TW202532639A - Engineered exosomes for treating hair loss and/or graying - Google Patents

Abstract

The present invention discloses an engineered exosome comprising (a) a KGF polypeptide fused to a first anchoring polypeptide, (b) a VEGF-A polypeptide fused to a second anchoring polypeptide, and (c) a Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway fused to a third anchoring polypeptide, wherein (a), (b), and (c) are anchored to the exosome membrane via the first, second, and third anchoring polypeptides, respectively, and wherein the KGF polypeptide, the VEGF-A polypeptide, and the Wnt family member polypeptide are exposed on the outer surface of the exosome membrane. Also disclosed are compositions, nucleic acid constructs, and uses of the exosomes for treating hair loss and/or graying.

Description

Engineered exosomes for the treatment of hair loss and/or graying

This disclosure relates to engineered exosomes, specifically exosomes carrying different fusion proteins anchored to the exterior of the exosome membrane. This disclosure also relates to compositions comprising the engineered exosomes, nucleic acid constructs comprising polynucleotides encoding the fusion proteins, and the use of engineered exosomes in preventing or treating hair loss and/or graying.

Hair loss, also known as alopecia or hair loss, refers to the loss of hair from the head or body. Hair loss can range in severity from a small area to a complete loss of the entire body. It can be triggered by psychological stress, medication, or other factors. Common types of hair loss include androgenic alopecia (including male and female pattern baldness), alopecia areata, and a thinning hair condition called telogen effluvium. The causes of male pattern baldness include a combination of genetics and androgen effects, while the causes of female pattern baldness are unknown. The cause of alopecia areata is autoimmune, while the cause of telogen effluvium is often a stressful event, either physically or psychologically.

The hair follicle is a regenerative organ where stem cells are capable of massive renewal. The hair follicle is composed of an outer root sheath, an inner root sheath, and the hair shaft. Proliferating undifferentiated stromal cells form the inner root sheath and hair shaft, which are surrounded by the dermal papilla, composed of specialized mesenchymal cells. The dermal papilla directs the formation of the hair follicle, but the characteristics of the hair follicle are acquired through epithelial information. The lower portion of the hair follicle undergoes a growth cycle consisting of the phases of anagen (active growth), catagen (destruction), and telogen (resting). These different phases last for different periods of time, depending on the location and function of the hair follicle. During the anagen phase, stromal cells rapidly proliferate, migrate upward, and then differentiate into the cell types of the inner root sheath and hair stem. During the catagen phase, the lower hair follicle undergoes apoptotic death, and the dermal papilla migrates upward until it reaches the area below the bulge. During the telogen phase, the dermal papilla remains in this area. Once the dermal papilla recruits stem cells from the bulge, the anagen phase resumes, and the hair follicle can regenerate through proliferation and differentiation.

Managing hair loss usually involves medication or surgery. Medications currently used to treat hair loss are minoxidil, finasteride, and dutasteride. Hair loss can be managed through surgical procedures, including hair transplantation. Hair transplantation is usually performed under local anesthesia. The surgeon transplants healthy hair from the back and sides of the head to the areas where hair is thinning. The procedure can take four to eight hours, and additional treatments may be performed to create thicker hair. Conventional hair transplantation methods are ineffective when there isn't enough hair in the non-bald areas. Stem cell therapy represents an emerging strategy for treating and managing hair loss. However, in the case of conventional hair transplantation, administering hair follicle stem cells to the balding scalp area is very inefficient because the skin in such areas does not have the growth factors necessary for new hair follicles to grow.

Graying (natural hair graying) is associated with a specific and progressive depletion of hair melanocytes, affecting both the melanocytes and melanocyte precursors of 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 this progressive and specific depletion of melanocytes and melanocyte precursors in the hair follicle remains unknown.

Therefore, there is a need for additional and improved methods and compositions for treating hair loss, including androgenic alopecia and alopecia areata. To prevent gray hair, it is also necessary to prevent the loss of melanocytes in human hair follicles, a process that affects both active melanocytes in the hair bulb and quiescent melanocytes in the upper region of the follicle.

In one aspect of the present invention, an engineered exosome is provided, comprising (a) a KGF polypeptide fused to a first anchoring polypeptide, (b) a VEGF-A polypeptide fused to a second anchoring polypeptide, and (c) a Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway, fused to a third anchoring polypeptide, wherein (a), (b) and (c) are anchored to the membrane of the exosome via the first, second and third anchoring polypeptides, respectively, and wherein the KGF polypeptide, the VEGF-A polypeptide and the Wnt family member polypeptide are exposed on the outer surface of the membrane of the exosome.

In some embodiments, the KGF polypeptide is a KGF-1 polypeptide. Preferably, the KGF-1 polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4.

In some embodiments, the VEGF-A polypeptide is a human VEGF ₂₀₆ isomer or an ortholog or paralog thereof. Preferably, the VEGF-A polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5.

In some embodiments, the Wnt family member polypeptide is a Wnt10a, Wnt10b, Wnt3a, Wnt1a, Wnt4, Wnt5a, Wnt7a, or Wnt7b polypeptide; preferably, the Wnt family member polypeptide is a Wnt10b polypeptide; more preferably, the Wnt10b polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 6.

In some embodiments, these anchoring polypeptides are exosomal membrane proteins, membrane targeting sequences, or anchoring functional fragments thereof. Exemplary exosomal membrane proteins include, but are not limited to, LAMP2b, tetraspanins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid-anchored proteins.

In preferred embodiments, the first, second, and third anchoring polypeptides comprise full-length CD63 or a truncated CD63 that retains the TM3 domain. In preferred embodiments, each of the first, second, and third anchoring polypeptides comprises the TM3 domain of CD63. In preferred embodiments, each of the first, second, and third anchoring polypeptides is the TM3 domain of CD63. In some embodiments, each of the first, second, and third anchoring polypeptides is the TM3 domain of CD63; preferably, the TM3 domain of CD63 comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 13.

In some embodiments, the KGF polypeptide, the VEGF-A polypeptide, and the Wnt family member polypeptide are optionally fused to the C-terminus of the first, second, and third anchor polypeptides, respectively, via a peptide linker. Preferably, the peptide linker consists of glycine and serine, such as (G4S)n, where n is an integer from 1 to 3.

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

In some embodiments, the exosomes promote hair regrowth and/or improve hair loss, such as androgenic alopecia, alopecia areata, or telogen effluvium. In some embodiments, the exosomes prevent and/or limit and/or stop the formation of gray hair and maintain and/or promote the natural re-pigmentation of hair and/or body hair. In some embodiments, the exosomes: (i) promote hair regrowth and/or improve hair loss, such as androgenic alopecia, alopecia areata, or telogen effluvium; and (ii) prevent and/or limit and/or stop the formation of gray hair and maintain and/or promote the natural re-pigmentation of hair and/or body hair.

Another aspect of the present disclosure relates to a composition comprising the exosomes according to any one of claims 1 to 8, and a carrier; preferably, the composition is a liquid preparation; more preferably, the composition is formulated for topical or subcutaneous administration.

In some embodiments, the composition does not contain KGF polypeptide (e.g., KGF-1 polypeptide), VEGF-A polypeptide, or Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway (e.g., Wnt10b polypeptide) that is not attached to the membrane of the exosome.

Another aspect of the present disclosure relates to a nucleic acid construct comprising a polynucleotide encoding: (a) a KGF polypeptide fused to a first anchoring polypeptide, (b) a VEGF-A polypeptide fused to a second anchoring polypeptide, and (c) a Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway fused to a third anchoring polypeptide.

In some embodiments, the KGF polypeptide is a KGF-1 polypeptide. Preferably, the KGF-1 polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4.

