Engineered exosomes for the treatment of alopecia and / or gray hair

Abstract

The present invention discloses engineered exosomes, said engineered exosomes comprising (a) a KGF polypeptide fused to a first anchor polypeptide, (b) a VEGF-A polypeptide fused to a second anchor polypeptide, and (c) a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway 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 KGF polypeptide, said VEGF-A polypeptide, and said Wnt family member polypeptide are exposed on the outer surface of said exosome membrane. The present invention further discloses the use of said exosomes in compositions, nucleic acid structures, and treatments for alopecia and / or gray hair.

Description

Technology Field

[0001] The present invention 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 the prevention or treatment of alopecia and / or gray hair. Background Technology

[0002] Hair loss, also known as alopecia or baldness, refers to the phenomenon of hair falling out from the head or body. The severity of hair loss varies from small areas to the entire body. Hair loss can be caused by psychological stress, medications, or other factors. Common types of hair loss include androgenetic alopecia (including male and female pattern baldness), alopecia areata, and telogen effluvium, a type of hair loss. The causes of male pattern baldness include the combined action of genetic factors and androgens; the cause of female pattern baldness is not yet clear; the cause of alopecia areata is autoimmune; and telogen effluvium is generally caused by physical or psychological stress.

[0003] The hair follicle is a regenerative organ capable of large-scale regeneration by stem cells. The hair follicle consists of an outer root sheath, an inner root sheath, and a hair shaft. Proliferating undifferentiated stromal cells form the inner root sheath and hair shaft, which are surrounded by dermal papillae composed of specialized mesenchymal cells. While the dermal papillae direct the formation of the hair follicle, the characteristics of the follicle are acquired through epithelial information. The lower part of the hair follicle undergoes a growth cycle that includes the growth phase (active growth), the regression phase (degeneration), and the resting phase (rest). These different phases last for varying durations depending on the location and function of the hair follicle. During the growth phase, stromal cells proliferate rapidly, migrate upward, and differentiate into cell types for the inner root sheath and hair shaft. During the regression phase, the lower hair follicle undergoes apoptosis, and the dermal papillae migrate upward until they reach the region beneath the process. During the resting phase, the dermal papillae remain in that region. Once the dermal papilla mobilizes stem cells from the projection, the growth phase resumes, and the hair follicle regenerates through proliferation and differentiation.

[0004] Hair loss control is generally achieved through medication or surgery. Currently, medications used to treat hair loss include minoxidil, finasteride, and dutasteride. Hair loss is also controlled through surgical procedures, including hair transplantation. Hair transplantation is typically performed under local anesthesia. Doctors transplant healthy hair from the back and sides of the head to areas with sparse hair. This process can take between four and eight hours, and additional treatments may be required to make the hair thicker. If there is insufficient hair in non-hair loss areas, conventional hair transplantation methods are less effective. Stem cell therapy is emerging as a new strategy for treating and controlling hair loss. However, in the case of conventional hair transplantation, administering follicular stem cells to the bald scalp is highly inefficient because the skin in that area lacks the growth factors necessary for the growth of new hair follicles.

[0005] Gray hair (natural hair whitening) is associated with the specific and progressive depletion of hair melanocytes, which affects both melanocytes and melanocyte precursor cells within the hair bulb. Other cell types present in the hair follicle are unaffected. Furthermore, this depletion of melanocytes is not observed in the epidermis. The cause of the progressive and specific depletion of melanocytes and melanocyte precursors within the hair follicle has not been identified to date.

[0006] Therefore, additional and improved methods and compositions are needed for the treatment of hair loss (including androgenetic alopecia and alopecia areata). To prevent gray hair, it is also necessary to prevent the loss of melanocytes within human hair follicles, and this process affects both active melanocytes within the hair bulb and quiescent melanocytes in the upper region of the hair follicle.

[0007] In one embodiment of the present invention, an engineered exosome is provided, said engineered exosome comprising (a) a KGF polypeptide fused to a first anchor polypeptide, (b) a VEGF-A polypeptide fused to a second anchor polypeptide, and (c) a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway 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, and said KGF polypeptide, the VEGF-A polypeptide and the Wnt family member polypeptide are exposed to the outer surface of the membrane of the exosome.

[0008] In some embodiments, the KGF polypeptide is a KGF-1 polypeptide, and preferably, the KGF-1 polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO. 4.

[0009] In some embodiments, the VEGF-A polypeptide is human VEGF 206 It is an isomer or a parallel homolog or a serial homolog thereof, and preferably, the VEGF-A polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 2 or SEQ ID NO. 5.

[0010] In some embodiments, the Wnt family member polypeptide is Wnt10a, Wnt10b, Wnt3a, Wnt1a, Wnt4, Wnt5a, Wnt7a, or Wnt7b polypeptide; preferably, the Wnt family member polypeptide is Wnt10b polypeptide; more preferably, the Wnt10b polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 3 or SEQ ID NO. 6.

[0011] 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.

[0012] In a preferred embodiment, the first, second, and third 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 comprises the TM3 domain of CD63. In a preferred embodiment, each of the first, second, and third anchor polypeptides comprises the TM3 domain of CD63. In some embodiments, each of the first, second, and third 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. 13.

[0013] In some embodiments, the KGF polypeptide, the VEGF-A polypeptide, and the Wnt family member polypeptide are each optionally fused to the C-terminus of the first, second, and third 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.

[0014] In some embodiments, the exosomes are not derived from mesenchymal stem cells; preferably, the exosomes are not derived from stem cells.

[0015] In some embodiments, the exosomes promote hair regeneration and / or improve hair loss such as androgenetic alopecia, alopecia areata, or telogen effluvium. In some embodiments, the exosomes prevent and / or limit and / or inhibit the formation of gray hair and maintain and / or promote the restoration of the natural color of hair and / or body hair. In some embodiments, the exosomes (i) promote hair regeneration and / or improve hair loss such as androgenetic alopecia, alopecia areata, or telogen effluvium; and (ii) prevent and / or limit and / or inhibit the formation of gray hair and maintain and / or promote the restoration of the natural color of hair and / or body hair.

[0016] Another aspect of the present disclosure relates to 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.

[0017] In some embodiments, the composition does not include a KGF polypeptide not attached to the membrane of the exosome (e.g., KGF-1 polypeptide), a VEGF-A polypeptide, or a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway (e.g., Wnt10b polypeptide).

[0018] 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) a KGF polypeptide fused with a first anchor polypeptide, (b) a VEGF-A polypeptide fused with a second anchor polypeptide, and (c) a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway fused with a third anchor polypeptide.

