Engineered exosomes for skin rejuvenation

Abstract

An engineered exosome comprising (a) a KGF polypeptide fused to a first anchor polypeptide, (b) an EGF polypeptide fused to a second anchor polypeptide, and (c) an FGF-2 polypeptide fused to a third anchor polypeptide, wherein (a), (b), and (c) are anchored to the membrane of the exosome by the first, second, and third anchor polypeptides, respectively, and wherein the KGF polypeptide, the EGF polypeptide, and the FGF-2 polypeptide are exposed on the outer surface of the membrane of the exosome. Also disclosed are compositions, nucleic acid constructs, and uses of the exosome for skin rejuvenation.

Description

Technical Field

[0001] This disclosure relates to engineered exosomes, and more specifically to exosomes carrying different fusion proteins anchored to the membrane exterior of the exosome. This disclosure also relates to compositions comprising engineered exosomes, nucleic acid constructs comprising polynucleotides encoding fusion proteins, and the use of engineered exosomes in skin rejuvenation. Background Technology

[0002] Sun exposure, skin conditions, aging, and even genetics can all cause abnormalities in the skin of the face and other parts of the body. These abnormalities include textural changes such as wrinkles and acne scars, pigmentation changes such as freckles and sunspots, or visible blood vessels. In addition, the skin may lose its luster, feel less firm, and lose the healthy glow of youthful skin.

[0003] Skin resurfacing refers to any process designed to improve the appearance of the skin. Different treatments can be used to treat different aspects of skin damage. Laser resurfacing, mechanical resurfacing, chemical resurfacing, and injectable products can improve the appearance of fine lines and wrinkles that appear on the entire face or specific areas of the face, such as the upper lip and around the eyes. These treatments can also be used to address pigmentation disorders such as sunspots and age spots, and they can be used to improve the appearance of acne scars or other skin conditions.

[0004] Regenerative medicine-based products can be divided into two categories: cell-based therapies and cell-free therapies. These can be autologous or allogeneic. The most common regenerative medicine-based products include cell-based approaches utilizing autologous adult stem cells (such as mesenchymal stem cells (MSCs) and fibroblasts), emerging blood-derived cell therapies (such as platelet-rich plasma (PRP) products), and the clinical application of induced pluripotent stem cells (iPSCs) is constantly developing. Cell therapies for various diseases are currently in clinical trials, especially those derived from iPSCs with lower risk profiles (such as iPSC-derived MSCs (iPSC-MSCs)). Cell-free products are primarily based on secreted components of MSCs, such as MSC-derived exosomes (MSC-exo), MSC-derived conditioned medium (MSC-CM), and MSC-derived extracellular vesicles (MSC-EVs). Autologous transplantation is performed by isolating and culturing MSCs derived from the patient's own tissues, such as adipose-derived MSCs (AD-MSCs), bone marrow MSCs (BM-MSCs), umbilical cord MSCs (UC-MSCs), fetal dermal MSCs (FD-MSCs), and endometrial and menstrual blood (eMSCs / MenSCs). Autologous PRP is obtained by collecting and centrifuging the patient's own blood. The molecular mechanisms underlying the therapeutic effects of regenerative medicine are still under investigation. However, the unique properties of MSCs, such as self-renewal, multi-lineage differentiation, inflammation regulation, immune regulation, angiogenesis, and the hematopoietic capacity of PRP manifested through the release of cytokines and various growth factors, are believed to play an important role in skin regeneration.

[0005] Therefore, there is a need for additional and improved methods and compositions for skin rejuvenation. Summary of the Invention

[0006] In one aspect of the invention, an engineered exosome is provided comprising (a) a KGF peptide fused to a first anchoring peptide, (b) an EGF peptide fused to a second anchoring peptide, and (c) an FGF-2 peptide fused to a third anchoring peptide, wherein (a), (b), and (c) are anchored to the membrane of the exosome by the first, second, and third anchoring peptides, respectively, and wherein the KGF peptide, the EGF peptide, and the FGF-2 peptide are exposed on the outer surface of the membrane of the exosome.

[0007] In some embodiments, the engineered exosome further comprises (d) a PDGF-BB peptide fused with a fourth anchoring peptide, and the PDGF-BB peptide is exposed on the outer surface of the exosome's membrane.

[0008] In some embodiments, the KGF polypeptide is a KGF-1 polypeptide, preferably, the KGF-1 polypeptide contains an amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1.

[0009] In some embodiments, the EGF polypeptide is a human EGF polypeptide or its ortholog or paralog, and preferably, the EGF polypeptide contains an amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 2.

[0010] In some embodiments, the FGF-2 polypeptide is human FGF-2 or its ortholog or paralog; more preferably, the FGF-2 polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 3.

[0011] In some embodiments, the PDGF-BB polypeptide is a human PDGF-BB dimer; more preferably, the PDGF-BB dimer comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 4.

[0012] In some embodiments, these anchoring peptides are exosome membrane proteins, membrane-targeting sequences, or their anchoring functional fragments. Exemplary exosome membrane proteins include, but are not limited to, lamp2b, tetraspan membrane proteins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactobacin (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.

[0013] In a preferred embodiment, these anchoring peptides comprise full-length CD63 or truncated CD63 retaining the TM3 domain. In a preferred embodiment, each of the first, second, third, and fourth anchoring peptides (when present) comprises the TM3 domain of CD63. In a preferred embodiment, each of the first, second, third, and fourth anchoring peptides (when present) is the TM3 domain of CD63. In some embodiments, each of these anchoring peptides is 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 shown in SEQ ID NO. 9.

[0014] In some embodiments, the KGF polypeptide, the EGF polypeptide, the FGF-2 polypeptide, and the PDGF-BB polypeptide (when present) are optionally fused to the N-terminus or C-terminus of the corresponding anchoring polypeptide via a peptide linker, preferably composed of glycine and serine, such as (G4S)n, where n is an integer from 1 to 3.

[0015] In some implementations, the exosome is not derived from mesenchymal stem cells; preferably, the exosome is not derived from stem cells.

[0016] In some implementations, the exosome (i) enhances fibroblast proliferation, (ii) upregulates the COL2A1, COL3A1, fibronectin, TIMP1 and / or COL1A1B genes, (iii) downregulates the MMP7, MMP9 and / or MMP16B genes, and / or (iv) downregulates melanin synthesis.

[0017] Another aspect of this disclosure relates to a composition comprising exosomes as described herein and a carrier; preferably, the composition is a liquid formulation; more preferably, the composition is formulated for topical or subcutaneous application.

[0018] In some embodiments, the composition does not contain KGF peptides (e.g., KGF-1 peptides), EGF peptides, FGF-2 peptides (e.g., human FGF-2 peptides), or PDGF-BB peptides (e.g., human PDGF-BB dimer peptides) that are not attached to the membrane of the exosome.

[0019] Another aspect of this disclosure relates to a nucleic acid construct comprising a polynucleotide encoding: (a) a KGF polypeptide fused with a first anchoring polypeptide, (b) an EGF polypeptide fused with a second anchoring polypeptide, and (c) an FGF-2 polypeptide fused with a third anchoring polypeptide.

[0020] In some implementations, the nucleic acid construct also includes a polynucleotide encoding a (d)PDGF-BB polypeptide fused with a fourth anchoring polypeptide.

[0021] In some embodiments, the KGF polypeptide is a KGF-1 polypeptide, preferably, the KGF-1 polypeptide contains an amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1.

[0022] In some embodiments, the EGF polypeptide is a human EGF polypeptide or its ortholog or paralog, and preferably, the EGF polypeptide contains an amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 2.

[0023] In some embodiments, the FGF-2 polypeptide is human FGF-2 or its ortholog or paralog; more preferably, the FGF-2 polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 3.

[0024] In some embodiments, the PDGF-BB polypeptide is a human PDGF-BB dimer; more preferably, the PDGF-BB dimer comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 4.

[0025] In some embodiments, the polynucleotide encoding the KGF polypeptide has a nucleotide sequence as shown in SEQ ID NO. 5 or a degenerate sequence thereof.

[0026] In some embodiments, the polynucleotide encoding the EGF polypeptide has a nucleotide sequence as shown in SEQ ID NO. 6 or a degenerate sequence thereof.

[0027] In some embodiments, the polynucleotide encoding the FGF-2 polypeptide has a nucleotide sequence as shown in SEQ ID NO. 7 or a degenerate sequence thereof.

[0028] In some embodiments, the polynucleotide encoding the PDGF-BB polypeptide has a nucleotide sequence as shown in SEQ ID NO. 8 or a degenerate sequence thereof.

[0029] In some embodiments, these anchoring peptides are exosome membrane proteins, membrane-targeting sequences, or their anchoring functional fragments. Exemplary exosome membrane proteins include, but are not limited to, lamp2b, tetraspan membrane proteins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactobacin (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.

[0030] In a preferred embodiment, these anchoring peptides comprise full-length CD63 or truncated CD63 retaining the TM3 domain. In a preferred embodiment, each of the first, second, third, and fourth anchoring peptides (when present) comprises the TM3 domain of CD63. In a preferred embodiment, each of the first, second, third, and fourth anchoring peptides (when present) is the TM3 domain of CD63. In some embodiments, the polynucleotide encoding the TM3 domain of CD63 has a nucleotide sequence as shown in SEQ ID NO. 10 or a degenerate sequence thereof.

[0031] In some embodiments, the polynucleotide is a single polynucleotide comprising a nucleotide fragment encoding polypeptides (a), (b), (c), and optionally (d), wherein each of polypeptides (a), (b), (c), and optionally (d) is linked to an anchoring polypeptide; preferably, polypeptides (a), (b), (c), and optionally (d) are separated by self-cleaving peptides; optionally, these self-cleaving peptides are 2A peptides, such as T2A, E2A, P2A, or any combination thereof.

[0032] Another aspect of this disclosure relates to a vector comprising any of the nucleic acid constructs described herein.

[0033] Another aspect of this disclosure relates to a cell transduced using the vector described herein, wherein the polynucleotide is integrated into the cell's genome.

[0034] 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.

[0035] Another aspect of this disclosure relates to a method for producing engineered exosomes as disclosed herein, the method comprising (a) transducing cells as described above using a vector as described above; (b) culturing the cells under conditions that allow exosomes to be secreted from the cells; and (c) collecting and purifying the exosomes.

[0036] In some implementations, the method further includes adapting the cell to serum-free conditions during step (b).

