Engineered extracellular vesicles and uses thereof

Abstract

Described herein are engineered extracellular vesicles (EVs) comprising at least one cyclin-dependent kinase (CDK) protein. Also provided are methods using the EVs to treat a wound, e.g., a diabetic wound.

Description

[0001] Atorney Docket No. 15670-0434WO1

[0002] ENGINEERED EXTRACELLULAR VESICLES AND USES THEREOF

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 694,374, filed on September 13, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety7. Said ASCII copy, created on September 1 1, 2025, is named 15670- 0434WOl_SL_ST26 and is 23,405 bytes in size.

[0007] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0008] This invention was made with Government support under Grant Nos. GM140137 and GM149245 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0009] TECHNICAL FIELD

[0010] Described herein are engineered extracellular vesicles (EVs) comprising at least one cyclin-dependent kinase (CDK) protein. Also provided are methods using the EVs to treat a wound, e.g., a diabetic wound.

[0011] BACKGROUND

[0012] Cutaneous wound healing is a complex tissue repair process comprising stages of inflammation and proliferation, followed by remodeling. In normal, healthy physiology, multiple cell types communicate and act across space and time to resolve an injury and repair tissue. In pathological conditions (both chronic and acute), these communications and actions can be disrupted, impairing wound healing processes. Such processes are impaired in diabetic patients. Up to 34% of diabetic patients experience impaired wound healing in their lifetime with chronic wounds that are characterized by dysregulated inflammation, decreased angiogenesis, and disrupted Atorney Docket No. 15670-0434WO1 keratinocyte migration. Full recovery from chronic wounds with treatment is below 50%. Currently there are approximately 78.000 amputations each year in patients with diabetic foot ulcers and the cost of such amputations can be over $43,000 per patient.

[0013] SUMMARY

[0014] Wound healing is a complex process involving numerous cell ty pes to accomplish sequential, yet overlapping phases of inflammation, proliferation and tissue remodeling. Although the impaired wound-healing phenotype in diabetes is multi-factorial, recent studies implicate the disruption of cell-cycle mediators, which are important molecular switches to control entry into the cell cycle. Progression through the cell-division cycle is regulated by the coordinated activities of cyclin / cyclin-dependent kinases (CDK) complexes. Indeed, CDKs are key players in regulatory mechanisms of the cell cycle and are shown to be downregulated in the wound site of diabetic patients. As such, a method of delivering a CDK protein to the wound site would be beneficial in increasing wound healing capacity7in the subject, particularly in a diabetic subject.

[0015] Increasing evidence suggests that extracellular vesicles (EVs) mediate indirect signaling between cell types that is pro-reparative in wound healing. Furthermore, EVs have been identified as important mediators of epidermal homeostasis and intercellular communication responsible for delivering their cargoes such as nucleic acids, proteins, and lipids in skin microenvironment. However, despite these advances in the EV field, few studies have show n EV-delivery of foreign cargoes consisting of bioactive molecules (e.g., a CDK protein) for therapeutic applications in skin disease research.

[0016] The present disclosure provides EVs engineered to express a CDK protein pay load to promote wound healing.

[0017] Provided herein are engineered extracellular vesicles (EVs) comprising an extracellular vesicle isolated from a biological cell; and a cyclin-dependent kinase (CDK) protein. In some embodiments, an EV disclosed herein can be engineered to express the CDK protein within the EV. In some embodiments, an EV disclosed herein can further comprise a membrane anchor. Atorney Docket No. 15670-0434WO1

[0018] In some embodiments, a CDK protein disclosed herein can be fused to the EV. In some embodiments, a CDK protein disclosed herein can be a releasable payload. In some embodiments, a CDK protein disclosed herein can be CDK1.

[0019] In some embodiments, a biological cell disclosed herein can be a primary mesenchymal stem cell, an embry onic kidney cell, an embry onic fibroblast cell, an alveolar basal epithelial cell, or a monocytic cell or an immortalized cell-line thereof. In some embodiments, a biological cell disclosed herein can be an embryonic kidney cell.

[0020] Provided herein are also pharmaceutical compositions comprising any of the engineered EVs disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition disclosed herein can be formulated for local delivery' or systemic delivery. In some embodiments, a pharmaceutical composition disclosed herein can be formulated for topical administration. In some embodiments, a pharmaceutical composition disclosed herein can be formulated for intravenous delivery. In some embodiments, a pharmaceutical composition disclosed herein can further comprise a hydrogel.

[0021] Provided herein are methods of treating a disease in a subject in need thereof, wherein the method comprises administering to the subject any one of the engineered EVs disclosed herein and / or any one of the pharmaceutical compositions disclosed herein, thereby treating the disease in the subject. In some embodiments, the disease can be an intractable wound. In some embodiments, the disease can be a skin disease. In some embodiments, the disease can comprise a diabetic wound. In some embodiments, the administering comprises topical administration. In some embodiments, the administering comprises intravenous administration.

[0022] Also provided herein are methods of accelerating tissue regeneration in a subject in need thereof, wherein the method comprises administering to the subject any one of the engineered EVs disclosed herein and / or any one of the pharmaceutical compositions disclosed herein, thereby accelerating tissue regeneration in the subject. In some embodiments, the subject in need thereof can have or be suspected of having a disease. In some embodiments, the disease can be an intractable wound. In some embodiments, the disease can be a skin disease. In some embodiments, the disease can comprise a diabetic wound. In some embodiments, the administering comprises Atorney Docket No. 15670-0434WO1 topical administration. In some embodiments, the administering comprises intravenous administration.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The term “about’' as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0024] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0025] DESCRIPTION OF DRAWINGS

[0026] FIGS. 1A-1G: Characterization of CDKl-loaded sEVs. FIG. 1A shows a schematic of engineering CDKl-loaded sEVs. FIG. IB shows determination of EV concentration of mock (empty vector) and CDKl-loaded sEVs (n = 7 each). FIG. 1C shows size distribution of mock (left panel) and CDKl-loaded (right panel) sEVs (n = 7). FIG. ID depicts representative vFC analyses of mock and CDKl-loaded sEVs (n = 7). MFI, mean fluorescent intensity. FIG. IE shows immunoblotting of sEVs using EV markers, CDK1 and calnexin. FIG. IF shows normalized CDK1 expression based on immunoblotting, region of interest values shown (n = 4; **p < 0.01). FIG. 1G shows immunoblotting of sEVs using phosphorylation of CDKlTyrl5 vs. pan- CDK1, and Alix as EV marker.