[0019] In some embodiments, the KGF polypeptide is a KGF-1 polypeptide, and preferably, the KGF-1 polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO. 4.

[0020] In some embodiments, the VEGF-A polypeptide is human VEGF 206 It is an isomer or a parallel homolog or a serial homolog thereof, and preferably, the VEGF-A polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 2 or SEQ ID NO. 5.

[0021] In some embodiments, the Wnt family member polypeptide is Wnt10a, Wnt10b, Wnt3a, Wnt1a, Wnt4, Wnt5a, Wnt7a, or Wnt7b polypeptide; preferably, the Wnt family member polypeptide is Wnt10b polypeptide; more preferably, the Wnt10b polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 3 or SEQ ID NO. 6.

[0022] In some embodiments, the polynucleotide encoding the KGF polypeptide has a nucleotide sequence indicated by SEQ ID NO. 7, SEQ ID NO. 10, or a degenerate sequence thereof.

[0023] In some embodiments, the polynucleotide encoding the VEGF-A polypeptide has a nucleotide sequence indicated by SEQ ID NO. 8, SEQ ID NO. 11, or a degenerate sequence thereof.

[0024] In some embodiments, the polynucleotide encoding the Wnt family member polypeptide has a nucleotide sequence indicated by SEQ ID NO. 9, SEQ ID NO. 12, or a degenerate sequence thereof.

[0025] 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.

[0026] In a preferred embodiment, the first, second, and third 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 comprises the TM3 domain of CD63. In a preferred embodiment, each of the first, second, and third anchor polypeptides is 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. 14 or a degenerate sequence thereof.

[0027] In some embodiments, the polynucleotide is a single polynucleotide comprising a nucleotide fragment encoding (a), (b) and (c); preferably, the polynucleotide is from 5' to 3',

[0028] (i) [1st anchor polypeptide]-[KGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[3rd anchor polypeptide]-[Wnt],

[0029] (ii) [1st anchor polypeptide]-[KGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[3rd anchor polypeptide]-[VEGF-A],

[0030] (iii) [1st anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[2nd anchor polypeptide]-[KGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[Wnt],

[0031] (iv) [1st anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[2nd anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[3rd anchor polypeptide]-[KGF],

[0032] (v) [1st anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[2nd anchor polypeptide]-[KGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[VEGF-A], or

[0033] (vi) comprising nucleotide fragments encoding substances such as [1st anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[2nd anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[3rd anchor polypeptide]-[KGF], etc.

[0034] Here, [] represents a single polypeptide, and ]-[ represents a linker or bond;

[0035] Here, Wnt represents a polypeptide of the Wnt family member that participates in the Wnt / β-catenin signaling pathway.

[0036] In some embodiments, the self-cleaving peptide is a 2A peptide, such as T2A, E2A, P2A, or any combination thereof.

[0037] In another embodiment, each of the first, second, and third anchor polypeptides is located at the C-terminus of Wnt, KGF, and / or VEGF to ensure that Wnt, KGF, and / or VEGF are exposed to 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., Wnt, KGF, and VEGF) may be located at the N-terminus of lamp2b. In some embodiments, if 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. For example, in one embodiment, a single polynucleotide comprises a nucleotide fragment encoding (a), (b) and (c), and the single polynucleotide comprises a nucleotide fragment encoding a substance such as [first anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[VEGF-A]-[second anchor polypeptide]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF] from 5' to 3', wherein the second anchor polypeptide is located at the C-terminus of the VEGF-A polypeptide.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] In some embodiments, the method further includes the step of adapting the cells to serum-free conditions during step (b).

[0043] Another aspect of the present disclosure relates to the use of engineered exosomes or compositions in the manufacture of drugs for treating alopecia and / or gray hair disclosed herein, preferably, said alopecia is androgenetic alopecia, alopecia areata, or telogen effluvium; and preferably, said gray hair is age-related hair graying, premature graying, or acute graying.

[0044] Another aspect of the present disclosure relates to a method for treating alopecia and / or gray hair; preferably, the alopecia is androgenetic alopecia, alopecia areata, or telogen effluvium; preferably, the gray hair is age-related hair graying, premature graying, or acute graying; and the method comprises the step of administering an effective amount of engineered exosomes or compositions disclosed herein to a subject.

[0045] Another aspect of the present disclosure relates to engineered exosomes or compositions disclosed herein used to treat alopecia and / or gray hair; preferably, the alopecia is androgenetic alopecia, alopecia areata, or telogen effluvium; preferably, the gray hair is age-related hair graying, premature graying, or acute graying.

[0046] These aspects and other advantages of the present disclosure will become apparent from the specific embodiments provided below. Brief explanation of the drawing

[0047] Fig. 1. Construction of a stable cell line capable of secreting engineered exosomes loaded with functional proteins Mammalian cells (e.g., HEK293 cells) were cultured and infected with a lentivirus packaged with the functional genes KGF, VEGFA, and Wnt10b. Next, stable cell lines were selected using blasticidin. After three subcultures and selections, expression of the functional genes was detected in both the cell precipitate and exosomes. Once expression was confirmed, 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. Assessment of engineered exosomes for androgenetic alopecia. . (A) The particle size and particle concentration of exosomes derived from stable cell lines were analyzed using NanoFCM. (B) Exosomes were examined using a transmission electron microscope (TEM). (C) Immunoblot analysis was performed on exosomes using an antibody against anti-CD63-TM3, a scaffold protein fused with exosomes. Fig. 3. In vivo evaluation of engineered exosome 35# for hair loss treatment . (A) This is a schematic diagram of a mouse model of androgenetic alopecia and a solution. After depilating the dorsal hair of the mice, the mouse skin was treated once every 3 days with 35# exosomes, control exosomes (Ctrl Exo.), PBS (negative control), or 5% minoxidil (positive control). After continuous observation during the 18-day treatment period, the animals were sacrificed on the 18th day for histological analysis. (B) The hair cover status was observed. Mice were divided into four groups (n=4), and representative images of the mice on day 15 were shown. (C)Histological analysis of follicle count, length, and growth stage ratio of the dorsal skin of mice. Data points represent the mean ± SD (n=4). Error bars represent the standard deviation (SD.); ns indicates no significant difference; *P<0.05, **P<0.01, and ***P<0.001. Fig. 4. In vivo evaluation of engineered exosome 35# in resistance to hair graying. . (A) This is a schematic diagram of the hydroquinone-induced hair graying model and the solution. After depilating the dorsal hair of mice, female C57BL / 6 mice were randomly divided into groups (n=4) and hydroquinone cream was applied topically twice daily. The negative model control group was treated topically with PBS. 35# exosome groups were treated via different routes of administration (e.g., topical application after microneedle inoculation, topical application after nanocrystal injection, subcutaneous injection). They were continuously observed for a treatment period of 28 days. (B) The hair on the back was observed. A representative image of the mouse on day 14 was shown. Specific details for implementing the invention

[0048] definition

[0049] When used with the word “include” in this specification, including claims, the words “one” and “one” indicate “one or more”.

