyveromyces, Cyberlindnera, Debaryomyces, Pichia, Saccharomyces, Kazachstania. In another aspect, examples of the family Schizosaccharomycetaceae include, but are not limited to, 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, 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.Specifically, in some embodiments of the present disclosure, the yeast belongs to Kluyveromyces lactis (also known as lactic acid yeast), Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, or Aspergillus niger.

[0013] In some embodiments of the present disclosure, the concentration of exosomes enriched in the exosome composition is greater than about 1×10 10 exosomes / ml, about 5×10 10 exosomes / ml, about 1×10 11 exosomes / ml, about 2×10 11 exosomes / ml, about 4×10 11 exosomes / ml, about 6×10 11 exosomes / ml, about 8×10 11 exosomes / ml, about 1×10 12 exosomes / ml, about 2×10 12 exosomes / ml, about 3×10 12 exosomes / ml, about 4×10 12 exosomes / ml, about 5×10 12 exosomes / ml, about 6×10 12 exosomes / ml, about 7×10 12 exosomes / ml, about 8×10 12 exosomes / ml, about 9×10 12 exosomes / mL or about 1×10 13 In some other embodiments, the concentration of exosomes enriched in the exosome composition is about 1×10 12 exosomes / mL to 2×10 12 exosomes / mL.

[0014] In some embodiments of the present disclosure, the exosome composition comprises an enriched exosome population 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.

[0015] In some embodiments of the present disclosure, the fungus is engineered with nucleotides encoding a Ganoderma lucidum immunomodulatory protein, a recombinant thereof, or a fragment thereof.

[0016] In some embodiments of the present disclosure, fungi are cultured with conditioned medium. Examples of conditioned medium include, but are not limited to, BMGY medium.

[0017] 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. Specifically, the exosome composition is obtained by performing at least one step of tangential flow filtration (TFF). Examples of TFF include, but are not limited to, performing at least one step of TFF using a TFF membrane having a pore size of about 0.8 μm to about 8 μm, or performing at least one step of TFF 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 depth filtration using a membrane having a pore size of about 0.22 μm to about 0.45 μm. In some other 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 cutoff 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.

[0018] In another aspect, the present disclosure provides a method for producing an exosome composition as disclosed herein, comprising culturing fungi and obtaining an enriched exosome population.

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

[0020] 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 as disclosed herein.

[0021] In another aspect, the present disclosure provides a method for treating and / or preventing a neurological disorder in a subject in need thereof, comprising administering to the subject an exosome composition as disclosed herein.

[0022] Examples of neurological disorders include, but are not limited to, neurodegenerative diseases, neurological conditions secondary to diseases, conditions, or therapies that primarily act outside the nervous system; damage to the nervous system caused by trauma; memory loss; or psychiatric disorders. Specifically, examples of neurological disorders include, but are not limited to, peripheral nerve damage caused by physical injury, ischemia, prolonged exposure to cold temperatures, damage to the central nervous system, dementia, Alzheimer's disease, Huntington's disease, or Parkinson's disease.

[0023] 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.

[0024] Simple diagram description

[0025] Figure 1 PCR analysis is shown confirming the presence of the gmi gene in the genome of strain mm106. In lanes V and E, a 339 bp fragment amplified by PCR using GMI-f / GMI-r primers was detected.

[0026] Figure 2 PCR analysis for yeast species identification is shown. The same approximately 300 bp fragment was amplified from mm166 and the host strain.

[0027] Figure 3 Representative histograms showing particle concentration and size distribution of isolated engineered exosomes measured by nanoparticle tracking analysis (NTA) are shown.

[0028] Figure 4 Representative cryo-EM images of engineered exosomes are shown. Scale bar: 100 nm.

[0029] Figure 5SDS-PAGE analysis of engineered exosomes subjected to different filtration processes is shown, visualized by Coomassie Brilliant Blue (CBR) staining. Lane M: protein marker with molecular weight indicated in kDa. Lanes 1-5: standard GMI protein at different concentrations (0.2, 0.3, 0.5, 0.6 and 0.8 mg / mL) used as protein concentration control. Lanes 6-8: supernatant (SUP) of engineered Pichia pastoris mm106 fermentation after removal of cells by centrifugation, loaded at different dilutions (3×, 4× and 5×). Lanes 9-11: fermentation after microfiltration (MF) for cell clarification, loaded at different dilutions (2×, 3× and 4×). Lanes 12-14: Fermentates further processed by ultrafiltration / nanofiltration (UF / NF) for engineered exosome isolation were loaded at different dilution factors (35×, 40×, and 45×). Implementation

[0031] It should be understood that the present disclosure is not limited to the specific materials and methods described herein. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments and is not intended to limit the scope of the present disclosure, which will be limited only by the appended claims.