[0037] Another aspect of this disclosure relates to the use of engineered exosomes or compositions as disclosed herein in the manufacture of medicaments for skin rejuvenation; preferably, the skin rejuvenation includes treating any of the following: sun damage; sensitive skin; scars such as acne or stretch marks such as stretch marks or puberty marks, chickenpox or wounds; wrinkles such as static or dynamic wrinkles; dark circles; hyperpigmentation such as darkening of the skin or freckles, loose skin, uneven skin tone or dullness, or any combination thereof.

[0038] Another aspect of this disclosure relates to a method for skin rejuvenation; preferably, the skin rejuvenation includes treating any of the following: sun damage; sensitive skin; scars such as acne or stretch marks such as stretch marks or puberty marks, chickenpox or wounds; wrinkles such as static or dynamic wrinkles; dark circles; hyperpigmentation such as darkening of the skin or freckles, loose skin, uneven skin tone or dullness, or any combination thereof.

[0039] Another aspect of this disclosure relates to engineered exosomes or compositions as disclosed herein for skin rejuvenation; preferably, the skin rejuvenation includes treating any of the following: sun damage; sensitive skin; scars such as acne or stretch marks such as stretch marks or puberty marks, chickenpox or wounds; wrinkles such as static or dynamic wrinkles; dark circles; hyperpigmentation such as darkening of the skin or freckles, loose skin, uneven skin tone or dullness, or any combination thereof.

[0040] These and other aspects and advantages of this disclosure will become apparent from the specific embodiments provided below. Attached Figure Description

[0041] Figure 1 Construction of stable cell lines capable of secreting engineered exosomes loaded with functional proteins. Mammalian cells (e.g., HEK293 cells) were cultured and infected with lentiviruses packaged with functional genes EGF, FGF-2, KGF-1, and PDGF-BB. Stable cell lines were then selected using antibiotics. After three passages of selection, expression of the functional genes was observed in both the cell pellet and the exosomes. Following confirmation of expression, the stable cell lines were adapted for serum-free culture. Finally, the culture supernatant from the serum-free stable cell lines was collected, and the exosomes purified by ultracentrifugation were identified.

[0042] Figure 2. Identification of engineered exosomes derived from stable cell lines. (A) Particle size and particle concentration of exosomes derived from stable cell lines (NO. 6 and NO. 8) were analyzed by NanoFCM. (B) Exosomes from stable cell lines (NO. 6 and NO. 8) were further examined by transmission electron microscopy (TEM). (C) Immunoblot analysis of exosomes using an anti-CD63 antibody, which is the scaffold protein fused to the exosomes.

[0043] Figure 3. Engineered exosomes exhibit enhanced cell proliferation activity against fibroblasts. Two fibroblast cell lines, HSF (human skin fibroblasts) (A) and HFF (human foreskin fibroblasts) (B), were cultured and incubated with different concentrations (0.5 μg, 1.0 μg, and 2.0 μg) of exosomes (NO. 6 and NO. 8). The functional protein KGF (KGF-1) was used as a positive control. Cell viability was assessed by CCK8 assay after 48 hours of incubation.

[0044] Figure 4. Engineered exosomes regulate skin regeneration-related genes on skin fibroblasts. Skin fibroblasts (HSFs) were cultured and incubated with exosomes (NO. 6 or NO. 8) for 48 hours. RNA was then extracted from the cell pellet, and skin regeneration-related genes, including COL2A1 (A), COL3A1 (B), fibronectin (C), TIMP1 (D), and MMP7 (E), were detected by RT-qPCR.

[0045] Figure 5. Regulation of skin regeneration-related genes in a zebrafish model by engineered exosomes. Zebrafish embryos (4 dpf, where dpf refers to the number of days post-fertilization) were prepared with yolk sacs. Exosomes (NO. 6 or NO. 8) were injected. Twenty-four hours after injection, gene expression analysis was performed on zebrafish using RT-qPCR to detect skin regeneration-related genes, including COL1A1B (A), MMP9 (B), and MMP16B (C).

[0046] Figure 6. Engineered exosomes facilitate the downregulation of melanin synthesis. (A) Human melanocytes were cultured and incubated with exosomes (NO. 6 or NO. 8) for 48 hours. The cell pellet was then used for melanin extraction, and melanin concentration was measured using a fluidometric assay kit. (B) In a zebrafish model, zebrafish embryos (4 hpf, where hpf refers to hours post-fertilization) were cultured in exosomes (NO. 8) at 28°C in the dark for 45 hours. Images were then captured and analyzed under a dissecting microscope to evaluate melanin. Whitening efficacy (%) = (Mock - NO. 8) / Mock * 100%. Detailed Implementation

[0047] definition

[0048] When used in this specification with the word "comprising," including the claims, the words "a" and "an" mean "one or more."

[0049] As used herein, the terms “or” and “and / or” are used to describe combinations or mutual exclusions of multiple components. 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 implementation scheme.

[0050] In this application, the term “about” is used in its common and general sense in the field of cell and molecular biology to indicate that a value includes the standard deviation of the error of the apparatus or method used to determine that value.

[0051] "Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be compared for comparative 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 with the number of matching or homologous positions the sequences have. An "irrelevant" or "non-homologous" sequence has less than 40% identity with one of the sequences in this disclosure, but preferably less than 25% identity.

[0052] "Sequence identity" of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence having a specific percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that when the two sequences are compared, the percentage of bases (or amino acids) is the same. This alignment and homology or sequence identity percentage can be determined using software programs known in the art.

[0053] As used in this article, the term "linker" refers to a short segment of an amino acid (AA) or a nucleotide sequence containing two or more identical or different amino acids or nucleotides.

[0054] As used herein, a “cell line” refers to a population of cells formed through one or more passages of a primary cell culture. Each round of passage is called a passage. After cells have been passaged, they are referred to as “passaged” cells. A particular cell population or cell line is sometimes indicated or characterized by its number of passages. For example, a cell population that has been passaged ten times may be called a P10 culture. A primary culture, that is, the first culture after cells are isolated from a tissue, is named P0. After the first passage, the cells are described as a second-generation culture (P1 or generation 1). After the second passage, the cells become a third-generation culture (P2 or generation 2), and so on. Those skilled in the art will understand that many population multiplications may occur during passage; therefore, the population multiplication rate of a culture is greater than the number of passages. Cell expansion during passage (e.g., population multiplication rate) depends on many factors, including but not limited to seeding density, substrate, culture medium, growth conditions, and passage interval.

[0055] When referring to any symptoms expressed by untreated subjects compared to treated subjects, the terms “reduced,” “suppressed,” “alleviated,” “lowered,” “decreased,” “prevented,” and their grammatical equivalents (including “lower,” “smaller,” etc.) mean that the number and / or severity of symptoms in the treated subject is less than the number and / or severity of symptoms in the untreated subject, and that this difference is clinically relevant to any medical professional. In one implementation, the number and / or severity of symptoms in the treated subject is at least 10%, at least 25%, at least 50%, at least 75%, and / or at least 90% lower than the number and / or severity of symptoms in the untreated subject.

[0056] As used herein, the term "therapeutic effective amount" is synonymous with "effective amount," "therapeutic effective dose," and / or "effective dose," and refers to the amount of compound that elicits the biological, cosmetic, or clinical response sought by the practitioner in an individual in need. As an example, an effective amount is an amount sufficient to reduce hair loss. For the specific application of the disclosed methods, the appropriate effective amount administered can be determined by those 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 in this specification. Those skilled in the art will recognize that the individual's condition can be monitored throughout the treatment process, and the effective amount of the exosomes or compositions disclosed herein administered can be adjusted accordingly.

[0057] As used herein, the term "treatment" refers to an intervention that attempts to alter the natural processes of the individual or cells being treated, and can be used to prevent or to proceed in the pathological process of a disease or condition. Treatment can achieve one or more of a variety of desired outcomes, including, for example, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological outcome of the disease, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis.

[0058] As used herein, the term “subject” may be used interchangeably with the terms “individual” or “patient”, and generally refers to an individual in need of treatment. Subjects may be mammals, such as humans, dogs, cats, horses, pigs, or rodents.

[0059] As used herein, "carrier" refers to a diluent, adjuvant, excipient, or medium administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as saline solutions and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions are preferred carriers when the composition is administered intravenously. Saline solutions, as well as glucose 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, skim milk powder, glycerin, propylene glycol, water, ethanol, and the like. If desired, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and similar forms. Such compositions will contain a therapeutically effective amount of the compound, preferably in a purified form, and a suitable amount of carrier to provide an appropriate form of administration to the patient. The formulation should be suitable for the mode of administration.

[0060] The term "N-terminal amino acid residue" or "N-terminus" refers to the first amino acid residue (amino acid number 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 number n, where n = the total number of residues in the peptide or polypeptide).

[0061] The term "KGF" refers to keratinocyte growth factor, a member of the epithelial mitogen and fibroblast growth factor (FGF) family. In vertebrates, the FGF family comprises 22 members essential for regulating numerous developmental processes. KGF includes two functionally similar variants: KGF-1 (also known as FGF-7) and KGF-2 (also known as FGF-10). The characterization of recombinant human fibroblast growth factor FGF-10 reveals its functional similarity to keratinocyte growth factor (FGF-7). Both growth factors interact with the same high-affinity receptor (a KGFR isoform of FGFR2), which differs from FGFR2 in the latter half of the third immunoglobulin loop and is encoded by a substitute exon. KGF-1 and KGF-2 distinguish themselves from other members of the FGF-10 family by their high-affinity binding to KGFRs. KGF-2 is highly correlated with KGF-1, binding to the same receptor as KGF-1, and they share 57% sequence homology. Human KGF-2 shares 96% identity with rat KGF-2 and specifically stimulates the growth of normal human epidermal keratinocytes. The amino acid sequence of human KGF-1 is available from UniProtKB / Swiss-Prot: P21781.1, and the mature chain of human KGF-1 is shown in SEQ ID NO. 1 of this disclosure. The amino acid sequence of human KGF-2 is available from UniProtKB / Swiss-Prot: O15520.1, and its mature chain is located between amino acids 38 and 208.

[0062] The term “FGF-2” is also known as basic FGF (“bFGF”). Both FGF1 and FGF2 are known to be released in large quantities by damaged endothelial cells and macrophages at the wound site, and wound angiogenesis is almost completely impaired if FGF2 activity is blocked. FGF2 is also known to induce scarless healing. However, due to the short half-life of free FGF, delivery systems have been proposed. Research on the application of FGFs in wound healing and skin regeneration has primarily focused on FGF2. Because acidic gelatin is highly negatively charged, it is able to retain FGF2 well by forming ionic complexes with gelatin. The amino acid sequence of human KGF-2 is available from UniProtKB / Swiss-Prot: P09038.3, and its mature chain lies between amino acids 143 and 288, which is shown in this disclosure as SEQ ID NO. 3.