[0050] 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.

[0051] 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.

[0052] “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.

[0053] 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 the two sequences, the percentage of bases (or amino acids) is the same. Such comparison and the percentage of homology or sequence identity may be determined using software programs known in the art.

[0054] 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.

[0055] As used herein, “cell line” refers to a population of cells formed through one or more subcultures of a primary cell culture. Each process of subculture is referred to as a primary subculture. After cells have been subcultured, they are referred to as “subcultured” cells. A specific cell population or cell line is sometimes indicated or characterized by its number of subcultures. For example, a cultured cell population that has been subcultured ten 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 subculture, the cells are described as a second-generation culture (P1 or the first generation). After the second subculture, the cells become a third-generation culture (P2 or the second generation), and this continues in this manner. Those skilled in the art will understand that many population duplications may occur during subculture; therefore, the number of population duplications of a culture is greater than the number of subcultures. 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] As used herein, the term “subject” may be used interchangeably with the terms “individual” or “patient,” and generally refers to an individual requiring treatment. The subject may be a human, dog, cat, horse, pig, or mammal such as a rodent. The subject may be, for example, a patient who suffers from or is suspected of having a disease or medical condition related to hair loss, or who is at risk of developing such disease or medical condition. In the case of a subject who suffers from or is suspected of having a medical condition directly or indirectly related to hair loss, said medical condition may be of one or more types.

[0060] 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.

[0061] 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).

[0062] The term “KGF” refers to keratinocyte growth factor, which is an epidermal cell division-promoting factor and a member of the fibroblast growth factor (FGF) family. In vertebrates, the FGF family includes 22 members essential for regulating many developmental processes. KGF includes two functionally similar variants: KGF-1 (also known as FGF-7) and KGF-2 (also known as FGF-10). The characteristics of recombinant human fibroblast growth factor FGF-10 demonstrate functional similarity to keratinocyte growth factor (FGF-7). Both growth factors interact with the same high-affinity receptor (KGFR, an isoform of FGFR2), which differs from FGFR2 in the latter part of the third immunoglobulin loop and is encoded by an alternative exon. The ability of KGF-1 and KGF-2 to bind to KGFR with high affinity distinguishes them from other members of the FGF-10 family. KGF-2 is highly related to KGF-1, and KGF-2 binds to the same receptor as KGF-1, and they have 57% sequence homology. Human KGF-2 has 96% identity with rat KGF-2 and specifically stimulates the growth of normal human epidermal keratinocytes.

[0063] The term “VEGFA,” or Vascular Endothelial Growth Factor A, is a major member of the vascular endothelial growth factor (VEGF) family of cytokines and plays a crucial role in angiogenesis, angiogenesis, and lymphangiogenesis, along with placental growth factor (PlGF), mammalian VEGF-B / C / D, and VEGF-E / F in other organisms. It was first named “VEGF” by N. Ferrara et al. in early 1989, and VEGF-A is the most extensively studied member of the VEGF family. Selective splicing at exon 8 of the Vegfa gene generates different VEGF-A isoforms, forming VEGFxxxa or VEGFxxxb isoforms. In addition to the recently discovered post-translational read-through phenomenon, selective splicing at exons 5–7 generates VEGF-A isoforms with varying bioavailability and interactions with the co-receptor Neuropilin-1. 16 different VEGFA isoforms from the 6 most common transcripts to date: VEGF111, VEGF121, VEGF145, VEGF165, VEGF189, and VEGF 206 This has been confirmed. A similar VEGF-A splice variant map has also been described in rats, with each variant encoding one fewer amino acid than in humans. The shorter isoforms, VEGF111 and VEGF121, both lack exons 6 and 7, so they are not linked to the extracellular matrix (ECM) and can diffuse freely. Conversely, the longer isoforms containing both exons 6a and 7, VEGF145, VEGF189, and VEGF 206 It can bind to heparan sulfate glycoproteins with high affinity.

[0064] The term “Wnt / β-catenin signaling pathway” refers to the typical Wnt pathway involving the nuclear translocation of β-catenin and the activation of target genes via TCF / LEF transcription factors. The typical Wnt pathway primarily controls cell proliferation. Atypical Wnt pathways do not rely on β-catenin-T-cell factor / lymph-enhanced factor-binding factor (TCF / LEF), and, for example, Wnt / Ca 2+ There are atypical Wnt planar cell polarity pathways. The Wnt / β-catenin signaling pathway is essential for embryonic development and the homeostatic regeneration of adult tissues. Abnormal regulation of the pathway is closely associated with various diseases, indicating that the Wnt / β-catenin signaling pathway is an attractive target for disease treatment. The term “Wnt family member polypeptides participating in the Wnt / β-catenin signaling pathway” refers to any Wnt family member or a functional fragment thereof participating in the Wnt / β-catenin signaling pathway, including but not limited to Wnt10a, Wnt10b, Wnt3a, Wnt1a, Wnt4, Wnt5a, Wnt7a, or Wnt7b polypeptides.

[0065] Members of the tetraspanin family (e.g., CD63, CD81, and CD9) are universally expressed in exosomes and are widely used as exosome biomarkers participating in physiological processes such as cell adhesion, cell migration, and signal transduction. CD63 (whose primary characteristic is that it is a tetraspanin) possesses 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.

[0066] The term “anchor polypeptide” is a polypeptide that anchors to the exosome membrane when exosomes are 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 wholly 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 KGF-1 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.

[0067] Engineered Exosomes

[0068] One aspect of the present disclosure relates to an engineered exosome, said engineered exosome comprising (a) a KGF polypeptide fused to a first anchor polypeptide, (b) a VEGF-A polypeptide fused to a second anchor polypeptide, and (c) a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway 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 KGF polypeptide, said VEGF-A polypeptide and said Wnt family member polypeptide are exposed to the outer surface of the membrane of the exosome.

[0069] In the present disclosure, the KGF polypeptide is a KGF-1 polypeptide or a KGF-2 polypeptide. In a preferred embodiment, the KGF polypeptide is a KGF-1 polypeptide. In some embodiments, the KGF-1 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.