In some embodiments, the VEGF-A polypeptide is a human VEGF ₂₀₆ isomer or an ortholog or paralog thereof. Preferably, the VEGF-A polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5.

In some embodiments, the Wnt family member polypeptide is a Wnt10a, Wnt10b, Wnt3a, Wnt1a, Wnt4, Wnt5a, Wnt7a, or Wnt7b polypeptide; preferably, the Wnt family member polypeptide is a Wnt10b polypeptide; more preferably, the Wnt10b polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 6.

In some embodiments, the polynucleotide encoding the KGF polypeptide has a nucleotide sequence as shown in SEQ ID NO: 7, SEQ ID NO: 10, or a degenerate sequence thereof.

In some embodiments, the polynucleotide encoding the VEGF-A polypeptide has a nucleotide sequence as shown in SEQ ID NO: 8, SEQ ID NO: 11, or a degenerate sequence thereof.

In some embodiments, the polynucleotide encoding the Wnt family member polypeptide has a nucleotide sequence as shown in SEQ ID NO: 9, SEQ ID NO: 12, or a degenerate sequence thereof.

In some embodiments, these anchoring polypeptides are exosomal membrane proteins, membrane targeting sequences, or anchoring functional fragments thereof. Exemplary exosomal membrane proteins include, but are not limited to, LAMP2b, tetraspanins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid-anchored proteins.

In preferred embodiments, the first, second, and third anchoring polypeptides comprise full-length CD63 or truncated CD63 that retains the TM3 domain. In preferred embodiments, each of the first, second, and third anchoring polypeptides comprises the TM3 domain of CD63. In preferred embodiments, each of the first, second, and third anchoring polypeptides is the TM3 domain of CD63. In some embodiments, the polynucleotide encoding the TM3 domain of CD63 has the nucleotide sequence set forth in SEQ ID NO: 14 or a degenerate sequence thereof.

In some embodiments, the polynucleotide is a single polynucleotide comprising nucleotide fragments encoding (a), (b) and (c); preferably, the polynucleotide comprises nucleotide fragments encoding the following substances from 5' to 3':

(i) [First anchoring polypeptide]-[KGF]-[self-cleaving peptide]-[Second anchoring polypeptide]-[VEGF-A]-[self-cleaving peptide]-[Third anchoring polypeptide]-[Wnt],

(ii) [first anchor polypeptide]-[KGF]-[self-cleaving peptide]-[second anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[third anchor polypeptide]-[VEGF-A],

(iii) [first anchoring polypeptide]-[VEGF-A]-[self-cleaving peptide]-[second anchoring polypeptide]-[KGF]-[self-cleaving peptide]-[third anchoring polypeptide]-[Wnt],

(iv) [first anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[second anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[third anchor 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, [] represents a single polypeptide, and ]-[ represents a linker or bond;

Wnt represents a Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway.

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

In other embodiments, each of the first, second, and third anchoring polypeptides is located at the C-terminus of Wnt, KGF, and/or VEGF to ensure exposure of Wnt, KGF, and/or VEGF on the surface of the exosomes. For example, when lamp2b is used as one of these anchoring polypeptides, the polypeptide to be presented on the exosome surface (e.g., Wnt, KGF, and VEGF) can be located at the N-terminus of lamp2b. In some embodiments, when different anchoring polypeptides are used, the polypeptide to be presented on the exosome surface can be located at the N-terminus or C-terminus of these anchoring polypeptides, depending on the type of anchoring polypeptide used. For example, in one embodiment, a single polynucleotide comprises nucleotide fragments encoding (a), (b), and (c), and the single polynucleotide comprises, from 5' to 3', a nucleotide fragment encoding the following substances: [first anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[VEGF-A]-[second anchor polypeptide]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF], wherein the second anchor polypeptide is located at the C-terminus of the VEGF-A polypeptide.

Another aspect of the present disclosure relates to a vector comprising a nucleic acid construct described in any one of the nucleic acid constructs described herein.

Another aspect of the present disclosure relates to a cell transduced with a vector described herein, wherein the polynucleotide is integrated into the genome of the cell.

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 a HEK293 or CHO cell.

Another aspect of the present disclosure relates to a method for producing engineered exosomes as disclosed herein, comprising (a) transducing 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.

In some embodiments, the method further comprises adapting the cells to serum-free conditions during step (b).

Another aspect of the present disclosure relates to the use of the engineered exosomes or compositions disclosed herein for the preparation of a medicament for treating hair loss and/or gray hair; preferably, the hair loss is androgenic alopecia, alopecia areata, or telogen effluvium; preferably, the gray hair is age-related graying of hair, premature graying of hair, or sudden graying of hair.

Another aspect of the present disclosure relates to a method for treating hair loss and/or gray hair; preferably, the hair loss is androgenic alopecia, alopecia areata, or telogen effluvium; preferably, the gray hair is age-related graying, premature graying, or sudden graying of hair; the method comprises administering to a subject an effective amount of the engineered exosomes or composition disclosed herein.

Another aspect of the present disclosure relates to the engineered exosomes or compositions disclosed herein for use in treating hair loss and/or gray hair; preferably, the hair loss is androgenic alopecia, alopecia areata, or telogen effluvium; preferably, the gray hair is age-related graying of hair, premature graying of hair, or sudden graying of hair.

These and other aspects and advantages of the present disclosure will be apparent from the detailed description provided below.

Figure 1 shows the construction of a stable cell line that secretes engineered exosomes loaded with functional proteins. Mammalian cells (e.g., HEK293 cells) are cultured and infected with lentivirus containing the functional genes KGF, VEGFA, and Wnt10b. Stable cell lines are then selected using blasticidin. After three passages, expression of the functional gene is detected in both cell pellets and exosomes. After confirming expression, the stable cell line is adapted to culture under serum-free conditions. Finally, the culture supernatant of the serum-free stable cell line is collected and the exosomes purified by ultracentrifugation for identification.

Figure 2 shows the identification of engineered exosomes for androgenic alopecia. Figure 2A shows the particle size and concentration of exosomes derived from a stable cell line analyzed by NanoFCM. Figure 2B shows the examination of exosomes by transmission electron microscopy (TEM). Figure 2C shows immunoblot analysis of exosomes using an antibody against CD63-TM3, a scaffold protein fused to the exosomes.

Figure 3 shows the in vivo evaluation of engineered exosomes 35# for the treatment of hair loss. Figure 3A shows a schematic diagram of the androgenic alopecia mouse model and protocol. After hair removal on the back of 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). Observation continued during the 18-day treatment period, and the animals were then sacrificed on the 18th day for histological analysis. Figure 3B shows the observation of hair coverage. The mice were divided into four groups (n=4), and representative images of the mice on the 15th day are shown. Figure 3C shows the histological analysis of the back skin of the mice regarding the number, length, and growth stage ratio of hair follicles. Data points represent mean ± SD (n=4). Error bars indicate standard deviation (SD); ns indicates no significant difference; *P<0.05, **P<0.01, and ***P<0.001.

Figure 4 shows an in vivo evaluation of engineered exosome 35# in combating hair graying. Figure 4A shows a schematic diagram of the hydroquinone-induced hair graying model and protocol. After dorsal hair removal, female C57BL/6 mice were randomly divided into groups (n=4) and topically applied with hydroquinone cream twice daily. A negative model control group was topically treated with PBS. The 35# exosome groups were treated using different application routes (e.g., microneedle vaccination followed by application, nanocrystal infusion followed by application, subcutaneous injection). Observations were continued over the 28-day treatment period. Figure 4B shows observations of dorsal hair. Representative images of mice on day 14 are shown.

