Engineered Exosomes for Improving Wound Healing

Abstract

The present invention discloses engineered exosomes, said engineered exosomes comprising (a) an EGF polypeptide fused to a first anchor polypeptide, (b) an NGF polypeptide fused to a second anchor polypeptide, and (c) a PDGF-BB polypeptide fused to a third anchor polypeptide, wherein (a), (b) and (c) are anchored to the membrane of said exosomes through the first, second, and third anchor polypeptides, respectively, said EGF polypeptide, said NGF polypeptide, and said PDGF-BB polypeptide are exposed on the outer surface of said exosomes membrane. The present invention further discloses the use of said exosomes in compositions, nucleic acid structures, and wound healing.

Description

Technology Field

[0001] The present disclosure relates to engineered exosomes, specifically to exosomes comprising different fusion proteins anchored outside the membrane of the exosome. The present disclosure also relates to compositions comprising engineered exosomes, nucleic acid structures comprising polynucleotides encoding fusion proteins, and the use of engineered exosomes in wound healing. Background Technology

[0002] Wound healing is a highly complex process that relies on the interactions of various cell types and media in a strictly regulated temporal and spatial sequence. Platelets, macrophages, fibroblasts, epithelial cells, and endothelial cells interact with the extracellular matrix, growth inhibitors, cytokines, and chemokines, which occur in a highly synchronized manner across four stages: hemostasis, inflammation, proliferation / migration, and tissue reorganization.

[0003] Hemostasis occurs immediately after injury. As a mechanism to stop bleeding from damaged blood vessels, hemostasis involves several interconnected stages. During the early stages of wound healing, vasoconstriction, platelet activation, and aggregation take place. During the hemostasis process, platelets release various signaling molecules, including cyclic adenosine monophosphate (cAMP), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). Platelets play a crucial role in hemostasis. They constitute a major component of the initial thrombus and also release signaling molecules that activate and mobilize fibroblasts, macrophages, and endothelial cells. PDGF is essential for the activation and mobilization of fibroblasts, which then migrate to the wound site and begin producing collagen and glycosaminoglycans.

[0004] The next stage of wound healing is inflammation, which occurs within 24 hours of skin injury. The inflammatory phase is characterized by the creation of a highly inflammatory environment through the secretion of various compounds (including inflammatory cytokines, proteases, reactive oxygen species, and growth factors). Macrophages play a crucial role in the inflammatory phase as they migrate to the site of injury and remove bacteria and necrotic tissue. Equally important as this phagocytic action is the role macrophages play in mediating the transition to the next stage of wound healing. Activated macrophages release soluble media, induce the process of angiogenesis, and mobilize and activate fibroblasts to synthesize new tissue matrix.

[0005] Once bleeding and inflammation are controlled, the proliferative phase begins. During the proliferative phase of wound healing, soluble media released during the inflammatory phase promotes the migration of epithelial cells and fibroblasts to the site of injury, supporting capillary growth and new tissue synthesis. Fibroblasts are the key cells in the proliferative phase of wound healing. In this phase, fibroblasts stabilize, begin to proliferate, and synthesize collagen, fibronectin, proteoglycans, and glycosaminoglycans, which are major components of the extracellular matrix (ECM). Fibroblasts not only produce the ECM but also secrete angiogenic signals in conjunction with macrophages, vascular endothelial cells, and epidermal cells. As a result of these two activities, granulation tissue, a metastatic substitute, is formed. This newly synthesized, highly vascularized tissue has significantly lower tensile strength compared to intact tissue.

[0006] Finally, in the final stage of wound healing, granulation tissue is reorganized until the degree of vascularization, matrix composition, and tissue tensile strength become similar to those of healthy tissue. However, tensile strength is generally reported to increase to a maximum of about 80% of normal tissue.

[0007] Acute wounds generally heal effectively by overlapping the four aforementioned stages. However, in certain situations, the skin's natural regenerative capacity can be severely impaired after injury. For example, changes in wound healing ability have been reported in patients experiencing severe burns, extensive skin loss, unhealed ulcers, post-surgical interventions, or prolonged immobility. Additionally, altered wound healing ability has been reported in patients suffering from conditions such as diabetes, neuropathy, and / or vascular disease. In these patients, the healing process is irregular, incomplete, and prolonged, resulting in a poor functional prognosis. Consequently, insufficient healing leads to alterations in the inflammation, proliferation, and reconstruction phases, and skin lesions may progress to a chronic state. Chronic wounds are characterized by the disruption of normal regeneration processes, an increased risk of infection, and a complex and slow healing process.

[0008] According to findings from various studies investigating the processes that cause abnormal wound healing, significant molecular differences have been identified within the bodies of patients with chronic wounds. It is important to note that both the molecular environment of chronic wounds and the ability of wound cells to respond to cytokines and growth factors appear to be altered. Furthermore, research results indicate that fibroblasts in unhealed skin ulcers fail to respond to growth factors and cannot divide into fibroblasts from healed wounds.

[0009] As various studies have confirmed that fibroblasts in chronic wounds exhibit abnormal phenotypes, research into therapies targeting fibroblasts is actively underway. Currently used methods include directly delivering viable fibroblasts to the wound site or stimulating fibroblast division or activity by applying growth factors (e.g., PDGF, TGF-β).

[0010] As the incidence of diseases such as diabetes, vascular disease, and obesity continues to rise, chronic wounds are becoming a major global issue due to limited treatment strategies, undesirable treatment outcomes, and high medical costs. Chronic wounds are particularly common among the elderly, affecting up to 3% of the population aged 65 and older. Consequently, chronic wounds are not only painful conditions that severely impact patients' mobility and quality of life, but also place a significant economic burden on the healthcare system.

[0011] In light of the aforementioned limitations, it is desirable to provide a method and composition that can improve wound healing in a simple, economical, and non-invasive manner. This is particularly important for promoting wound healing after surgical incisions, damage caused by radiation therapy, diabetic foot ulcers, burns, and other acute or chronic wounds.

[0012] Therefore, there is a need for methods to improve wound healing, including but not limited to severe burns, extensive skin loss, unhealed ulcers, damage from surgery or radiation therapy and / or other acute or chronic wounds.

[0013] In one embodiment of the present invention, an engineered exosome is provided, said engineered exosome comprising (a) an EGF polypeptide fused to a first anchor polypeptide, (b) an NGF polypeptide fused to a second anchor polypeptide, and (c) a PDGF-BB polypeptide fused to a third anchor polypeptide, wherein (a), (b) and (c) are anchored to the membrane of the exosome through the first, second, and third anchor polypeptides, respectively, said EGF polypeptide, said NGF polypeptide and said PDGF-BB polypeptide are exposed to the outer surface of the membrane of the exosome.

[0014] In some embodiments, the engineered exosome further comprises (d) an IDR-1018 polypeptide fused with a fourth anchor polypeptide comprising an amino acid sequence indicated by SEQ ID NO. 4, and the IDR-1018 polypeptide is exposed on the outer surface of the membrane of the exosome.

[0015] In some embodiments, the EGF polypeptide is a human EGF polypeptide or a parallel homolog or a serial homolog thereof, and preferably, the EGF polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 1.

[0016] In some embodiments, the NGF polypeptide is a human NGF polypeptide or a parallel homolog or a serial homolog thereof, and preferably, the NGF polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 2.

[0017] In some embodiments, the PDGF-BB polypeptide is a human PDGF-BB dimer or a parallel homolog or a serial homolog thereof; more preferably, the PDGF-BB dimer comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 3.

[0018] In some embodiments, these anchor polypeptides are exosome membrane proteins, membrane targeting sequences, or fragments of their anchoring function. Exemplary exosome membrane proteins include, but are not limited to, tetraspanins such as lamp2b, CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative modulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid anchor proteins.

[0019] In a preferred embodiment, these anchor polypeptides comprise full-length CD63 or truncated CD63 in which the TM3 domain is preserved. In a preferred embodiment, each of the first, second, and third anchor polypeptides and the fourth anchor polypeptide (if present) comprises the TM3 domain of CD63. In a preferred embodiment, each of the first, second, and third anchor polypeptides and the fourth anchor polypeptide (if present) comprises the TM3 domain of CD63. In some embodiments, each of these anchor polypeptides comprises the TM3 domain of CD63; preferably, the TM3 domain of CD63 comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 9.

[0020] In some embodiments, the EGF polypeptide, the NGF polypeptide, the PDGF-BB polypeptide and the IDR-1018 polypeptide (if present) are optionally fused to the N-terminus or C-terminus of the corresponding anchor polypeptide via a peptide linker, preferably, the peptide linker is composed of glycine and serine, for example, (G4S)n, where n is an integer from 1 to 3.

[0021] In some embodiments, the exosomes are not derived from mesenchymal stem cells; and the exosomes are not derived from stem cells.

[0022] Another aspect of the present disclosure relates to a composition comprising an exosome and a carrier according to the present specification; preferably, the composition is a liquid formulation; more preferably, the composition is prepared for topical or subcutaneous administration; and more preferably, the composition is a pharmaceutical composition.

[0023] In some embodiments, the composition does not include an EGF polypeptide (e.g., human EGF polypeptide), an NGF polypeptide (e.g., human NGF polypeptide), a PDGF-BB polypeptide (e.g., human PDGF-BB polypeptide), or an IDR-1018 polypeptide that is not attached to the membrane of the exosome.

[0024] Another aspect of the present disclosure relates to a nucleic acid structure, said nucleic acid structure comprising a polynucleotide encoding a substance such as (a) an EGF polypeptide fused with a first anchor polypeptide, (b) an NGF polypeptide fused with a second anchor polypeptide, and (c) a PDGF-BB polypeptide fused with a third anchor polypeptide.

[0025] In some embodiments, the nucleic acid structure also codes for an IDR-1018 polypeptide fused with a fourth anchor polypeptide comprising an amino acid sequence indicated by (d) SEQ ID NO. 4.

[0026] In some embodiments, the EGF polypeptide is a human EGF polypeptide or a parallel homolog or a serial homolog thereof, and preferably, the EGF polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 1.

[0027] In some embodiments, the NGF polypeptide is a human NGF polypeptide or a parallel homolog or a serial homolog thereof, and preferably, the NGF polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 2.

[0028] In some embodiments, the PDGF-BB polypeptide is a human PDGF-BB dimer; more preferably, the PDGF-BB dimer comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 3.

[0029] In some embodiments, the polynucleotide encoding the EGF polypeptide has a nucleotide sequence indicated by SEQ ID NO. 5 or a degenerate sequence thereof.

[0030] In some embodiments, the polynucleotide encoding the NGF polypeptide has a nucleotide sequence indicated by SEQ ID NO. 6 or a degenerate sequence thereof.

[0031] In some embodiments, the polynucleotide encoding the PDGF-BB polypeptide has a nucleotide sequence indicated by SEQ ID NO. 7 or a degenerate sequence thereof.

[0032] In some embodiments, the polynucleotide encoding the IDR-1018 polypeptide has a nucleotide sequence indicated by SEQ ID NO. 8 or a degenerate sequence thereof.