[0070] In a preferred embodiment, the KGF-1 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. 4. In some embodiments, the KGF-1 polypeptide comprises the amino acid sequence indicated by SEQ ID NO. 4, and the polypeptide is a human KGF-1 polypeptide. In some embodiments, the amino acid sequence of the KGF-1 polypeptide is indicated by SEQ ID NO. 4.

[0071] In the present disclosure, the VEGF-A polypeptide may be any VEGF-A isomer found in the art. In a preferred embodiment, the VEGF-A polypeptide is human VEGF 206 It is an isomer or a parallel homolog or a series homolog thereof. In some embodiments, the VEGF-A 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.

[0072] In a preferred embodiment, the VEGF-A 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. 5. In some embodiments, the VEGF-A polypeptide comprises the amino acid sequence indicated by SEQ ID NO. 5, and the polypeptide is human VEGF 206 It is an isomer. In some embodiments, human VEGF 206 The amino acid sequence of the isomer is indicated by SEQ ID NO. 5.

[0073] In the present disclosure, the Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway may be any Wnt family member participating in the Wnt / β-catenin signaling pathway. In a preferred embodiment, the Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway is Wnt10a, Wnt10b, Wnt3a, Wnt1a, Wnt4, Wnt5a, Wnt7a, or Wnt7b polypeptide.

[0074] In a preferred embodiment, the Wnt family member polypeptide is Wnt10b. In a preferred embodiment, the Wnt10b 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 a preferred embodiment, the Wnt10b 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. 6. In some embodiments, the Wnt10b polypeptide is SEQ ID NO. The polypeptide comprises an amino acid sequence indicated by 6, and said polypeptide is a human Wnt10b polypeptide. In some embodiments, the amino acid sequence of said Wnt10b polypeptide is indicated by SEQ ID NO. 6.

[0075] 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, the first, second, and third anchor polypeptides each include the TM3 domain of CD63. In a preferred embodiment, each of the first, second, and third anchor polypeptides includes the TM3 domain of CD63. In some embodiments, this anchor polypeptide is a full-length CD63 protein, such as full-length human CD63 (see, for example, UniProtKB / Swiss-Prot:F8VZE2, P08962, Q5TZP3, Q8N6Z9, or Q9UCG6). In some embodiments, this anchor polypeptide comprises a TM3 domain and at least one truncated CD63 protein among the TM1, TM2, and TM4 domains. For example, this anchor polypeptide may consist of TM2 and TM3 of CD63; TM3 and TM4 of CD63; or TM1, TM2, and TM3 of CD63. In some embodiments, this anchor polypeptide consists of the TM3 domain of CD63. In the present disclosure, the first, second, and third anchor polypeptides may be different or identical. In a preferred embodiment, the first, second, and third anchor polypeptides are identical and consist of the TM3 domain of CD63.

[0076] 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. 13. In a preferred embodiment, the TM3 domain of the CD63 comprises the amino acid sequence indicated by SEQ ID NO. 13. In a preferred embodiment, the amino acid sequence of the TM3 domain of the CD63 is indicated by SEQ ID NO. 13.

[0077] Accordingly, in a preferred embodiment, an engineered exosome is provided, said engineered exosome comprising (a) a KGF polypeptide fused to a first anchor polypeptide, (b) a VEGF-A polypeptide fused to a second anchor polypeptide, and (c) a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway fused to a third anchor polypeptide, wherein each of the first, second, and third anchor polypeptides comprises the 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 KGF-1 polypeptide, said VEGF-A polypeptide, and said Wnt10b polypeptide are exposed to the outer surface of the membrane of said exosome.

[0078] In a preferred embodiment, an engineered exosome is provided, said engineered exosome comprising (a) a human KGF-1 polypeptide fused to a first anchor polypeptide, (b) a human VEGF-A polypeptide fused to a second anchor polypeptide, and c) a human Wnt10b 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 KGF-1 polypeptide, said human VEGF-A polypeptide, and said human Wnt10b polypeptide are exposed to the outer surface of the membrane of said exosome.

[0079] In a preferred embodiment, an engineered exosome is provided, wherein the engineered exosome comprises: (a) a KGF-1 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. 4; (b) a VEGF-A 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. 5; and (c) SEQ ID NO. A Wnt10b 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 6, wherein each of the first, second, and third anchor polypeptides includes the 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 KGF-1 polypeptide, the VEGF-A polypeptide, and the Wnt10b polypeptide are exposed to the outer surface of the membrane of the exosome.

[0080] In a preferred embodiment, an engineered exosome is provided, wherein the engineered exosome comprises: (a) a KGF-1 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. 4; (b) a VEGF-A 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. 5; and (c) SEQ ID NO. A Wnt10b 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 6, 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 13, wherein (a), (b) and (c) are anchored to the membrane of the exosome through the first, second, and third anchor polypeptides, respectively, wherein the KGF-1 polypeptide, the VEGF-A polypeptide, and the Wnt10b polypeptide are exposed on the outer surface of the membrane of the exosome.

[0081] In a preferred embodiment, an engineered exosome is provided, said engineered exosome comprising (a) a KGF-1 polypeptide fused with a first anchor polypeptide having an amino acid sequence indicated by SEQ ID NO. 4, (b) a VEGF-A polypeptide fused with a second anchor polypeptide having an amino acid sequence indicated by SEQ ID NO. 5, and (c) a Wnt10b polypeptide fused with a third anchor polypeptide having an amino acid sequence indicated by SEQ ID NO. 6, wherein each of the first, second, and third anchor polypeptides is composed of a TM3 domain of CD63, said domain being SEQ ID NO. Having an amino acid sequence indicated by 13, where (a), (b) and (c) are anchored to the membrane of the exosome through the first, second, and third anchor polypeptides, respectively, where the KGF-1 polypeptide, the VEGF-A polypeptide, and the Wnt10b polypeptide are exposed on the outer surface of the exosome membrane.

[0082] In any of the above embodiments, the KGF polypeptide, the VEGF-A polypeptide, and the Wnt family member polypeptide are each fused to the C-terminus of the first, second, and third 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.

[0083] 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.

[0084] In some embodiments, the exosomes provided in the present disclosure promote hair regeneration and / or improve hair loss such as androgenetic alopecia, alopecia areata, or telogen effluvium. For example, the exosomes provided in the present disclosure increase the number of hair follicles, hair follicle length, and / or the growth phase / telogen ratio.

[0085] In some embodiments, the exosomes provided in the present disclosure prevent and / or limit and / or inhibit the formation of gray hair and maintain and / or promote the restoration of the natural color of hair and / or body hair.