Definition

When used with the word "comprising" in this specification, including the claims, the words "a" and "an" mean "one or more."

As used herein, the terms "or" and "and/or" are used to describe combinations of multiple components or mutual exclusions. For example, "x, y, and/or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." Specifically, x, y, or z can be explicitly excluded from the embodiment.

In this application, the term "about" is used according to its ordinary and customary meaning in the fields of cellular and molecular biology to indicate that a value includes the standard deviation of error for the device or method employed to determine the value.

"Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences varies depending on the number of matching or homologous positions the sequences have. An "unrelated" or "nonhomologous" sequence has less than 40% identity, but preferably less than 25% identity, to one of the disclosed sequences.

When a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a particular percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, it means that when aligned, the percentage of bases (or amino acids) in the two sequences is the same. Such alignment and homology or sequence identity percentage can be determined using software programs known in the art.

As used herein, the term "linker" refers to a short stretch of amino acids (AA) or a nucleotide sequence containing two or more identical or different amino acids or nucleotides.

As used herein, a "cell line" refers to a cell population formed by one or more subcultures of a primary cell culture. Each round of subculture is called a passage. After cells have been subcultured, they are referred to as "passaged" cells. A particular cell population or cell line is sometimes referred to or characterized by the number of passages it has been subcultured. For example, a cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture after isolation of cells from tissue, is designated P0. After the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a third-passage culture (P2 or passage 2), and so on. Those skilled in the art will appreciate that there may be many population doublings during the subculture period; thus, the number of population doublings of a culture is greater than the number of passages. The expansion of cells during subculture (e.g., the number of population doublings) depends on many factors, including but not limited to the seeding density, substrate, culture medium, growth conditions, and the time between subcultures.

The terms "reduce," "inhibit," "alleviate," "reduce," "decrease," "prevent," and their grammatical equivalents (including "lower," "smaller," etc.), when referring to any symptom in an untreated subject compared to a treated subject, mean that the amount and/or extent of the symptom in the treated subject is less than the amount and/or extent of the symptom in the untreated subject, and that this difference is clinically relevant in the eyes of any medical professional. In one embodiment, the amount and/or extent of the symptom in the treated subject is at least 10% less, at least 25% less, at least 50% less, at least 75% less, and/or at least 90% less than the amount and/or extent of the symptom in the untreated subject.

As used herein, the term "therapeutically effective amount" is synonymous with "effective amount," "therapeutically effective dose," and/or "effective dose," and refers to the amount of a compound that elicits the biological, cosmetic, or clinical response sought by the practitioner in a subject in need thereof. As an example, an effective amount is an amount sufficient to reduce hair loss. For a particular application of the disclosed methods, the appropriate effective amount to administer can be determined by one skilled in the art using the guidance provided herein. For example, the effective amount can be inferred from in vitro and in vivo assays as described herein. One skilled in the art will recognize that the subject's condition can be monitored throughout treatment and the effective amount of the exosomes or compositions disclosed herein administered can be adjusted accordingly.

As used herein, the term "treatment" refers to intervention intended to alter the natural course of the body or cells being treated, and may be performed either prophylactically or during the pathological course of a disease or condition. Treatment may achieve one or more of a variety of desired outcomes, including, for example, preventing the occurrence or recurrence of the disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, reducing the rate of progression of the disease, ameliorating or lessening the disease state, or providing remission or improved prognosis.

As used herein, the term "subject" is used interchangeably with the term "individual" or "patient" and generally refers to an individual in need of treatment. The subject can be a mammal, such as a human, dog, cat, horse, pig, or rodent. The subject can be, for example, a patient who has, is suspected of having, or is at risk of having a disease or medical condition related to hair loss. For a subject who has, or is suspected of having, a medical condition directly or indirectly related to hair loss, the medical condition can be of one or more types.

As used herein, "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as aqueous saline solutions and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the composition is administered intravenously, aqueous saline solutions are preferred carriers. Aqueous saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, defatted milk powder, glycerin, propylene glycol, water, ethanol, and the like. If desired, the composition may also contain a small amount of a wetting or emulsifying agent or a pH buffering agent. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, and a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should be suitable for the mode of administration.

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 = the total number of residues in the peptide or polypeptide) of a polypeptide or peptide.

The term "KGF" refers to keratinocyte growth factor, which is a member of the epithelial mitogen and fibroblast growth factor (FGF) family. In vertebrates, the FGF family contains 22 members that are 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). Characterization of recombinant human fibroblast growth factor FGF-10 revealed its 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 second half of the third immunoglobulin loop and is encoded by an alternative exon. The ability of KGF-1 and KGF-2 to bind KGFR with high affinity distinguishes them from other members of the FGF-10 family. KGF-2 is highly related to KGF-1, binding to the same receptor as KGF-1 and sharing 57% sequence homology. Human KGF-2 is 96% identical to rat KGF-2 and specifically stimulates the growth of normal human epidermal keratinocytes.

The term "VEGFA," or vascular endothelial growth factor A, is a key member of the vascular endothelial growth factor (VEGF) family of cytokines. Along with placental growth factor (PlGF), VEGF-B/C/D in mammals, and VEGF-E/F in other organisms, it plays a key role in angiogenesis, vasculogenesis, and lymphangiogenesis. First named "VEGF" in early 1989 by N. Ferrara et al., VEGF-A is the most extensively studied member of the VEGF family. Alternative splicing of exon 8 of the Vegfa gene generates distinct VEGF-A isoforms, forming either VEGFxxxa or VEGFxxxb. In addition to the recently discovered post-translational readthrough events, alternative splicing events within exons 5-7 generate VEGF-A isoforms that differ in their bioavailability and interaction with the co-receptor pilin-1. To date, 16 different VEGFA isoforms have been identified from the six most common transcripts: VEGF _-111 , VEGF _-121 , VEGF _-145 , VEGF-165, VEGF _{-189 ,} and VEGF _-206 . In rats, a similar spectrum of VEGF-A splice variants has been described, with each variant encoding one less amino acid than in humans. The shorter isoforms, VEGF ₁₁₁ and VEGF ₁₂₁ , lack both exon 6 and exon 7 and are therefore not associated with the extracellular matrix (ECM) and diffuse freely. In contrast, the longer isoforms, VEGF ₁₄₅ , VEGF ₁₈₉ , and VEGF ₂₀₆ , which contain both exon 6a and exon 7, bind to heparan sulfate glycoproteins with high affinity.

The term "Wnt/β-catenin signaling pathway" refers to the canonical Wnt pathway, which involves nuclear translocation of β-catenin and activation of target genes through TCF/LEF transcription factors. The canonical Wnt pathway primarily controls cell proliferation. Atypical Wnt pathways, such as the Wnt/Ca2 ⁺ pathway and atypical Wnt planar cell polarity, are independent of β-catenin-T-cytokine/lymphoid enhancer-binding factor (TCF/LEF) regulation. The Wnt/β-catenin signaling pathway is essential for embryonic development and homeostatic tissue regeneration in adults. Aberrant regulation of this pathway is closely associated with various diseases, making the Wnt/β-catenin signaling pathway an attractive target for disease treatment. The term "Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway" refers to any Wnt family member or functional fragment thereof involved in the Wnt/β-catenin signaling pathway, including but not limited to Wnt10a, Wnt10b, Wnt3a, Wnt1a, Wnt4, Wnt5a, Wnt7a or Wnt7b polypeptides.