[0063] The term "EGF" refers to epidermal growth factor, a growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR. It has been shown to act as a potent mitogen by stimulating the synthesis of mRNA, DNA, and proteins in epithelial cells. It is a single-chain polypeptide composed of 53 amino acids derived from the cleavage of a large precursor protein, EGF. EGF is now known to be the primitive form of the Group I EGF family, which also includes transforming growth factor-α (TGF-α), heparin-bound EGF (HB-EGF), bimodalin, β-cytokinin, epidermal regulatory factor, and epidermal growth factor. Structurally, they all contain one or more EGF repeat sequences (EGF motifs) in their extracellular domain. These repeat sequences are sequences of 35 to 40 amino acids separated by six conserved cysteine ​​residues in the following pattern: CX7CX3–5CX10–12CXCX5GXRC (C, cysteine; G, glycine; R, arginine; X, other amino acid). One glycine and one arginine in this sequence are conserved in all EGF-associated growth factors, but not in proteins containing the EGF motif but lacking growth factor activity. These six cysteine ​​residues pair to form three intramolecular disulfide bonds with the following interactions: C1–C3, C2–C4, and C5–C6 (numbered sequentially in the sequence), which are essential for maintaining their biological activity. Functionally, these growth factors have the ability to bind to the same receptor, the EGF receptor (EGFR, ErbB1), activate their intrinsic tyrosine kinase activity, and couple this receptor to downstream signaling pathways controlling cell proliferation, differentiation, survival, or motility. The amino acid sequence of human EGF is available from GenBank: AAS83395.1, and is also shown in this disclosure as SEQ ID NO.2.

[0064] The term "PDGF" refers to platelet-derived growth factors, which are potent mitogens and chemotherapeutic agents for many mesenchymal-derived cells, such as fibroblasts, activated macrophages, and smooth muscle cells. PDGF plays a crucial role in angiogenesis, embryonic development, inflammation, and cell differentiation; therefore, it can benefit wound healing. The PDGF family of growth factors consists of four different polypeptide chains encoded by four different genes: the classic PDGF-A and PDGF-B chains, and the more recently discovered PDGF-C and PDGF-D chains. These four PDGF chains assemble into disulfide dimers through homodimerization or heterodimerization, and five different dimer isoforms have been described to date: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. Two different PDGF receptors (α and β) mediate the action of PDGF on target cells. PDGF-A and PDGF-C chains selectively bind to α receptors, while PDGF-D preferentially binds to β receptors, and PDGF-B exhibits similar affinity for both receptors. Receptor activation requires PDGF-induced receptor dimerization, leading to transphosphorylation of tyrosine residues. PDGF AA induces only α / α receptor dimers, PDGF AB induces both α / α and α / β dimers, and PDGF BB induces all three combinations. Although PDGF-C binds only to α receptors, it can generate α-β heterodimers through transactivation of β receptors. Potential applications of PDGF-BB protein include inducing wound healing in diabetic patients. Furthermore, compositions containing PDGF can promote the healing of damaged or degenerated bone by promoting connective tissue growth, bone growth, and stimulating collagen synthesis in damaged bone regions. The amino acid sequence of human PDGF-B is available from GenBank: CAA02294.1, where amino acids 82 to 190 represent the mature peptide, which is also shown in SEQ ID NO. 4 of this disclosure.

[0065] The COL2A1 (collagen type II α1) gene provides the instructions for the production of a component of type II collagen called the precursor α1(II) chain. Type II collagen strengthens the structure and strength of connective tissue that supports the body's muscles, joints, organs, and skin. Type II collagen is primarily found in cartilage, a strong yet flexible tissue that forms the majority of the skeleton during early development. Most cartilage later transforms into bone, except for the cartilage that continues to cover and protect the ends of bones and is present in the nose and outer ear. Type II collagen is also part of the transparent gel that fills the eyeball (vitreous humor), the inner ear, and the central portion (nucleus pulposus) of the intervertebral discs between the vertebrae. To build type II collagen, three precursor α1(II) chains are intertwined to form a triple-chained, rope-like precursor collagen molecule. The precursor collagen molecule is then enzymatically processed within the cell. After processing, these molecules leave the cell and align themselves into elongated fibrils that connect (crosslink) with each other in the space surrounding the cell. Crosslinking results in the formation of very strong, mature type II collagen fibers.

[0066] The COL3A1 (collagen type III α1 chain) gene provides the instructions for producing type III collagen. Collagen is a family of proteins that strengthen and support many tissues in the body. Type III collagen is found in the skin, lungs, intestinal walls, and blood vessel walls. The component of type III collagen called the precursor α1(III) chain is produced by the COL3A1 gene. Each molecule of type III procollagen consists of three copies of this chain. The three-chained rope-like procollagen molecules are enzymatically processed extracellularly to produce mature type III collagen. The collagen molecules then align themselves into elongated fibrils, which form stable interactions (crosslinks) with each other and with other types of collagen in the space between cells. Crosslinking results in the formation of very strong collagen fibers.

[0067] The term "fibronectin" refers to the fibronectin 1 gene or its products. This gene encodes fibronectin, a glycoprotein that exists in plasma as a soluble dimer and in dimer or multimer forms on the cell surface and in the extracellular matrix. The encoded protoprotein is processed by proteolysis to produce the mature protein. Fibronectin is involved in cell adhesion and migration processes, including embryonic development, wound healing, coagulation, host defense, and metastasis. The gene has three regions undergoing alternative splicing, potentially producing 20 different transcript variants, at least one of which encodes the isotype that has undergone proteolytic processing.

[0068] TIMP1, or TIMP metallopeptidase inhibitor 1, belongs to the TIMP gene family. Proteins encoded by this gene family are natural inhibitors of matrix metalloproteinases (MMPs), a group of peptidases involved in the degradation of the extracellular matrix. In addition to its inhibitory effect on most known MMPs, this encoded protein promotes cell proliferation in various cell types and also possesses anti-apoptotic functions. Transcription of this gene is highly inducible in response to many cytokines and hormones. Furthermore, partial, but not complete, inactivation of expression on the X chromosome indicates that gene inactivation is polymorphic in human females.

[0069] The COL1A1B gene is zebrafish specific. In tetrapods, collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by the COL1A1 and COL1A2 genes, respectively. In contrast, zebrafish possess three type I collagen genes encoding α1(I), α3(I), and α2(I) chains: col1a1a, col1a1b, and col1a2. During embryonic and larval development, these three collagen type I genes exhibit similar spatial-temporal expression patterns, indicating co-regulation and interdependence at these stages. The presence of three α(I) chains has been confirmed in both embryonic and adult tissues, although α1(I) exists in two distinct glycosylation states in the embryo, suggesting a developmentally specific collagen composition. Although equal amounts of α1(I), α3(I), and α2(I) chains are present in adult bones, skin, and scales, the data presented indicate tissue-specific stoichiometry and / or post-translational modification status of collagen type I.

[0070] Matrix metalloproteinases (MMPs) belong to the zinc-dependent extracellular matrix (ECM) remodeling endopeptidases, a family capable of degrading almost every component of the ECM. ECM degradation is crucial because it is involved in embryonic development and angiogenesis. It is also involved in cell repair and tissue remodeling. When MMP expression is altered, it leads to abnormal degradation that produces the ECM. This is the initial cause of the development of chronic degenerative diseases and vascular complications, such as diabetes. Furthermore, this process is associated with neurodegeneration and cancer progression. In the ECM, tissue inhibitors of MMPs (TIMPs) suppress the proteolytic activity of MMPs. TIMPs are important regulators of ECM renewal, tissue remodeling, and cellular behavior. Therefore, TIMPs (similar to MMPs) regulate angiogenesis, cell proliferation, and apoptosis. Disruption of the balance between MMPs and TIMPs is associated with the pathophysiology and progression of several diseases. MMPs belong to the endopeptidases family, which comprises 23 members. They contain zinc, are calcium-dependent, and are capable of degrading and remodeling proteins that form the ECM. They are also involved in various biological and physiological processes regulated by hormones, growth factors, and cytokines. Based on their subcellular distribution and specificity to ECM components, MMPs are classified into membrane-type matrix metalloproteinases (MT-MMPs), collagenases, gelatinases, matrix-degrading proteins, and matrix lysins. Collagenases (MMP-1, MMP-8, MMP-13, and MMP-18) degrade triple-helical fibrous collagen, a major component of bone and ligaments. Gelatinases (MMP-2 and MMP-9) are involved in various cellular processes, including angiogenesis and neurogenesis; these proteases alter the molecules of the basement membrane, subsequently leading to cell death. Matrix-degrading proteins (MMP-3, MMP-10, and MMP-11) are small proteases that degrade ECM fragments. Matrix lysins (MMP-7 and MMP-26) process cell surface molecules and digest ECM components.

[0071] Members of the tetraspanic membrane protein family (such as CD63, CD81, and CD9) are widely expressed on exosomes and are extensively used as exosomal biomarkers involved in physiological processes such as cell adhesion, cell motility, and signal transduction. CD63 (characterized primarily as a tetraspanic membrane protein) possesses two extracellular loops of unequal size and two short cytoplasmic domains, participating in signal transduction processes in various types of immune cells. The loss of the continuous domain has indicated that transmembrane helix 3 (TM3) is necessary and sufficient for membrane anchoring and exosome targeting. The amino acid sequence of human CD63 is available, for example, from GenBank: AHI51903.1, and the amino acid sequence of its TM3 corresponds to amino acids 70 to 133, which is also shown in this disclosure as SEQ ID NO. 9.

[0072] The term "anchored polypeptide" is a polypeptide that is anchored to the exosome membrane when exosomes are produced by cells. Transmembrane proteins are typical anchored polypeptides in this disclosure. "Anchored" or its grammatical variations refer to at least one fragment of a polypeptide being embedded in the exosome membrane. Anchored polypeptides may be fully or partially embedded in the exosome membrane. In this disclosure, anchored polypeptides are fused with polypeptides heterologous to naturally produced exosomes from the same cell, such as the KGF-1 polypeptide. Exemplary anchored polypeptides are exosome membrane proteins, membrane-targeting sequences, or their anchoring functional fragments. Exemplary exosome membrane proteins include, but are not limited to, lamp2b, tetraspan membrane proteins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactin (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. Detailed reviews of GPI anchors in exosomes are available, for example, 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 this disclosure, the anchoring polypeptide comprises or is composed of the transmembrane helix 3 (TM3) of the CD63 protein.