[0086] In some embodiments, the exosomes provided in the present disclosure promote hair regeneration and / or improve hair loss such as androgenetic alopecia, alopecia areata or telogen effluvium; prevent and / or limit and / or inhibit the production of gray hair, and maintain and / or promote the restoration of the natural color of hair and / or body hair.

[0087] Method and composition for treating or preventing hair loss

[0088] Each aspect of the present disclosure relates to hair loss, gray hair, or methods and compositions for the treatment or prevention thereof. Hair loss (hair shedding) refers to the phenomenon of hair falling out from the head or any part of the body of a subject. Various types of hair loss are known in the art and are considered herein, including but not limited to androgenetic alopecia (e.g., male pattern baldness and female pattern baldness), alopecia areata, and telogen effluvium. In some embodiments, a method for treating alopecia areata is disclosed. Gray hair (natural hair whitening) is associated with the specific and progressive depletion of hair melanocytes, which affects both the melanocytes of the hair bulb and the precursor cells of the melanocytes. Various types of gray hair are known in the art and are considered herein, including but not limited to age-related hair graying, premature graying, or acute graying. In some embodiments, a method for treating age-related hair graying is disclosed.

[0089] As disclosed herein, the engineered exosomes provided in this disclosure may be used for the treatment and prevention of hair loss, gray hair, or both. Accordingly, embodiments of this disclosure relate to a method for the treatment or prevention of hair loss, gray hair, or both, said method comprising the step of providing an effective amount of engineered exosomes to a subject. In some embodiments, the method of this disclosure comprises the step of administering engineered exosomes to a subject for the treatment or prevention of hair loss, gray hair, or both. Administration of such compositions includes, for example, topical administration, transdermal administration, and intradermal administration.

[0090] Engineered exosomes may be provided to a subject in combination with one or more therapeutic agents or regimens. When said engineered exosomes are used for the treatment of hair loss, in one example, the engineered exosomes of the present disclosure are administered to a subject in combination with diphenylcyclopropenone (e.g., simultaneously, prior, or after). In one example, the engineered exosomes of the present disclosure are administered to a subject in combination with baricitinib (e.g., simultaneously, prior, or after). In one example, the engineered exosomes of the present disclosure are administered to a subject in combination with minoxidil (e.g., simultaneously, prior, or after). In one example, the engineered exosomes of the present disclosure are administered to a subject in combination with finasteride (e.g., simultaneously, prior, or after). In one example, the engineered exosomes of the present disclosure are administered to a subject in combination with spironolactone or dutasteride (e.g., simultaneously, prior, or after). In one example, the engineered exosomes of the present disclosure are administered to a subject in combination with hair transplant surgery (e.g., simultaneously, before, or after).

[0091] When the above-mentioned engineered exosomes are used for the treatment of gray hair, in one example, the engineered exosomes of the present disclosure are administered to a subject in combination with dyeing (e.g., simultaneously, before, or after).

[0092] 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. As a composition comprising the engineered exosomes, examples such as soaps, shampoos, ointments, and other such formulations may be considered.

[0093] In a preferred embodiment, the composition does not include a KGF polypeptide (e.g., KGF-1 polypeptide), a VEGF-A polypeptide, or a Wnt family member polypeptide (e.g., Wnt10b polypeptide) that participates in the Wnt / β-catenin signaling pathway 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 KGF polypeptide (e.g., KGF-1 polypeptide), a VEGF-A polypeptide, or a Wnt family member polypeptide (e.g., Wnt10b polypeptide) that participates in the Wnt / β-catenin signaling pathway is added to the composition.

[0094] 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. In some embodiments, the composition comprises other active agents for treating hair loss. Preferably, other active agents for treating hair loss are selected from diphenylcyclopropenone, baricitinib, minoxidil, finasteride, spironolactone, dutasteride, or any combination thereof. In a preferred embodiment, other active agents for treating hair loss are diphenylcyclopropenone, baricitinib, or minoxidil. In some embodiments, the composition comprises other active agents for treating gray hair.

[0095] Nucleic acid constructs, vectors, cells, and methods of generation

[0096] Each aspect of the present disclosure also relates to a nucleic acid structure encoding a polypeptide anchored to an exosome.

[0097] In some embodiments, a nucleic acid structure is provided, said nucleic acid structure comprises a polynucleotide encoding a substance such as (a) a KGF polypeptide fused to a first anchor polypeptide, (b) a VEGF-A polypeptide fused to a second anchor polypeptide, and (c) a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway fused to a third anchor polypeptide.

[0098] 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.

[0099] In some embodiments, a set of two nucleic acid structures is provided, wherein one nucleic acid structure comprises a polynucleotide encoding (a) and (b), and the other nucleic acid structure comprises a polynucleotide encoding (c), wherein (a), (b) and (c) are as defined above.

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

[0101] In some embodiments, a set of two nucleic acid structures is provided, wherein one nucleic acid structure comprises a polynucleotide encoding (b) and (c), and the other nucleic acid structure comprises a polynucleotide encoding (a), wherein (a), (b) and (c) are as defined above.

[0102] 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.

[0103] 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.

[0104] In some embodiments, each of the first, second, and third anchor polypeptides is located at the N-terminus of Wnt, KGF, and / or VEGF to ensure that Wnt, KGF, and / or VEGF are exposed to the surface of the exosome. In some embodiments, each of the first, second, and third anchor polypeptides is located at the C-terminus of Wnt, KGF, and / or VEGF to ensure that Wnt, KGF, and / or VEGF are exposed to the surface of the exosome. For example, if lamp2b is used as one of these anchor polypeptides, the polypeptides to be presented on the exosome surface (e.g., Wnt, KGF, and VEGF) may be located at the N-terminus of lamp2b. For example, if the TM3 domain of CD63 is used as one of these anchor polypeptides, the polypeptides to be presented on the exosome surface (e.g., Wnt, KGF, and VEGF) 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 the anchor polypeptide, which is determined by the type of anchor polypeptide used.

[0105] In a preferred embodiment, the first, second, and third 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 comprises the TM3 domain of CD63. In a preferred embodiment, each of the first, second, and third anchor polypeptides comprises the TM3 domain of CD63.