[0033] In some embodiments, these anchor polypeptides are exosome membrane proteins, membrane targeting sequences, or fragments of their anchoring function. Exemplary exosome membrane proteins include, but are not limited to, tetraspanins such as lamp2b, CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative modulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid anchor proteins.

[0034] In a preferred embodiment, these anchor polypeptides comprise full-length CD63 or truncated CD63 in which the TM3 domain is preserved. In a preferred embodiment, each of the first, second, and third anchor polypeptides and the fourth anchor polypeptide (if present) comprises the TM3 domain of CD63. In a preferred embodiment, each of the first, second, and third anchor polypeptides and the fourth anchor polypeptide (if present) comprises the TM3 domain of CD63. In some embodiments, the polynucleotide encoding the TM3 domain of CD63 has a nucleotide sequence indicated by SEQ ID NO. 10 or a degenerate sequence thereof.

[0035] In some embodiments, the polynucleotide is a single polynucleotide comprising a nucleotide fragment encoding polypeptides (a), (b), (c) and optionally (d), wherein each of polypeptides (a), (b), (c) and optionally (d) is linked to an anchor polypeptide; preferably, polypeptides (a), (b), (c) and optionally (d) are separated by a self-cleaving peptide; optionally, such self-cleaving peptide is a 2A peptide, such as T2A, E2A, P2A, or any combination thereof.

[0036] Another aspect of the present disclosure relates to a vector, said vector comprising a nucleic acid structure according to any one of the nucleic acid structures according to the present specification.

[0037] Another aspect of the present disclosure relates to a cell transfected with a vector according to the present specification, wherein the polynucleotide is incorporated into the genome of the cell.

[0038] In some embodiments, the cell is not a mesenchymal stem cell; preferably, the cell is not a stem cell; preferably, the cell is a mammalian cell; or more preferably, the cell is an HEK293 or CHO cell.

[0039] Another aspect of the present disclosure relates to a method for generating engineered exosomes disclosed herein, the method comprising: (a) transfecting a cell as described above with a vector as described above; (b) culturing the cell under conditions that allow exosomes to be secreted from the cell; and (c) collecting and purifying the exosomes.

[0040] In some embodiments, the method further includes the step of adapting the cells to serum-free conditions during step (b).

[0041] Another aspect of the present disclosure relates to the use of engineered exosomes or compositions disclosed herein in the manufacture of drugs for improving wound healing or treating wounds; preferably, said wounds are chronic wounds, preferably diabetic wounds; more preferably, said wounds include diabetic foot ulcers.

[0042] Another aspect of the present disclosure relates to a method for improving wound healing or treating a wound; preferably, the wound is a chronic wound, preferably a diabetic wound; more preferably, the wound includes a diabetic foot ulcer.

[0043] Another aspect of the present disclosure relates to engineered exosomes or compositions disclosed herein used to improve wound healing or treat wounds; preferably, the wound is a chronic wound, preferably a diabetic wound; more preferably, the wound comprises a diabetic foot ulcer.

[0044] Another aspect of the present disclosure relates to the use of engineered exosomes or compositions as disclosed herein in the manufacture of drugs that inhibit Propionibacterium acnes (also known as Cutibacterium acnes, or Propionibacterium acnes) infection (e.g., on the skin); preferably, said Propionibacterium acnes infection is acne or acne-prone skin, such as scars or skin damage caused by acne.

[0045] These aspects and other advantages of the present disclosure will become apparent from the specific embodiments provided below. Brief explanation of the drawing

[0046] Fig. 1. Construction of a stable cell line secreting engineered exosomes loaded with functional proteins. Mammalian cells (e.g., HEK293 cells) were cultured and infected with a lentivirus containing the functional genes EGF, NGF, PDGF-BB, and IDR-1018. Stable cell lines were selected using the following antibiotics. After three subcultures and selection, expression of the functional genes was detected in both the cell sediment and the exosomes. After confirming expression, the stable cell lines were adaptively cultured under serum-free conditions. Finally, the culture supernatant of the serum-free stable cell lines was collected, and the purified exosomes were analyzed via ultracentrifugation. Fig. 2. Emotion of engineered exosomes derived from stable cell lines. (a) The particle size and particle concentration of exosomes derived from stable cell lines were analyzed using NanoFCM. (b) Exosomes were further examined using a transmission electron microscope (TEM). (c) Immunoblot analysis was performed on exosomes using an antibody against anti-CD63, a scaffold protein fused with exosomes. Fig. 3.Preparation of HSF cells (human skin fibroblasts) used for scratch damage measurement. Once aggregated with HSF cells, a cell layer was scraped in a straight line using a 1 mm pipette tip. After scraping, the cell monolayer was gently washed to remove the detached cells, and then fresh culture medium (Mock) or exosomes (No. 1, No. 37, No. 39) were added, respectively. Images were then captured at various time intervals (0 hours, 24 hours, 48 ​​hours) using a 10x magnification microscope. Fig. 4. Construction of a rat wound healing model for diabetic foot ulcers (DFU). A DFU (diabetic foot ulcer) model was constructed using Sprague-Dawley rats via streptozotocin and skin biopsy punch. (a) This is a schematic diagram of the DFU rat model construction and intradermal administration design. Blood glucose levels from animals of different groups (n=5) within the DFU model. (b) and weight (c) It is illustrated at different time points. These groups include a healthy control group (Health-Vehicle), a DFU model group (Model-Vehicle), and a DFU model group treated with exosomes (NO. 38) or (NO. 40). IP, intraperitoneal injection; ID, intradermal injection. Fig. 5. Wound healing status at various time points after treatment with exosomes (NO. 38) or (NO. 40). Wound healing rates over 13 days were calculated for each group (n=5), including a healthy control group (Health-Vehicle), a DFU model group (Model-Vehicle), and a DFU model group treated with exosomes (NO. 38) or (NO. 40). Data are expressed as mean ± SD. * indicates p<0.05 compared to the control group. Fig. 6.Promotion of vascular and nerve regeneration by engineered exosomes in a rat DFU model. Wound tissues from DFU rats were collected at the end of the study. HE staining was analyzed in the DFU model group (Model-Vehicle) and the DFU model group treated with exosomes (NO. 40). The number of blood vessels (AC) was determined using CD31, and nerve regeneration was indicated using PGP9.5 as the detection marker (DF). Fig. 7. Normal wound healing status at different time points after treatment with exosomes (NO. 40). (A) The normal wound healing rate over 16 days was calculated for each group (n=6), including the model group (Vehicle) and the model group treated with exosomes (NO. 40). (BC) Tissue sections from the injured area were prepared at the end of the study (day 16) and used for HE staining, and scabs and inflammation-inducing cells were analyzed. Data are expressed as mean ± SD. * indicates p<0.05 compared to the control group. Specific details for implementing the invention

[0047] definition

[0048] When used with the word “include” in this specification, including claims, the words “one” and “one” indicate “one or more”.

[0049] As used herein, the terms “or” and “and / or” are used to describe combinations or mutual exclusivity of various components. For example, “x, y and / or z” may mean alone “x,” alone “y,” alone “z,” “x, y and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” Specifically, x, y, or z may be explicitly excluded from the embodiments.

[0050] In this application, the term “about” is used in accordance with the ordinary and general meaning in the field of cell and molecular biology and refers to a value including the standard deviation of error of the device or method used to determine the said value.

[0051] “Homology,” “identity,” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions of each comparable sequence for comparison purposes. If the positions of the sequences being compared are occupied by the same base or amino acid, the molecules have homology at those positions. The degree of homology between sequences varies depending on the number of matching or identical positions the sequences have. “Unrelated” or “non-homologous” sequences have less than 40% identity with one of the sequences of the present disclosure, but preferably less than 25% identity.

[0052] Having “sequence identity” of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence at a specific percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that when comparing two sequences, the percentage of bases (or amino acids) is the same. Such comparison and percentage of homology or sequence identity may be determined using software programs known in the art.

[0053] As used herein, the term “linker” means a short fragment of an amino acid (AA) or a sequence of nucleotides containing two or more identical or different amino acids or nucleotides.

[0054] As used herein, “cell line” means a population of cells formed through one or more passages of a primary cell culture. Each passage is referred to as a passage. After cells have been passaged, they are referred to as “passed” cells. A specific cell population or cell line is sometimes indicated or characterized by the number of passages. For example, a cultured cell population that has been passaged 10 times may be referred to as a P10 culture. The primary culture, that is, the first culture after the cells are isolated from the tissue, is designated as P0. After the first passage, the cells are described as a second-generation culture (P1 or the first generation). After the second passage, the cells become a third-generation culture (P2 or the second generation), and so on. Those skilled in the art will understand that many population doublings may occur during passage; therefore, the number of population doublings of the culture is greater than the number of passages. Cell amplification during subculture (e.g., population doubling) is determined by many factors, including but not limited to inoculation density, substrate, culture medium, growth conditions, and subculture interval time.

[0055] When comparing any symptoms appearing in an untreated subject and a treated subject, the terms “reduction,” “suppression,” “relief,” “decrease,” “descent,” “prevention,” and their grammatical equivalents (“lower,” “smaller,” etc.) mean that the number and / or severity of symptoms in the treated subject is lower than the number and / or severity of symptoms in the untreated subject, and that this difference is clinically relevant to all healthcare professionals. In one embodiment, the number and / or severity of symptoms in the treated subject is at least 10%, at least 25%, at least 50%, at least 75%, and / or at least 90% lower than the number and / or severity of symptoms in the untreated subject.

[0056] As used herein, the term “therapeutic effective dose” is synonymous with “effective dose,” “therapeutic effective dose,” and / or “effective dose,” and refers to an amount of compound that induces a desired biological, cosmetic, or clinical response in the subject in need. For example, an effective dose is an amount sufficient to reduce hair loss. In a specific application of the method of this disclosure, the appropriate effective dose to be administered may be determined by a person skilled in the art using the guidelines provided herein. For example, the effective dose may be inferred from in vitro and in vivo measurements as described herein. A person skilled in the art will understand that the subject’s condition may be monitored throughout the course of treatment, and the effective dose of the exosome or composition disclosed herein may be adjusted accordingly.

[0057] As used herein, the term “treatment” means an intervention that is an attempt to alter the natural processes of the individual or cell being treated, and may be performed for prevention or treatment during the pathological process of a disease or pathology. Treatment may achieve one or more of various desirable outcomes, including prevention of disease onset or recurrence, alleviation of symptoms, reduction of the direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement or alleviation of the disease state, alleviation or improvement of the prognosis, etc.

[0058] As used herein, the term “subject” may be used interchangeably with the terms “entity” or “patient” and generally refers to an entity requiring treatment. The subject may be a human, dog, cat, horse, pig, or mammal such as a rodent.