[0027] FIGS. 2A-2G: Testing of the activity of CDKl-loaded sEVs in impaired wound healing. FIG. 2A shows a schematic of CDKl-loaded sEVs used in the single-dose treatment of the wound bed of diabetic obese mice. FIG. 2B shows representative images of w ound bed following topical treatment with PBS, mock sEVs, or CDKl-loaded sEVs. FIG. 2C depicts quantification of wound-closure kinetics (n = 6 per group; *p < 0.05; ****p < 0.0001). FIG. 2D shows representative Atorney Docket No. 15670-0434WO1

[0028] H&E-stained section of wounds collected on day 3 post-treatment with sEVs (top row: low magnification; bottom row: high magnification). FIG. 2E depicts quantification of epithelial thickness based on imaging analysis of H&E-stained sections (n = 6; ****p < 0.0001). FIG. 2F shows localization of Ki67+ cells by immunohistochemistry on day 3 post-treatment with sEVs (top row: low magnification; bottom row: high magnification; brown arrows indicate Ki67+ staining). FIG. 2G depicts quantification of Ki67+ cells, showing the number of Ki67+ cells per unit area (n = 6; **p < 0.01; ***p < 0.001).

[0029] FIGS. 3A-3F : Testing the activity of CDKl-loaded sEVs upon human keratinocytes in vitro. FIG. 3A shows immunofluorescence staining of cells with an anti-CDKl antibody (inside dashed lines), and counterstained with a nuclear stain post-sEV treatment (scale bar: 50 pm). FIG. 3B shows proliferation of human keratinocytes following EV treatment using CCK-8 assay (n = 10; **p < 0.01; ****p < 0.0001). FIG. 3C shows representative imaging of an in vitro scratch assay in the presence of the proliferation inhibitor mitomycin C following treatment with sEVs and controls (scale bar: 200 pm). FIG. 3D shows quantification of closure kinetics (n = 4; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). FIG. 3E shows the effect of PBS (left panel) Mock sEV (middle panel) CDK1 sEV (right panel) treatment on cell cycle using a cell-permeable DNA dye and analysis by flow cytometry. FIG. 3F shows quantification of G2, M phase from sEV -treated cells (n = 3, ***p < 0.001; ****p < 0.0001).

[0030] FIGS. 4A-4I: Downstream signaling mediated by treatment with CDKl- loaded sEVs in human keratinocytes. FIGS. 4A and 4B show immunofl uores cent localization of p-AKTSer473(FIG. 4A) and quantification (FIG. 4B). FIGS. 4C and 4D show immunofluorescent localization of p-ERK11"202 1'1'204(FIG. 4C) and quantification (FIG. 4D). FIGS. 4E and 4F show immunofluorescent localization of phospho-4E-BPlThr37 / 46(FIG. 4E) and quantification (FIG. 4F). For FIGS. 4A-4F. scale bar: 50 pm. n = 8; *p < 0.05; *** ? < 0.001; **** ? < 0.0001). FIGS. 4G-4I show immunoblotting for: (FIG. 4G) pan-AKT and p-AKTSer473; (FIG. 4H) pan-ERK and phospho-ERKThr202 / Tyr204; and (FIG. 41) pan-4E-BPl and phospho-4E-BPlThr3746and levels normalized to -actin. Atorney Docket No. 15670-0434WO1

[0031] FIGS. 5A-5D: Analysis of histone phosphorylation following treatment with CDKl-loaded sEVs onto human keratinocytes. FIG. 5A shows immunofluorescent imaging to localize p-Histone H3Serl° and counterstained with acetyl-a-tubulin and DAPI for nuclei following sEV treatment (top row: low magnification; bottom row: high magnification) (scale bars: 50 and 20 pm). FIG. 5B shows quantification of p- Histone H3Serl° following EV treatment (n = 8; **** ? < 0.0001). FIG. 5C shows representative immunofluorescent images of cell-cycle progression following sEV (scale bar: 20 pm). FIG. 5D shows distribution of mitotic phases based on each sEV treatment (n = 50 for each treatment).

[0032] FIG. 6 depicts a model for driving entry into the mitotic cycle by treatment of cells with CDKl-loaded sEVs that promote cytosolic signaling and phosphorylation of nuclear histones on chromatin.

[0033] FIG. 7 : Controls for single vesicle flow cytometry (vFC) based on concentration of samples by polymeric precipitation. Representative controls showing event distribution in a running buffer only (Blank), negative control that has running buffer with fluorescent lipophilic membrane dye vFRed without sample. Mock sEVs and CDKl-loaded sEVs with vFRed dye. The left column contains plots of size based on calibration with Nanorainbow beads vs Violet SSC-H. The middle column shows diameter vs. count to obtain a size distribution and further supporting the -110 nm population. The right column is a distribution of vFRed positive events vs. Violet SSC-H with concentration calculated based on events, rate, and volume.

[0034] FIGS. 8A-8C: Retention time of CDKl-loaded sEVs in wound area. FIG. 8A shows representative immunofluorescent images of wound margin with an anti- CDK1 antibody and counter-stained with a nuclear stain post-sEV treatment (Scale bar: 200 pm) and FIG. 8B shows their quantification (n = 4, ****p < 0.0001). FIG. 8C shows immunoblotting of CDK1 in the wound area.

[0035] FIGS. 9A-9C: EV cargo release occurred from lysosomes. FIG. 9A shows immunofluorescent images stained with lysotracker incubated with GFP-loaded sEVs for 3, 6, 12, and 24 hours. Particles represent co-localization of lysosome and GFP and arrows point escaped GFP (Scale bar: 50 pm). FIG. 9B shows quantified fluorescence intensity7of GFP for 24 hours (n = 6, ****p < 0.0001, **p < 0.01). FIG. 9C shows quantification of co-localization of lysosome and GFP (n = 6, **p < 0.01). Atorney Docket No. 15670-0434WO1

[0036] FIGS. 10 A- 10C: Release kinetics of CDK1 in recipient keratinocytes from sEVs. FIG. 10A shows immunofluorescent images stained with CDK1 and counter stained with DAPI for nuclei after CDK1 -loaded sEV treatment (Scale bar: 50 gm), and FIG. 10B shows their quantification (n = 5, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05). FIG. 10C shows an immunoblot of CDK1 in recipient whole cell lysate after CDK1 -loaded sEV treatment.