[0032] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0033] As used herein, "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If the use of a term is not clear to one of ordinary skill in the art in the context in which it is used, "about" will mean plus or minus 10% of the particular term.

[0034] As used herein, the term "exosome" refers to a small membrane extracellular vesicle with a diameter of about 30-300 nm that is secreted from a producing cell into the extracellular environment. The surface of the exosome comprises a lipid bilayer from the membrane of the donor cell, and the lumen of the exosome is topologically identical to the cytosol of the cell producing the exosome. Exosomes contain proteins, RNA, lipids, and carbohydrates of the producing cell, but some of these may be modified or added to the exosome after the exosome is released from the cell by natural processes or by experimental manipulation.

[0035] The term "genetically engineered" or "genetically modified" of a cell refers to the use of genetic material to manipulate genes to change the gene copies and / or gene expression levels in a cell. The genetic material can be in the form of DNA or RNA. The genetic material can be transferred to the cell by various means including viral transduction and non-viral transfection. After genetic engineering, the expression levels of certain genes in the cell can be permanently or temporarily changed.

[0036] As used herein, the terms "treatment", "treating" and similar terms encompass any treatment of a disease in mammals, especially humans, and include: (a) preventing the onset of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, that is, curbing its development; and (c) ameliorating the disease, that is, causing regression of the disease.

[0037] The terms "preventing" or "prevention" are recognized in the art and when used in conjunction with a condition, include administering an agent prior to the onset of the condition to reduce the frequency or severity of symptoms of a medical condition in a subject, or to delay the onset of the condition, relative to a subject not receiving the agent.

[0038] As used interchangeably herein, the terms "individual," "subject," "subject," and "patient" refer to mammals, including but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, sheep, pigs, goats), etc. In particular, an individual is vaccinated.

[0039] As used herein, the term "in need of treatment" refers to the judgment made by a caregiver (e.g., a physician, nurse, care practitioner, or individual in the case of humans; a veterinarian in the case of animals (including non-human mammals)) that the individual requires or will benefit from treatment. This judgment is made based on a variety of factors within the caregiver's area of ​​expertise and includes the knowledge that the individual is or will become ill due to a condition that can be treated with the compounds of the present disclosure.

[0040] As used herein, the term "neuron" includes a neuron and one or more parts thereof (e.g., a neuronal cell body, axon, or dendrite). The term "neuron" refers to a nervous system cell that includes a central cell body or soma and two types of extensions or projections: dendrites, by which most neuronal signals are generally transmitted to the cell body; and axons, by which most neuronal signals are generally transmitted from the cell body to effector cells, such as target neurons or muscles.

[0041] As used herein, "neuropathy" means any physiological dysfunction or death of neurons present in the central nervous system or peripheral nervous system or caused by abnormal glial cell function. The non-limiting list of such diseases includes multiple sclerosis, sciatic nerve defect, brain or spinal cord injury, dementia, frontotemporal lobe dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, prion disease, neuron disease and motor neuron disease. "Neuropathy" is characterized by the death of neuronal cells of motor neurons or sensory neurons, and therefore neuron disease can be subdivided into motor neuron disease and sensory neuron disease.

[0042] The present disclosure provides a novel exosome composition comprising an enriched exosome population of a fungus engineered with a Ganoderma lucidum immunomodulatory protein.

[0043] Therefore, the present disclosure provides an exosome composition, which comprises an enriched exosome population of fungi or a mixture derived from an enriched exosome population, wherein the enriched exosomes of fungi contain a Ganoderma lucidum immunomodulatory protein, a recombinant thereof, or a fragment thereof.

[0044] In one embodiment, the immunomodulatory protein or its recombinant is derived from red Ganoderma lucidum, Ganoderma pine, Ganoderma microspore or Ganoderma purpurogenum. Preferably, the immunomodulatory protein is LZ-8 derived from red Ganoderma lucidum, FIP-gts derived from Ganoderma pine, GMI derived from Ganoderma microspore or FIP-gja derived from Ganoderma purpurogenum or its recombinant.