[0073] engineered exosomes

[0074] One aspect of this disclosure relates to an engineered exosome comprising (a) a KGF peptide fused to a first anchoring peptide, (b) an EGF peptide fused to a second anchoring peptide, and (c) an FGF-2 peptide fused to a third anchoring peptide, wherein (a), (b), and (c) are anchored to the membrane of the exosome via the first, second, and third anchoring peptides, respectively, and wherein the KGF peptide, the EGF peptide, and the FGF-2 peptide are exposed on the outer surface of the membrane of the exosome.

[0075] In a preferred embodiment, the engineered exosome provided in this disclosure comprises (a) a KGF peptide fused to a first anchoring peptide, (b) an EGF peptide fused to a second anchoring peptide, (c) an FGF-2 peptide fused to a third anchoring peptide, and (d) a PDGF-BB peptide fused to a fourth anchoring peptide, wherein (a), (b), (c), and (d) are anchored to the membrane of the exosome via the first, second, third, and fourth anchoring peptides, respectively, and wherein the KGF peptide, the EGF peptide, the FGF-2 peptide, and the PDGF-BB peptide are exposed on the outer surface of the exosome membrane.

[0076] In this disclosure, the KGF polypeptide can be 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 is a human EGF polypeptide or an ortholog or paralog thereof. 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 shown in SEQ ID NO. 1. In some embodiments, the KGF-1 polypeptide comprises the amino acid sequence shown in SEQ ID NO. 1. In some embodiments, the KGF-1 polypeptide consists essentially of the amino acid sequence shown in SEQ ID NO. 1.

[0077] In a preferred embodiment, the EGF polypeptide is a human EGF polypeptide or its ortholog or paralog. In some embodiments, the EGF polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO. 2. In some embodiments, the EGF polypeptide comprises the amino acid sequence shown in SEQ ID NO. 2. In some embodiments, the EGF polypeptide consists essentially of the amino acid sequence shown in SEQ ID NO. 2.

[0078] In a preferred embodiment, the FGF-2 polypeptide is a human FGF-2 polypeptide or an ortholog or paralog thereof. In some embodiments, the EGF polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO. 3. In some embodiments, the FGF-2 polypeptide comprises the amino acid sequence shown in SEQ ID NO. 3. In some embodiments, the FGF-2 polypeptide consists essentially of the amino acid sequence shown in SEQ ID NO. 3.

[0079] In a preferred embodiment, the PDGF-BB polypeptide is a human PDGF-BB dimer or its ortholog or paralog. In some embodiments, the PDGF-BB polypeptide is a human PDGF-BB dimer. In some embodiments, the PDGF-BB polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO. 4. In some embodiments, the PDGF-BB polypeptide comprises the amino acid sequence shown in SEQ ID NO. 4. In some embodiments, the PDGF-BB polypeptide consists essentially of the amino acid sequence shown in SEQ ID NO. 4.

[0080] In some embodiments, these anchoring peptides are exosome membrane proteins, membrane-targeting sequences, or their anchoring functional fragments. Exemplary exosome membrane proteins include, but are not limited to, lamp2b, four-span membrane proteins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactobacin (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 a preferred embodiment of this disclosure, the first, second, third, and fourth anchoring peptides comprise the TM3 domain of CD63. In a preferred embodiment, each of the first, second, third, and fourth anchoring peptides comprises the TM3 domain of CD63. In some embodiments, these anchoring peptides are full-length CD63 proteins, such as full-length human CD63 (see, for example, UniProtKB / Swiss-Prot: F8VZE2, P08962, Q5TZP3, Q8N6Z9, or Q9UCG6). In some embodiments, these anchoring peptides are truncated CD63 proteins comprising a TM3 domain and at least one of the TM1, TM2, and TM4 domains. For example, these anchoring peptides may consist of TM2 and TM3 of CD63; TM3 and TM4 of CD63; or TM1, TM2, and TM3 of CD63. In some embodiments, these anchoring peptides consist of the TM3 domain of CD63. In this disclosure, the first, second, third, and fourth anchoring peptides may be different or the same. In a preferred embodiment, the first, second, third, and fourth anchoring peptides are the same and consist of the TM3 domain of CD63.

[0081] In a preferred embodiment, the TM3 domain of 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 shown in SEQ ID NO. 9. In a preferred embodiment, the TM3 domain of CD63 comprises the amino acid sequence shown in SEQ ID NO. 9. In a preferred embodiment, the TM3 domain of CD63 is substantially composed of the amino acid sequence shown in SEQ ID NO. 9.

[0082] Therefore, in a preferred embodiment, an engineered exosome is provided comprising (a) a KGF-1 peptide fused to a first anchoring peptide, (b) an EGF peptide fused to a second anchoring peptide, and (c) an FGF-2 peptide fused to a third anchoring peptide, wherein each of the first, second, and third anchoring peptides comprises a TM3 domain of CD63, wherein (a), (b), and (c) are anchored to the membrane of the exosome via the first, second, and third anchoring peptides, respectively, and wherein the KGF-1 peptide, the EGF peptide, and the FGF-2 peptide are exposed on the outer surface of the membrane of the exosome.

[0083] In a preferred embodiment, an engineered exosome is provided comprising (a) a human KGF-1 peptide fused to a first anchoring peptide, (b) a human EGF peptide fused to a second anchoring peptide, and (c) a human FGF-2 peptide fused to a third anchoring peptide, wherein each of the first, second, and third anchoring peptides comprises a TM3 domain of CD63, wherein (a), (b), and (c) are anchored to the membrane of the exosome via the first, second, and third anchoring peptides, respectively, and wherein the human KGF-1 peptide, the human EGF peptide, and the human FGF-2 peptide are exposed on the outer surface of the membrane of the exosome.

[0084] In a preferred embodiment, an engineered exosome is provided comprising (a) a KGF-1 polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1, the KGF-1 polypeptide being fused to a first anchoring polypeptide; (b) an EGF 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 shown in SEQ ID NO. 2, the EGF polypeptide being fused to a second anchoring polypeptide; and (c) an FGF-2 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 shown in SEQ ID NO. 1, the EGF-1 ... first anchoring polypeptide; and (d) an FGF-2 polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1, the EGF-1 polypeptide being fused to a first anchoring polypeptide; and (e) an FGF-2 polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1, the EGF-1 polypeptide being fused to a first anchoring polypeptide; and (e) an FGF-2 polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence having The amino acid sequence shown in 3 has an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. The FGF-2 polypeptide is fused with a third anchoring polypeptide, wherein each of the first, second, and third anchoring polypeptides contains a TM3 domain of CD63. (a), (b), and (c) are anchored to the membrane of the exosome by the first, second, and third anchoring polypeptides, respectively, and the KGF-1 polypeptide, the EGF polypeptide, and the FGF-2 polypeptide are exposed on the outer surface of the membrane of the exosome.

[0085] In a preferred embodiment, an engineered exosome is provided comprising (a) a KGF-1 polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1, the KGF-1 polypeptide being fused to a first anchoring polypeptide; (b) an EGF 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 shown in SEQ ID NO. 2, the EGF polypeptide being fused to a second anchoring polypeptide; and (c) an FGF-2 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 shown in SEQ ID NO. 1, the EGF-1 ... first anchoring polypeptide; and (d) an FGF-2 polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1, the EGF-1 polypeptide being fused to a first anchoring polypeptide; and (e) an FGF-2 polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1, the EGF-1 polypeptide being fused to a first anchoring polypeptide; and (e) an FGF-2 polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence having The amino acid sequence shown in SEQ ID NO. 9 has 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 shown in SEQ ID NO. 9. The FGF-2 polypeptide is fused with a third anchoring polypeptide, wherein each of the first, second, and third anchoring polypeptides contains a CD63 TM3 domain containing 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 shown in SEQ ID NO. 9. (a), (b), and (c) are anchored to the membrane of the exosome by the first, second, and third anchoring polypeptides, respectively, and wherein the KGF-1 polypeptide, the EGF polypeptide, and the FGF-2 polypeptide are exposed on the outer surface of the membrane of the exosome.

[0086] In a preferred embodiment, an engineered exosome is provided comprising (a) a KGF-1 polypeptide having the amino acid sequence shown in SEQ ID NO. 1, fused with a first anchoring polypeptide; (b) an EGF polypeptide having the amino acid sequence shown in SEQ ID NO. 2, fused with a second anchoring polypeptide; and (c) an FGF-2 polypeptide having the amino acid sequence shown in SEQ ID NO. 3, fused with a third anchoring polypeptide, wherein each of the first, second, and third anchoring polypeptides comprises a TM3 domain of CD63 having the amino acid sequence shown in SEQ ID NO. 9, 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 EGF polypeptide, and the FGF-2 polypeptide are exposed on the outer surface of the membrane of the exosome.

[0087] In a preferred embodiment, an engineered exosome is provided comprising (a) a KGF-1 peptide fused to a first anchoring peptide, (b) an EGF peptide fused to a second anchoring peptide, (c) an FGF-2 peptide fused to a third anchoring peptide, and (d) a PDGF-BB peptide fused to a fourth anchoring peptide, wherein each of the first, second, third, and fourth anchoring peptides comprises a TM3 domain of CD63, wherein (a), (b), (c), and (d) are anchored to the membrane of the exosome via the first, second, third, and fourth anchoring peptides, respectively, and wherein the KGF-1 peptide, the EGF peptide, the FGF-2 peptide, and the PDGF-BB peptide are exposed on the outer surface of the exosome membrane.

[0088] In a preferred embodiment, an engineered exosome is provided comprising (a) a human KGF-1 peptide fused to a first anchoring peptide, (b) a human EGF peptide fused to a second anchoring peptide, (c) a human FGF-2 peptide fused to a third anchoring peptide, and (d) a human PDGF-BB peptide fused to a fourth anchoring peptide, wherein each of the first, second, third, and fourth anchoring peptides comprises a TM3 domain of CD63, wherein (a), (b), (c), and (d) are anchored to the membrane of the exosome via the first, second, third, and fourth anchoring peptides, respectively, and wherein the human KGF-1 peptide, the human EGF peptide, the FGF-2 peptide, and the PDGF-BB peptide are exposed on the outer surface of the exosome membrane.