[0037] FIGS. 11A-11C: In vitro migration assay by CDK1 delivered to human keratinocyte by sEV treatment. FIG. 11A shows proliferation inhibitory efficacy of mitomycin C in human keratinocyte (n = 6, ****p < 0.0001). FIG. 11B shows quantification of cell migration without proliferation arrest by mitomycin C, and their representative images are shown in FIG. 11C. (Scale bar: 100 pm, ****p < 0.0001, ***p < 0.001, *p < 0.05).

[0038] FIGS. 12 A and 12B: Repression of p27Kipl by activated AKT and ERK signaling. FIG. 12A shows immunofluorescent images stained with p27Kipl and counter stained with DAPI for nuclei after CDK1 -loaded sEV treatment, and FIG. 12B shows their quantification. (Scale bar: 50 pm, n = 8, ***p < 0.001).

[0039] FIG. 13 shows a schematic of the LSL-tdTomato reporter system of the transgenic mice controlled by Cre recombinase.

[0040] FIG. 14 shows a schematic of the treatment protocol using intratracheal administration (i.t.) administration of bleomycin to induce ROS in the lungs followed by intravenous (i.v) injection of EVs,

[0041] FIG. 15 shows immunoblots of liver (left panels) and lung (right panels) tissues harvested from ROS-induced LSL-tdTomato transgenic mice.

[0042] DETAILED DESCRIPTION

[0043] Cutaneous wound healing is a complex tissue repair process comprising stages of inflammation and proliferation, followed by remodeling. This process is impaired in diabetic patients who experience chronic wounds characterized by dysregulated inflammation, decreased angiogenesis, and disrupted keratinocyte migration. Although the impaired wound-healing phenotype in diabetes is multi-factorial, recent studies implicate the disruption of cell-cycle mediators - including CDKs - which are important molecular switches that control entry into the cell cycle. Atorney Docket No. 15670-0434WO1

[0044] Given the pathophysiological significance of wound healing and the multiple levels of CDK regulation, one or more of these kinases can affect the wound repair process. This is in contrast to its complex partner, cyclins. For example, CDK1 has been shown to be downregulated in the wound site of diabetic patients. This impaired capacity' for cell-cycle progression underlies the reduced proliferation and migration of resident stem cells that is characteristic of the diabetic wound. In preclinical models, the loss of CDK1 reduced cell proliferation and lipid metabolism, which resulted in insulin resistance. Although a role for CDKs, particularly CDK1, as regulators of cutaneous wound healing has been supported by the reduced kinetics of wound closure in transgenic mouse studies (e.g., CDK1 knockout mice), the specific mechanism remained unclear. In the present disclosure, a mechanism for a CDK protein (i.e., CDK1) in the regulation of wound healing is demonstrated and the potential for CDK-based therapeutics is exemplified by loading small EVs (sEVs) with CDK1 protein to deliver stable, biologically active, pro-reparative EVs that modulate defined molecular endpoints relevant to CDK action in the cytoplasm and nucleus and accelerate wound healing.

[0045] CDK-loaded EVs provided herein are able to take advantage of both the protein sorting machinery' of EV formation in the multi vesicular body and the protection of cargoes to maintain bioactivity. In general, EV cargoes are released into the cytoplasm as a result of endosomal maturation influenced by luminal pH and cholesterol, which was confirmed with GFP-loaded sEV studies as shown in the present disclosure. As demonstrated herein, the CDK-loaded EVs of the present disclosure were internalized into recipient cells and escaped the endosomal pathway to activate effector molecules in the cytosol and nucleus.

[0046] Herein, delivery of CDKl-loaded sEVs stimulated the phosphorylation of cytosolic 4E-BP1 and nuclear histone H3. These effectors were downstream of AKT and ERK activation, which are important regulators of proliferation and wound healing. CDK1 -mediates increases in 4E-BP1 phosphorylation to regulate global translation downstream, coupling cell proliferation with protein synthesis, as identified in the CDK1 -induced proliferation of human keratinocytes herein. CDK1 is also associated with the regulation of other pathways, such as its interactions with cyclin B and its repression of lysosomal degradation. In this example, CDK1 is also known to inhibit mTOR complex 1 (mTORCl). a regulator of autophagy. It is Atorney Docket No. 15670-0434WO1 interesting that CDK1 expression repressed mTORCl trafficking to lysosomes. In the studies herein, data suggested that non-specific mitosis initiated by EV treatment led to the CDK 1 -dependent arrest of lysosomal degradation that facilitated further EV endosomal escape and the dose-dependent CDK1 activity identified in the present disclosure.

[0047] Provided herein are methods of loading biologically active protein payloads (e.g., a CDK protein, a CDK1 protein) in EVs to reverse the impaired wound healing observed in diabetes, infection, and aging. Chronic wounds, particularly in diabetic patients, are highly prevalent and carry' significant morbidity and mortality. EV -based therapy has emerged as a promising strategy in promoting the kinetics and durability of wound healing, in part because distinct EVs can promote the various stages of hemostasis, inflammation, proliferation, and remodeling. As demonstrated herein, a single dose of CDKl-sEVs were administered in the inflammatory phase during wound healing to evaluate the effect on cell proliferation of resident cells in the wound area. The potential of EVs as therapeutics is further supported by low immunogenicity and the ability to harness endogenous EV release pathways from donor cells and uptake pathways in recipient cells to deliver functional payloads. With relatively few studies using engineered EVs to deliver biologically active protein payloads that accelerate wound healing in chronic models, development of the CDK (e.g.. CDK1) delivery strategy as detailed herein that focuses on well-defined molecular endpoints like cell proliferation is groundbreaking in this field and can be potentially expanded to consider other relevant pro-reparative EV pay loads. For example, CDK-related factors, such as CDK1. are thought to be dysregulated in diabetic patients based in part on changes in PI3K / AKT signaling that is impaired in various tissues due to the insulin resistance of diabetic obese patients. In support of this, the CDK1 -regulated signaling factor histone H3 is directly phosphorylated at SerlO by CDK1 via KimH3 phosphory lation or indirectly via the ERK signaling pathway. During cell division, histone H3Ser10phosphorylation localizes to the centromeric heterochromatin in late G2 phase, then spreads along the chromosomal arms and throughout the whole chromosome in prophase. In other examples, high glucose, transforming growth factor , or angiotensin II induce CDK inhibitors such as p21c,pland p27K,p1that can result in cell-cycle arrest. Dy sfunction of AKT signaling in diabetic wounds can reduce the phosphorylation of 4E-BP1 and the Atorney Docket No. 15670-0434WO1 expression of growth factor. These examples provide insights into the various pathways that CDK (e.g., CDK1) and its effectors can regulate to restore healthy wound closure.