[0045] In one embodiment, the immunomodulatory protein is derived from Ganoderma microsporum (GMI) or its recombinant (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 of MSDTALIFTLAWNVKQLAFDYTPNWGRGRPSSFIDTVTFPTVLTDKAYTYRVVVSGKDLGVRPSYAVESDGSQKINFLEYNSGYGIADTNTIQVYVIDPDTGNNFIVAQWN (SEQ ID NO: 3) or EEAEAEFMSDTALIFTLAWNVKQLAFDYTPNWGRGRPSSFIDTVTFPTVLTDKAYTYRVVVSGKDLGVRPSYAVESDGSQKINFLEYNSGYGIADTNTIQVYVIDPDTGNNFIVAQWN (SEQ ID NO: 4). The amino acid sequence of NNFIVAQWNYLEQKLISEEDLNSAVDHHHHHH (SEQ ID NO: 4).

[0046] The choice of host cells is a key consideration for exosome production, as different cell types have different exosome production capabilities. In some embodiments, the fungi are listed in Table 1.

[0047] Table 1

[0048]

[0049]

[0050] Specifically, in some embodiments of the present disclosure, the fungus belongs to Kluyveromyces lactis (also known as lactic acid yeast), Pichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe.

[0051] Since the mid-1980s, Pichia pastoris has been widely used in recombinant protein production with a well-documented safety profile in the pharmaceutical, food and cosmetic industries. 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 a lipase preparation expressed in Pichia pastoris, further validating the reliability of the strain in industrial applications. In addition, in 1993, the US Food and Drug Administration (FDA) approved the use of Pichia pastoris dry yeast as a food additive in animal feed and drinking water, strengthening its regulatory safety profile.

[0052] In some embodiments, engineered fungal cells are suitable for exosome production. These eukaryotic species are selected due to their high yield and stability of protein production. The safety of using these eukaryotic species has been recognized. Without being limited by theory, it is believed that compared with mammalian cells, when considering the use of exosomes for human therapy, these mammalian cells have the potential for zoonotic infectious diseases, and the use of eukaryotic cells provides a potentially safer alternative for exosome production. In some embodiments, appropriate safety measures are taken when handling and producing these cells to minimize any potential risks.

[0053] Host yeast and fungal cell lines are screened for endogenous exosome production using standard protocols for exosome enrichment and characterization. In some embodiments, briefly, exosomes are isolated using centrifugation and size exclusion filtration at a sub-mL scale, while filtration methods are applied to exosome enrichment at a sub-hundred-liter scale. Genetic (target-specific) engineering of host cell lines is achieved by modified homologous recombination using linear DNA fragments containing homologous regions, desired modifications, and selection markers. In the engineering of host cells for exosome production, parameters including transformation strategy / efficiency, genetic engineering targets, and selection or screening strategies are all taken into account.

[0054] In some embodiments, the fungus is engineered with nucleotides encoding a Ganoderma lucidum immunomodulatory protein, a recombinant thereof, or a fragment thereof. The transformation strategy depends on the number of samples processed in a single process and the type of host cell. Physical and chemical methods for gene transformation of fungal cells are established. In some embodiments, transformation includes physical methods, electroporation, rather than chemical methods due to its transformation efficiency and condition optimization potential.

[0055] The engineering target (cargo loading) depends on the desired application of the exosomes. For example, exosomes are engineered to load biologically active proteins such as antibodies, cytokines, or growth factors - any designed coding sequence. The target protein is selected based on its function and application.

[0056] To select cells with the highest exosome expression, a selection or screening strategy is used. In some embodiments, antibiotic / nutrient deficiency selection is applied to cells with stable integration of genetic material. Antibiotic / nutrient deficiency selection is used to identify successfully engineered cells. In some embodiments, quantitative polymerase chain reaction (qPCR) or Western blotting are used to specifically characterize the engineered host and corresponding exosomes for the expression of one or more target molecules. NTA is used to evaluate the purity, size and yield of engineered exosomes. Analysis is also used to evaluate the cargo load of engineered exosomes, such as assessing the amount of protein.

[0057] In general, the transfection strategy, genetic engineering target, and selection or screening strategy are optimized based on the specific application of the engineered exosomes. By carefully selecting the target molecule and optimizing the transformation and selection strategies, exosomes with high expression and activity of the desired protein molecule are engineered.

[0058] In some embodiments, the culture conditions of fungal cells are crucial for producing high yields of recombinant proteins and corresponding exosomes. The culture medium usually includes 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.

[0059] A carbon source is an essential component of the culture, with glucose being the most commonly used. Other carbon sources such as maltose, galactose, fructose, and glycerol are also used.