[0089] In a preferred embodiment, an engineered exosome is provided comprising (a) a KGF-1 polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1, the KGF-1 polypeptide being fused to a first anchoring polypeptide; (b) an EGF 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 shown in SEQ ID NO. 2, the EGF polypeptide being fused to a second anchoring polypeptide; (c) an FGF-2 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 shown in SEQ ID NO. 3, the FGF-2 polypeptide being fused to a third anchoring polypeptide; and (d) The PDGF-BB polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO. 4, the PDGF-BB polypeptide being fused with a fourth anchoring polypeptide, wherein each of the first, second, third, and fourth anchoring polypeptides comprises a TM3 domain of CD63, wherein (a), (b), (c), and (d) are anchored to the membrane of the exosome via the first, second, third, and fourth anchoring polypeptides, respectively, and wherein the KGF-1 polypeptide, the EGF polypeptide, the FGF-2 polypeptide, and the PDGF-BB polypeptide are exposed on the outer surface of the membrane of the exosome.

[0090] In a preferred embodiment, an engineered exosome is provided comprising (a) a KGF-1 polypeptide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1, the KGF-1 polypeptide being fused to a first anchoring polypeptide; (b) an EGF 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 shown in SEQ ID NO. 2, the EGF polypeptide being fused to a second anchoring polypeptide; (c) an FGF-2 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 shown in SEQ ID NO. 3, the FGF-2 polypeptide being fused to a third anchoring polypeptide; and (d) A PDGF-BB 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 shown in SEQ ID NO. 4, wherein the PDGF-BB polypeptide is fused with a fourth anchoring polypeptide, wherein each of the first, second, third, and fourth anchoring polypeptides comprises a TM3 domain of CD63, the domain 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 shown in SEQ ID NO. 4, and wherein the PDGF-BB polypeptide is fused with a fourth anchoring polypeptide, wherein each of the first, second, third, and fourth anchoring polypeptides comprises a TM3 domain of CD63 The amino acid sequence shown in 9 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity, wherein (a), (b), (c), and (d) are anchored to the membrane of the exosome by the first, second, third, and fourth anchoring peptides, respectively, and wherein the KGF-1 peptide, the EGF peptide, the FGF-2 peptide, and the PDGF-BB peptide are exposed on the outer surface of the membrane of the exosome.

[0091] In a preferred embodiment, an engineered exosome is provided comprising (a) a KGF-1 polypeptide having the amino acid sequence shown in SEQ ID NO. 1, fused to a first anchoring polypeptide; (b) an EGF polypeptide having the amino acid sequence shown in SEQ ID NO. 2, fused to a second anchoring polypeptide; (c) an FGF-2 polypeptide having the amino acid sequence shown in SEQ ID NO. 3, fused to a third anchoring polypeptide; and (d) a PDGF-BB polypeptide comprising the amino acid sequence shown in SEQ ID NO. 4, fused to a fourth anchoring polypeptide, wherein each of the first, second, third, and fourth anchoring polypeptides comprises a TM3 domain of CD63 having the amino acid sequence shown in SEQ ID NO. 1. The amino acid sequence shown in 9, wherein (a), (b), (c) and (d) are anchored to the membrane of the exosome by the first, second, third and fourth anchoring peptides, respectively, and wherein the KGF-1 peptide, the EGF peptide, the FGF-2 peptide and the PDGF-BB peptide are exposed on the outer surface of the membrane of the exosome.

[0092] In any of the above embodiments, the KGF peptide, the EGF peptide, the FGF-2 peptide, and the PDGF-BB peptide (when present) are fused directly or via peptide linkers to the C-terminus of the first, second, third, and fourth anchoring peptides, respectively. The peptide linker can be any peptide linker available in the art for linking different domains or functional regions 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.

[0093] In this 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 by the present invention are derived from non-stem cells, such as CHO or HEK293 cells. In this disclosure, preferably, the exosomes are purified and / or isolated from the cells from which they are derived.

[0094] In some embodiments, the exosomes provided in this disclosure enhance fibroblast proliferation. In some embodiments, the exosomes provided in this disclosure upregulate the COL2A1, COL3A1, fibronectin, TIMP1, and / or COL1A1B genes. In some embodiments, the exosomes provided in this disclosure downregulate the MMP7, MMP9, and / or MMP16B genes. In some embodiments, the exosomes provided in this disclosure downregulate melanin synthesis. In some embodiments, the exosomes provided in this disclosure enhance fibroblast proliferation; upregulate the COL2A1, COL3A1, fibronectin, TIMP1, and / or COL1A1B genes; downregulate the MMP7, MMP9, and / or MMP16B genes; and downregulate melanin synthesis.

[0095] Methods and compositions for skin rejuvenation

[0096] Various aspects of this disclosure relate to methods and compositions for skin rejuvenation. In a preferred embodiment, the skin rejuvenation includes treating any of the following: sun damage; sensitive skin; scars such as acne or stretch marks such as stretch marks or puberty marks, chickenpox, or wounds; wrinkles such as static or dynamic wrinkles; dark circles; hyperpigmentation such as darkening of the skin or freckles, loose skin, uneven skin tone or dullness, or any combination thereof; the method includes administering to a subject in need a pharmaceutically or cosmetically effective amount of the engineered exosomes or compositions disclosed herein.

[0097] In a preferred embodiment, an anti-wrinkle method is provided, the wrinkles including, for example, static and dynamic wrinkles on the neck and / or face, the method comprising administering to a subject in need a pharmaceutically or cosmetically effective amount of the engineered exosomes or compositions disclosed herein. In a preferred embodiment, a method for treating hyperpigmentation, for example on the neck and / or face, is provided.

[0098] In a preferred embodiment, a method for treating scars, including scars arising from acne or stretch marks such as stretch marks or puberty marks, for example on the abdomen and / or legs, is provided, the method comprising administering to a subject in need a pharmaceutically or cosmetically effective amount of the engineered exosomes or compositions disclosed herein.

[0099] In this disclosure, the provided composition comprises the engineered exosome and a carrier. In a preferred embodiment, the composition is a liquid formulation. In a preferred embodiment, the composition is formulated for topical or subcutaneous application. Compositions comprising the engineered exosome are contemplated for use, including, for example, soaps, shampoos, ointments, and other such formulations.

[0100] In a preferred embodiment, the composition does not contain KGF peptides (e.g., KGF-1 peptides), EGF peptides, FGF-2 peptides, or PDGF peptides (such as PDGF-BB peptides) that are not attached to the membrane of the exosome (i.e., free peptides). For example, no additional KGF peptides (e.g., KGF-1 peptides), EGF peptides, FGF-2 peptides, or PDGF peptides (such as PDGF-BB peptides) are added to or supplemented to the composition other than peptides anchored to the membrane of the engineered exosome.

[0101] 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.

[0102] Nucleic acid constructs, vectors, cells, and production methods

[0103] Various aspects of this disclosure also relate to nucleic acid constructs encoding peptides anchored to exosomes.

[0104] In some embodiments, a nucleic acid construct is provided comprising a polynucleotide encoding: (a) a KGF polypeptide fused to a first anchoring polypeptide, (b) an EGF polypeptide fused to a second anchoring polypeptide, and (c) an FGF-2 polypeptide fused to a third anchoring polypeptide.

[0105] In some embodiments, a nucleic acid construct is provided comprising a polynucleotide encoding: (a) a KGF polypeptide fused to a first anchoring polypeptide, (b) an EGF polypeptide fused to a second anchoring polypeptide, (c) an FGF-2 polypeptide fused to a third anchoring polypeptide, and (d) a PDGF-BB polypeptide fused to a fourth anchoring polypeptide.

[0106] In some implementations, a set of three nucleic acid constructs is provided, wherein the first nucleic acid construct contains a polynucleotide encoding (a), the second nucleic acid construct contains a polynucleotide encoding (b), and the third nucleic acid construct contains a polynucleotide encoding (c), wherein (a), (b), and (c) are as defined above.

[0107] In some implementations, a set of four nucleic acid constructs is provided, wherein the first nucleic acid construct contains a polynucleotide encoding (a), the second nucleic acid construct contains a polynucleotide encoding (b), the third nucleic acid construct contains a polynucleotide encoding (c), and the fourth nucleic acid construct contains a polynucleotide encoding (d), wherein (a), (b), (c), and (d) are as defined above.

[0108] In some implementations, a set of two nucleic acid constructs is provided, one of which contains a polynucleotide encoding two of (a), (b), and (c), and the other contains a polynucleotide encoding the remaining polypeptide, wherein (a), (b), and (c) are as defined above.

[0109] In some implementations, a set of two nucleic acid constructs is provided, one of which contains a polynucleotide encoding two of (a), (b), (c) and (d), and the other contains a polynucleotide encoding the other two, wherein (a), (b), (c) and (d) are as defined above.

[0110] In some implementations, a set of three nucleic acid constructs is provided, wherein the first nucleic acid construct contains a polynucleotide encoding two of (a), (b), (c) and (d), the second nucleic acid construct contains a polynucleotide encoding one of the remaining polypeptides, and the third nucleic acid construct contains a polynucleotide encoding the other of the remaining polypeptides, wherein (a), (b), (c) and (d) are as defined above.

[0111] 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.

[0112] In a preferred embodiment, a single nucleic acid construct is provided comprising a polynucleotide encoding (a), (b), (c), and (d), wherein (a), (b), (c), and (d) are as defined above.

[0113] In some embodiments, these anchoring peptides are exosome membrane proteins, membrane-targeting sequences, or their anchoring functional fragments. Exemplary exosome membrane proteins include, but are not limited to, lamp2b, tetraspan membrane proteins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactobacin (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.

[0114] In some embodiments, each of these anchoring peptides may be located at the N-terminus of the peptide (e.g., EGF, KGF, FGF-2, and PDGF-BB) to be presented to the exosome surface, ensuring that the peptide to be presented is exposed on the exosome surface. In some embodiments, each of these anchoring peptides may be located at the C-terminus of the peptide to be presented to the exosome surface, ensuring that the peptide to be presented is exposed on the exosome surface. For example, when lamp2b is used as one of these anchoring peptides, the peptide to be presented to the exosome surface (e.g., EGF, KGF, FGF-2, or PDGF-BB) may be located at the N-terminus of lamp2b. For example, when the TM3 domain of CD63 is used as one of these anchoring peptides, the peptide to be presented to the exosome surface (e.g., EGF, KGF, FGF-2, or PDGF-BB) may be located at the C-terminus of the TM3 domain of CD63. In some implementations, when different anchoring peptides are used, the peptide to be presented to the surface of the exosome may be located at the N-terminus or C-terminus of these anchoring peptides, depending on the type of anchoring peptide used.