Members of the tetraspanin family (such as CD63, CD81, and CD9) are ubiquitously expressed on exosomes and widely used as exosome biomarkers. They participate in physiological processes such as cell adhesion, cell motility, and signal transduction. CD63 (first characterized as a tetraspanin) has two extracellular loops of varying sizes and two short cytoplasmic domains, and is involved in signal transduction processes in various immune cell types. Sequential domain deletions have demonstrated that transmembrane helix 3 (TM3) is necessary and sufficient for membrane anchoring and exosome targeting.

The term "anchor polypeptide" refers to a polypeptide that is anchored to the exosome membrane when the exosome is produced by a cell. Transmembrane proteins are typical anchor polypeptides in this disclosure. "Anchor" or its grammatical variants refer to at least one fragment of a polypeptide being embedded in the exosome membrane. The anchor polypeptide can be fully or partially embedded in the exosome membrane. In this disclosure, the anchor polypeptide is fused to a polypeptide that is heterologous to the exosome and is naturally produced in the same cell, such as a KGF-1 polypeptide. Exemplary anchor polypeptides are exosome membrane proteins, membrane targeting sequences, or anchoring-functional fragments thereof. Exemplary exosomal membrane proteins include, but are not limited to, LAMP2b, tetraspanins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid-anchored proteins. A detailed review of GPI anchors in exosomes can be found, for example, in Michel Vidal, "Exosomes and GPI-anchored proteins: Judicious pairs for investigating biomarkers from body fluids," Advanced Drug Delivery Reviews , vol. 161-162, 2020 (incorporated herein by reference in its entirety). In a preferred embodiment of the present disclosure, the anchoring polypeptide comprises or consists of transmembrane helix 3 (TM3) of the CD63 protein.

Engineered exosomes

One aspect of the present disclosure relates to an engineered exosome comprising (a) a KGF polypeptide fused to a first anchoring polypeptide, (b) a VEGF-A polypeptide fused to a second anchoring polypeptide, and (c) a Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway fused to a third anchoring polypeptide, wherein (a), (b), and (c) are anchored to the membrane of the exosome via the first, second, and third anchoring polypeptides, respectively, and wherein the KGF polypeptide, the VEGF-A polypeptide, and the Wnt family member polypeptide are exposed on the outer surface of the exosome membrane.

In the present disclosure, the KGF polypeptide is a KGF-1 polypeptide or a KGF-2 polypeptide. In preferred embodiments, the KGF polypeptide is a KGF-1 polypeptide. In some embodiments, the KGF-1 polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1.

In preferred embodiments, the KGF-1 polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the KGF-1 polypeptide comprises the amino acid sequence set forth in 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 set forth in SEQ ID NO:4.

In the present disclosure, the VEGF-A polypeptide can be any VEGF-A isomer discovered in the art. In preferred embodiments, the VEGF-A polypeptide is human VEGF ₂₀₆ isomer or an ortholog or paralog thereof. In some embodiments, the VEGF-A polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2.

In preferred embodiments, the VEGF-A polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the VEGF-A polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5, which is human VEGF ₂₀₆ isomer. In some embodiments, the amino acid sequence of human VEGF ₂₀₆ isomer is set forth in SEQ ID NO: 5.

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

In preferred embodiments, the Wnt family member polypeptide is Wnt10b. In preferred embodiments, the Wnt10b polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:3. In preferred embodiments, the Wnt10b polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the Wnt10b polypeptide comprises the amino acid sequence set forth in SEQ ID NO:6 and is a human Wnt10b polypeptide. In some embodiments, the amino acid sequence of the Wnt10b polypeptide is shown in SEQ ID NO: 6.

In some embodiments, these anchoring polypeptides are exosomal membrane proteins, membrane targeting sequences, or anchoring functional fragments thereof. Exemplary exosomal membrane proteins include, but are not limited to, LAMP2b, tetraspanins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid anchoring proteins. In preferred embodiments, the first, second, and third anchoring polypeptides comprise the TM3 domain of CD63. In preferred embodiments, each of the first, second, and third anchoring polypeptides comprises the TM3 domain of CD63. In some embodiments, the anchoring polypeptides are full-length CD63 proteins, such as full-length human CD63 (see, e.g., UniProtKB/Swiss-Prot: F8VZE2, P08962, Q5TZP3, Q8N6Z9, or Q9UCG6). In some embodiments, the anchoring polypeptides are truncated CD63 proteins comprising the TM3 domain and at least one of the TM1, TM2, and TM4 domains. For example, the anchoring polypeptides can consist of TM2 and TM3 of CD63; TM3 and TM4 of CD63; or TM1, TM2, and TM3 of CD63. In some embodiments, the anchoring polypeptides consist of the TM3 domain of CD63. In the present disclosure, the first, second, and third anchoring polypeptides can be different or the same. In a preferred embodiment, the first, second and third anchoring polypeptides are identical and consist of the TM3 domain of CD63.

In preferred embodiments, the TM3 domain of CD63 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13. In preferred embodiments, the TM3 domain of CD63 comprises the amino acid sequence set forth in SEQ ID NO: 13. In preferred embodiments, the amino acid sequence of the TM3 domain of CD63 is set forth in SEQ ID NO: 13.

Therefore, in a preferred embodiment, an engineered exosome is provided, comprising (a) a KGF-1 polypeptide fused to a first anchoring polypeptide, (b) a VEGF-A polypeptide fused to a second anchoring polypeptide, and (c) a Wnt10b polypeptide fused to a third anchoring polypeptide, wherein each of the first, second, and third anchoring polypeptides comprises the TM3 domain of CD63, wherein (a), (b), and (c) are anchored to the membrane of the exosome via the first, second, and third anchoring polypeptides, respectively, and 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.

In a preferred embodiment, an engineered exosome is provided, comprising (a) a human KGF-1 polypeptide fused to a first anchoring polypeptide, (b) a human VEGF-A polypeptide fused to a second anchoring polypeptide, and (c) a human Wnt10b polypeptide fused to a third anchoring polypeptide, wherein each of the first, second, and third anchoring polypeptides comprises the TM3 domain of CD63, wherein (a), (b), and (c) are anchored to the membrane of the exosome via the first, second, and third anchoring polypeptides, respectively, and wherein the human KGF-1 polypeptide, the human VEGF-A polypeptide, and the human Wnt10b polypeptide are exposed on the outer surface of the membrane of the exosome.

In a preferred embodiment, an engineered exosome is provided, comprising: (a) a KGF-1 polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 4, wherein the KGF-1 polypeptide is fused to a first anchor polypeptide; (b) a VEGF-A polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5, wherein the VEGF-A polypeptide is fused to a second anchor polypeptide; and (c) a Wnt10b polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 5, wherein the VEGF-A polypeptide is fused to a second anchor polypeptide. The amino acid sequence shown in NO: 6 has an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, the Wnt10b polypeptide is fused to a third anchoring polypeptide, wherein each of the first, second and third anchoring polypeptides comprises the TM3 domain of CD63, wherein (a), (b) and (c) are anchored to the membrane of the exosome via the first, second and third anchoring polypeptides, respectively, and 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.

In a preferred embodiment, an engineered exosome is provided, comprising (a) a KGF-1 polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 4, wherein the KGF-1 polypeptide is fused to a first anchor polypeptide, (b) a VEGF-A polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 5, wherein the VEGF-A polypeptide is fused to a second anchor polypeptide, and (c) a Wnt10b polypeptide comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 6, wherein the VEGF-A polypeptide is fused to a second anchor polypeptide. NO:6, wherein the Wnt10b polypeptide is fused to a third anchoring polypeptide, wherein each of the first, second, and third anchoring polypeptides comprises a TM3 domain of CD63, which domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:13; wherein (a), (b), and (c) are anchored to the exosome membrane via the first, second, and third anchoring polypeptides, respectively, and wherein the KGF-1 polypeptide, the VEGF-A polypeptide, and the Wnt10b polypeptide are exposed on the outer surface of the exosome membrane.