[0106] In a preferred embodiment, the polynucleotide is from 5' to 3',

[0107] (i) [1st anchor polypeptide]-[KGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[3rd anchor polypeptide]-[Wnt],

[0108] (ii) [1st anchor polypeptide]-[KGF]-[self-cleaving peptide]-[2nd anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[3rd anchor polypeptide]-[VEGF-A],

[0109] (iii) [1st anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[2nd anchor polypeptide]-[KGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[Wnt],

[0110] (iv) [1st anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[2nd anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[3rd anchor polypeptide]-[KGF],

[0111] (v) [1st anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[2nd anchor polypeptide]-[KGF]-[self-cleaving peptide]-[3rd anchor polypeptide]-[VEGF-A], or

[0112] (vi) comprising nucleotide fragments encoding substances such as [1st anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[2nd anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[3rd anchor polypeptide]-[KGF], etc.

[0113] Here, [] represents a single polypeptide, and ]-[ represents a linker or bond; where each of the polypeptides is arranged from the N-terminus to the C-terminus; where Wnt represents a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway.

[0114] 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 produces three independent fusion proteins, each comprising a single polypeptide and a single anchor polypeptide, which are cleaved and presented on the surface of the exosome.

[0115] In another embodiment, a single polynucleotide comprises a nucleotide fragment encoding (a), (b) and (c), and the single polynucleotide comprises a nucleotide fragment encoding a substance such as [first anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[VEGF-A]-[second anchor polypeptide]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF] from 5' to 3', wherein the second anchor polypeptide is located at the C-terminus of the VEGF-A polypeptide. This modification applies to any polynucleotide listed in (i) through (vi) above. In some embodiments, since each of the first, second, and third anchor polypeptides is different, such anchor polypeptides may be located at the N-terminus or C-terminus of polypeptides Wnt, KGF, and VEGF-A.

[0116] In a preferred embodiment, a single nucleic acid structure is provided comprising polynucleotides encoding (a), (b) and (c), wherein (a), (b) and (c) are as defined above, wherein each of the first, second, and third anchor polypeptides comprises a TM3 domain of CD63, and the polynucleotides are from 5' to 3',

[0117] (i) [TM3]-[KGF]-[T2A]-[TM3]-[VEGF-A]-[T2A]-[TM3]-[Wnt],

[0118] (ii) [TM3]-[KGF]-[T2A]-[TM3]-[Wnt]-[T2A]-[TM3]-[VEGF-A];

[0119] (iii) [TM3]-[VEGF-A]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[Wnt],

[0120] (iv) [TM3]-[VEGF-A]-[T2A]-[TM3]-[Wnt]-[T2A]-[TM3]-[KGF],

[0121] (v) [TM3]-[Wnt]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[VEGF-A], and

[0122] (vi) May include a nucleotide fragment encoding one of the substances such as [TM3]-[Wnt]-[T2A]-[TM3]-[VEGF-A]-[T2A]-[TM3]-[KGF], and

[0123] Here, [] represents a single polypeptide, and ]-[ represents a linker or bond; where each of the polypeptides is arranged from the N-terminus to the C-terminus; where, Wnt represents a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway; TM3 represents the TM3 domain of CD63; and T2A represents the self-cleaving peptide T2A.

[0124] In this section, the Wnt family member polypeptides participating in the KGF, VEGF-A, and Wnt / β-catenin signaling pathways have the meaning and preferred embodiments presented in the section titled “Engineered Exosomes”.

[0125] In some embodiments, the polynucleotide encoding the KGF polypeptide has a nucleotide sequence indicated by SEQ ID NO. 7, SEQ ID NO. 10, or a degenerate sequence thereof.

[0126] In some embodiments, the polynucleotide encoding the VEGF-A polypeptide has a nucleotide sequence indicated by SEQ ID NO. 8, SEQ ID NO. 11, or a degenerate sequence thereof.

[0127] In some embodiments, the polynucleotide encoding the Wnt family member polypeptide has a nucleotide sequence indicated by SEQ ID NO. 9, SEQ ID NO. 12, or a degenerate sequence thereof.

[0128] In some embodiments, the polynucleotide encoding the TM3 domain of the CD63 has a nucleotide sequence indicated by SEQ ID NO. 14 or a degenerate sequence thereof.

[0129] 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.

[0130] 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 engineered exosomes.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] Ranking table

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142] Examples

[0143] Example 1. Construction of engineered exosomes derived from a stable cell line.

[0144] Materials and Methods

[0145] 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).

[0146] pGOI Plasmid Construction: The amino acid sequences of all target genes (including KGF-1, VEGF-A, and Wnt10b) were all derived from Uniprot, and the corresponding DNA sequences (see SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12, respectively) were synthesized into the plasmid pCDH-CMV-MCS-EF1a-GFP+BSD (System Biosciences) by General Biotechnology (Chuzhou, China). T2A peptides (see SEQ ID NO. 17 and SEQ ID NO. 18) 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 for lentivirus generation, where pCDH-CMV-MCS-EF1a-GFP+BSD (blasticidin resistance) was used as the lentivirus packaging plasmid. The lentivirus was packaged by WZ Biotechnology (Jinan, China). Payload gene constructed in pGOI plasmid: CD63-TM3-linker- KGF-1 -T2A-CD63-TM3-Linker- VEGF-A -T2A-CD63-TM3-Linker- Wnt10b In this case, each CD63-TM3 is the TM3 domain of CD63 and is encoded by the DNA sequence labeled SEQ ID NO. 14, each linker is encoded by the DNA sequence labeled SEQ ID NO. 16, and each T2A is encoded by the DNA sequence labeled SEQ ID NO. 18.

[0147] Generation of stable cell lines: Stable HEK293 cell lines expressing target proteins (including KGF-1, VEGF-A, and Wnt10b) were generated by infection with the corresponding lentivirus. 48 hours after infection, cells were selected by adding an antibiotic such as 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.

[0148] Adaptation of cell cultures to SFM (serum-free medium): Stable cell lines were first subcultured three times in FM (complete medium), and then began to be adapted to serum-free culture starting from the fourth subculture. Cells were subcultured using the medium composition of Table 1. To establish a culture fully adapted to serum-free culture, cells must be subcultured at least three times in SFM (HyClone™ peak expression, SH31193.02, Cytiva Life Sciences).

[0149] Adaptive media step Gradual adaptation 0 100% FM 1 50% FM + 50% SFM 2 25% FM + 75% SFM 3 12.5% ​​FM + 87.5% SFM 4 5% FM + 95% SFM 5 2.5% FM + 97.5% SFM 6 100% SFM

[0150] 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 culture, 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, dead 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, and the obtained exosomes are referred to as #35 exosomes in the following examples.

[0151] 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.