[0115] In a preferred embodiment, these anchoring peptides comprise full-length CD63 or truncated CD63 retaining the TM3 domain. In a preferred embodiment, each of these anchoring peptides comprises the TM3 domain of CD63. In a preferred embodiment, each of the first, second, third, and fourth anchoring peptides comprises the TM3 domain of CD63.

[0116] In a preferred embodiment, the polynucleotide from 5' to 3' may contain a nucleotide fragment encoding the following substances:

[0117] (i) [First anchoring peptide]-[KGF]-[autocleaving peptide]-[Second anchoring peptide]-[EGF]-[autocleaving peptide]-[Third anchoring peptide]-[FGF-2],

[0118] (ii) [First anchoring peptide]-[KGF]-[autocleaving peptide]-[Second anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Third anchoring peptide]-[EGF],

[0119] (iii) [First anchoring peptide]-[EGF]-[autocleaving peptide]-[Second anchoring peptide]-[KGF]-[autocleaving peptide]-[Third anchoring peptide]-[FGF-2],

[0120] (iv) [First anchoring peptide]-[EGF]-[autocleaving peptide]-[Second anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Third anchoring peptide]-[KGF],

[0121] (v) [First anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Second anchoring peptide]-[KGF]-[autocleaving peptide]-[Third anchoring peptide]-[EGF], or

[0122] (vi) [First anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Second anchoring peptide]-[EGF]-[autocleaving peptide]-[Third anchoring peptide]-[KGF],

[0123] In this context, [] represents a single polypeptide, and ]-[ represents a linker or bond.

[0124] 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. Cleavage of the self-cleaving peptide after translation of the polynucleotide yields three or four independent fusion proteins, each containing a single polypeptide and a single anchoring polypeptide to be presented to the surface of an exosome.

[0125] In a preferred embodiment, a single nucleic acid construct is provided comprising polynucleotides 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 a TM3 domain of CD63, the polynucleotide from 5' to 3' comprising a nucleotide fragment encoding one of the following:

[0126] (i) [TM3]-[KGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[FGF-2],

[0127] (ii) [TM3]-[KGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[EGF],

[0128] (iii) [TM3]-[EGF]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[FGF-2],

[0129] (iv) [TM3]-[EGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[KGF],

[0130] (v) [TM3]-[FGF-2]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[EGF], and

[0131] (vi) [TM3]-[FGF-2]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[KGF],

[0132] Wherein, [] represents a single polypeptide, and ]-[ represents a linker or bond; and where TM3 represents the TM3 domain of CD63; and T2A represents the self-cleaving peptide T2A.

[0133] In a preferred embodiment, the polynucleotide from 5' to 3' may contain a nucleotide fragment encoding the following substances:

[0134] (a) [First anchoring peptide]-[KGF]-[autocleaving peptide]-[Second anchoring peptide]-[EGF]-[autocleaving peptide]-[Third anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Fourth anchoring peptide]-[PDGF-BB],

[0135] (b) [First anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Second anchoring peptide]-[KGF]-[autocleaving peptide]-[Third anchoring peptide]-[EGF]-[autocleaving peptide]-[Fourth anchoring peptide]-[FGF-2],

[0136] (c) [First anchoring peptide]-[KGF]-[autocleaving peptide]-[Second anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Third anchoring peptide]-[EGF]-[autocleaving peptide]-[Fourth anchoring peptide]-[FGF-2],

[0137] (d) [First anchoring peptide]-[KGF]-[autocleaving peptide]-[Second anchoring peptide]-[EGF]-[autocleaving peptide]-[Third anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Fourth anchoring peptide]-[FGF-2],

[0138] (e) [First anchoring peptide]-[KGF]-[autocleaving peptide]-[Second anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Third anchoring peptide]-[EGF]-[autocleaving peptide]-[Fourth anchoring peptide]-[PDGF-BB],

[0139] (f) [First anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Second anchoring peptide]-[KGF]-[autocleaving peptide]-[Third anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Fourth anchoring peptide]-[EGF],

[0140] (g) [First anchoring peptide]-[KGF]-[autocleaving peptide]-[Second anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Third anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Fourth anchoring peptide]-[EGF],

[0141] (h) [First anchoring peptide]-[KGF]-[autocleaving peptide]-[Second anchoring peptide]-[FGF2]-[autocleaving peptide]-[Third anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Fourth anchoring peptide]-[EGF],

[0142] (i) [First anchoring peptide]-[EGF]-[autocleaving peptide]-[Second anchoring peptide]-[KGF]-[autocleaving peptide]-[Third anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Fourth anchoring peptide]-[PDGF-BB],

[0143] (j) [First anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Second anchoring peptide]-[EGF]-[autocleaving peptide]-[Third anchoring peptide]-[KGF]-[autocleaving peptide]-[Fourth anchoring peptide]-[FGF-2],

[0144] (k) [First anchoring peptide]-[EGF]-[autocleaving peptide]-[Second anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Third anchoring peptide]-[KGF]-[autocleaving peptide]-[Fourth anchoring peptide]-[FGF-2],

[0145] (l) [First anchoring peptide]-[EGF]-[autocleaving peptide]-[Second anchoring peptide]-[KGF]-[autocleaving peptide]-[Third anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Fourth anchoring peptide]-[FGF-2],

[0146] (m) [First Anchored Peptide]-[EGF]-[Autocleaving Peptide]-[Second Anchored Peptide]-[FGF-2]-[Autocleaving Peptide]-[Third Anchored Peptide]-[KGF]-[Autocleaving Peptide]-[Fourth Anchored Peptide]-[PDGF-BB],

[0147] (n) [First anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Second anchoring peptide]-[EGF]-[autocleaving peptide]-[Third anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Fourth anchoring peptide]-[KGF],

[0148] (o) [First anchoring peptide]-[EGF]-[autocleaving peptide]-[Second anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Third anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Fourth anchoring peptide]-[KGF],

[0149] (p) [First anchoring peptide]-[EGF]-[autocleaving peptide]-[Second anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Third anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Fourth anchoring peptide]-[KGF],

[0150] (q) [First anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Second anchoring peptide]-[KGF]-[autocleaving peptide]-[Third anchoring peptide]-[EGF]-[autocleaving peptide]-[Fourth anchoring peptide]-[PDGF-BB],

[0151] (r) [First anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Second anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Third anchoring peptide]-[KGF]-[autocleaving peptide]-[Fourth anchoring peptide]-[EGF],

[0152] (s) [First anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Second anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Third anchoring peptide]-[KGF]-[autocleaving peptide]-[Fourth anchoring peptide]-[EGF],

[0153] (t) [First anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Second anchoring peptide]-[KGF]-[autocleaving peptide]-[Third anchoring peptide]-[PDGF-BB] [autocleaving peptide]-[Fourth anchoring peptide]-[EGF],

[0154] (u) [First Anchored Peptide]-[FGF-2]-[Autocleaving Peptide]-[Second Anchored Peptide]-[EGF]-[Autocleaving Peptide]-[Third Anchored Peptide]-[KGF]-[Autocleaving Peptide]-[Fourth Anchored Peptide]-[PDGF-BB],

[0155] (v) [First anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Second anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Third anchoring peptide]-[EGF]-[autocleaving peptide]-[Fourth anchoring peptide]-[KGF],

[0156] (w) [First anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Second anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Third anchoring peptide]-[EGF]-[autocleaving peptide]-[Fourth anchoring peptide]-[KGF], or

[0157] (x) [First anchoring peptide]-[FGF-2]-[autocleaving peptide]-[Second anchoring peptide]-[EGF]-[autocleaving peptide]-[Third anchoring peptide]-[PDGF-BB]-[autocleaving peptide]-[Fourth anchoring peptide]-[KGF],

[0158] In this context, [] represents a single polypeptide, and ]-[ represents a linker or bond.

[0159] 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.

[0160] In other embodiments, a single polynucleotide encodes at least one anchoring polypeptide located at the C-terminus of a polypeptide to be presented to the surface of an exosome. In some embodiments, each of the first, second, third, and fourth anchoring polypeptides is different, and thus these anchoring polypeptides may be located at the N-terminus or C-terminus of polypeptides KGF, EGF, FGF-2, and optionally PDGF-BB.

[0161] In a preferred embodiment, a single nucleic acid construct is provided comprising polynucleotides encoding (a), (b), (c), and (d), wherein (a), (b), (c), and (d) are as defined above, and wherein each of the first, second, third, and fourth anchoring polypeptides comprises a TM3 domain of CD63, and the polynucleotide from 5' to 3' may comprise a nucleotide fragment encoding one of the following:

[0162] [TM3]-[KGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[PDGF-BB],

[0163] [TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[FGF-2],

[0164] [TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[FGF-2],

[0165] [TM3]-[KGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[FGF-2],

[0166] [TM3]-[KGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB],

[0167] [TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[EGF],

[0168] [TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[EGF],

[0169] [TM3]-[KGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF],

[0170] [TM3]-[EGF]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[PDGF-BB],

[0171] [TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[FGF-2],

[0172] [TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[FGF-2],

[0173] [TM3]-[EGF]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[FGF-2],

[0174] [TM3]-[EGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB],

[0175] [TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[KGF],

[0176] [TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[KGF],

[0177] [TM3]-[EGF]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF],

[0178] [TM3]-[FGF-2]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB],

[0179] [TM3]-[PDGF-BB]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[EGF],

[0180] [TM3]-[FGF-2]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[EGF],

[0181] [TM3]-[FGF-2]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF],

[0182] [TM3]-[FGF-2]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[KGF]-[T2A]-[TM3]-[PDGF-BB],

[0183] [TM3]-[PDGF-BB]-[T2A]-[TM3]-[FGF-2]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[KGF],

[0184] [TM3]-[FGF-2]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[KGF], or

[0185] [TM3]-[FGF-2]-[T2A]-[TM3]-[EGF]-[T2A]-[TM3]-[PDGF-BB]-[T2A]-[TM3]-[KGF]

[0186] Wherein, [] represents a single polypeptide, and ]-[ represents a linker or bond; and where TM3 represents the TM3 domain of CD63; and T2A represents the self-cleaving peptide T2A.