[0060] Cells require nitrogen to synthesize amino acids, the building blocks of proteins. In some embodiments, ammonium sulfate is the most commonly used nitrogen source in culture. Other sources, such as yeast extract and peptone, are also used.

[0061] 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 necessary 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.

[0062] In some embodiments, the culture medium further contains other additives, such as buffers, pH adjusters (acid / base), and antibiotics to prevent contamination. In some embodiments, the selection and concentration of these components are optimized.

[0063] In some embodiments, other environmental factors including temperature, aeration, and dissolved oxygen content are also optimized. The optimal pH for cell growth and the quality of the final product vary depending on the strain and the target being genetically engineered. In some embodiments, the temperature is maintained at a level that promotes cell growth while also allowing for optimal protein folding and stability and exosome integrity. In some embodiments, aeration is controlled to affect oxygen availability and cell growth rate.

[0064] In some embodiments, chemically defined media or animal-origin-free (AOF) media containing well-defined amounts of each component are used to ensure reproducibility and consistency of production quality. A fed-batch culture strategy with continuous or intermittent nutrient addition is also employed to further improve productivity.

[0065] Without wishing to be bound by theory, it is believed that optimizing culture conditions for fungal cells involves careful control of media components, pH, temperature, aeration, and other factors that may affect the yield, quality, and stability of recombinant proteins and exosomes.

[0066] In some embodiments, physical-based methods, specifically tangential flow filtration (TFF), are used for exosome isolation to ensure the ability to scale up. In some embodiments, a three-step TFF method followed by nanofiltration is used to isolate engineered exosomes from engineered cell culture media. First, cell clarification is performed by microfiltration of the fermentation broth using a 0.45 μm membrane with 0.22 μm filtered reverse osmosis (RO) water as the dialysis buffer. Further engineered exosome isolation is then achieved by ultrafiltration using a 10 kD 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 a nanofiltration step using a 0.22 μm membrane.

[0067] Without wishing to be bound by theory, it is believed that these physics-based methods offer several advantages over ultracentrifugation for exosome enrichment in scale-down and scale-up models, especially for subsequent industrial applications. The methods are optimized to provide high yields of pure engineered exosomes and are easily scaled up for large-scale production.

[0068] 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-related disease in a subject in need thereof, comprising administering to the subject an exosome composition as disclosed herein.

[0069] 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 disorders, lupus, MS, inflammatory bowel disease, Crohn's disease, ulcerative colitis, stenosis, restenosis, atherosclerosis, metabolic syndrome, endothelial dysfunction, vasospasm, diabetes, aging, chronic fatigue, coronary heart disease, cardiac fibrosis, myocardial infarction, hypertension, angina, Prizmetal's angina, ischemia, angioplasty, hypoxia, Keshan disease, disease), glucose-6-phosphate dehydrogenase deficiency, favism, ischemic reperfusion injury, rheumatoid arthritis and osteoarthritis, asthma, chronic obstructive pulmonary disease (such as 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, cataract formation, etc.

[0070] 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 as disclosed herein.

[0071] Many other disorders involve oxidative stress and inflammation in affected tissues, including inflammatory bowel disease; inflammatory skin diseases; mucositis associated with radiation therapy and chemotherapy; eye diseases, such as uveitis, glaucoma, macular degeneration, and various forms of retinopathy; transplant failure and rejection; ischemia-reperfusion injury; chronic pain; degenerative conditions of the bones and joints, including osteoarthritis and osteoporosis; asthma and cystic fibrosis; epileptic disorders; and neuropsychiatric conditions, including schizophrenia, depression, bipolar disorder, post-traumatic stress disorder, attention deficit disorder, autism spectrum disorders, and eating disorders such as anorexia nervosa.

[0072] In another aspect, the present disclosure provides a method for treating and / or preventing a neurological disorder in a subject in need thereof, comprising administering to the subject an exosome composition as disclosed herein.

[0073] Examples of neurological disorders include, but are not limited to, neurodegenerative diseases, neurological conditions secondary to diseases, conditions, or therapies that primarily act outside the nervous system; damage to the nervous system caused by trauma; memory loss; or psychiatric disorders. Specifically, examples of neurological disorders include, but are not limited to, peripheral nerve damage caused by physical injury, ischemia, prolonged exposure to cold temperatures, damage to the central nervous system, dementia, Alzheimer's disease, Huntington's disease, or Parkinson's disease.

[0074] The following examples are provided to help those skilled in the art to implement the present disclosure.