In a preferred embodiment, an engineered exosome is provided, comprising (a) a KGF-1 polypeptide having an amino acid sequence as shown in SEQ ID NO: 4, fused to a first anchoring polypeptide, (b) a VEGF-A polypeptide having an amino acid sequence as shown in SEQ ID NO: 5, fused to a second anchoring polypeptide, and (c) a Wnt10b polypeptide having an amino acid sequence as shown in SEQ ID NO: 6, fused to a third anchoring polypeptide, wherein each of the first, second, and third anchoring polypeptides consists of the TM3 domain of CD63 having an amino acid sequence as shown in SEQ ID NO: 7. The amino acid sequence shown in NO: 13, wherein (a), (b) and (c) are anchored to the exosome membrane via the first, second and third anchoring polypeptides, respectively, and wherein the KGF-1 polypeptide, the VEGF-A polypeptide and the Wnt10b polypeptide are exposed on the outer surface of the exosome membrane.

In any of the above embodiments, the KGF polypeptide, the VEGF-A polypeptide, and the Wnt family member polypeptide are fused directly or via a peptide linker to the C-termini of the first, second, and third anchor polypeptides, respectively. The peptide linker can be any peptide linker available in the art for linking different structural or functional domains in a 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.

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 herein 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.

In some embodiments, the exosomes provided herein promote hair regrowth and/or improve hair loss, such as androgenic alopecia, alopecia areata, or telogen effluvium. For example, the exosomes provided herein increase the number of hair follicles, the length of hair follicles, and/or the anagen/telogen ratio.

In some embodiments, the exosomes provided by the present disclosure prevent and/or limit and/or stop the formation of gray hair, and maintain and/or promote the natural restoration of hair and/or body hair.

In some embodiments, the exosomes provided herein promote hair regrowth and/or improve hair loss, such as androgenic alopecia, alopecia areata, or telogen effluvium; and prevent and/or limit and/or stop the formation of gray hair, and maintain and/or promote the natural restoration of hair and/or body hair.

Methods and compositions for treating or preventing hair loss

Various aspects of the present disclosure relate to methods and compositions for treating or preventing hair loss, gray hair, or both. Alopecia (also referred to as "hair loss") describes the loss of hair from any part of a subject's head or body. Various types of hair loss are known in the art and contemplated herein, including, but not limited to, androgenic alopecia (e.g., male pattern baldness, female pattern baldness), alopecia areata, and telogen effluvium. In some embodiments, methods for treating alopecia areata are disclosed. Gray hair (natural graying of hair) is associated with a 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 contemplated herein, including, but not limited to, gray hair that is age-related graying, premature graying, or sudden graying of hair. In some embodiments, methods for treating age-related graying of hair are disclosed.

As disclosed herein, the engineered exosomes provided herein can be used to treat and prevent hair loss, gray hair, or both. Accordingly, embodiments of the present disclosure relate to methods for treating or preventing hair loss, gray hair, or both, comprising providing an effective amount of engineered exosomes to a subject. In some embodiments, the methods of the present disclosure comprise administering engineered exosomes to a subject for treating or preventing hair loss, gray hair, or both. Administration of such compositions includes, for example, topical administration, transdermal administration, and intradermal administration.

The engineered exosomes can be provided to a subject in combination with one or more therapeutic agents or treatments. When the engineered exosomes are intended for the treatment of hair loss, in one example, the engineered exosomes disclosed herein are administered to a subject in combination with diphenylcyclopropenone (e.g., simultaneously, before, or after). In one example, the engineered exosomes disclosed herein are administered to a subject in combination with baricitinib (e.g., simultaneously, before, or after). In one example, the engineered exosomes disclosed herein are administered to a subject in combination with minoxidil (e.g., simultaneously, before, or after). In one example, the engineered exosomes disclosed herein are administered to a subject in combination with finasteride (e.g., simultaneously, before, or after). In one example, the engineered exosomes disclosed herein are administered to a subject in combination with (e.g., simultaneously with, before, or after) spironolactone or dutasteride. In one example, the engineered exosomes disclosed herein are administered to a subject in combination with (e.g., simultaneously with, before, or after) hair transplant surgery.

When the engineered exosomes are intended for use in treating gray hair, in one example, the engineered exosomes of the present disclosure are administered to a subject in combination with hair dye (e.g., simultaneously, before, or after).

The present disclosure provides compositions comprising the engineered exosomes and a carrier. In preferred embodiments, the composition is a liquid formulation. In preferred embodiments, the composition is formulated for topical or subcutaneous administration. Compositions comprising the engineered exosomes are contemplated for use in, for example, soaps, shampoos, ointments, and other such formulations.

In preferred embodiments, the composition does not contain KGF polypeptides (e.g., KGF-1 polypeptides), VEGF-A polypeptides, or Wnt family member polypeptides involved in the Wnt/β-catenin signaling pathway (e.g., Wnt10b polypeptides) that are not attached to the exosome membrane. For example, other than the polypeptides anchored to the membrane of the engineered exosomes, no additional KGF polypeptides (e.g., KGF-1 polypeptides), VEGF-A polypeptides, or Wnt family member polypeptides involved in the Wnt/β-catenin signaling pathway (e.g., Wnt10b polypeptides) are added to the composition.

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 an additional active agent for treating hair loss. Preferably, the additional active agent for treating hair loss is selected from diphenylcyclopropenone, baricitinib, minoxidil, finasteride, spironolactone, dutasteride, or any combination thereof. In a preferred embodiment, the additional active agent for treating hair loss is diphenylcyclopropenone, baricitinib, or minoxidil. In some embodiments, the composition comprises an additional active agent for treating gray hair.

Nucleic acid constructs, vectors, cells, and production methods

Various aspects of the present disclosure also relate to nucleic acid constructs encoding polypeptides anchored to exosomes.

In some embodiments, a nucleic acid construct is provided, comprising a polynucleotide encoding: (a) a KGF polypeptide fused to a first anchoring polypeptide, (b) a VEGF-A polypeptide fused to a second anchoring polypeptide, and (c) a Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway fused to a third anchoring polypeptide.

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

In some embodiments, a set of two nucleic acid constructs is provided, wherein one nucleic acid construct comprises polynucleotides encoding (a) and (b), and the other nucleic acid construct comprises a polynucleotide encoding (c), wherein (a), (b), and (c) are as defined above.

In some embodiments, a set of two nucleic acid constructs is provided, wherein one nucleic acid construct comprises polynucleotides encoding (a) and (c), and the other nucleic acid construct comprises a polynucleotide encoding (b), wherein (a), (b), and (c) are as defined above.

In some embodiments, a set of two nucleic acid constructs is provided, wherein one nucleic acid construct comprises polynucleotides encoding (b) and (c), and the other nucleic acid construct comprises a polynucleotide encoding (a), wherein (a), (b), and (c) are as defined above.

In a preferred embodiment, a single nucleic acid construct is provided comprising a polynucleotide encoding (a), (b) and (c), wherein (a), (b) and (c) are as defined above.

In some embodiments, these anchoring polypeptides are exosomal membrane proteins, membrane targeting sequences, or anchoring functional fragments thereof. Exemplary exosomal membrane proteins include, but are not limited to, LAMP2b, tetraspanins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof. Exemplary membrane targeting sequences include, but are not limited to, glycosylphosphatidylinositol (GPI) anchors and lipid-anchored proteins.