[0187] In this section, KGF, EGF, FGF-2, and PDGF-BB peptides have the meanings and preferred embodiments given in the section entitled "Engineered Exosomes" above.

[0188] In some embodiments, the polynucleotide encoding the KGF polypeptide has a nucleotide sequence as shown in SEQ ID NO. 5 or a degenerate sequence thereof.

[0189] In some embodiments, the polynucleotide encoding the EGF polypeptide has a nucleotide sequence as shown in SEQ ID NO. 6 or a degenerate sequence thereof.

[0190] In some embodiments, the polynucleotide encoding the FGF-2 polypeptide has a nucleotide sequence as shown in SEQ ID NO. 7 or a degenerate sequence thereof.

[0191] In some embodiments, the polynucleotide encoding the PDGF-BB polypeptide has a nucleotide sequence as shown in SEQ ID NO. 8 or a degenerate sequence thereof.

[0192] In some embodiments, the polynucleotide encoding the TM3 domain of CD63 has a nucleotide sequence as shown in SEQ ID NO. 10 or a degenerate sequence thereof.

[0193] A vector containing the aforementioned nucleic acid construct is also 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.

[0194] The vector provided in this paper facilitates the integration of the following polynucleotide into the cellular genome that produces exosomes, which encodes a polypeptide anchored to the membrane of the engineered exosome.

[0195] Cells transduced using this vector are also 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.

[0196] This disclosure also provides a method for generating the engineered exosomes provided herein, the method comprising transducing the cells, such as HEK293 cells, with the aforementioned vector; culturing the cells under conditions that allow the engineered exosomes to be secreted from the cells; and collecting and purifying the engineered exosomes.

[0197] In some embodiments, the method includes adapting cells from serum-containing conditions to serum-free conditions during culture. This adaptation may include a stepwise adaptation, i.e., gradually reducing the proportion of complete culture medium while increasing the proportion of serum-free culture medium.

[0198] sequence list Example

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

[0200] Materials and methods

[0201] Materials: HEK293 cell line (human embryonic kidney 293 cells, CRL-1573™) was 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 cell line (Chinese hamster ovary cells) was purchased from BeNa Culture Collection Center (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. HSF (human skin fibroblasts) and HFF (human foreskin fibroblasts) were purchased from Mingzhou Biotechnology Co., Ltd. (Ningbo, China) and cultured in DMEM (high glucose) containing 10% FBS. Cells were incubated at 37°C in humidified air containing 5% CO2. The antibody used in this study was an anti-CD63 antibody (catalog number MA5-32085, Invitrogen).

[0202] pGOI plasmid construction: The amino acid sequences of all target genes (including KGF-1, EGF, FGF-2, and PDGF-BB) were derived from Uniprot, and the corresponding DNA sequences (see SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively) were synthesized by General Biotechnology (Chuzhou, China) using plasmid pCDH-CMV-MCS-EF1a-GFP+BSD (System Biosciences). The T2A peptide (see SEQ ID NO. 13 and SEQ ID NO. 14) was used to dissociate all target proteins into single proteins post-translation. Four plasmids from the third-generation system (i.e., pGOI, pGag / pol, pRev, and pVSV-G) were used to generate lentiviruses, with pCDH-CMV-MCS-EF1a-GFP+BSD (blast fungicide resistance) serving as the lentivirus packaging plasmid. The lentiviruses were packaged by WZ Biotechnology (Jinan, China).

[0203] The payload genes constructed in two pGOI plasmids:

[0204] No. 6: CD63-TM3-linker-KGF-1-T2A-CD63-TM3-linker-EGF-T2A-CD63-TM3-linker-FGF-2, wherein each CD63-TM3 is the TM3 domain of CD63 encoded by the DNA sequence shown in SEQ ID NO. 10, each linker is encoded by the DNA sequence shown in SEQ ID NO. 12, and each T2A is encoded by the DNA sequence shown in SEQ ID NO. 14.

[0205] No. 8: CD63-TM3-linker-KGF-1-T2A-CD63-TM3-linker-EGF-T2A-CD63-TM3-linker-FGF-2-T2A-CD63-TM3-linker-PDGF-BB, wherein each CD63-TM3 is the TM3 domain of CD63 encoded by the DNA sequence shown in SEQ ID NO. 10, each linker is encoded by the DNA sequence shown in SEQ ID NO. 12, and each T2A is encoded by the DNA sequence shown in SEQ ID NO. 14.

[0206] Generation of stable cell lines: Stable HEK293 cell lines expressing target proteins (including KGF-1, EGF, FGF-2, and PDGF-BB) were generated by plasmid transfection or infection with the corresponding lentiviruses. Forty-eight hours post-transfection or infection, cells were selected by adding an antibiotic, such as blastomycin (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 blastomycin. The expression of GFP and corresponding target proteins in the stable cell lines was monitored.

[0207] To acclimate cell cultures to SFM (serum-free medium): After three initial passages in FM (complete medium), the stable HEK293 cell line should be acclimatized to serum-free culture starting from the fourth passage. Passage cells using the medium compositions in Table 1. To establish a fully serum-free culture, cells should be passaged at least three times in SFM (HyClone™ peak expression, SH31193.02, Cytiva Life Sciences).

[0208] Table 1. Adaptation Culture Media

[0209] FM: Complete culture medium; SFM: Serum-free culture medium

[0210] Exosome isolation: Stable cell lines were seeded in T150 flasks and cultured for 24 hours. After thorough washing with PBS, the cells were incubated in SFM for another 48 hours. Cells were removed by centrifugation at 300×g for 10 minutes, yielding cell-free extracellular culture medium containing exosomes. Dead cells and cell debris were then removed by centrifugation at 10000×g for 30 minutes. Finally, the clear supernatant was centrifuged at 100000×g for 70 minutes to precipitate the exosomes twice. The exosome precipitate was then resuspended. All centrifugation steps were performed at 4°C.

[0211] Figure 1 A flowchart illustrating the exosome generation process derived from HEK293 cells is shown schematically according to an exemplary embodiment of the present disclosure.

[0212] Example 2. Characterization of exosomes

[0213] Analysis of exosome particle concentration and size distribution

[0214] The particle concentration and size distribution of exosomes from stable cell lines NO. 6 or NO. 8 (transduced with pGOI No. 6 and 8, respectively) were analyzed using NanoFCM (NanoFCM Corporation, Xiamen, China). NanoFCM analysis employed two single-photon counting avalanche photodiodes (APDs) to simultaneously detect side scattering (SSC) and fluorescence of individual particles. First, exosome precipitates for analysis were prepared. Then, 200 nm PE and AF488 fluorophore-conjugated polystyrene beads were used for particle concentration, and a mixture of silica nanospheres (NanoFCM Corporation, Xiamen, China) was used for particle size distribution. The detector recorded particles passing through at 1-minute intervals in each test. Each sample was diluted to an optimal particle count range of 3000 to 9000 particles / min. Flow rate and side scattering intensity were converted to vesicle concentration and size using NanoFCM software (NanoFCM Pro V2.0).

[0215] Figure 2A The particle size and particle concentration of exosomes derived from stable cell lines No. 6 and 8 are shown by NanoFCM analysis. Exosomes from No. 6 have an average particle size of 71.3 nm, and exosomes from No. 8 have an average particle size of 72.9 nm.

[0216] Identification of target proteins on exosomes using Western blotting (WB) or ELISA

[0217] 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 phosphatase inhibitor (Beyotime), followed by heat denaturation, separation by SDS-PAGE, and transfer to a PVDF membrane (Millipore, MA, USA). Protein detection was performed by incubation with a primary antibody against CD63 (the scaffold protein of exosomes), followed by incubation with an HRP-conjugated secondary antibody (Invitrogen). The membrane was then visualized using an enhanced chemiluminescence reagent (Millipore, MA, USA). To determine target protein concentration, purified exosomes were prepared and analyzed for target proteins on exosomes using appropriate ELISA kits (Solarbio, SEKH-0051; SEKH-0220; SEKH-0052; SEKH-0290) according to the manufacturer's instructions.

[0218] Transmission electron microscopy (TEM) analysis of exosomes

[0219] TEM was used to confirm the presence of exosomes. Approximately 20 μL of exosomes were added individually to a copper grid. All excess liquid was removed using filter paper, and the sample was negatively stained with 2% uranium dioxide for 30 seconds. The grid was rinsed with deionized water and allowed to dry overnight. The sample was then air-dried under an incandescent lamp and observed using an electron microscope (Hitachi, S-3000N).

[0220] Figure 2B Transmission electron microscopy (TEM) images of exosomes produced by stable cell lines No. 6 and 8 are shown. Figure 2C The immunoblotting analysis of exosomes from cell line No. 8 using an antibody against CD63-TM3 (a scaffold protein fused to exosomes) is shown. As expected, engineered exosome-associated target proteins KGF (KGF-1), FGF-2, PDGF-BB, and EGF were present in the purified exosomes derived from the stable cell line No. 8.

[0221] The concentrations of functional proteins in exosomes were measured using an ELISA kit. Table 2 shows the results of different protein concentrations as determined by ELISA.

[0222] Table 2. Effective factor concentrations as determined by ELISA

[0223] Example 3. Engineered exosomes exhibit enhanced cell proliferation activity against fibroblasts.

[0224] Exosomes incubated with fibroblast HSF or HFF

[0225] HSF or HFF cells at a density of 4000 cells / well were incubated with exosomes from NO. 6 or NO. 8 at different concentrations (0.5 μg, 1.0 μg, 2.0 μg) for 48 hours in 96-well plates. KGF was used as a positive control. After 48 hours, cell viability was determined using a cell counting kit (CCK8) (MCE, HY-K0301).

[0226] Figure 3 shows that engineered exosomes exhibited enhanced cell proliferation activity in both HSF and HFF fibroblasts. Moreover, the cell proliferation activity was enhanced in a dose-dependent manner, especially when HSF cells were treated with exosome NO. 8.

[0227] Example 4. After treatment with exosomes, skin regeneration-related genes in fibroblasts were quantified by RT-qPCR.

[0228] Total RNA was isolated from fibroblasts treated as described above using TRIzol LS reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. The tested RNA was reverse transcribed using the Hifair® III One Step RT-qPCR SYBR Green kit (Yeasen, HB220624) and measured by qPCR. RNA copy numbers were normalized to the cellular 18S rRNA copy number. Specific primers for skin regeneration-related genes are shown in Table 3.