[0075] Analytical methods for characterization and quality control

[0076] Nanoparticle Tracking Analysis (NTA)

[0077] Using NanoSight TM NS300(Malvern UK) for NTA. Samples were diluted in particle-free phosphate-buffered saline (PBS) to a concentration between 1 × 10 8 With 1×10 9 For each sample, five 60-second videos were recorded and analyzed using NTA 3.0 software. Measurements were performed at room temperature.

[0078] Cryo-electron microscopy (cryo-EM)

[0079] Exosome samples (1-3 μL) were applied to glow-discharged holey carbon grids and incubated at 98% humidity and 4°C using a Vitrobot TM Mark IV The grids were vitrified by blotting for 4.5 seconds and then plunged into liquid ethane.

[0080] SDS-PAGE analysis

[0081] Protein samples from different stages of the filtration process were mixed with 4× Laemmli TM Sample buffer (Bio- ) and heated at 95°C for 5 minutes. The samples (20 μg total protein per lane) were loaded onto a 12% SDS-polyacrylamide gel along with protein molecular weight markers. Mini-PROTEAN TM Tetra Cell System Electrophoresis was performed at 100 V for 90 min. The gel was stained with Coomassie Brilliant Blue R-250 TM Stain for 1 hour and destain overnight in a solution of 40% methanol and 10% acetic acid. TM III Imaging System Capture gel images.

[0082] Protein quantification

[0083] BCA (Pierce TM ) was used to determine the protein concentration, with bovine serum albumin (BSA) as the standard.

[0084] Example 1. Host cell selection, engineering, and screening of engineered strains

[0085] The engineered strain selected and named Pichia pastoris strain mm106 was derived from the commercially available Pichia pastoris KM71H (aox1::ARG, arg4) using recombinant DNA technology. Strain Derivation This strain was engineered to express the proteins of SEQ ID NOs. 1 to 4 as described in US Pat. No. 7,601,808.

[0086] Construction of GMI performance carrier

[0087] The gmi gene was cloned from G. microsporum using primers with EcoRI / XbaI restriction sites as described in US Patent No. 7,601, 808. The PCR-amplified DNA fragment was purified by agarose gel electrophoresis, digested with EcoRI and XbaI, and ligated into the corresponding sites of pPICZαA to generate the GMI expression vector.

[0088] Construction and selection of strain mm106

[0089] Based on Pichia performance kit Manual preparation of competent Pichia pastoris KM71H cells. The GMI expression vector was linearized with PmeI and transformed into competent cells. Potential strains were selected based on antibiotic resistance, where higher resistance was associated with higher recombinant GMI expression. The strain named mm106 that showed the highest production level was selected as the production strain.

[0090] Confirmation of the identity of the gmi gene in the production strain Ey166

[0091] PCR analysis confirmed the presence of the gmi gene in the genome of strain mm106. Primers GMI-f (SEQ ID NO: 5, GAATTCATGTCCGACACTGCCTT) and GMI-r (SEQ ID NO: 6, GTTCCACTGGGCAACAATGAAA) designed based on the 5' and 3' ends of the gmi gene were used to amplify a 339 bp fragment ( Figure 1 ). This fragment was visualized by agarose gel electrophoresis, and no signal from the host strain was detected.

[0092] In addition, the internal transcribed spacer primers ITS1 (SEQ ID NO. 7, TCCGTAGGTGAACCTGCGG) and ITS4 (SEQ ID NO. 8, TCCTCCGCTTATTGATATGC) used for yeast species identification were used to amplify the same approximately 300 bp fragment from mm166 and the host strain ( Figure 2 ). Sequencing of these fragments (data not shown) confirmed that both strains belonged to the species Pichia pastoris.

[0093] Example 2. Manufacturing process and process control

[0094] Fermentation:

[0095] A frozen culture (stored in 15% glycerol and stored at -80°C) was prepared from a batch of pure mm106 cultures and inoculated into 400 mL YPD medium to obtain a preculture. The preculture (OD600>0.4) was inoculated into 3LYPD medium (initial OD600=0.05-0.1) to obtain a seed culture. The seed culture (OD600=12-15) was inoculated into 40 L BMGY medium (initial OD600=0.6-0.9) for batch fermentation (agitation: 200-500 rpm; aeration: air+O2, 0.5-0.7 vvm; pressure: 0.1-0.3 kg / cm 2; temperature: 30°C; pH: 5) or fed-batch fermentation (DO peak; 5 kg 50% glycerol fed in 2 hours). Production fed-batch fermentation was performed as follows: DO peak; methanol fed at a flow rate of 60-80 g / h in 44 hours.