[0048] Pro-reparative EVs can stimulate the proliferation of basal keratinocytes, as determined by Ki67 immunohistochemistry. Furthermore, the activity' of these engineered EVs can be demonstrated on isolated human keratinocytes using cell proliferation and signaling readouts. The studies herein show that CDK1 -loaded sEVs promote keratinocyte migration, proliferation, and signaling, which are critical roles for the keratinocyte in regulating skin homeostasis and inflammation. Kinetic studies of wound closure suggest that keratinocytes migrate upon a layer of fibroblasts that provide extracellular matrix to establish a viable proliferative epithelial barrier. The effects of CDK1 -loaded EVs upon both cell migration and proliferation suggest that there may be overlapping pathways or that the effects may have different kinetics. CDK1 activity influences not only cell proliferation but also adhesion processes relevant to migration, such as phosphorylation of keratin 5, which are critical for the formation of the basal layer of stratified squamous epithelia.

[0049] Overall, the present disclosure demonstrated that the delivery of a well- defined kinase (e.g., a CDK, a CDK1) by EVs can drive the proliferation and migration of keratinocytes and enhance wound closure. The present disclosure also demonstrated the downstream signaling pathways utilized in this process, expanding our molecular and biochemical understanding of tissue repair, and identifying a novel therapeutic strategy' for treating non-healing wounds, a great clinical need.

[0050] The disclosure generally relates to an extracellular vesicle (EV) composition comprising at least one cyclin-dependent kinase (CDK) protein, wherein the CDK protein can promote wound healing and / or tissue regeneration.

[0051] Cyclin Dependent Kinases (CDKs)

[0052] Cyclin Dependent Kinases (CDKs) are closely connected to the regulation of cell cycle progression. The human genome contains 20 different CDKs, which can be divided in at least three different sub-family with different functions, mechanisms of regulation, expression patterns and subcellular localization. See. e.g., Pellarin et al., Signal Transduct Target Ther. 2025 Jan 13; 10(1): 11. Atorney Docket No. 15670-0434WO1

[0053] The present disclosure provides engineered extracellular vesicle (EVs) comprising at least one CDK protein and methods of used thereof. In some embodiments, a CDK protein suitable for use herein can be any known CDK protein (e.g., CDK 1-20). See, e.g., Pellarin et al.; Malumbres, Genome Biol. 2014;15(6):122; Lukasik et al., IntJMol Sci. 2021 Mar 13;22(6):2935. In some embodiments, a CDK protein suitable for use herein can be a CDK known to affect tissue repair and / or the wound healing mechanism. In some embodiments, a CDK protein suitable for use herein can be a CDK that is downregulated or otherwise dysregulated in diabetic wounds or other models of impaired wound healing (e.g., models related to ischemia, obesity, infection, and aging). In some embodiments, a CDK protein suitable for use herein can be a CDK that promotes entry into at least one cell-cycle that is required for wound healing. In some embodiments, a CDK protein suitable for use herein can be a CDK that promotes anti-inflammatory activity.

[0054] Non-limiting examples of CDK proteins contemplated for use herein can include CDK1. CDK4. CDK5. CDK6. CDK13, or any isoform or variant thereof. In some embodiments, a CDK protein suitable for use herein can be CDK1, a variant of CDK1, or any isoform of CDK1. In some embodiments, a CDK protein suitable for use herein can comprise at least about or about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence identity to the sequences provided in Table 1.

[0055] TABLE 1 : CDK Proteins Atorney Docket No. 15670-0434WO1 Atorney Docket No. 15670-0434WO1

[0056] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring Atorney Docket No. 15670-0434WO1 matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0057] Also provided in the disclosure herein are CDK proteins comprising at least one anchoring moiety (i.e., a membrane anchor). Addition of at least one membrane anchor can be used to anchor the CDK protein payload (or any other moieties of interest) to the EVs disclosed herein. In some embodiments, the CDK protein payload is linked directly to the membrane anchor. In some embodiments, the CDK protein payload is linked to the membrane anchor via a linker, such as those described herein. In some embodiments, the CDK protein payload can be attached to a membrane anchor and / or linker via reaction between a ‘‘reactive group” (RG; e.g., amine, thiol, hydroxy, carboxylic acid, or azide) with a “reactive moiety” (RM; e.g., maleimide. succinate, NHS). In some embodiments, the membrane anchor can be conjugated to a CDK protein payload directly or indirectly via a linker combination, at any chemically feasible location, e.g., at the 5' and / or 3' end of the CDK protein. Nonlimiting examples of membrane anchors can include, S-palmitoylation. N- palmitoylation, N-myristoylation, O-acylation, famesylation, geranylgeranylation, cholesterol, or any combination thereof. In some embodiments, the CDK protein is myristoylated. While not wishing to be bound by any specific theory', it is believed that myristoylation can, among other things, promote anchoring of the CDK protein to the membrane, such that the CDK protein may be loaded / trafficked / shuttled / anchored (e.g., anchored to an EV membrane) into the EVs described herein. Using this myristoylation fusion approach, as demonstrated herein, membrane-targeted CDK- loaded EVs were released into conditioned media from EV donor cells, demonstrating loading of a specific protein pay load into the EVs, which was subsequently released into recipient cells.