[0229] Table 3. Primers for RT-qPCR in fibroblasts

[0230] Figures 4A to 4E The changes in gene copy number of the tested genes are shown. Both exosome treatments No. 6 and No. 8 significantly increased the expression of proteins involved in the maintenance of the extracellular matrix, such as collagen (COL1A2, COL3A1), fibronectin, and TIMP1, and inhibited the expression of MMP7.

[0231] Example 5. Evaluation of anti-wrinkle efficacy in a zebrafish model

[0232] Zebrafish embryos (4 dpf, where dpf refers to the number of days post-fertilization) were prepared to have yolk sacs. Exosomes (NO. 6 or NO. 8) were injected. Twenty-four hours after injection, gene expression analysis of the zebrafish was performed by RT-qPCR to detect genes related to skin regeneration (Hunter Biotech). Specific primers are shown in Table 4 below.

[0233] Table 4. RT-qPCR primers in zebrafish

[0234] Figure 5 shows that engineered exosomes regulate skin regeneration-related genes in a zebrafish model. Treatment with exosomes NO. 8 significantly increased in vivo collagen (COL1A1B) synthesis and inhibited the expression of MMP9 and MMP16B, which can degrade the extracellular matrix. Exosomes NO. 6 significantly increased in vivo collagen (COL1A1B) synthesis and inhibited MMP9 expression.

[0235] Example 6. Engineered exosomes help downregulate melanin synthesis.

[0236] In vitro melanin synthesis analysis

[0237] Human melanocytes were cultured and incubated with exosomes derived from stable cell lines of NO. 6 or NO. 8 for 48 hours. The cell pellet was then used for melanin extraction, and melanin concentration was measured using a melanin assay kit (Fluorometric) (ABN-KA6030, Abnova).

[0238] Evaluation of efficacy in inhibiting hyperpigmentation in zebrafish models

[0239] Zebrafish embryos (4 hpf, where hpf refers to hours post-fertilization) were cultured at 28°C for 45 hours in 6-well plates containing 3 ml of exosomes (NO. 8). All wells (15 zebrafish embryos per well) were cultured in the dark. Images were then captured and analyzed under a dissecting microscope to assess the inhibitory efficacy against hyperpigmentation. The whitening efficacy (%) was calculated as follows: (Mock - NO. 8) / Mock * 100%.

[0240] Figure 6A and Figure 6B The results showed that engineered exosome NO.8 significantly downregulated melanin synthesis in both the melanocyte and zebrafish models. Furthermore, in the zebrafish model, treatment with NO.8 resulted in a whitening effect of 48% (P < 0.001).

Claims

1. An engineered exosome, said engineered exosome comprising... (a) A KGF peptide, wherein the KGF peptide is fused to a first anchoring peptide. (b) An EGF peptide, wherein the EGF peptide is fused with a second anchoring peptide, and (c) FGF-2 peptide, wherein the FGF-2 peptide is fused with a third anchoring peptide. Wherein (a), (b), and (c) are anchored to the membrane of the exosome via the first, second, and third anchoring peptides, respectively, and The KGF polypeptide, the EGF polypeptide, and the FGF-2 polypeptide are exposed on the outer surface of the membrane of the exosome.

2. The engineered exosome of claim 1, wherein the engineered exosome further comprises (d) a PDGF-BB polypeptide fused with a fourth anchoring polypeptide, and the PDGF-BB polypeptide is exposed on the outer surface of the membrane of the exosome.

3. 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.

4. The engineered exosome according to claim 1, wherein the EGF polypeptide is human EGF or its ortholog or paralog, preferably, the EGF polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO.

2.

5. The engineered exosome according to claim 1, wherein the FGF-2 polypeptide is human FGF-2 or its ortholog or paralog; more preferably, the FGF-2 polypeptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO.

3.

6. The engineered exosome according to claim 2, wherein the PDGF-BB polypeptide is a human PDGF-BB dimer or its ortholog or paralog; more preferably, the PDGF-BB dimer comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO.

4.

7. The engineered exosomes according to claim 1 or 2, wherein the anchoring polypeptide is an exosome membrane protein, a membrane targeting sequence, or an anchoring functional fragment thereof; preferably, the exosome membrane protein includes lamp2b, tetraspan membrane proteins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactobacin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof; preferably, the membrane targeting sequence includes a glycosylphosphatidylinositol (GPI) anchor and a lipid anchoring protein; preferably, the anchoring polypeptide comprises full-length CD63 or a truncated CD63 retaining the TM3 domain; more preferably, each of the first, second, third, and optionally fourth anchoring polypeptides comprises the TM3 domain of CD63; more preferably, each of the anchoring polypeptides is the TM3 domain of CD63; preferably, the TM3 domain of CD63 comprises, as shown in SEQ ID NO. The amino acid sequence shown in 9 has at least 80% amino acid sequence identity.

8. The engineered exosome according to claim 1 or 2, wherein the KGF polypeptide, the EGF polypeptide, the FGF-2 polypeptide and the PDGF-BB polypeptide are optionally fused to the C-terminus of the first, second, third and fourth anchoring polypeptides via peptide linkers, preferably, the peptide linkers are composed of glycine and serine, for example (G4S)n, where n is an integer from 1 to 3.

9. 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.

10. The engineered exosomes according to claim 1, wherein the engineered exosomes: (i) Enhances fibroblast proliferation (ii) Upregulation of COL2A1, COL3A1, fibronectin, TIMP1, and / or COL1A1B genes (iii) Downregulation of MMP7, MMP9 and / or MMP16B genes, and / or (iv) Downregulate melanin synthesis.

11. A composition comprising an exosome according to any one of claims 1 to 10, and a carrier; preferably, the composition is a liquid formulation; more preferably, the composition is formulated for topical or subcutaneous application; the composition is optionally a cosmetic composition or a non-cosmetic composition; and wherein the composition is optionally a pharmaceutical composition.

12. The composition of claim 11, wherein the composition does not contain KGF polypeptides (e.g., KGF-1 polypeptides), EGF polypeptides, FGF-2 polypeptides (e.g., human FGF-2 polypeptides) or PDGF-BB polypeptides (e.g., human PDGF-BB dimer polypeptides) that are not attached to the membrane of the exosomes.

13. A nucleic acid construct comprising a polynucleotide encoding the following substances: (a) A KGF peptide, wherein the KGF peptide is fused to a first anchoring peptide. (b) An EGF peptide, wherein the EGF peptide is fused with a second anchoring peptide, and (c) FGF-2 peptide, wherein the FGF-2 peptide is fused with a third anchoring peptide.

14. The nucleic acid construct of claim 11, wherein the polynucleotide further encodes (d) PDGF-BB polypeptide, the PDGF-BB polypeptide being fused with a fourth anchoring polypeptide.

15. The nucleic acid construct according to claim 13 or 14, wherein (i) The KGF polypeptide is a KGF-1 polypeptide, preferably, the KGF-1 polypeptide contains an amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1; (ii) The EGF polypeptide is human EGF or its ortholog or paralog, preferably, the EGF polypeptide contains an amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 2; (iii) The FGF-2 polypeptide is human FGF-2 or its ortholog or paralog; more preferably, the FGF-2 polypeptide contains an amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 3; (iv) The PDGF-BB polypeptide is a human PDGF-BB dimer; more preferably, the PDGF-BB dimer comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 4; (v) The polynucleotide encoding the KGF polypeptide has a nucleotide sequence as shown in SEQ ID NO. 5 or a degenerate sequence thereof; (vi) The polynucleotide encoding the EGF polypeptide has a nucleotide sequence as shown in SEQ ID NO. 6 or a degenerate sequence thereof; (vii) The polynucleotide encoding the FGF-2 polypeptide has a nucleotide sequence as shown in SEQ ID NO. 7 or a degenerate sequence thereof; (viii) The polynucleotide encoding the PDGF-BB polypeptide has a nucleotide sequence as shown in SEQ ID NO. 8 or a degenerate sequence thereof; (ix) The anchoring polypeptide is an exosome membrane protein, a membrane targeting sequence, or an anchoring functional fragment thereof; preferably, the exosome membrane protein includes lamp2b, tetraspan membrane proteins such as CD63, CD9, and CD81, platelet-derived growth factor receptor (PDGFR), lactobacin (C1C2 domain), vesicular stomatitis virus glycoprotein (VSVG), prostaglandin F2 receptor negative regulator (PTGFRN), and any combination thereof; preferably, the membrane targeting sequence includes a glycosylphosphatidylinositol (GPI) anchor and a lipid anchoring protein; preferably, the polypeptide comprises full-length CD63 or a truncated CD63 retaining the TM3 domain; more preferably, each of the first, second, third, and optional fourth anchoring polypeptides comprises the TM3 domain of CD63; more preferably, each of the first, second, third, and optional fourth anchoring polypeptides is the TM3 domain of CD63; and / or (x) The polynucleotide encoding the TM3 domain of CD63 has a nucleotide sequence as shown in SEQ ID NO. 10 or a degenerate sequence thereof.

16. The nucleic acid construct according to any one of claims 13 to 15, wherein the polynucleotide is a single polynucleotide comprising a nucleotide fragment encoding the polypeptides (a), (b), (c) and optionally (d), wherein each of the polypeptides (a), (b), (c) and optionally (d) is linked to the anchoring polypeptide; preferably, the polypeptides (a), (b), (c) and optionally (d) are separated by self-cleaving peptides; optionally, the self-cleaving peptides are 2A peptides, such as T2A, E2A, P2A or any combination thereof.

17. A vector comprising a nucleic acid construct according to any one of claims 13 to 16; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector or an adeno-associated virus vector.

18. A cell transduced using the vector according to claim 17, 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.

19. A method for producing engineered exosomes according to any one of claims 1 to 10, the method comprising: (a) Transducing the cells according to claim 18 using the vector according to claim 17; (b) The cells are cultured under conditions that allow exosomes to be secreted from the cells; as well as (c) Collect and purify the exosomes.

20. The method of claim 19, further comprising adapting the cells to serum-free conditions during step (b).

21. Use of engineered exosomes according to any one of claims 1 to 10 or the composition according to claim 11 or 12 in the manufacture of a medicament for skin rejuvenation; preferably, the skin rejuvenation includes treating any of the following: sun damage; sensitive skin; scars such as acne or stretch marks such as stretch marks or puberty marks, chickenpox or wounds; wrinkles such as static or dynamic wrinkles; dark circles; hyperpigmentation such as darkening of the skin or freckles, loose skin, uneven skin tone or dullness, or any combination thereof.

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