[0059] As used herein, “carrier” means a diluent, adjuvant, excipient, or medium administered together with the therapeutic agent. Such drug carriers may be sterile liquids such as physiological saline and oils including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, and analogs thereof. When the composition is administered by intravenous injection, a physiological saline solution is a preferred carrier. In addition to physiological saline solutions, glucose and glycerol solutions may also be used as liquid carriers and are particularly suitable for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc powder, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol, and analogs thereof. Optionally, the composition may further contain small amounts of a wetting agent, emulsifier, or pH buffer. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and similar forms. These compositions contain a therapeutically effective amount of a compound, preferably in tablet form, and an appropriate amount of a carrier to provide the patient with a suitable mode of administration. The formulation must be suitable for the mode of administration.

[0060] The term “N-terminal amino acid residue” or “N-terminus” refers to the first amino acid residue (amino acid 1) of a polypeptide or peptide. The term “C-terminal amino acid residue” or “C-terminus” refers to the last amino acid residue (amino acid n, where n is the total number of residues in the peptide or polypeptide).

[0061] The term “EGF” stands for Epidermal Growth Factor and is a growth factor that stimulates cell growth, proliferation, and differentiation by binding to the receptor EGFR. It has been proven to be used as an effective mitotic promoter by stimulating the synthesis of mRNA, DNA, and proteins in epithelial cells. It is a single-strand polypeptide composed of 53 amino acids derived from the cleavage of the large precursor protein EGF. Currently, EGF is known as the primitive form of the Group I EGF family, which further includes transforming growth factor-α (TGF-α), heparin-bound EGF (HB-EGF), amphiregulin, betacellulin, epiregulin, and epidermal growth factor. Structurally, they all contain one or more EGF repeat sequences (EGF motifs) in their extracellular domains, and these repeat sequences are 35 to 40 amino acid sequences with six conserved cysteines spaced apart in the pattern CX7CX3-5CX10-12CXCX5GXRC (C, cysteine; G, glycine; R, arginine; X, other amino acid). One glycine and one arginine in the sequence are conserved in all EGF-related growth factors, but are not conserved in proteins containing EGF motifs that lack growth factor activity. These six cysteines pair to form three intramolecular disulfide bonds with C1-C3, C2-C4, and C5-C6 interactions (numbered according to sequence order), which are important for maintaining their biological activity. Functionally, these growth factors have the ability to bind to the same receptor, namely the EGF receptor (EGFR, ErbB1), activate their intrinsic tyrosine kinase activity, and link said receptor to downstream signaling pathways that control cell proliferation, differentiation, survival, or motility. The amino acid sequence of human EGF can be obtained from GenBank: AAS83395.1, which is also SEQ ID NO. in this disclosure.It is displayed as 1.

[0062] The term “NGF” refers to Nerve Growth Factor (NGF), the first discovered member of the neurotrophic factor family, collectively known as Neurotrophins, which includes Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), and Neurotrophin 4 / 5. These factors possess significant structural homology and originate from a common ancestral gene. Studies have shown that NGF not only has a protective effect on the survival of degenerative peripheral neurons but also on the regulation of neurotransmitter and neuropeptide synthesis in sympathetic and sensory neurons. The administration of exogenous NGF influences neuronal plasticity, enabling the adult nervous system to alter its structure and function in response to stimuli. Furthermore, studies have shown that constitutive synthesis of NGF in adult tissues correlates with phenotypic characteristics of peripheral nervous system (PNS) neurons, such as neural distribution density, cell body size, axon formation, dendritic growth, and the induction and / or inhibition of neuropeptides, neurotransmitters, or neurotransmitter-producing enzymes. NGF plays a crucial role in the growth and survival of many brain neurons, as well as peripheral sensory and sympathetic neurons, particularly basal forebrain cholinergic neurons (BFCNs), which are one of the major target cells of NGF in the central nervous system (CNS). The amino acid sequence of human NGF is available from GenBank: AAA59931.1, where amino acids 122–239 represent the mature peptide, which is also indicated as part of SEQ ID NO. 2 in this disclosure.

[0063] The term “PDGF” stands for Platelet-Derived Growth Factor, which is an effective mitotic promoter and chemical attractant used in various mesenchymal-derived cells (e.g., fibroblasts, activated macrophages, and smooth muscle cells). PDGF plays a crucial role in angiogenesis, embryonic development, inflammation, and cell differentiation; therefore, it may be beneficial for wound healing. The growth factor PDGF family consists of four distinct polypeptide chains encoded by four different genes: the classic PDGF-A and PDGF-B chains, and the recently discovered PDGF-C and PDGF-D. These four PDGF chains assemble into disulfide-linked dimers through homomerization or heteromerization, and to date, five distinct dimeric isoforms have been described: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. Two distinct PDGF receptors (α and β) mediate the action of PDGF on target cells. PDGF-A and PDGF-C chains selectively bind to α receptors, whereas PDGF-D binds preferentially to β receptors, and PDGF-B exhibits similar affinity for both receptors. Receptor activation requires PDGF-induced receptor dimerization, which results in the transphosphorylation of tyrosine. PDGF-AA induces only α / α receptor dimers, PDGF-AB induces α / α and α / β dimers, and PDGF-BB induces all three combinations. PDGF-C binds only to α receptors but can generate α-β heteromers through the transactivation of β receptors. A potential application of the PDGF-BB protein is to induce wound healing in diabetic patients. Additionally, compositions containing PDGF can promote the healing of damaged or deteriorated bones by stimulating connective tissue growth, bone growth, and collagen synthesis in damaged bone sites. The amino acid sequence of human PDGF-B is GenBank: CAA02294.It can be obtained from 1, wherein amino acids 82 to 190 represent mature peptides, which are also indicated by SEQ ID NO. 3 of the present disclosure.

[0064] The term “innate defense regulatory peptide 1018 (IDR-1018)” is a cationic peptide with 12 amino acid residues (VRLIVAVRIWRR-NH2). Due to its similarity to innate defense regulators, its antimicrobial and immunomodulatory activities have been studied. IDR-1018 possesses anti-infectious, anti-inflammatory, wound healing (in rats), and anti-biometabolic activities and has been demonstrated, for example, to reduce the tumor necrosis factor-α response induced by lipopolysaccharides.

[0065] Unless otherwise specified, as used herein, the term “wound healing improvement” means an improved degree of wound healing compared to wound healing without the use of the method and / or composition. In some embodiments, wound healing improvement includes improved fibroblast proliferation and / or fibroblast migration. In some other embodiments, wound healing improvement includes increased wound closure and / or reduced time required for wound healing completion. Those skilled in the art are well aware of other methods for evaluating wound healing improvement, and improved wound healing according to the present invention can be demonstrated through cell proliferation measurements, such as scratch measurements and / or epithelial cell proliferation measurements, for example.

[0066] Members of the tetraspanin family (e.g., CD63, CD81, and CD9) are universally expressed in exosomes and are widely used as exosome biomarkers involved in physiological processes such as cell adhesion, cell migration, and signal transduction. CD63 (whose primary characteristic is that it is a tetraspanin) has two extracellular loops of different sizes and two short cytoplasmic domains and participates in signal transduction processes in various types of immune cells. Deletion of the continuous domain demonstrates that transmembrane helix 3 (TM3) is necessary and sufficient for membrane anchoring and exosome targeting. The amino acid sequence of human CD63 is available from GenBank: AHI51903.1, and the amino acid sequence of TM3 corresponds to amino acids 70 through 133, which is also indicated by SEQ ID NO. 9 of the present disclosure.

[0067] The term “anchor polypeptide” is a polypeptide that anchors to the exosome membrane when the exosome is produced by a cell. Transmembrane proteins are typical anchor polypeptides in the specification of this disclosure. “Anchor” or grammatical variants thereof mean that at least one fragment of the polypeptide is inserted into the exosome membrane. The anchor polypeptide may be inserted into the exosome membrane completely or partially. In this disclosure, the anchor polypeptide is fused with a polypeptide that is heterogeneous to the exosome naturally produced in the same cell, such as the EGF polypeptide. An exemplary anchor polypeptide is an exosome membrane protein, a membrane targeting sequence, or a fragment of its anchor function. Exemplary exosome membrane proteins include, but are not limited to, tetraspanins such as lamp2b, CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative modulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid anchor proteins. Detailed descriptions of GPI anchors within exosomes can be obtained, for example, from the literature of Michel Vidal, “Exosomes and GPI-anchored proteins: Judicious pairs for investigating biomarkers from body fluids,” Advanced Drug Delivery Reviews, Vol. 161–162, 2020 (the full text of which is incorporated herein by reference). In a preferred embodiment of the present disclosure, the anchor polypeptide comprises or is composed of transmembrane helix 3 (TM3) of the CD63 protein.

[0068] Engineered Exosomes

[0069] One aspect of the present disclosure relates to an engineered exosome, said engineered exosome comprising (a) an EGF polypeptide fused to a first anchor polypeptide, (b) an NGF polypeptide fused to a second anchor polypeptide, and (c) a PDGF-BB polypeptide fused to a third anchor polypeptide, wherein (a), (b) and (c) are anchored to the membrane of the exosome through the first, second, and third anchor polypeptides, respectively, said EGF polypeptide, said NGF polypeptide and said PDGF-BB polypeptide are exposed to the outer surface of the membrane of the exosome.

[0070] In a preferred embodiment, the engineered exosome provided in the present disclosure comprises (a) an EGF polypeptide fused to a first anchor polypeptide, (b) an NGF polypeptide fused to a second anchor polypeptide, (c) a PDGF-BB polypeptide fused to a third anchor polypeptide, and (d) an IDR-1018 polypeptide fused to a fourth anchor polypeptide, wherein (a), (b), (c) and (d) are anchored to the membrane of the exosome through the first, second, third, and fourth anchor polypeptides, respectively, and wherein the EGF polypeptide, the NGF polypeptide, the PDGF-BB polypeptide and the IDR-1018 polypeptide are exposed to the outer surface of the membrane of the exosome.

[0071] In the present disclosure, the EGF polypeptide is a human EGF polypeptide or a parallel homolog or a serial homolog thereof. In some embodiments, the EGF polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence represented by SEQ ID NO. 1. In some embodiments, the EGF polypeptide comprises the amino acid sequence represented by SEQ ID NO. 1. In some embodiments, the EGF polypeptide is basically composed of the amino acid sequence represented by SEQ ID NO. 1.

[0072] In a preferred embodiment, the NGF polypeptide is a human NGF polypeptide or a parallel homolog or a serial homolog thereof. In some embodiments, the NGF polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by SEQ ID NO. 2. In some embodiments, the NGF polypeptide comprises the amino acid sequence indicated by SEQ ID NO. 2. In some embodiments, the NGF polypeptide is basically composed of the amino acid sequence indicated by SEQ ID NO. 2.

[0073] In a preferred embodiment, the PDGF-BB polypeptide is a human PDGF-BB dimer or a parallel homolog or a serial homolog thereof. In some embodiments, the PDGF-BB polypeptide is a human PDGF-BB dimer. In some embodiments, the PDGF-BB polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by SEQ ID NO. 3. In some embodiments, the PDGF-BB polypeptide comprises the amino acid sequence indicated by SEQ ID NO. 3. In some embodiments, the PDGF-BB polypeptide is basically composed of the amino acid sequence indicated by SEQ ID NO. 3.