[0152] Example 2. Characterization of exosomes

[0153] Analysis of Exosome Particle Concentration and Size Distribution

[0154] The particle concentration and size distribution of exosomes derived from stable cell lines were analyzed using NanoFCM (NanoFCM, Xiamen, China). The NanoFCM assay utilized two single-photon counting avalanche photodiodes (APDs) to simultaneously detect the side scattering (SSC) and fluorescence of single particles. First, exosome precipitates were prepared for analysis. Next, 200 nm PE and AF488 fluorescent dye-conjugated polystyrene beads were used for particle concentration measurements, while 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 rates and side scattering intensities were converted into vesicle concentration and size using NanoFCM software (NanoFCM Professional V2.0).

[0155] Figure 2a illustrates the analysis of particle size and particle concentration of exosomes derived from a stable cell line using NanoFCM. The exosomes have an average particle size of 75.4 nm.

[0156] Identification of exosome target proteins using immune blot (WB)

[0157] To assess the expression of target proteins in exosomes, purified exosomes were lysed in RIPA lysis buffer (Beyotime) 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 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).

[0158] Transmission Electron Microscopy (TEM) Analysis of Exosomes

[0159] 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).

[0160] Figure 2b shows a transmission electron microscope (TEM) image of exosomes. Figure 2c shows an immunoblot analysis of exosomes using an anti-CD63-TM3 antibody (a scaffold protein fused to exosomes). As expected, engineered exosome-associated target proteins (Wnt10b, VEGF-A, and KGF-1) were present in purified exosomes derived from stable cell lines.

[0161] Example 3. Evaluation of Engineered Exosomes for Hair Loss Treatment in a Mouse Model

[0162] Animal and in vivo studies

[0163] All experiments were performed using 6-week-old male C57BL / 6 mice. The animals were purchased from Cavens (Changzhou, China). Figure 3a illustrates a schematic diagram of the androgenetic alopecia mouse model and the solution. After depilating the dorsal hair of the mice, the mouse skin was treated once every 3 days with 35# exosomes, control exosomes (Ctrl Exo.), PBS (negative control), or 5% minoxidil (positive control). After continuous observation during the 18-day treatment period, the animals were sacrificed on the 18th day for histological analysis. In the in vivo treatment experiment, the mice were first shaved, hair was removed using a depilatory cream, and pink skin was observed. The day after depilation, a 30 mg / EA DHT (dihydrotestosterone) suspension was subcutaneously injected into five points on the dorsal depilatory site. The animals were randomly divided into four groups (n=4) to study hair regeneration. After depilating the mice, the dorsal skin of the mice was treated every 3 days with 35# exosomes, control exosomes, and PBS (negative control) or 5% minoxidil (positive control). Exosome treatment dose: 8.0×10⁻⁶ in 100 μl PBS 9 Exosomes were subcutaneously injected into five sites on the dorsal skin (20 μl at each site). 200 μl of minoxidil was applied topically daily. The dorsal skin was photographed on days 0, 9, 12, and 15. The depilated and hair growth areas of the mice were analyzed using Image J. Hair coverage (%) = (New hair area / Depilated area) × 100%. After continuous observation during the 18-day treatment period, the animals were sacrificed for further analysis.

[0164] skin histology

[0165] Mice were euthanized, and the entire dorsal skin was excised. The skin tissue was fixed in 4% paraformaldehyde, then cut into sections using a cryostat, and stained with hematoxylin and eosin (HE). Five fields were randomly selected from each section for observation. The number of hair follicles in the dermis was statistically analyzed, and the ratio of growing to resting follicles was calculated. The average follicle length was calculated by measuring the distance from the hair follicle to the epidermis.

[0166] Figure 3B illustrates the results of hair cover observations. Mice were divided into four groups (n=4), and representative images of the mice on day 15 are shown. The results demonstrated that 35# exosome treatment could restore hair cover almost completely on day 15, which is similar to the efficacy of the positive control, minoxidil. Figure 3c illustrates the histological analysis of the number of hair follicles, length, and growth phase ratio of the dorsal skin of mice. Data points represent the mean ± SD (n=4). Error bars represent the SD; ns indicates no significant difference; and *P<0.05, **P<0.01, and ***P<0.001. 35# exosome treatment significantly increased the number and length of hair follicles and the extended growth / resting phase ratio, which suggests that it can promote the normalization of hair follicles and further enhance the transition from the resting to the growth phase.

[0167] Example 4. Evaluation of Engineered Exosomes for Gray Hair Treatment in a Mouse Model

[0168] All experiments were performed using 6-week-old female C57BL / 6 mice. Figure 4a is a schematic diagram of the hydroquinone-induced hair graying model and the solution. In the in vivo treatment experiments, the mice were first shaved, and then the hair was removed using a depilatory cream. The animals were randomly divided into four groups (n=4) to study hair graying. The day after depilation, a 2.5% concentration of hydroquinone cream was applied topically to the depilated area on the back. The negative model control group was topically treated with PBS. The 35# exosome groups were treated via different routes of administration (e.g., topical application after microneedle inoculation, topical application after nanocrystal injection, subcutaneous injection). Exosome treatment dose: A volume of 200 μl of 1E11 exosomes was administered via different routes. The back hair of the mice was continuously monitored during the 28-day treatment period. Table 2 shows a summary of the treatment solution.

[0169] Summary of therapeutic solutions for different groups army therapy Administration route Frequency Capacity (particles) Volume (mL) G1 PBS embrocation Q3D*10 / 0.2mL G2 35#Exosome Microneedle inoculation + application Q3D*10 1E11 0.2mL G3 35#Exosome Nanocrystal injection + coating Q3D*10 1E11 0.2mL G4 35#Exosome subcutaneous injection Q3D*10 1E11 0.2mL

[0170] Figure 4b illustrates the observation results for the dorsal hair. Representative images of mice on day 14 are shown. The results showed that mice treated with 35# exosomes via different administration routes (e.g., topical application after microneedle inoculation, topical application after nanocrystal injection, and subcutaneous injection) had hair with darker coloration in all treatment groups compared to the mock treatment.

Claims

Claim 1 An engineered exosome, wherein the engineered exosome comprises (a) a KGF polypeptide fused with a first anchor polypeptide, (b) a VEGF-A polypeptide fused with a second anchor polypeptide, and (c) a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway 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 KGF polypeptide, the VEGF-A polypeptide, and the Wnt family member polypeptide are exposed to the outer surface of the membrane of the exosome. Claim 2 The engineered exosome according to claim 1, wherein the KGF polypeptide is a KGF-1 polypeptide, and preferably, the KGF-1 polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO.

4. Claim 3 In claim 1, the VEGF-A polypeptide is human VEGF 206 An engineered exosome that is an isomer or a parallel homolog or a series homolog thereof, preferably, said VEGF-A polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 2 or SEQ ID NO.