[0058] The CDK proteins contemplated herein can further comprise a tag. Protein tags are generally know n in the art and can have a multitude of uses including (but not limited to) purification, detection, solubilization, localization, or protease protection. In some embodiments, a CDK protein can be linked with one or more of the protein tags provided in Table 2. In some embodiments, the tag can be linked, for example, to the N-terminus and / or C-terminus of the CDK protein. Atorney Docket No. 15670-0434W01

[0059] TABLE 2: Protein Tags

[0060] Provided herein are methods of generating the CDK proteins disclosed (e g., a CDK1 protein, a CDK protein fused to at least one tag). Methods disclosed herein can include introducing a CDK protein in a nucleic acid that encodes them into a cell (e g-, a target cell). In order to express a fusion protein disclosed herein, a sequence encoding the CDK protein can be subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al.. MOLECULAR CLONING, A LABORATORY MANUAL (3d ed. 2001); Kriegler, GENE TRANSFER AND EXPRESSION: A LABORATORY MANUAL (1990); and CU RENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al., eds., 2010). Bacterial expression systems for expressing the fusion Atorney Docket No. 15670-0434WO1 proteins are available in, e.g., E. co / i. Bacillus sp., and Salmonella (see. e.g., Palva et al., 1983. Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. Standard transfection methods can be used herein to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g.. Colley et al.. 1989. J. Biol. Chem.. 264: 17619-22; GUIDE TO PROTEIN PURIFICATION, IN METHODS IN ENZYMOLOGY, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokary otic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacterial. 132:349-351; Clark- Curtiss & Curtiss, Methods in Enzymology 101 :347-362 (Wu et al., eds, 1983). Any of the known procedures for introducing foreign nucleotide sequences into host cells can be used in the present disclosure. Examples include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (3d ed. 2001); Kriegler, GENE TRANSFER A D EXPRESSION: A LABORATORY MANUAL (1990)).

[0061] Extracellular Vesicles (EVs)

[0062] Naturally occurring EVs promote epidermal homeostasis based on the intercellular exchange of biochemically active payloads, such as nucleic acids, proteins, and lipids. Previous studies have identified specific pro-reparative EV payloads that stimulate angiogenesis, timely resolution of inflammation, and proliferation of specific cell types in the skin. For example, functional studies of specific microRNAs (miRNAs) or circular RNAs. either through passive or active EV loading, have shown beneficial effects on wound closure, including promoting cell proliferation, migration, and signaling. However, the efficacy of protein-loaded EVs in promoting wound healing is limited because of their relatively low abundance based on stoichiometry studies, and the underappreciated importance of active loading for therapeutic testing. To address these present limitations, this disclosure provides Atorney Docket No. 15670-0434WO1 developed and validated specific protein-loaded EV payloads to deliver specific proteins (e.g., CDK proteins) with wound healing properties.

[0063] An "‘extracellular vesicle’’ can refer to a cell-derived vesicle comprising a membrane that encloses an internal space. EVs contemplated herein can comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. Generally, extracellular vesicles can range in diameter from 20 nm to 1000 nm and can comprise various types of macromolecular payloads (e.g., at least one CDK protein) either within the internal space, displayed on the external surface of the extracellular vesicle, and / or spanning the membrane. By way of example and without limitation, extracellular vesicles can include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane). Extracellular vesicles may be derived from a living or dead organism, explanted tissues or organs, and / or cultured cells.

[0064] EVs disclosed herein can be generated by various methods (e.g., in vivo, using the methods described herein, or synthetically) and may have various morphologies. For example, they may comprise either one lipid bilayer (unilamellar vesicle) or a series of concentric bilayers separated by narrow aqueous compartments (multi- lamellar vesicle or MLV). Engineered EVs are also contemplated herein. In some embodiments, the EVs contemplated herein are substantially homogeneous in size and density distribution. In some embodiments, the EVs used herein can have a diameter (mean particle diameter) from about 15 to about 500 nm, from about 50 nm to about 250 nm, from 80 nm to about 100 nm, or from about 80 nm to about 100 nm. The size of the EVs contemplated herein can be determined by any suitable method known in the art including single particle optical sizing (SPOS), scanning electron microscopy , light scattering, laser diffraction, coulter counter (electrical zone sensing), and digital image analysis. EVs of a desired size can be isolated using methods known in the art including size exclusion chromatography and density gradient centrifugation.

[0065] In some embodiments, EVs disclosed herein are isolated from a biological cell. In some embodiments, the biological cell can be a primary mesenchymal stem cell, an embryonic kidney cell, an embryonic fibroblast cell, an alveolar basal Atorney Docket No. 15670-0434WO1 epithelial cell, or a monocytic cell or an immortalized cell-line thereof. Methods for isolating and purifying extracellular vesicles EVs from different biological cells are generally known in the art (see. e.g., Chen et al., (2024) Methods for the Isolation of Extracellular Vesicles. In: Wang, Q., Zheng, L. (eds) EXTRACELLULAR VESICLES. Springer, Singapore, doi.org / 10. 1007 / 978-981-99-8365-0_6) and are suitable for use herein.

[0066] In some embodiments, a biological cell can be a stem cell. Non-limiting examples of stem cells that can be used to generate EVs for use herein can include a mesenchymal stem cell (MSC), a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), an adipose tissue derived stem cell, and a neural stem cell (NSC). In some embodiments, the EVs disclosed herein can be prepared from at least one iPSC. As used herein, an “induced pluripotent stem cell” refers to a type of pluripotent stem cell that is derived from adult somatic cells but has been reprogrammed through induction of certain genes and factors to be pluripotent. iPSC- derived EVs are of particular interest as these EVs can also tone down inflammation, alter the immune system, and induce tissue repair (see, e.g., Wang et al., IntJMol Sci. 2021;22: 1769). Numerous human iPSC lines are well established and available in the art, non-limiting examples of which include 19-11-1, 19-9-7 or 6-9-9 cells (see, e.g., Yu et al., (2009) Science 324:797-801; and Wang et al., Burns Trauma. 2025 Feb 11 ; 13 :tkafO 13). Culture media and culture conditions for maintaining and expanding iPSCs are also well established and commercially available in the art. Preparation of EVs from iPSCs is generally well known in the art (see e.g., Jeske et al., (2020) Tissue Eng. Part B: Reviews 26: 129-144; Syromiatnikova et al., Int J Mol Sci. 2022 Sep 10;23(18): 10522; Wang et al., Burns Trauma. 2025 Feb 11 ; 13:tkafO 13) and is suitable for use herein. For example, a method of producing EVs from iPSCs suitable for use herein can comprise (i) culturing the iPSCs by dynamic culture such as by spinner flask bioreactor, stirred tank bioreactor or orbital shaker; and (ii) collecting the EVs from culture medium of the iPSCs.