[0074] In some embodiments, these anchor polypeptides are exosome membrane proteins, membrane targeting sequences, or fragments of their anchoring function. Exemplary exosome membrane proteins include, but are not limited to, tetraspanins such as lamp2b, CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative modulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid anchor proteins. In a preferred embodiment of the present disclosure, the first, second, third, and fourth anchor polypeptides include the TM3 domain of CD63. In a preferred embodiment, each of the first, second, third, and fourth anchor polypeptides includes the TM3 domain of CD63. In some embodiments, these anchor polypeptides are full-length CD63 proteins, such as full-length human CD63 (e.g., UniProtKB / Swiss-Prot: F8VZE2, P08962, Q5TZP3, Q8N6Z9, or Q9UCG6). In some embodiments, these anchor polypeptides comprise a TM3 domain and at least one truncated CD63 protein among the TM1, TM2, and TM4 domains. For example, the anchor polypeptides may consist of TM2 and TM3 of CD63; TM3 and TM4 of CD63; or TM1, TM2, and TM3 of CD63. In some embodiments, these anchor polypeptides consist of the TM3 domain of CD63. In the present disclosure, the first, second, third, and fourth anchor polypeptides may be different or identical. In a preferred embodiment, the first, second, third, and fourth anchor polypeptides are identical and consist of the TM3 domain of CD63.

[0075] In a preferred embodiment, the TM3 domain of the CD63 comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by SEQ ID NO. 9. In a preferred embodiment, the TM3 domain of the CD63 comprises the amino acid sequence indicated by SEQ ID NO. 9. In a preferred embodiment, the TM3 domain of the CD63 is basically composed of the amino acid sequence indicated by SEQ ID NO. 9.

[0076] Accordingly, in a preferred embodiment, an engineered exosome is provided, said engineered exosome comprising (a) an EGF polypeptide fused to a first anchor polypeptide, (b) an NGF polypeptide fused to a second anchor polypeptide, and (c) a PDGF-BB polypeptide fused to a third anchor polypeptide, wherein each of the first, second, and third anchor polypeptides comprises a TM3 domain of CD63, said engineered exosome comprising (a), (b), and (c) respectively anchored to the membrane of said exosome through the first, second, and third anchor polypeptides, said EGF polypeptide, said NGF polypeptide, and said PDGF-BB polypeptide are exposed to the outer surface of the membrane of said exosome.

[0077] In a preferred embodiment, an engineered exosome is provided, said engineered exosome comprising (a) a human EGF polypeptide fused to a first anchor polypeptide, (b) a human NGF polypeptide fused to a second anchor polypeptide, and (c) a human PDGF-BB polypeptide fused to a third anchor polypeptide, wherein each of the first, second, and third anchor polypeptides comprises a TM3 domain of CD63, said engineered exosome comprising (a), (b), and (c) respectively anchored to the membrane of said exosome through the first, second, and third anchor polypeptides, said human EGF polypeptide, said human NGF polypeptide, and said human PDGF-BB polypeptide are exposed to the outer surface of the membrane of said exosome.

[0078] In a preferred embodiment, an engineered exosome is provided, wherein the engineered exosome comprises: (a) an EGF polypeptide fused with a first anchor polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 1; (b) an NGF polypeptide fused with a second anchor polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by SEQ ID NO. 2; and (c) SEQ ID NO. A PDGF-BB polypeptide fused with a third anchor polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by 3, wherein each of the first, second, and third anchor polypeptides comprises a TM3 domain of CD63, wherein (a), (b), and (c) are anchored to the membrane of the exosome through the first, second, and third anchor polypeptides, respectively, wherein the EGF polypeptide, the NGF polypeptide, and the PDGF-BB polypeptide are exposed to the outer surface of the exosome membrane.

[0079] In a preferred embodiment, an engineered exosome is provided, wherein the engineered exosome comprises: (a) an EGF polypeptide fused with a first anchor polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 1; (b) an NGF polypeptide fused with a second anchor polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by SEQ ID NO. 2; and (c) SEQ ID NO. A PDGF-BB polypeptide fused with a third anchor polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by 3, wherein each of the first, second, and third anchor polypeptides comprises a TM3 domain of CD63, and said domain is SEQ ID NO. It includes an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by 9, wherein (a), (b), and (c) are anchored to the membrane of the exosome through the first, second, and third anchor polypeptides, respectively, wherein the EGF polypeptide, the NGF polypeptide, and the PDGF-BB polypeptide are exposed on the outer surface of the membrane of the exosome.

[0080] In a preferred embodiment, an engineered exosome is provided, said engineered exosome comprising (a) an EGF polypeptide fused with a first anchor polypeptide having an amino acid sequence indicated by SEQ ID NO. 1, (b) an NGF polypeptide fused with a second anchor polypeptide having an amino acid sequence indicated by SEQ ID NO. 2, and (c) a PDGF-BB polypeptide fused with a third anchor polypeptide having an amino acid sequence indicated by SEQ ID NO. 3, wherein each of the first, second, and third anchor polypeptides is composed of a TM3 domain of CD63, said domain having an amino acid sequence indicated by SEQ ID NO. 9, said anchor polypeptides are anchored to the membrane of the exosome through the first, second, and third anchor polypeptides, said anchor polypeptides are each exposed to the outer surface of the exosome membrane.

[0081] In a preferred embodiment, an engineered exosome is provided, said engineered exosome comprising (a) an EGF polypeptide fused to a first anchor polypeptide, (b) an NGF polypeptide fused to a second anchor polypeptide, (c) a PDGF-BB polypeptide fused to a third anchor polypeptide, and (d) an IDR-1018 polypeptide fused to a fourth anchor polypeptide, wherein each of the first, second, third, and fourth anchor polypeptides comprises a TM3 domain of CD63, said (a), (b), (c), and (d) are anchored to the membrane of said exosome through the first, second, third, and fourth anchor polypeptides, said EGF polypeptide, said NGF polypeptide, said PDGF-BB polypeptide, and said IDR-1018 polypeptide are exposed to the outer surface of said exosome membrane.

[0082] In a preferred embodiment, an engineered exosome is provided, said engineered exosome comprising (a) a human EGF polypeptide fused to a first anchor polypeptide, (b) a human NGF polypeptide fused to a second anchor polypeptide, (c) a human PDGF-BB polypeptide fused to a third anchor polypeptide, and (d) a human IDR-1018 polypeptide fused to a fourth anchor polypeptide, wherein each of the first, second, third, and fourth anchor polypeptides comprises a TM3 domain of CD63, said (a), (b), (c), and (d) are anchored to the membrane of said exosome through the first, second, third, and fourth anchor polypeptides, said human EGF polypeptide, said human NGF polypeptide, said PDGF-BB polypeptide, and said IDR-1018 polypeptide are exposed to the outer surface of said exosome membrane.

[0083] In a preferred embodiment, an engineered exosome is provided, wherein the engineered exosome comprises: (a) an EGF polypeptide fused with a first anchor polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 1; (b) an NGF polypeptide fused with a second anchor polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by SEQ ID NO. 2; and (c) SEQ ID NO. A PDGF-BB polypeptide fused with a third anchor polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by 3, and (d) SEQ ID NO. It comprises an IDR-1018 polypeptide fused with a fourth anchor polypeptide having an amino acid sequence indicated by 4, wherein each of the first, second, third, and fourth anchor polypeptides comprises a TM3 domain of CD63, wherein (a), (b), (c) and (d) are anchored to the membrane of the exosome through the first, second, third, and fourth anchor polypeptides, respectively, wherein the EGF polypeptide, the NGF polypeptide, the PDGF-BB polypeptide, and the IDR-1018 polypeptide are exposed to the outer surface of the membrane of the exosome.

[0084] In a preferred embodiment, an engineered exosome is provided, wherein the engineered exosome comprises: (a) an EGF polypeptide fused with a first anchor polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 1; (b) an NGF polypeptide fused with a second anchor polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by SEQ ID NO. 2; and (c) SEQ ID NO. (d) a PDGF-BB polypeptide fused with a third anchor polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by 3, and an IDR-1018 polypeptide fused with a fourth anchor polypeptide comprising an amino acid sequence indicated by SEQ ID NO. 4, wherein each of the first, second, third, and fourth anchor polypeptides comprises a TM3 domain of CD63, said domain being SEQ ID NO. It includes an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence indicated by 9, wherein (a), (b), (c), and (d) are anchored to the membrane of the exosome through the first, second, third, and fourth anchor polypeptides, respectively, wherein the EGF polypeptide, the NGF polypeptide, the PDGF-BB polypeptide, and the IDR-1018 polypeptide are exposed on the outer surface of the membrane of the exosome.

[0085] In a preferred embodiment, an engineered exosome is provided, said engineered exosome comprising (a) an EGF polypeptide fused with a first anchor polypeptide having an amino acid sequence indicated by SEQ ID NO. 1, (b) an NGF polypeptide fused with a second anchor polypeptide having an amino acid sequence indicated by SEQ ID NO. 2, (c) a PDGF-BB polypeptide fused with a third anchor polypeptide having an amino acid sequence indicated by SEQ ID NO. 3, and (d) an IDR-1018 polypeptide comprising an amino acid sequence indicated by SEQ ID NO. 4, wherein each of the first, second, third, and fourth anchor polypeptides is composed of a TM3 domain of CD63, said domain being SEQ ID NO. Having an amino acid sequence indicated by 9, where (a), (b), (c) and (d) are anchored to the membrane of the exosome through the first, second, third, and fourth anchor polypeptides, respectively, where the EGF polypeptide, the NGF polypeptide, the PDGF-BB polypeptide and the IDR-1018 polypeptide are exposed on the outer surface of the membrane of the exosome.

[0086] In any of the above embodiments, the EGF polypeptide, the NGF polypeptide, the PDGF-BB polypeptide, and the IDR-1018 polypeptide (if present) are each fused to the C-terminus of the first, second, third, and fourth anchor polypeptides, either directly or through a peptide linker. The peptide linker may be any peptide linker available in the art used to link different domains or functional regions within the fusion protein. In a preferred embodiment, the peptide linker is composed of glycine and serine, for example, (G4S)n, where n is an integer from 1 to 3.

[0087] In the present disclosure, preferably, the exosomes are not derived from mesenchymal stem cells. More preferably, the exosomes are not derived from stem cells. In a preferred embodiment, the exosomes provided in the present invention are derived from non-stem cells such as CHO or HEK293 cells. In the present disclosure, preferably, the exosomes are purified and / or isolated from the cells from which they are derived.