5. Claim 4 The engineered exosome according to claim 1, wherein the Wnt family member polypeptide is Wnt10a, Wnt10b, Wnt3a, Wnt1a, Wnt4, Wnt5a, Wnt7a, or Wnt7b polypeptide; preferably, the Wnt family member polypeptide is Wnt10b polypeptide; more preferably, the Wnt10b polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 3 or SEQ ID NO.

6. Claim 5 In claim 1, 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 glycosylphosphatidylinositol (GPI) anchor and lipid anchor proteins; preferably, the first, second, and third anchor polypeptides comprise full-length CD63 or truncated CD63 with the TM3 domain preserved; more preferably, each of the first, second, and third anchor polypeptides comprises the TM3 domain of CD63; More preferably, each of the first, second, and third anchor polypeptides is a TM3 domain of CD63; more 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. 13, an engineered exosome. Claim 6 In claim 1, the KGF polypeptide, the VEGF-A polypeptide, and the Wnt family member polypeptide are each optionally fused to the C-terminus of the first, second, and third 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, engineered exosome. Claim 7 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 8 The engineered exosome of claim 1, wherein the exosome is (i) promoting hair regeneration and / or improving hair loss such as androgenetic alopecia, alopecia areata or telogen effluvium; and / or (ii) preventing and / or limiting and / or inhibiting the production of gray hair, and maintaining and / or promoting the restoration of the natural color of hair and / or body hair. Claim 9 A composition comprising an exosome and a carrier according to any one of claims 1 to 8; preferably, the composition is prepared for topical or subcutaneous administration; the composition is optionally a cosmetic composition or a non-cosmetic composition; and the composition is optionally a pharmaceutical composition. Claim 10 In claim 9, the composition does not include a KGF polypeptide not attached to the membrane of the exosome (e.g., KGF-1 polypeptide), a VEGF-A polypeptide, or a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway (e.g., Wnt10b polypeptide). Claim 11 A nucleic acid structure comprising a polynucleotide encoding a substance such as (a) a KGF polypeptide fused with a first anchor polypeptide, (b) a VEGF-A polypeptide fused with a second anchor polypeptide, and (c) a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway fused with a third anchor polypeptide. Claim 12 In claim 11, (i) the KGF polypeptide is a KGF-1 polypeptide, preferably, the KGF-1 polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO. 1 or SEQ ID NO. 4; and (ii) the VEGF-A polypeptide is human VEGF 206 (iii) the isomer or its parallel or serial homolog, and preferably, the VEGF-A polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 2 or SEQ ID NO. 5; (iii) the Wnt family member polypeptide is Wnt10a, Wnt10b, Wnt3a, Wnt1a, Wnt4, Wnt5a, Wnt7a, or Wnt7b polypeptide; preferably, the Wnt family member polypeptide is Wnt10b polypeptide; more preferably, the Wnt10b polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence indicated by SEQ ID NO. 3 or SEQ ID NO. 6; and (iv) the polynucleotide encoding the KGF polypeptide is SEQ ID NO. 7, SEQ ID NO. (v) the polynucleotide encoding the VEGF-A polypeptide has the nucleotide sequence indicated by SEQ ID NO. 8, SEQ ID NO. 11, or the degenerate sequence thereof; (vi) the polynucleotide encoding the Wnt family member polypeptide has SEQ ID NO. 9, SEQ ID NO.It has a nucleotide sequence indicated by 12 or a degenerate sequence thereof; (vii) 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 first, second, and third anchor polypeptides comprise a full-length CD63 or a truncated CD63 with a preserved TM3 domain; More preferably, each of the first, second, and third anchor polypeptides comprises the TM3 domain of CD63; more preferably, each of the first, second, and third anchor polypeptides comprises the TM3 domain of CD63; and / or (viii) a nucleic acid structure in which the polynucleotide encoding the TM3 domain of CD63 has the nucleotide sequence indicated by SEQ ID NO. 14 or a degenerate sequence thereof. Claim 13 In claim 11 or 12, the polynucleotide is a single polynucleotide comprising a nucleotide fragment encoding (a), (b) and (c); preferably, the polynucleotide is a single polynucleotide comprising a nucleotide fragment encoding (a), (b) and (c) from 5' to 3'; Preferably, the polynucleotide is arranged from 5' to 3', (i) [1st anchor polypeptide]-[KGF]-[self-cleavage peptide]-[2nd anchor polypeptide]-[VEGF-A]-[self-cleavage peptide]-[3rd anchor polypeptide]-[Wnt], (ii) [1st anchor polypeptide]-[KGF]-[self-cleavage peptide]-[2nd anchor polypeptide]-[Wnt]-[self-cleavage peptide]-[3rd anchor polypeptide]-[VEGF-A], (iii) [1st anchor polypeptide]-[VEGF-A]-[self-cleavage peptide]-[2nd anchor polypeptide]-[KGF]-[self-cleavage peptide]-[3rd anchor polypeptide]-[Wnt], (iv) [1st anchor polypeptide]-[VEGF-A]-[self-cleavage peptide]-[2nd anchor polypeptide]-[Wnt]-[self-cleavage peptide]-[3rd anchor A nucleic acid structure comprising a nucleotide fragment encoding a substance such as [polypeptide]-[KGF], (v) [first anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[second anchor polypeptide]-[KGF]-[self-cleaving peptide]-[third anchor polypeptide]-[VEGF-A], or (vi) [first anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[second anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF], wherein [ ] indicates a single polypeptide, and ]-[ indicates a linker or bond; Wnt indicates a Wnt family member polypeptide participating in the Wnt / β-catenin signaling pathway; optionally, the self-cleaving peptide is a 2A peptide, e.g., T2A, E2A, P2A, or any combination thereof. Claim 14 A vector, wherein the vector comprises a nucleic acid structure according to any one of claims 11 to 13; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector or an adeno-associated virus vector. Claim 15 Cells transfected with a vector according to claim 14, 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 16 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 15 with a vector according to claim 14; (b) culturing the cell under conditions that allow exosomes to be secreted from the cell; and (c) collecting and purifying the exosomes. Claim 17 A method for generating engineered exosomes according to claim 16, wherein the method further comprises the step of adapting the cells to serum-free conditions during step (b). Claim 18 The 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 treating alopecia and / or gray hair, wherein, preferably, the alopecia is androgenetic alopecia, alopecia areata, or telogen effluvium; and preferably, the gray hair is age-related hair graying, premature graying, or acute graying.

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