[0067] EVs provided herein can be loaded with at least one pay load that results in delivery of at least one CDK protein to the targeted cell. In some embodiments, an EV contemplated herein can comprise a nucleic acid sequence (e.g., naked mRNA or DNA) encoding for any of the CDK proteins contemplated herein (e.g.. encoding for a CDK1 protein). In some embodiments, an EV contemplated herein can comprise at Atorney Docket No. 15670-0434WO1 least one expression construct comprising a nucleic acid sequence encoding for any of the CDK proteins contemplated herein (e.g.. encoding for a CDK1 protein).

[0068] Expression constructs comprising sequences encoding a CDK protein can include viral vectors, including recombinant retroviruses, adenovirus, adeno-associated virus (AAV), lenti virus, and herpes simplex virus- 1, or recombinant bacterial or eukary otic plasmids. In some embodiments, an EV contemplated herein can comprise at least one CDK protein. Methods of loading EVs with nucleic acids, expression vectors, and / or proteins are generally known in the art (e g., Busatto et al., Pharmaceuticals (Basel). 2021 Apr 13;14(4):356; Li et al., Pharmaceutics. 2022 Dec 2;14(12):2699; Brezgin et al. Int. J. Mol. Sci. 2024, 25, 10401) and are suitable for use herein.

[0069] In some embodiments, EVs disclosed herein can further comprise at least one RNA barcode. Addition of a barcode to the EVs contemplated herein can allow for one of skill in the art to track and / or estimate the amount of EVs released by the host cell. Further, EVs comprising the at least one mRNA barcode disclosed herein can be recovered using PCR techniques. Methods of preparing and loading mRNA barcodes into EVs are generally known in the art (see, e.g., Luo et al., Small Methods . 2022 Nov;6(l l):e2200881; Kunitake et al., Nat Commun. 2024 Nov 19; 15(1):9777; Ivanova et al., Adv Sci (Weinh). 2025 Mar; 12(10):e2407850) and are suitable for use herein.

[0070] In some embodiments, a CDK protein payload can be loaded into an EV contemplated herein by at least one releasable linker. A releasable linker as understood herein comprises a chemical linker between the EV and CDK protein that can be cleaved by enzymatic reaction, a reactive oxygen species (ROS), and / or under reductive conditions. As such, a CDK protein can be a "‘releasable payload” when loaded into an EV contemplated herein by at least one releasable linker. In some embodiments, a releasable linker disclosed herein can comprise at least one linker sensitive to ROS such as, but not limited to, thioether, selenide, diselenide, telluride, thioketal. arylboronic ester, aminoacrylate, oligoproline, peroxalate ester, and / or mesoporous silicon. In some embodiments, at least one CDK protein and at least one EV are linked by a ROS-sensitive linker. In some embodiments, a ROS-sensitive linker can be introduced to a membrane anchor. In some embodiments, a ROS- sensitive linker can be introduced to a myristylation tag. In some embodiments, the ROS-sensitive linker used herein can be an oligo-proline linker (ROSL). Atorney Docket No. 15670-0434WO1

[0071] Pharmaceutical Compositions and Methods of Administration

[0072] The methods described herein include the use of pharmaceutical compositions comprising or consisting of at least one of the engineered EVs comprising a CDK protein (e.g., CDK1 protein) disclosed herein, and / or compositions comprising a plurality of engineered EVs comprising a CDK protein disclosed herein. In some embodiments, compositions provided herein comprise at least one CDK expressing iPSC.

[0073] Pharmaceutical compositions ty pically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary' active compounds (e.g., an anti-inflammatory', an agent to treat diabetes) can also be incorporated into the compositions. A listing of active compounds and specific drugs suitable for use herein as supplementary active compounds can be found in The Merck Index Online; Royal Society of Chemistry, 2025; rsc.org / merck-index (accessed March 12, 2025), and the United States Pharmacopeia-47 / National Formulary-47, published by the United States Pharmacopeial Convention, Inc.. Rockville Md., 2024. In some embodiments, an optional supplementary active compound can be one used for treating acute wounds, chronic wounds, bums, and / or diabetic foot ulcers. See, e.g., Cwajda-Bialasik et al., Postepy Dermatol Alergol. 2022 Feb;39(l): 141-151; Powers et al., J Am Acad Dermatol. 2016 Apr;74(4):607-25).

[0074] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous (i.v.), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0075] In some embodiments, a pharmaceutical composition of the present disclosure is formulated for systemic delivery'. In some embodiments, a pharmaceutical composition of the present disclosure is formulated for intravenous delivery. In some embodiments, a pharmaceutical composition of the present disclosure is formulated for local delivery. In some embodiments, a pharmaceutical composition of the present disclosure is formulated for local delivery at the site of the wound. In some Atorney Docket No. 15670-0434WO1 embodiments, a pharmaceutical composition of the present disclosure is formulated for topical administration.

[0076] Methods of formulating suitable pharmaceutical compositions are know n in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for topical, parenteral, intravenous, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite: chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0077] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it can be preferable to include isotonic agents, for example, sugars, polyalcohols such as Atorney Docket No. 15670-0434WO1 mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0078] Pharmaceutical compositions disclosed herein can be formulated for controlled release of the payload of interest (e g., CDK, CDK1). For example, controlled release of the pay load of interest can be achieved through embedding at least one of the CDK-loaded EVs disclosed herein in an extracellular matrix-forming polymer, i.e., a hydrogel. Hydrogel-forming natural polymers can include proteins such as collagen and gelatine and polysaccharides such as starch, alginate, and agarose. Synthetic polymers that form hydrogels are traditionally prepared using chemical polymerization methods. In some embodiments, the extracellular matrix forming polymer is selected from the group consisting of polyethylene glycol (PEG), dextran, dextran sulfate, dextran acetate, polyvinyl alcohol, polyvinyl acetate, or polyvinyl sulfate, polyvinylpyrrolidon, hyaluronic acid, hydroxy ethyl starch.