[0088] The engineered exosomes provided in the present invention demonstrated significant improvements in wound healing, and some exosome treatments exhibited effects similar to those of healthy controls. These results are surprising because, compared to acute wounds, chronic wounds such as diabetic foot ulcers are very difficult to heal due to changes in wound healing dynamics. These results are expected to be achieved, at least in part, through the combination of polypeptides presented on the exosomes. In the present disclosure, at least three polypeptides (i.e., EGF, NGF, and PDGF-BB) are presented on the surface of exosomes via anchor polypeptides. When this combination of polypeptides is displayed on the surface of exosomes, it is expected to exhibit superior effects compared to any single polypeptide. For example, in terms of wound treatment or improvement of wound healing, engineered exosomes displaying EGF, NGF, and PDGF-BB are more effective than engineered exosomes displaying EGF, NGF, or PDGF-BB alone.

[0089] Method and composition for improving wound healing

[0090] Each aspect of the present disclosure relates to a method and composition for improving wound healing or treating a wound. In some embodiments, the wound is an acute wound, such as a severe burn, extensive skin loss, damage resulting from post-surgical intervention or radiation therapy. In a preferred embodiment, the wound is a chronic wound. In a preferred embodiment, the chronic wound is an unhealed ulcer or a diabetic wound; more preferably, the wound includes a diabetic foot ulcer.

[0091] In a preferred embodiment, a method for treating chronic wounds is provided, said method comprising the step of administering a therapeutically effective amount of engineered exosomes or compositions disclosed herein to a subject in need thereof.

[0092] In a preferred embodiment, a method for treating diabetic foot ulcers is provided, the method comprising the step of administering a therapeutically effective amount of engineered exosomes or compositions disclosed herein to a subject in need thereof.

[0093] In the present disclosure, the provided composition comprises the engineered exosomes and a carrier. In a preferred embodiment, the composition is a liquid formulation. In a preferred embodiment, the composition is prepared for topical or subcutaneous administration. In a preferred embodiment, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In a preferred embodiment, the composition is a cosmetic composition comprising a cosmetically acceptable carrier.

[0094] In a preferred embodiment, the composition does not contain EGF polypeptide, NGF polypeptide, PDGF-BB polypeptide, or IDR-1018 polypeptide (i.e., free polypeptide) that is not attached to the membrane of the exosome. For example, in addition to the polypeptide anchored to the membrane of the engineered exosome, no additional EGF polypeptide, NGF polypeptide, PDGF-BB polypeptide, or IDR-1018 polypeptide is added to or supplemented to the composition.

[0095] In some embodiments, the composition is a cosmetic composition. In some embodiments, the composition is a non-cosmetic composition. In some embodiments, the composition is a pharmaceutical composition.

[0096] Nucleic acid constructs, vectors, cells, and methods of generation

[0097] Each aspect of the present disclosure also relates to a nucleic acid structure encoding a polypeptide anchored to an exosome.

[0098] In some embodiments, a nucleic acid structure is provided, said nucleic acid structure comprises a polynucleotide encoding a substance such as (a) an EGF polypeptide fused with a first anchor polypeptide, (b) an NGF polypeptide fused with a second anchor polypeptide, and (c) a PDGF-BB polypeptide fused with a third anchor polypeptide.

[0099] In some embodiments, a nucleic acid structure is provided, said nucleic acid structure comprises a polynucleotide encoding a substance such as (a) an EGF polypeptide fused to a first anchor polypeptide, (b) an NGF polypeptide fused to a second anchor polypeptide, (c) a PDGF-BB polypeptide fused to a third anchor polypeptide, and (d) an IDR-1018 polypeptide fused to a fourth anchor polypeptide.

[0100] In some embodiments, a set of three nucleic acid structures is provided, wherein the first nucleic acid structure comprises a polynucleotide encoding (a), the second nucleic acid structure comprises a polynucleotide encoding (b), and the third nucleic acid structure comprises a polynucleotide encoding (c), wherein (a), (b) and (c) are as defined above.

[0101] In some embodiments, a set of four nucleic acid structures is provided, wherein the first nucleic acid structure comprises a polynucleotide encoding (a), the second nucleic acid structure comprises a polynucleotide encoding (b), the third nucleic acid structure comprises a polynucleotide encoding (c), and the fourth nucleic acid structure comprises a polynucleotide encoding (d), wherein (a), (b), (c) and (d) are as defined above.

[0102] In some embodiments, a set of two nucleic acid structures is provided, wherein one nucleic acid structure comprises a polynucleotide encoding two of (a), (b) and (c), and the other nucleic acid structure comprises a polynucleotide encoding the remaining polypeptide, wherein (a), (b) and (c) are as defined above.

[0103] In some embodiments, a set of two nucleic acid structures is provided, wherein one nucleic acid structure comprises a polynucleotide encoding two of (a), (b), (c) and (d), and the other nucleic acid structure comprises a polynucleotide encoding the remaining two, wherein (a), (b), (c) and (d) are as defined above.

[0104] In some embodiments, a set of three nucleic acid structures is provided, wherein the first nucleic acid structure comprises a polynucleotide encoding two of (a), (b), (c) and (d), the second nucleic acid structure comprises a polynucleotide encoding one of the remaining polypeptides, and the third nucleic acid structure comprises a polynucleotide encoding another of the remaining polypeptides, wherein (a), (b), (c) and (d) are as defined above.

[0105] In a preferred embodiment, a single nucleic acid structure comprising polynucleotides encoding (a), (b) and (c) is provided, wherein (a), (b) and (c) are as defined above.

[0106] In a preferred embodiment, a single nucleic acid structure comprising polynucleotides encoding (a), (b), (c) and (d) is provided, wherein (a), (b), (c) and (d) are as defined above.

[0107] In some embodiments, these anchor polypeptides are exosome membrane proteins, membrane targeting sequences, or fragments of their anchoring function. Exemplary exosome membrane proteins include, but are not limited to, tetraspanins such as lamp2b, CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative modulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid anchor proteins.

[0108] In some embodiments, each of these anchor polypeptides may be located at the N-terminus of the polypeptide to be presented on the exosome surface (e.g., NGF, EGF, PDGF-BB, and IDR-1018) to ensure that the polypeptide to be presented is exposed on the surface of the exosome. In some embodiments, each of these anchor polypeptides may be located at the C-terminus of the polypeptide to be presented on the exosome surface to ensure that the polypeptide to be presented is exposed on the surface of the exosome. For example, if lamp2b is used as one of these anchor polypeptides, the polypeptide to be presented on the exosome surface (e.g., NGF, EGF, PDGF-BB, or IDR-1018) may be located at the N-terminus of lamp2b. For example, when the TM3 domain of CD63 is used as one of these anchor polypeptides, the polypeptide to be presented on the exosome surface (e.g., NGF, EGF, PDGF-BB, or IDR-1018) may be located at the C-terminus of the TM3 domain of CD63. In some embodiments, when different anchor polypeptides are used, the polypeptide to be presented on the exosome surface may be located at the N-terminus or C-terminus of these anchor polypeptides, which is determined by the type of anchor polypeptide used.

[0109] In a preferred embodiment, these anchor polypeptides comprise full-length CD63 or truncated CD63 in which the TM3 domain is preserved. In a preferred embodiment, each of these anchor polypeptides comprises the TM3 domain of CD63. In a preferred embodiment, each of the first, second, third, and fourth anchor polypeptides is the TM3 domain of CD63.

[0110] In a preferred embodiment, the polynucleotide is from 5' to 3',

[0111] (i) [1st anchor polypeptide]-[EGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[NGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[PDGF-BB],

[0112] (ii) [1st anchor polypeptide]-[EGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[3rd anchor polypeptide]-[NGF],

[0113] (iii) [1st anchor polypeptide]-[NGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[EGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[PDGF-BB],

[0114] (iv) [1st anchor polypeptide]-[NGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[3rd anchor polypeptide]-[EGF],

[0115] (v) [1st anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[2nd anchor polypeptide]-[EGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[NGF], or

[0116] (vi) May include nucleotide fragments encoding substances such as [1st anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[2nd anchor polypeptide]-[NGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[EGF], and

[0117] Here, [] represents a single polypeptide, and ]-[ represents a linker or bond.

[0118] In a preferred embodiment, the self-cleaving peptide is a 2A peptide, such as T2A, E2A, P2A or any combination thereof. For example, the self-cleaving peptide is a T2A peptide. After translation of the polynucleotide, the self-cleaving peptide is cleaved to produce three or four independent fusion proteins each comprising a single polypeptide to be presented on the exosome surface and a single anchor polypeptide.

[0119] In a preferred embodiment, there is provided a single nucleic acid construct comprising a polynucleotide encoding (a), (b) and (c), wherein (a), (b) and (c) are as defined above, wherein each of said first, second and third anchor polypeptides comprises the TM3 domain of CD63, and said polynucleotide, from 5' to 3',

[0120] (i) [TM3]-[EGF]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[PDGF-BB],

[0121] (ii) [TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[NGF],

[0122] (iii) [TM3]-[NGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB],

[0123] (iv) [TM3]-[NGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF],

[0124] (v) [TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[NGF], and

[0125] (vi) [TM3]-[PDGF-BB]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[EGF], may comprise a nucleotide fragment encoding one of the substances such as

[0126] Here, [] represents a single polypeptide, and ]-[ represents a linker or bond; here, TM3 represents the TM3 domain of CD63; and T2A represents the self-cleaving peptide T2A.

[0127] In a preferred embodiment, the polynucleotide is from 5' to 3',

[0128] (a) [1st anchor polypeptide]-[EGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[NGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[4th anchor polypeptide]-[IDR-1018],

[0129] (b) [1st anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[2nd anchor polypeptide]-[EGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[NGF]-[self-cleaving peptide]-[4th anchor polypeptide]-[PDGF-BB],

[0130] (c) [1st anchor polypeptide]-[EGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[3rd anchor polypeptide]-[NGF]-[self-cleaving peptide]-[4th anchor polypeptide]-[PDGF-BB],

[0131] (d) [1st anchor polypeptide]-[EGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[NGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[4th anchor polypeptide]-[PDGF-BB],

[0132] (e) [1st anchor polypeptide]-[EGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[3rd anchor polypeptide]-[NGF]-[self-cleaving peptide]-[4th anchor polypeptide]-[IDR-1018],

[0133] (f) [1st anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[2nd anchor polypeptide]-[EGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[4th anchor polypeptide]-[NGF],

[0134] (g) [1st anchor polypeptide]-[EGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[3rd anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[4th anchor polypeptide]-[NGF],

[0135] (h) [1st Anchor Polypeptide]-[EGF]-[Self-cleaving Peptide]-[2nd Anchor Polypeptide]-[PDGF-BB]-[Self-cleaving Peptide]-[3rd Anchor Polypeptide]-[IDR-1018]-[Self-cleaving Peptide]-[4th Anchor Polypeptide]-[NGF],

[0136] (i) [1st anchor polypeptide]-[NGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[EGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[4th anchor polypeptide]-[IDR-1018],

[0137] (j) [1st anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[2nd anchor polypeptide]-[NGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[EGF]-[self-cleaving peptide]-[4th anchor polypeptide]-[PDGF-BB],