In some embodiments, each of the first, second, and third anchoring polypeptides is located at the N-terminus of Wnt, KGF, and/or VEGF to ensure that Wnt, KGF, and/or VEGF are exposed on the surface of exosomes. In some embodiments, each of the first, second, and third anchoring polypeptides is located at the C-terminus of Wnt, KGF, and/or VEGF to ensure that Wnt, KGF, and/or VEGF are exposed on the surface of exosomes. For example, when lamp2b is used as one of these anchoring polypeptides, the polypeptide to be presented on the surface of the exosomes (e.g., Wnt, KGF, and VEGF) can be located at the N-terminus of lamp2b. For example, when the TM3 domain of CD63 is used as one of these anchoring polypeptides, the polypeptide to be presented on the surface of the exosomes (e.g., Wnt, KGF, and VEGF) can be located at the C-terminus of the TM3 domain of CD63. In some embodiments, when different anchoring polypeptides are used, the polypeptide to be presented on the surface of the exosomes can be located at the N-terminus or C-terminus of these anchoring polypeptides, depending on the type of anchoring polypeptide used.

In preferred embodiments, the first, second, and third anchoring polypeptides comprise full-length CD63 or truncated CD63 that retains the TM3 domain. In preferred embodiments, each of the first, second, and third anchoring polypeptides comprises the TM3 domain of CD63. In preferred embodiments, each of the first, second, and third anchoring polypeptides is the TM3 domain of CD63.

In a preferred embodiment, the polynucleotide may comprise a nucleotide fragment encoding the following substances from 5' to 3':

(i) [First anchoring polypeptide]-[KGF]-[self-cleaving peptide]-[Second anchoring polypeptide]-[VEGF-A]-[self-cleaving peptide]-[Third anchoring polypeptide]-[Wnt],

(ii) [first anchoring polypeptide]-[KGF]-[self-cleaving peptide]-[second anchoring polypeptide]-[Wnt]-[self-cleaving peptide]-[third anchoring polypeptide]-[VEGF-A],

(iii) [first anchoring polypeptide]-[VEGF-A]-[self-cleaving peptide]-[second anchoring polypeptide]-[KGF]-[self-cleaving peptide]-[third anchoring polypeptide]-[Wnt],

(iv) [first anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[second anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[third anchor 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 [] represents a single polypeptide, and ]-[ represents a linker or bond; wherein each of the polypeptides is arranged from N-terminus to C-terminus; and wherein Wnt represents a Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway.

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. Following translation of the polynucleotide, the self-cleaving peptide is cleaved to produce three separate fusion proteins, each comprising a single polypeptide to be presented on the surface of the exosome and a single anchoring polypeptide.

In other embodiments, a single polynucleotide comprises nucleotide segments encoding (a), (b), and (c), and the single polynucleotide comprises, from 5' to 3', a nucleotide segment encoding the following: [first anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[VEGF-A]-[second anchor polypeptide]-[self-cleaving peptide]-[third anchor polypeptide]-[KGF], wherein the second anchor polypeptide is located at the C-terminus of the VEGF-A polypeptide. This variation also applies to any of the polynucleotides listed in (i) to (vi) above. In some embodiments, each of the first, second, and third anchor polypeptides is different, and thus these anchor polypeptides can be located at the N-terminus or C-terminus of the Wnt, KGF, and VEGF-A polypeptides.

In a preferred embodiment, a single nucleic acid construct is provided comprising a polynucleotide encoding (a), (b) and (c), wherein (a), (b) and (c) are as defined above, and wherein each of the first, second and third anchoring polypeptides comprises the TM3 domain of CD63, and the polynucleotide may comprise, from 5' to 3', a nucleotide fragment encoding one of the following substances:

(i)

[TM3]-[KGF]-[T2A]-[TM3]-[VEGF-A]-[T2A]-[TM3]-[Wnt],

(ii)

[TM3]-[KGF]-[T2A]-[TM3]-[Wnt]-[T2A]-[TM3]-[VEGF-A],

(iii)

[TM3]-[VEGF-A]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[Wnt],

(iv)

[TM3]-[VEGF-A]-[T2A]-[TM3]-[Wnt]-[T2A]-[TM3]-[KGF],

(v)

[TM3]-[Wnt]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[VEGF-A], and

(vi)

[TM3]-[Wnt]-[T2A]-[TM3]-[VEGF-A]-[T2A]-[TM3]-[KGF],

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

In this section, KGF, VEGF-A and Wnt family member polypeptides involved in the Wnt/β-catenin signaling pathway have the meanings and preferred embodiments given above in the section entitled " Engineering Exosomes ".

In some embodiments, the polynucleotide encoding the KGF polypeptide has a nucleotide sequence as shown in SEQ ID NO: 7, SEQ ID NO: 10, or a degenerate sequence thereof.

In some embodiments, the polynucleotide encoding the VEGF-A polypeptide has a nucleotide sequence as shown in SEQ ID NO: 8, SEQ ID NO: 11, or a degenerate sequence thereof.

In some embodiments, the polynucleotide encoding the Wnt family member polypeptide has a nucleotide sequence as shown in SEQ ID NO: 9, SEQ ID NO: 12, or a degenerate sequence thereof.

In some embodiments, the polynucleotide encoding the TM3 domain of CD63 has a nucleotide sequence as shown in SEQ ID NO: 14 or a degenerate sequence thereof.

Also provided are vectors comprising the above-described nucleic acid constructs. In some embodiments, the vector is a viral vector. In preferred embodiments, the vector is a lentiviral vector or an adeno-associated viral vector.

The vectors provided herein facilitate the integration of a polynucleotide encoding a polypeptide anchored on the membrane of the engineered exosome into the genome of an exosome-producing cell.

Also provided are cells transduced with the vector. In preferred embodiments, the cells are not mesenchymal stem cells. In preferred embodiments, the cells are not stem cells. In preferred embodiments, the cells are non-stem cells, such as HEK293 or CHO cells.

The present disclosure also provides a method for producing the engineered exosomes provided herein, comprising transducing the above-mentioned cells, such as HEK293 cells, with the above-mentioned vector; culturing the cells under conditions that allow the engineered exosomes to be secreted from the cells; and collecting and purifying the engineered exosomes.

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

Sequence Listing

Implementation Examples

Example 1. Construction of engineered exosomes derived from stable cell lines.

Materials and methods

Materials: HEK293 cells (human embryonic kidney 293 cells, CRL-1573 ^™ ) were purchased from ATCC and maintained in DMEM (high glucose) containing 10% (v/v) FBS supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin. CHO-K1 cells (Chinese hamster ovary cells) were purchased from the BeNa Culture Collection (Beijing, China). CHO-K1 cells were maintained in F-12K (31765035, Thermo Fisher Scientific, USA) containing 10% (v/v) FBS supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO _2. The antibody used in this study was anti-CD63 antibody (Catalog No. MA5-32085, Invitrogen).

pGOI plasmid construction: The amino acid sequences of all target genes (including KGF-1, VEGF-A, and Wnt10b) were 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 by General Biotechnology (Chuzhou, China) using the plasmid pCDH-CMV-MCS-EF1a-GFP+BSD (System Biosciences). A T2A peptide (see SEQ ID NO: 17 and SEQ ID NO: 18) was used to separate all target proteins into single proteins after translation. Four plasmids from the third-generation system (pGOI, pGag/pol, pRev, and pVSV-G) were used to produce lentivirus, with pCDH-CMV-MCS-EF1a-GFP+BSD (blasticidin-resistant) serving as the lentiviral packaging plasmid. Lentivirus was packaged by WZ Biotechnology (Jinan, China). The payload gene constructed in the pGOI plasmid was: CD63-TM3-Linker-KGF-1-T2A-CD63-TM3-Linker-VEGF-A-T2A-CD63-TM3-Linker-Wnt10b, where each CD63-TM3 is the TM3 domain of CD63, encoded by the DNA sequence set forth in SEQ ID NO: 14, each linker is encoded by the DNA sequence set forth in SEQ ID NO: 16, and each T2A is encoded by the DNA sequence set forth in SEQ ID NO: 18.