[0079] The pharmaceutical compositions disclosed herein can be included in a container, pack, or dispenser together with instructions for administration. A kit comprising the plasmids encoding the CDK protein payload disclosed herein (e.g., CDK1) and instructions for preparing and loading at least one EV with the CDK protein payload for use in the methods disclosed herein is also contemplated in the present disclosure. Such kits can further include at least one material for use in preparing the CDK-EV embedded hydrogels. Kits can further comprise one or more mRNA barcodes for use in the EVs contemplated herein. Atorney Docket No. 15670-0434WO1

[0080] Methods of Treatment

[0081] The compositions (e.g., CDK-loaded EVs) described herein can be used to treat subjects with a wound e.g., a diabetic wound. As used herein, the terms “treatment"’ and “treating” can refer to obtaining a desired physiologic and / or dermatological effect by use of the compositions according to the methods disclosed herein. The effect may be prophylactic in terms of completely or partially preventing a disease, disorder, or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease, disorder, and / or symptom attributable to the disease or disorder. Thus, the terms can cover any treatment of a disorder or disease in a subject, such as: (a) preventing a wound from occurring in a subject that may be predisposed to developing the wound but has not yet been diagnosed as having it; (b) inhibiting a wound, e.g., arresting its development; and (c) relieving, alleviating, or ameliorating a wound by, for example, causing regression of the wound.

[0082] A “wound” refers to an injury to the body, including but not limited to an injury' from trauma, violence, accident, or surgery. A wound can occur due to laceration or breaking of a membrane (such as the skin) and usually damage to underlying tissues. A wound can occur in a topical location or internally. Chronic wounds can be caused by diseases, including but not limited to diabetes; diseases of internal organs, including but not limited to diseases of the liver, kidneys or lungs; cancer; or any other condition that slows the healing process. In some embodiments, the subject to be treated according to the methods disclosed herein can have at least one chronic wound. In some embodiments, the subject to be treated according to the methods disclosed herein can have at least one underlying condition and / or disease known to slow wound healing (e.g., diabetes) and have at least one wound.

[0083] As used herein, the terms “healing” and “heal” can refer to improving the natural cellular processes and humoral substances of tissue repair such that healing is faster, and / or the resulting healed area has less scaring, and / or the wounded area possesses tissue strength that is closer to that of uninjured tissue, and / or the wounded tissue attains some degree of functional recovery. The terms can additionally or alternatively refer to the physiological process wherein a wounded area returns to an effectively normal state. When the wound is an open wound, for example, healing can refer to the process whereby the skin or mucosa re-forms a continuous barrier. The Atorney Docket No. 15670-0434WO1 skilled artisan will appreciate that, after healing, the area of the wound may comprise scar tissue that is not identical to the surrounding tissue. In some embodiments, methods of the present disclosure can completely heal a wound (e.g., the integrity and function of the skin and the underlying tissue is completely restored equal to that comparable before formation of the wound). In some embodiments, methods of the present disclosure can partially heal a wound (e.g., some of the integrity and / or function of the skin and / or the underlying tissue is restored as compared to that before formation of the wound). In some embodiments, methods of the present disclosure can partially heal a wound by at least about or about 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, or about 99%.

[0084] The methods described herein can be used to treat a number of different types of wounds, and in particular, diabetic wounds. Non-limiting examples of diabetic wounds can include a livedoid vasculopathy, a diabetic ulcer, e.g., a peripheral arterial disease ulcer, a venous stasis ulcer, a chronic non-healing ulcer, a pressure ulcer, decubitus ulcers, chronic foot ulcers, etc. A wound subject to treatment according to the methods disclosed herein can also be a combination of one or more of the abovelisted wounds. A subject (e.g., a diabetic subject) in need of treatment or suspected to be in need of treatment according to the methods disclosed herein can have one or multiple wounds to be treated. Subjects include any mammal such as human beings, rats, mice, cats, dogs, goats, sheep, horses, monkeys, apes, rabbits, cattle, etc. The subject (e.g., mammal) can be in any stage of development including adults, and juveniles. In some embodiments, methods disclosed herein can treat an intractable wound. In some embodiments, methods disclosed herein can treat a skin disease.

[0085] In some embodiments, methods provided herein can accelerate the rate of tissue regeneration in a subject after administration of a composition disclosed compared to the rate of tissue regeneration in the subject before the administration of the composition. In some embodiments, methods provided herein can accelerate the rate of tissue regeneration in a subject after administration of a composition disclosed compared to the rate of tissue regeneration in a subject having the same disease severity' who has not been administered the composition. Atorney Docket No. 15670-0434WO1

[0086] EXAMPLES

[0087] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0088] Materials and Methods

[0089] The following materials and methods were used in the Examples below.

[0090] Cell culture: HEK 293T cells (catalog no. 632180, Takara Bio) were used for transient transfection and EV production. HaCaT cells were used for in vitro proliferation assay, cell-cycle assay, and phosphorylation assay. The HEK 293T and HaCaT cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; catalog no. 12430054, Gibco) supplemented with 10% (v / v) fetal bovine serum (FBS; catalog no. F0926, Sigma- Aldrich) and 1 x antibiotic-antimycotic (AA; catalog no. 15240062, Gibco) in humid air with 5% CO2 at 37°C. Cell counting was performed using a hemocytometer with 0.4% trypan blue (catalog no. T10282. Thermo Fisher). Cloning Construction of CDKl-EVs was based on the XPack CMV-Myr vector (System Biosciences [SBI]) that contained the EV signal peptide as an N-terminal fusion with a multiple cloning site (XPack CMV-Myr-MCS, catalog no. XPAK510PA-1, SBI). Primers designed by TAKARA tools (takarabio.com / leaming- centers / cloning / primer-design-and-other-tools) and amplified CDK1 genes from cDNA (catalog no. SC111605, Origene) encoding human CDK1 (NM_001786). PCR product was designed to contain Xho I (catalog no. R0146, New England Biolabs [NEB]) and Notl (catalog no. R0189S, NEB) restriction enzyme sites at the end of the insert gene. The following primers were used for CDK1-F (5'- GCAAAGATGCCTCGAGGATGGAAGATTATACCAAAATAGA-3' (SEQ ID NO: 21)) and CDK1-R (5'-AGAATTCTCGCGGCCGCCTACATCTTCTTAATCTGATTGT-3' (SEQ ID NO: 22)). After transformation into stable competent Escherichia coli (catalog no. C3040I, NEB), followed by ampicillin selection, recombinant plasmids were eluted (catalog no. D4203, Zymo Research).