[0138] (k) [1st anchor polypeptide]-[NGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[3rd anchor polypeptide]-[EGF]-[self-cleaving peptide]-[4th anchor polypeptide]-[PDGF-BB],

[0139] (l) [1st Anchor Polypeptide]-[NGF]-[Self-cleaving Peptide]-[2nd Anchor Polypeptide]-[EGF]-[Self-cleaving Peptide]-[3rd Anchor Polypeptide]-[IDR-1018]-[Self-cleaving Peptide]-[4th Anchor Polypeptide]-[PDGF-BB],

[0140] (m) [1st Anchor Polypeptide]-[NGF]-[Self-cleaving Peptide]-[2nd Anchor Polypeptide]-[PDGF-BB]-[Self-cleaving Peptide]-[3rd Anchor Polypeptide]-[EGF]-[Self-cleaving Peptide]-[4th Anchor Polypeptide]-[IDR-1018],

[0141] (n) [1st anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[2nd anchor polypeptide]-[NGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[4th anchor polypeptide]-[EGF],

[0142] (o) [1st Anchor Polypeptide]-[NGF]-[Self-cleaving Peptide]-[2nd Anchor Polypeptide]-[IDR-1018]-[Self-cleaving Peptide]-[3rd Anchor Polypeptide]-[PDGF-BB]-[Self-cleaving Peptide]-[4th Anchor Polypeptide]-[EGF],

[0143] (p) [1st Anchor Polypeptide]-[NGF]-[Self-cleaving Peptide]-[2nd Anchor Polypeptide]-[PDGF-BB]-[Self-cleaving Peptide]-[3rd Anchor Polypeptide]-[IDR-1018]-[Self-cleaving Peptide]-[4th Anchor Polypeptide]-[EGF],

[0144] (q) [1st Anchor Polypeptide]-[PDGF-BB]-[Self-cleaving Peptide]-[2nd Anchor Polypeptide]-[EGF]-[Self-cleaving Peptide]-[3rd Anchor Polypeptide]-[NGF]-[Self-cleaving Peptide]-[4th Anchor Polypeptide]-[IDR-1018],

[0145] (r) [1st anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[2nd anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[3rd anchor polypeptide]-[EGF]-[self-cleaving peptide]-[4th anchor polypeptide]-[NGF],

[0146] (s) [1st Anchor Polypeptide]-[PDGF-BB]-[Self-cleaving Peptide]-[2nd Anchor Polypeptide]-[IDR-1018]-[Self-cleaving Peptide]-[3rd Anchor Polypeptide]-[EGF]-[Self-cleaving Peptide]-[4th Anchor Polypeptide]-[NGF],

[0147] (t) [1st anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[2nd anchor polypeptide]-[EGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[4th anchor polypeptide]-[NGF],

[0148] (u) [1st Anchor Polypeptide]-[PDGF-BB]-[Self-cleaving Peptide]-[2nd Anchor Polypeptide]-[NGF]-[Self-cleaving Peptide]-[3rd Anchor Polypeptide]-[EGF]-[Self-cleaving Peptide]-[4th Anchor Polypeptide]-[IDR-1018],

[0149] (v) [1st Anchor Polypeptide]-[IDR-1018]-[Self-cleaving Peptide]-[2nd Anchor Polypeptide]-[PDGF-BB]-[Self-cleaving Peptide]-[3rd Anchor Polypeptide]-[NGF]-[Self-cleaving Peptide]-[4th Anchor Polypeptide]-[EGF],

[0150] (w) [1st anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[2nd anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[3rd anchor polypeptide]-[NGF]-[self-cleaving peptide]-[4th anchor polypeptide]-[EGF], or

[0151] (x) May include nucleotide fragments encoding substances such as [1st anchor polypeptide]-[PDGF-BB]-[self-cleaving peptide]-[2nd anchor polypeptide]-[NGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[IDR-1018]-[self-cleaving peptide]-[4th anchor polypeptide]-[EGF], and

[0152] Here, [] represents a single polypeptide, and ]-[ represents a linker or bond.

[0153] In a preferred embodiment, the self-cleaving peptide is a 2A peptide, such as T2A, E2A, P2A, or any combination thereof. For example, the self-cleaving peptide is a T2A peptide.

[0154] In another embodiment, a single polynucleotide codes for at least one anchor polypeptide located at the C-terminus of the polypeptide to be presented on the surface of the exosome. In some embodiments, since each of the first, second, third, and fourth anchor polypeptides is different, such anchor polypeptides may be located at the N-terminus or C-terminus of polypeptide EGF, NGF, PDGF-BB, and optionally IDR-1018.

[0155] In a preferred embodiment, a single nucleic acid structure is provided comprising polynucleotides encoding (a), (b), (c) and (d), wherein (a), (b), (c) and (d) are as defined above, wherein each of the first, second, third, and fourth anchor polypeptides comprises the TM3 domain of CD63, and the polynucleotides are from 5' to 3',

[0156] [TM3]-[EGF]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[IDR-1018],

[0157] [TM3]-[IDR-1018]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[PDGF-BB],

[0158] [TM3]-[EGF]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[PDGF-BB],

[0159] [TM3]-[EGF]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[PDGF-BB],

[0160] [TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[IDR-1018],

[0161] [TM3]-[IDR-1018]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[NGF],

[0162] [TM3]-[EGF]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[NGF],

[0163] [TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[NGF],

[0164] [TM3]-[NGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[IDR-1018],

[0165] [TM3]-[IDR-1018]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB],

[0166] [TM3]-[NGF]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB],

[0167] [TM3]-[NGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[PDGF-BB],

[0168] [TM3]-[NGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[IDR-1018],

[0169] [TM3]-[IDR-1018]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF],

[0170] [TM3]-[NGF]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF],

[0171] [TM3]-[NGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[EGF],

[0172] [TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[IDR-1018],

[0173] [TM3]-[IDR-1018]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[NGF],

[0174] [TM3]-[PDGF-BB]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[NGF],

[0175] [TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[NGF],

[0176] [TM3]-[PDGF-BB]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[IDR-1018],

[0177] [TM3]-[IDR-1018]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[EGF],

[0178] [TM3]-[PDGF-BB]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[EGF], or

[0179] It may include a nucleotide fragment encoding one of the substances such as [TM3]-[PDGF-BB]-[T2A]-[TM3]-[NGF]-[T2A]-[TM3]-[IDR-1018]-[T2A]-[TM3]-[EGF], and

[0180] Here, [] represents a single polypeptide, and ]-[ represents a linker or bond; here, TM3 represents the TM3 domain of CD63; and T2A represents the self-cleaving peptide T2A.

[0181] In this section, EGF, NGF, PDGF-BB, and IDR-1018 polypeptides have the meaning and preferred embodiments presented in the section titled “Engineered Exosomes”.

[0182] In some embodiments, the polynucleotide encoding the EGF polypeptide has a nucleotide sequence indicated by SEQ ID NO. 5 or a degenerate sequence thereof.

[0183] In some embodiments, the polynucleotide encoding the NGF polypeptide has a nucleotide sequence indicated by SEQ ID NO. 6 or a degenerate sequence thereof.

[0184] In some embodiments, the polynucleotide encoding the PDGF-BB polypeptide has a nucleotide sequence indicated by SEQ ID NO. 7 or a degenerate sequence thereof.

[0185] In some embodiments, the polynucleotide encoding the IDR-1018 polypeptide has a nucleotide sequence indicated by SEQ ID NO. 8 or a degenerate sequence thereof.

[0186] In some embodiments, the polynucleotide encoding the TM3 domain of the CD63 has a nucleotide sequence indicated by SEQ ID NO. 10 or a degenerate sequence thereof.

[0187] A vector comprising the above nucleic acid structure is further provided. In some embodiments, the vector is a viral vector. In a preferred embodiment, the vector is a lentiviral vector or an adeno-associated virus vector.

[0188] The vector provided in this specification helps to incorporate the following polynucleotide into the cell genome that generates exosomes, said polynucleotide encoding a polypeptide that is anchored to the membrane of an engineered exosome.

[0189] Cells transfected with the above vector are further provided. In a preferred embodiment, the cells are not mesenchymal stem cells. In a preferred embodiment, the cells are not stem cells. In a preferred embodiment, the cells are non-stem cells such as HEK293 or CHO cells.

[0190] The present disclosure further provides a method for generating engineered exosomes provided herein, the method comprising: transfecting said cells, such as HEK293 cells, with said vector; culturing said cells under conditions that allow said engineered exosomes to be secreted from said cells; and collecting and purifying said engineered exosomes.

[0191] In some embodiments, the method includes the step of adapting cells during culture from serum-containing conditions to serum-free conditions. The adaptation may include a stepwise adaptation, i.e., increasing the proportion of serum-free culture medium while gradually decreasing the proportion of complete culture medium.

[0192] Ranking table

[0193]

[0194]

[0195]

[0196]

[0197] AA: Amino acid sequence

[0198] Examples

[0199] Example 1. Construction of engineered exosomes derived from a stable cell line.

[0200] Materials and Methods

[0201] ingredient: HEK293 cell lines (human embryonic kidney 293 cells, CRL-1573™) were purchased from ATCC and maintained in DMEM (high glucose) supplemented with 10% (volume-to-volume) FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. CHO-K1 cell lines (Chinese hamster ovary cells) were purchased from the BeNa Strain Preservation Center (Beijing, China). CHO-K1 cells were maintained in F-12K (31765035, Thermo Fisher Scientific, USA) supplemented with 10% (volume-to-volume) FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were cultured at 37°C in humid air containing 5% CO2. The antibody used in this study was the anti-CD63 antibody (catalog number MA5-32085, Invitrogen).

[0202] pGOI Plasmid Construction: The amino acid sequences of all target genes (including EGF, NGF, PDGF-BB, and IDR-1018 derived from humans and rats) were all derived from Uniprot, and the corresponding DNA sequences (see SEQ ID NO. 18-20 and SEQ ID NO. 8, respectively, for rat genes) were synthesized into the plasmid pCDH-CMV-MCS-EF1a-GFP+BSD (System Biosciences) by General Biotechnology (Chuzhou, China). T2A peptides (see SEQ ID NO. 13 and SEQ ID NO. 14) were used to dissociate all target proteins into single proteins after translation. Four plasmids from the third-generation system (i.e., pGOI, pGag / pol, pRev, and pVSV-G) were used to generate lentiviruses, where pCDH-CMV-MCS-EF1a-GFP+BSD (blasticidin resistance) was used as the lentivirus packaging plasmid. The lentiviruses were packaged by WZ Biotechnology (Jinan, China).

[0203] Payload genes composed of 2 pGOI plasmids:

[0204] No. 1: CD63-TM3-linker-EGF, where CD63-TM3 is the TM3 domain of CD63, said CD63-TM3 is encoded by the DNA sequence indicated by SEQ ID NO. 10, and EGF is encoded by human EGF DNA indicated by SEQ ID NO. 5.

[0205] No. 2: CD63-TM3-linker-EGF, where CD63-TM3 is the TM3 domain of CD63, said CD63-TM3 is encoded by the DNA sequence indicated by SEQ ID NO. 10, and EGF is encoded by rat EGF DNA indicated by SEQ ID NO. 18.