[0096] Process after fermentation:

[0097] The cells were dialyzed using 0.22 μm filtered RO water as a dialysis buffer at 0.45 μm, 0.6-0.8 μm 2 Microfiltration of membrane. Through 10kD, 0.5m 2 The vesicles were isolated by ultrafiltration of membranes and 0.22 μm filtered phosphate buffered saline, PBS (pH 7.4) dialysis buffer. 2 Membrane nanofiltration performs the final filtration.

[0098] Isolation and characterization of engineered exosomes

[0099] Exosome isolation was performed on engineered Pichia pastoris mm106 fermentation filtrates using tangential flow filtration (TFF). During this process, the supernatant was concentrated approximately 10-fold. Nanoparticle tracking analysis (NTA) of the concentrated samples revealed the presence of particles with diameters ranging from approximately 50 to 250 nm. This size distribution is consistent with the expected size distribution of exosomes and small extracellular vesicles ( Figure 3 and Table 2).

[0100] Table 2

[0101]

[0102]

[0103] Cryo-electron microscopy (cryo-EM) was used to further characterize these particles. Cryo-EM images confirmed the presence of round structures with diameters matching those observed by NTA (50-250 nm). Importantly, these particles exhibited characteristic membrane structures, including monolayer and bilayer lipid bilayers, providing strong evidence for their identity as extracellular vesicles / exosomes. Within these vesicles, gray areas were observed, indicating encapsulated proteins, presumably GMI cargo.

[0104] To analyze the protein content of the engineered exosomes, samples from different stages of the filtration process were subjected to SDS-PAGE. The gel was visualized using Coomassie Brilliant Blue (CBR) staining. The SDS-PAGE results showed that the major cargo in the engineered exosomes was in fact the GMI protein.

[0105] Notably, the TFF ultrafiltration process not only achieved a 10-fold concentration of exosomes, but also maintained a high recovery of GMI protein (>85%). This high recovery emphasizes the efficiency of the TFF method in concentrating engineered exosomes while maintaining their protein cargo.

[0106] Together, these findings provide solid evidence for the successful generation and isolation of engineered exosomes containing GMI as their primary cargo. The consistency of multiple analytical techniques (NTA, cryo-EM, and SDS-PAGE) reinforced the identification of these particles as exosomes and confirmed their expected protein content.

[0107] Although the present disclosure has been described in conjunction with the specific embodiments described, many alternatives, modifications and variations thereof will be apparent to those skilled in the art. All such alternatives, modifications and variations are deemed to fall within the scope of the present disclosure.

Claims

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

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

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

3.

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

4. 5 . The exosome composition of claim 1 , wherein the fragment of the Ganoderma lucidum immunomodulatory protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 2.

6. The exosome composition of claim 1, wherein the fungus belongs 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, Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces cerevisiae cerevisiae, Saccharomyces florentinus, Kazachstania unispora (also known as Saccharomyces unisporus), Kazachstaniaturicensis (also known as Saccharomyces florentinus), Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica, or Aspergillus niger.

7. The exosome composition of claim 1, wherein the concentration of the enriched exosomes in the exosome composition is greater than about 1×10 10 exosomes / mL.

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

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

10. The exosome composition as claimed in claim 1, wherein the exosome composition is obtained by microfiltration, ultrafiltration and nanofiltration.

11. The exosome composition of claim 1, wherein the exosome composition is obtained by performing at least one tangential flow filtration (TFF) step. 12 . 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. 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 molecular weight cutoff of about 10 kDa to about 100 kDa. 14 . The exosome composition of claim 1 , wherein the exosome composition is obtained by depth filtration using a membrane having a pore size of about 0.22 μm to about 0.45 μm.

15. 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 cutoff 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. Use of the exosome composition of claim 1 for the manufacture of a medicament for treating and / or preventing an oxidative stress-related disease in a subject in need thereof.

17. Use of the exosome composition of claim 1 for the manufacture of a medicament for treating and / or preventing inflammation in a subject in need thereof.

18. Use of the exosome composition of claim 1 for the manufacture of a medicament for treating and / or preventing a neurological disorder in a subject in need thereof, comprising administering the exosome composition of claim 1 to the subject.

19. The method of claim 18, wherein the neurological disorder is a neurodegenerative disease; a neurological condition secondary to a disease, condition, or therapy that primarily acts outside the nervous system; damage to the nervous system caused by trauma; memory loss; or a psychiatric disorder.

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

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