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. Forty-eight hours after infection, cells were selected by adding antibiotics, such as blasticidin (Solarbio Life Sciences), to a final concentration of 6 μg/ml. Single-cell colonies expressing green fluorescent protein (GFP) were selected and cultured in complete medium containing 6 μg/ml blasticidin. Stable cell lines were monitored for GFP and corresponding target protein expression.

Adapting cell cultures to SFM (serum-free medium): After three initial passages in FM (complete medium), stable cell lines were adapted to serum-free culture starting at passage 4. Cells were subcultured using the medium compositions listed in Table 1. To establish complete serum-free-adapted cultures, cells should be subcultured at least three times in SFM (HyClone ^™ peak expression, SH31193.02, Cytiva Life Sciences).

Table 1. Adaptation medium

FM: complete medium; SFM: serum-free medium

Exosome isolation: Stable cell lines were inoculated in T150 flasks and cultured for 24 hours. The culture medium was then rinsed thoroughly with PBS and incubated in SFM for an additional 48 hours. Cells were removed by centrifugation at 300 × g for 10 minutes, and the cell-free extracellular medium containing exosomes was harvested. Dead cells and cellular debris were then removed by centrifugation at 10,000 × g for 30 minutes. Finally, the clarified supernatant was centrifuged twice at 100,000 × g for 70 minutes to precipitate the exosomes. The exosome pellet was then resuspended. All centrifugation steps were performed at 4°C. The resulting exosomes are referred to as exosomes #35 in the following examples.

Figure 1 schematically shows a flow chart of the process for producing exosomes from HEK293 cells according to an exemplary embodiment of the present disclosure.

Example 2. Characterization of Exosomes

Analysis of exosome particle concentration and size distribution

The particle concentration and size distribution of exosomes from stable cell lines were analyzed using NanoFCM (NanoFCM, Xiamen, China). NanoFCM analysis uses two single-photon counting avalanche photodiodes (APDs) to simultaneously detect side scatter (SSC) and fluorescence from individual particles. First, an exosome pellet was prepared for analysis. Then, 200 nm polystyrene beads conjugated with PE and AF488 fluorophores were used for particle concentration, and a silica nanosphere mixture (NanoFCM, Xiamen, China) was used for fine distribution. The detector recorded the number of particles passing through during a 1-minute interval in each test. Each sample was diluted to an optimal particle count range of 3,000 to 9,000 particles/min. Flow rate and side scatter intensity were converted to vesicle concentration and size using NanoFCM software (NanoFCM Professional V2.0).

Figure 2A shows the particle size and concentration of exosomes derived from a stable cell line analyzed by NanoFCM. The exosomes had an average particle size of 75.4 nm.

Identification of target proteins on exosomes by immunoblotting (WB)

To identify target protein expression on exosomes, purified exosomes were lysed with RIPA lysis buffer (Beyotime) supplemented with 1 mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Beyotime) and a phosphatase inhibitor (Beyotime), followed by heat denaturation, separation by SDS-PAGE, and transfer to a PVDF membrane (Millipore, MA, USA). Proteins were detected by incubation with a primary antibody against CD63 (an exosomal scaffold protein) followed by incubation with an HRP-conjugated secondary antibody (Invitrogen). The membrane was then visualized using enhanced chemiluminescence reagent (Millipore, MA, USA).

Transmission electron microscopy (TEM) analysis of exosomes

TEM was used to confirm the presence of exosomes. Approximately 20 μL of exosomes were added individually to a copper grid. Any excess liquid was removed using filter paper, and the sample was counterstained with 2% acetic acid uranium dioxide for 30 seconds. The grid was rinsed with deionized water and allowed to dry overnight. The sample was then air-dried using an incandescent lamp and observed using an electron microscope (Hitachi, S-3000N).

Figure 2B shows a transmission electron microscopy (TEM) image of exosomes. Figure 2C shows immunoblot analysis of exosomes using antibodies against CD63-TM3, the scaffold protein fused to the exosomes. As expected, engineered exosome-associated target proteins (Wnt10b, VEGF-A, and KGF-1) were present in purified exosomes derived from stable cell lines.

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

Animal and in vivo studies

All experiments were performed using 6-week-old male C57BL/6 mice. Animals were purchased from Cavens (Changzhou, China). Figure 3A shows a schematic diagram of the androgenic alopecia mouse model and protocol. After depilation of the dorsal hair of mice, the skin of the mice was treated once every 3 days with 35# exosomes, control exosomes (Ctrl Exo.), PBS (negative control), or 5% minoxidil (positive control). Observation was continued during the 18-day treatment period, and then the animals were sacrificed on the 18th day for histological analysis. For in vivo treatment experiments, the mice were first shaved and then the hair was removed with a depilatory cream, and the pink skin was observed. On the second day of hair removal, a 30mg/EA DHT (dihydrotestosterone) suspension was subcutaneously injected into five sites on the back of the depilated area. Animals were randomly divided into four groups (n=4) to study hair regrowth. After hair removal, the dorsal skin of mice was treated once every three days with 35# exosomes, control exosomes, and PBS (negative control) or 5% minoxidil (positive control). Exosome treatment dose: 8.0× ¹⁰⁹ exosomes in 100μl PBS were subcutaneously injected into five sites on the dorsal skin (20μl per site). 200μl of minoxidil was applied topically daily. The dorsal skin was photographed on days 0, 9, 12, and 15. Image J was used to analyze areas of hair loss and hair growth in mice. Hair cover (%) = (new hair area / hair loss area) × 100%. Observation was continued throughout the 18-day treatment period, after which the animals were sacrificed for further analysis.

Dermatology

Mice were euthanized, and the entire dorsal skin was removed. Skin tissue was fixed in 4% paraformaldehyde, then sliced 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 counted, and the ratio of anagen to telogen follicles was calculated. The distance from the dermis to the epidermis was measured to calculate the average follicle length.

Figure 3B shows observations of hair coverage. Mice were divided into four groups (n=4), and representative images of mice on day 15 are shown. The results demonstrate that treatment with 35# exosomes nearly completely restored hair coverage on day 15, with efficacy comparable to that of the positive control, minoxidil. Figure 3C shows histological analysis of the dorsal skin of mice, analyzing hair follicle number, length, and growth stage ratio. Data points represent mean ± SD (n=4). Error bars indicate SD.; ns indicates no significant difference; *P < 0.05, **P < 0.01, and ***P < 0.001. Exosome treatment significantly increased the number and length of hair follicles and prolonged the anagen/telogen ratio, which may promote hair follicle normalization and further enhance the telogen-to-anagen transition.

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

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 protocol. In the in vivo treatment experiment, the mice were first shaved and then the hair was removed with a depilatory cream. The animals were randomly divided into 4 groups (n=4) to study anti-hair graying. On the second day of depilation, a 2.5% concentration of hydroquinone cream was topically applied to the depilated area on the back. The negative model control group was treated with PBS by topical application. The 35# exosome group was treated with different application routes (e.g., application after microneedle vaccination, application after nanocrystal infusion, subcutaneous injection). Exosome treatment dose: 1E11 exosomes in a volume of 200μl were administered through different routes. The hair on the back of the mice was continuously observed during the 28-day treatment period. Table 2 shows a summary of the treatment regimen.