[0206] No. 37: CD63-TM3-Linker- EGF-T2A-CD63-TM3-Linker- NGF -T2A-CD63-TM3-Linker- PDGF-BB , where each CD63-TM3 is the TM3 domain of CD63 and is coded by the DNA sequence labeled SEQ ID NO. 10, each linker is coded by the DNA sequence labeled EQ ID NO. 12, and each T2A is coded by the DNA sequence labeled SEQ ID NO. 14; EGF, NGF, and PDGF-BB are coded by human EGF DNA labeled SEQ ID NO. 5, human NGF DNA labeled SEQ ID NO. 6, and human PDGF-BB DNA labeled SEQ ID NO. 7, respectively.

[0207] No. 38: CD63-TM3-Linker- EGF -T2A-CD63-TM3-Linker- NGF -T2A-CD63-TM3-Linker- PDGF-BB , where each CD63-TM3 is the TM3 domain of CD63 and is encoded by the DNA sequence labeled SEQ ID NO. 10, each linker is encoded by the DNA sequence labeled EQ ID NO. 12, and each T2A is encoded by the DNA sequence labeled SEQ ID NO. 14; EGF, NGF, and PDGF-BB are encoded by rat EGF DNA labeled SEQ ID NO. 18, rat NGF DNA labeled SEQ ID NO. 19, and rat PDGF-BB DNA labeled SEQ ID NO. 20, respectively.

[0208] No. 39: CD63-TM3-Linker- EGF -T2A-CD63-TM3-Linker- NGF -T2A-CD63-TM3-Linker- PDGF-BB -T2A-CD63-TM3-Linker- IDR-1018, where each CD63-TM3 is the TM3 domain of CD63 and is coded by the DNA sequence indicated by SEQ ID NO. 10, each linker is coded by the DNA sequence indicated by EQ ID NO. 12, and each T2A is coded by the DNA sequence indicated by SEQ ID NO. 14; EGF, NGF, PDGF-BB and IDR-1018 are coded by human EGF DNA indicated by SEQ ID NO. 5, human NGF DNA indicated by SEQ ID NO. 6, human PDGF-BB DNA indicated by SEQ ID NO. 7, and IDR-1018 DNA indicated by SEQ ID NO. 8, respectively.

[0209] No. 40: CD63-TM3-Linker- EGF -T2A-CD63-TM3-Linker- NGF -T2A-CD63-TM3-Linker- PDGF-BB -T2A-CD63-TM3-Linker- IDR-1018 , where each CD63-TM3 is the TM3 domain of CD63 and is encoded by the DNA sequence labeled SEQ ID NO. 10, each linker is encoded by the DNA sequence labeled EQ ID NO. 12, and each T2A is encoded by the DNA sequence labeled SEQ ID NO. 14; EGF, NGF, PDGF-BB, and IDR-1018 are encoded by rat EGF DNA labeled SEQ ID NO. 18, rat NGF DNA labeled SEQ ID NO. 19, rat PDGF-BB DNA labeled SEQ ID NO. 20, and IDR-1018 DNA labeled SEQ ID NO. 8, respectively.

[0210] Generation of stable cell lines: Stable HEK293 cell lines expressing target proteins (including EGF, NGF, PDGF-BB, and / or IDR-1018) were generated by plasmid transfection or infection with the corresponding lentivirus. Cells were selected 48 hours after transfection or infection by adding blasticidin (Solarbio Life Sciences) at a final concentration of 6 μg / ml. Single-cell colonies exhibiting green fluorescent protein (GFP) expression were selected and cultured in complete medium containing 6 μg / ml blasticidin. GFP and corresponding target protein expression in the stable cell lines were monitored.

[0211] Adaptation of cell cultures to SFM (serum-free medium): The stable HEK293 cell line was first subcultured three times in FM (complete medium), and then acclimated to serum-free culture starting from the fourth subculture. Cells were subcultured using the medium composition of Table 1. To establish a culture fully acclimated to serum-free culture, cells must be subcultured at least three times in SFM (HyClone™ peak expression, SH31193.02, Cytiva Life Sciences).

[0212] Adaptive media step Gradual adaptation 0 100% FM 1 75% FM + 25% SFM 2 50% FM + 50% SFM 3 25% FM + 75% SFM 4 12.5% ​​FM + 87.5% SFM 5 5% FM + 95% SFM 6 100% SFM

[0213] FM: Complete medium; SFM: Serum-free medium Exosome Separation: Stable cell lines were inoculated into T150 flasks, thoroughly washed with PBS after 24 hours of incubation, and cultured again in SFM for 48 hours. Cells were removed by centrifugation at 300×g for 10 minutes, and cell-free extracellular medium containing exosomes was obtained. Next, apoptotic cells and cell debris were removed by centrifugation at 10,000×g for 30 minutes. Finally, the clear supernatant was centrifuged at 100,000×g for 70 minutes to precipitate the exosomes twice. Subsequently, the exosome precipitate was resuspended. All centrifugation steps were performed at a temperature of 4°C.

[0214] FIG. 1 illustrates an exemplary flowchart of the process of generating exosomes derived from HEK293 cells according to an exemplary embodiment of the present disclosure.

[0215] Example 2. Characterization of exosomes

[0216] Analysis of Exosome Particle Concentration and Size Distribution

[0217] The particle concentration and size distribution of exosomes derived from stable cell lines NO. 38 or NO. 40 (transfected with pGOI NO. 38 and 40, respectively) were analyzed using NanoFCM (NanoFCM, Xiamen, China). The NanoFCM assay utilized two single-photon counting avalanche photodiodes (APDs) to simultaneously detect the side-scattered scattering (SSC) and fluorescence of single particles. First, exosome precipitates were prepared in PBS. Next, 200 nm PE and AF488 fluorescent dye-conjugated polystyrene beads were used for particle concentration measurements, and a silicon dioxide nanosphere mixture (NanoFCM, Xiamen, China) was used for particle size distribution measurements. The detector recorded passing particles at 1-minute intervals during each test. Each sample was diluted to achieve an optimal particle count range of 3,000 to 9,000 particles / minute. Flow velocity and lateral scattering intensity were converted into vesicle concentration and size using NanoFCM software (NanoFCM Professional V2.0).

[0218] Figure 2a illustrates the analysis of particle size and particle concentration of exosomes derived from stable cell lines No. 38 and 40 using NanoFCM. The exosomes of No. 38 have an average particle size of 70.8 nm, and the exosomes of No. 40 have an average particle size of 73.2 nm.

[0219] Identification of exosome target proteins using immunoblot (WB) or ELISA

[0220] To assess the expression of target proteins in exosomes, purified exosomes were lysed in RIPA lysis buffer supplemented with 1 mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Beyotime) and phosphatase inhibitor (Beyotime), then heat-denatured, separated by SDS-PAGE, and transferred to a PVDF membrane (Millipore, MA, USA). Proteins were detected by incubation with an anti-CD63 (exosome-fused scaffold protein) primary antibody, followed by incubation with an HRP-conjugated secondary antibody (Invitrogen). Subsequently, the membrane was visualized using an enhanced chemiluminescent reagent (Millipore, MA, USA). To detect the concentration of target proteins, purified exosomes were prepared, and the target proteins in the exosomes were analyzed using appropriate ELISA kits (Solarbio, SEKR-0010; SEKR-0036; SEKH-0290; SEKH-0050) according to the manufacturer's instructions.

[0221] Transmission Electron Microscopy (TEM) Analysis of Exosomes

[0222] TEM was used to confirm the presence of exosomes. Approximately 20 μL of exosomes were added alone to a copper grid. All excess liquid was removed using filter paper, and the sample was negatively stained with 2% uranyl acetate for 30 seconds. The grid was washed with deionized water and dried overnight. Next, the sample was air-dried using an incandescent lamp and observed with an electron microscope (Hitachi, S-3000N).

[0223] Figure 2b shows transmission electron microscopy (TEM) images of exosomes generated by stable cell lines No. 38 and 40. Figure 2c shows immunoblot analysis of exosomes derived from cell lines No. 38 and 40 using an anti-CD63-TM3 (scaffold protein fused to exosomes) antibody. As expected, engineered exosome-associated target proteins NGF, PDGF-BB, and EGF were present in the purified exosomes derived from stable cell lines No. 38 and 40. At the same time, IDR-1018 was present in the purified exosomes derived from stable cell line No. 40.

[0224] The concentration of functional proteins in exosomes was measured using an ELISA kit. Table 2 shows the results of different protein concentrations measured by ELISA.

[0225] Effective factor concentration measured by ELISA Concentration of active factor (pg / ml exosomes) Ra-NGF Ra-EGF Ra-PDGF-BB IDR-1018 No. 38 ND 85.16 2943.45 ND Hu-NGF Hu-EGF Hu-PDGF-BB IDR-1018 No. 40 ND 98.84 3078.27 ND

[0226] Example 3. Measurement of scratch damage HSF cells (human skin fibroblasts) used for scratch damage measurement were prepared. Once aggregated with HSF cells, the cell layer was scraped in a straight line using a 1 mm pipette tip. After scraping, the cell monolayer was gently washed to remove the detached cells, and then fresh culture medium (Mock) or exosomes (No. 1, No. 37, No. 39) were added, respectively. Images were then captured at various time intervals (0 hours, 24 hours, 48 ​​hours) using a 10x magnification microscope.

[0227] The image is as shown in Fig. 3, where exosomes were generated by stable cell lines No. 1, No. 37, or No. 39. The in vitro wound closure effect of engineered exosomes derived from stable cell lines No. 1, No. 37, or No. 39 on HSF was evaluated using the scratch measurement method. After 24 hours, treatment with exosomes from stable cell lines No. 37 or No. 39 showed significant wound closure compared to treatment with No. 1 or Mock. In particular, the exosomes from No. 39 showed a maximum degree of wound closure. Additionally, when treated only with exosomes from No. 39, complete closure of the scratch in the HSF culture was observed within 48 hours. This indicates that the exosome with four functional proteins (No. 39) has superior recovery efficacy for wound healing compared to the exosome with three functional proteins (No. 37) or the exosome with one functional protein (No. 1).

[0228] Example 4. Construction of a rat wound healing model for diabetic foot ulcers (DFU).

[0229] All animal experiments were conducted in accordance with procedures approved by the Animal Care and Use Committee of WuXi AppTec (Wuxi, China). The experiments were designed in accordance with animal welfare guidelines and the 3R principles. Male Sprague-Dawley (SD) rats (8 weeks old) were purchased from Charles River Laboratories (Beijing, China) and housed in a sterile (SPF) environment as an animal model for diabetic wound healing. Each rat was housed in a separate cage to prevent biting and fighting with other rats. All rats were free to consume water and standard laboratory food.

[0230] Diabetes was induced in rats by administering a single intraperitoneal injection of 60 mg / kg of streptozotocin (STZ). Blood glucose and body weight were measured to confirm whether the diabetes model had been successfully established. After 8 weeks, rats were anesthetized with 3% isoflurane, and a full-thickness circular wound was created on the left paw.

[0231] To observe the wound healing process, rats were randomly divided into four groups: (1) Health-vehicle group: healthy rats that naturally heal their wounds after PBS treatment; (2) Model-vehicle group: diabetic foot wound model rats treated with PBS; (3) NO. 38 group: diabetic foot wound model rats treated with NO. 38 exosomes; and (4) NO. 40 group: diabetic foot wound model rats treated with NO. 40 exosomes. 10 μg of exosomes dissolved in 100 μl of PBS was subcutaneously injected into the wound sites of the diabetic model rats a total of five times over days 1, 4, 7, 10, and 13. Wound dynamics were recorded using a camera before each injection. Healing efficacy was evaluated by quantifying the wound healing process through the calculation of wound area using image analysis software (ImageJ). The calculation method is as follows. Inhibition rate (%) = 100% × (Initial wound area - Current wound area) / Initial wound area.

[0232] Figure 4a illustrates a schematic diagram of the construction of a DFU rat model and the design of intradermal administration. Figures 4b and 4c illustrate the blood glucose and body weight of these animals at different time points.

[0233] Figure 5 showed that engineered exosomes exhibited enhanced wound healing effects in a rat DFU model. Compared to the model vehicle group, both the NO. 38 exosome treatment group and the NO. 40 exosome treatment group improved the wound healing rate on day 13. The NO. 40 exosome treatment group was more effective, significantly superior to the NO. 38 exosome treatment group, and nearly similar to the healthy vehicle group.

[0234] Wound tissues from DFU rats were collected at the end of the study. HE staining was analyzed in the DFU model vehicle and in the DFU model treated with exosomes (NO. 40). The number of blood vessels was confirmed using CD31, and nerve regeneration was indicated using PGP9.5 as a detection marker. CD31 is an endothelial cell marker in normal tissues, secreted by blood endothelial cells, while PGP9.5 is expressed in neurons and neuroendocrine cells and is used to identify central and peripheral nerves.

[0235] The results shown in Fig. 6 demonstrated that engineered exosomes promote vascular and nerve regeneration in a rat DFU model. Immunohistochemistry (IHC) results indicated that when treated five times with exosomes (NO. 40) at the end of the study, the expression of CD31 (Fig. 6A) and PGP9.5 (Fig. 6D) in diabetic wounds showed a significantly increasing trend.

[0236] Normal wound healing rates over 16 days were calculated for each group (n=6), including the model group (Vehicle) and the model group treated with exosomes (NO. 40). At the end of the study (day 16), tissue sections from the injured area were prepared for HE staining, and scabs and inflammation-inducing cells were analyzed. Data are expressed as mean ± SD. * indicates p<0.05 compared to the control group.

[0237] The results illustrated in Fig. 7 demonstrated that engineered exosomes promote normal wound healing at different time points. In a normal wound healing model, the healing rate of wounds treated with exosomes (NO. 40) was faster compared to the model group (Fig. 7A). Downregulation of pro-inflammatory cells (Figs. 7B and 7C) showed that in the late stages of healing, the aggressive pro-inflammatory or “M1” phenotype transitioned to a healing-promoting or “M2” phenotype, which can facilitate final healing. Reduction of scabs (Figs. 7B and 7C) may help remove obstacles to the healing process and accelerate re-epithelialization.

Claims

Claim 1 An engineered exosome, wherein the engineered exosome comprises (a) an EGF polypeptide fused with a first anchor polypeptide, (b) an NGF polypeptide fused with a second anchor polypeptide, and (c) a PDGF-BB polypeptide fused with a third anchor polypeptide, wherein (a), (b) and (c) are anchored to the membrane of the exosome through the first, second, and third anchor polypeptides, respectively, and the EGF polypeptide, the NGF polypeptide, and the PDGF-BB polypeptide are exposed to the outer surface of the membrane of the exosome. Claim 2 In claim 1, the engineered exosome further comprises (d) an IDR-1018 polypeptide comprising an amino acid sequence denoted by SEQ ID NO. 4, fused with a fourth anchor polypeptide, wherein the IDR-1018 polypeptide is exposed to the outer surface of the membrane of the exosome. Claim 3 The engineered exosome according to claim 1, wherein the EGF polypeptide is human EGF or a parallel homolog or a serial homolog thereof, and preferably, the EGF polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO.

1. Claim 4 The engineered exosome according to claim 1, wherein the NGF polypeptide is human NGF or a parallel homolog or a serial homolog thereof, and preferably, the NGF polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO.

2. Claim 5 The engineered exosome according to claim 1, wherein the PDGF-BB polypeptide is a human PDGF-BB dimer or a parallel homolog or a serial homolog thereof; more preferably, the PDGF-BB dimer comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO.

3. Claim 6 In claim 1 or 2, the anchor polypeptide is an exosome membrane protein, a membrane targeting sequence, or a fragment of the anchor function thereof; preferably, the exosome membrane protein comprises tetraspanins such as lamp2b, CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative modulator (PTGFRN), and any combination thereof; preferably, the membrane targeting sequence comprises a glycosylphosphatidylinositol (GPI) anchor and a lipid anchor protein; preferably, the anchor polypeptide comprises full-length CD63 or truncated CD63 with a preserved TM3 domain; more preferably, each of the first, second, and third anchor polypeptides and optionally the fourth anchor polypeptide comprises the TM3 domain of CD63; More preferably, each of the anchor polypeptides is a TM3 domain of CD63; preferably, the TM3 domain of CD63 comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 9, an engineered exosome. Claim 7 Engineered exosomes according to claim 1 or 2, wherein the EGF polypeptide, the NGF polypeptide, the PDGF-BB polypeptide and the IDR-1018 polypeptide are each optionally fused to the C-terminus of the first, second, third, and fourth anchor polypeptides through a peptide linker, preferably, the peptide linker is composed of glycine and serine, for example (G4S)n, where n is an integer from 1 to 3. Claim 8 In claim 1, the exosome is not derived from mesenchymal stem cells; the exosome is an engineered exosome that is not derived from stem cells. Claim 9 A composition comprising an exosome and a carrier according to any one of claims 1 to 8; preferably, the composition is a liquid formulation; more preferably, the composition is prepared for topical or subcutaneous administration; and preferably, the composition is a pharmaceutical composition. Claim 10 In claim 9, the composition does not include an EGF polypeptide (e.g., human EGF polypeptide), an NGF polypeptide (e.g., human NGF polypeptide), a PDGF-BB polypeptide (e.g., human PDGF-BB polypeptide), or an IDR-1018 polypeptide that is not attached to the membrane of the exosome. Claim 11 A nucleic acid structure comprising a polynucleotide encoding a substance such as (a) an EGF polypeptide fused with a first anchor polypeptide, (b) an NGF polypeptide fused with a second anchor polypeptide, and (c) a PDGF-BB polypeptide fused with a third anchor polypeptide. Claim 12 In claim 11, the polynucleotide is also (d) a nucleic acid construct encoding an IDR-1018 polypeptide comprising an amino acid sequence indicated by SEQ ID NO. 4, fused with a fourth anchor polypeptide. Claim 13 In claim 11 or 12, (i) the EGF polypeptide is human EGF or a parallel or serial homolog thereof, preferably, the EGF polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 1; (ii) the NGF polypeptide is human NGF or a parallel or serial homolog thereof, preferably, the NGF polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 2; (iii) the PDGF-BB polypeptide is a human PDGF-BB dimer; more preferably, the PDGF-BB dimer comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 3; and (iv) the polynucleotide encoding the EGF polypeptide has a nucleotide sequence indicated by SEQ ID NO. 5 or a degenerate sequence thereof; (v) The polynucleotide encoding the NGF polypeptide has the nucleotide sequence indicated by SEQ ID NO. 6 or its degenerate sequence; (vi) The polynucleotide encoding the PDGF-BB polypeptide has the nucleotide sequence indicated by SEQ ID NO. 7 or its degenerate sequence; (vii) The polynucleotide encoding the IDR-1018 polypeptide has SEQ ID NO.It has a nucleotide sequence indicated by 8 or a degenerate sequence thereof; (viii) the anchor polypeptide is an exosome membrane protein, a membrane targeting sequence, or a fragment of the anchor function thereof; preferably, the exosome membrane protein comprises tetraspanins such as lamp2b, CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative modulator (PTGFRN), and any combination thereof; preferably, the membrane targeting sequence comprises a glycosylphosphatidylinositol (GPI) anchor and a lipid anchor protein; preferably, the anchor polypeptide comprises a full-length CD63 or a truncated CD63 with a preserved TM3 domain; More preferably, each of the first, second, and third anchor polypeptides and optionally the fourth anchor polypeptide comprises the TM3 domain of CD63; more preferably, each of the first, second, and third anchor polypeptides and optionally the fourth anchor polypeptide comprises the TM3 domain of CD63; and / or (ix) a nucleic acid structure having a nucleotide sequence or a degenerate sequence thereof, wherein the polynucleotide encoding the TM3 domain of CD63 is represented by SEQ ID NO.

10. Claim 14 In any one of claims 11 to 13, the polynucleotide is a single polynucleotide comprising a nucleotide fragment encoding polypeptides (a), (b), (c) and optionally (d), wherein each of polypeptides (a), (b), (c) and optionally (d) is connected to the anchor polypeptide; preferably, polypeptides (a), (b), (c) and optionally (d) are separated by a self-cleaving peptide; optionally, the self-cleaving peptide is a 2A peptide, such as T2A, E2A, P2A, or any combination thereof, a nucleic acid structure. Claim 15 A vector, wherein the vector comprises a nucleic acid structure according to any one of claims 11 to 14; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector or an adeno-associated virus vector. Claim 16 Cells transfected with a vector according to paragraph 15, wherein the polynucleotide is incorporated into the genome of the cell; preferably, the cell is not a mesenchymal stem cell; preferably, the cell is not a stem cell; preferably, the cell is a mammalian cell; or more preferably, the cell is a HEK293 or CHO cell. Claim 17 A method for generating engineered exosomes according to any one of claims 1 to 8, wherein the method comprises: (a) transfecting a cell according to claim 16 with a vector according to claim 15; (b) culturing the cell under conditions that allow exosomes to be secreted from the cell; and (c) collecting and purifying the exosomes. Claim 18 A method for generating engineered exosomes according to claim 17, wherein the method further comprises the step of adapting the cells to serum-free conditions during step (b). Claim 19 Use of engineered exosomes according to any one of claims 1 to 8 or a composition according to claim 9 or 10 in the manufacture of a drug for improving wound healing or treating wounds or treating Propionibacterium acnes infection, wherein the wound is a chronic wound, preferably a diabetic wound; more preferably, the wound comprises a diabetic foot ulcer; preferably, the Propionibacterium acnes infection occurs on the skin; and preferably, the Propionibacterium acnes infection is acne or acne-prone skin, such as a scar or skin damage caused by acne.

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