Table 2. Summary of treatment regimens for different groups.

Figure 4B shows the results of observations on the back hair. Representative images of mice on day 14 are shown. The results show that mice treated with 35# exosomes through different administration routes (e.g., microneedle vaccination followed by application, nanocrystal infusion followed by application, and subcutaneous injection) had darker hair in all treatment groups compared to mock treatment.

TW202532639A_114100610_SEQ.XML

Claims (18)

An engineered exosome comprising (a) a KGF polypeptide, wherein the KGF polypeptide is fused to a first anchoring polypeptide, (b) a VEGF-A polypeptide fused to a second anchoring polypeptide, and (c) A Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway, wherein the Wnt family member polypeptide is fused with a third anchor polypeptide. wherein (a), (b) and (c) are anchored to the exosome membrane via the first, second and third anchoring polypeptides, respectively, and The KGF polypeptide, the VEGF-A polypeptide, and the Wnt family member polypeptide are exposed on the outer surface of the membrane of the exosome. The engineered exosome according to claim 1, wherein 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 shown in SEQ ID NO: 1 or SEQ ID NO: 4. The engineered exosomes according to claim 1, wherein the VEGF-A polypeptide is a human VEGF ₂₀₆ isomer or a direct homolog or paralog thereof, preferably, the VEGF-A polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5. The engineered exosome according to claim 1, wherein the Wnt family member polypeptide is a Wnt10a, Wnt10b, Wnt3a, Wnt1a, Wnt4, Wnt5a, Wnt7a, or Wnt7b polypeptide; preferably, the Wnt family member polypeptide is a Wnt10b polypeptide; more preferably, the Wnt10b polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6. The engineered exosomes according to claim 1, wherein the anchoring polypeptide is an exosome membrane protein, a membrane targeting sequence, or an anchoring functional fragment thereof; preferably, the exosome membrane protein comprises LAMP2b, tetraspanins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof; preferably, Preferably, the membrane targeting sequence comprises a glycosylphosphatidylinositol (GPI) anchor and a lipid anchor protein; preferably, the first, second, and third anchor polypeptides comprise full-length CD63 or a truncated CD63 retaining the 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 is the TM3 domain of CD63; more preferably, the TM3 domain of CD63 comprises an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 13. The engineered exosome according to claim 1, wherein the KGF polypeptide, the VEGF-A polypeptide, and the Wnt family member polypeptide are optionally fused to the C-termini of the first, second, and third anchor polypeptides respectively via a peptide linker. Preferably, the peptide linker is composed of glycine and serine, for example (G4S)n, wherein n is an integer from 1 to 3. The engineered exosomes according to claim 1, wherein the exosomes are not derived from mesenchymal stem cells; preferably, the exosomes are not derived from stem cells. The engineered exosome according to claim 1, wherein the exosome: (i) promoting hair regrowth and/or improving hair loss, such as androgenic alopecia, alopecia areata or telogen effluvium; and/or (ii) Prevent and/or limit and/or stop the formation of grey hair and maintain and/or promote the natural re-colouration of hair and/or body hair. A composition comprising the exosomes according to any one of claims 1 to 8, and a carrier; preferably, the composition is a liquid preparation; more preferably, the composition is formulated for topical or subcutaneous administration; the composition is optionally a cosmetic composition or a non-cosmetic composition; and wherein the composition is optionally a pharmaceutical composition. The composition according to claim 9, wherein the composition does not contain KGF polypeptide (e.g., KGF-1 polypeptide), VEGF-A polypeptide, or Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway (e.g., Wnt10b polypeptide) that is not attached to the membrane of the exosome. A nucleic acid construct comprising a polynucleotide encoding: (a) a KGF polypeptide, wherein the KGF polypeptide is fused to a first anchoring polypeptide, (b) a VEGF-A polypeptide fused to a second anchoring polypeptide, and (c) A Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway, wherein the Wnt family member polypeptide is fused to a third anchor polypeptide. The nucleic acid construct according to claim 11, wherein (i) The KGF polypeptide is a KGF-1 polypeptide, preferably, the KGF-1 polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4; (ii) the VEGF-A polypeptide is a human VEGF ₂₀₆ isomer or an ortholog or paralog thereof, preferably, the VEGF-A polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5; (iii) the Wnt family member polypeptide is a Wnt10a, Wnt10b, Wnt3a, Wnt1a, Wnt4, Wnt5a, Wnt7a, or Wnt7b polypeptide; preferably, the Wnt family member polypeptide is a Wnt10b polypeptide; more preferably, the Wnt10b polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 6; (iv) The polynucleotide encoding the KGF polypeptide has the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 10, or a degenerate sequence thereof; (v) The polynucleotide encoding the VEGF-A polypeptide has a nucleotide sequence as shown in SEQ ID NO: 8, SEQ ID NO: 11, or a combined sequence thereof; (vi) The polynucleotide encoding the Wnt family member polypeptide has a nucleotide sequence as shown in SEQ ID NO: 9, SEQ ID NO: 12, or a degenerate sequence thereof; (vii) the anchoring polypeptide is an exosomal membrane protein, a membrane targeting sequence or an anchoring functional fragment thereof; preferably, the exosomal membrane protein includes lamp2b, tetraspanins such as CD63, CD9 and CD81, platelet-derived growth factor receptor (PDGFR), lactadherin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (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 full-length CD63 or a truncated CD63 retaining the 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 is the TM3 domain of CD63; and/or (viii) The polynucleotide encoding the TM3 domain of CD63 has the nucleotide sequence shown in SEQ ID NO: 14 or a simplification thereof. The nucleic acid construct according to claim 11 or 12, wherein the polynucleotide is a single polynucleotide comprising nucleotide segments encoding (a), (b) and (c); preferably, the polynucleotide comprises, from 5' to 3', a nucleotide segment encoding the following substances: (i) [First anchoring polypeptide]-[KGF]-[self-cleaving peptide]-[Second anchoring polypeptide]-[VEGF-A]-[self-cleaving peptide]-[Third anchoring polypeptide]-[Wnt], (ii) [first anchoring polypeptide]-[KGF]-[self-cleaving peptide]-[second anchoring polypeptide]-[Wnt]-[self-cleaving peptide]-[third anchoring polypeptide]-[VEGF-A], (iii) [first anchoring polypeptide]-[VEGF-A]-[self-cleaving peptide]-[second anchoring polypeptide]-[KGF]-[self-cleaving peptide]-[third anchoring polypeptide]-[Wnt], (iv) [first anchor polypeptide]-[VEGF-A]-[self-cleaving peptide]-[second anchor polypeptide]-[Wnt]-[self-cleaving peptide]-[third anchor 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, [] represents a single polypeptide, and ]-[ represents a linker or bond; Among them, Wnt represents a Wnt family member polypeptide involved in the Wnt/β-catenin signaling pathway; Optionally, the self-cleaving peptide is a 2A peptide, such as T2A, E2A, P2A, or any combination thereof. A vector comprising the nucleic acid construct 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 viral vector. A cell transduced with the vector according to claim 14, wherein the polynucleotide is integrated 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. A method for producing an engineered exosome according to any one of claims 1 to 8, the method comprising: (a) transducing the cell according to claim 15 with the 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. The method of claim 16, further comprising adapting the cells to serum-free conditions during step (b). Use of the engineered exosomes according to any one of claims 1 to 8 or the composition according to claim 9 or 10 in the preparation of a medicament for treating hair loss and/or graying of hair; preferably, the hair loss is androgenic alopecia, alopecia areata, or telogen effluvium; preferably, the graying of hair is age-related graying of hair, premature graying of hair, or sudden graying of hair.