[0091] Plasmid transfection: HEK 293T cells were seeded into 100-mm tissue culture-treated dishes, with the numbers of dishes decided by each experiment. Lipofectamine 2000 transfection reagent (catalog no. 11668019, Thermo Fisher) was used for transient transfection of HEK 293T cells to express CDK1 into EVs. The transient transfection was carried out according to the manufacturer’s protocol. Atorney Docket No. 15670-0434WO1

[0092] Briefly. 1 day before transfection, 5 * 106cells were seeded into each dish with 10 mL culture media. When cell confluence reached 80%, transfection reagent complexes were added. For preparing transfection reagent complexes, 15 pg plasmids (XPack CMV-Myr-MCS as mock, XPack CMV-Myr-CDKl and XPack CMV-Myr- GFP [catalog no. XPAK530CL-1, SBI]) in the Opi-MEM up to 750 pL (catalog no. 31985062, Gibco) was mixed with 75 pL Lipofectamine reagent in 675 pL Opi-MEM for each dish. Before adding transfection reagent complexes, a plasmids-transfection reagent mixture was incubated at room temperature for complex formation (10 min).

[0093] Animal model: All animal experiments were conducted with protocols approved by the Institutional Animal Care and Use Committee of the University of California, San Diego (UCSD). 12- to 16-week-old db / db mice (B6.VJS(D)-Leprdb / J mice; J AX# 000697, The Jackson Laboratory) were used which had a blood glucose level of >300 mg / dL and a body weight >45 g, the criteria for the diabetic obese model. The mice were maintained on a 12-h light / dark cycle. sEV preparation: EV studies herein addressed the methodological recommendations of the Minimal Information for Studies of Extracellular Vesicles 202351and reporting that are archived at EV -TRACK. EVs were produced by transient transfection. After 2 days of adding plasmid-transfection reagent complexes, the medium was changed to DMEM supplemented with 10% (v / v) exosome-depleted FBS (catalog no. EXO-FBS-250A-1, SBI) and l x AA. After 72 h, the conditioned medium (CM) was harvested and centrifuged at 10,000 x g for 10 min, 2 times. The supernatant was filtered through a syringe filter (PES membrane, 0.22 pm; catalog no. 25-244, GenClone). sEVs from the filtered CM were concentrated using Exoquick reagent (catalog no. EXOTC50A-1) following the manufacturer’s protocol. The ratio of Exoquick reagent to CM was 1:5 (v / v) in this study. After adding the Exoquick reagent to CM, mixtures were incubated overnight at 4°C and centrifuged at 1,500 x g for 30 min. The supernatant was gently aspirated and residues were centrifuged at 1,500 x g for 5 min and discarded. Precipitated sEVs were resuspended by PBS.

[0094] Immunoblotting: After harvesting CM, transfected cells and sEVs were lysis by radioimmunoprecipitation assay lysis buffer (catalog no. 89901, Thermo Fisher) supplemented with lx protease and phosphatase inhibitor cocktail (Halt protease and phosphatase single-use inhibitor cocktail [100x], catalog no. 78442, Thermo Fisher). The whole-cell lysates (WCLs) and sEVs subjected to immunoblotting were Atorney Docket No. 15670-0434WO1 normalized by protein quantification by bicinchoninic acid assay (catalog no. 23225, Thermo Fisher). Samples were prepared in lithium dodecyl sulfate sample buffer (catalog no. NP0008, Thermo Fisher) with 5 mM dithiothreitol (catalog no. 15508013, Thermo Fisher). 6 pg protein were loaded into 12% Bis-Tris Mini Gel (catalog no. NP0342BOX, Thermo Fisher) to separate proteins and transferred it to polyvinylidene fluoride membrane (0.45 pm, catalog no. LC2005, Thermo Fisher). 5% non-fat dry milk (catalog no. 9999, Cell Signaling Technology [CST]) was used in Tris-buffered saline with 0.05% Tween 20 (catalog no. 9997, CST) was used for blocking and primary antibodies (Alix, 1:2,000, catalog no. 92880, CST; Calnexin, 1 :2,000, catalog no. 2679, CST; CD63, 1:2,000, catalog no. PA5-92370, Thermo Fisher; CDK1, 1:2.000, catalog no. 9116, CST; CD81, 1 :2,000. catalog no. 56039, CST; p-CDKl [Tyrl5], 1:2,000, catalog no. 9111, CST; Akt, 1: 1,000, catalog no. 4691, CST; ^-Akt [Ser473], 1 :2,000, catalog no. 4060, CST; 4E-BP1, 1 : 1,000, catalog no. 9644, CST; p-4E-BPl [Thr37 / 46], 1 :2,000, catalog no. 2855, CST; Erk 1 / 2, 1 : 1,000, catalog no. 4695, CST; -Erk 1 / 2 [Thr202 / 204], 1 :2,000. catalog no. 4370, CST; P-actin, 1 :2,000, catalog no. 3700, CST) incubated overnight at 4°C. Secondary antibodies (1 : 10,000) (anti-rabbit immunoglobulin G [IgG], horseradish peroxidase [HRP]-linked, catalog no. 7074, CST, or anti-mouse IgG, HRP-linked, catalog no. 7076. CST) incubated for 45 min at room temperature with gentle agitation. Immunoblots were detected with HRP-conjugated secondary, incubated with enhanced chemiluminescent reagent (SignalFire Elite ECL reagent, catalog no. 12757, CST), and detected with an IVIS-Lumina Imager (PerkinElmer).

[0095] Single vFC. sEV concentration, size distribution, and identification of transmembrane and fluorescent proteins were measured by single vFC using lipophilic fluorescent dye, vFRed (vFC EV Analysis assay kit, catalog no. CBS4HP, Cellarcus Biosciences), using Cytoflex S flow cytometer (Beckman Coulter). The flow cytometer was calibrated for vesicle size using fluorescent intensity standard beads (vCal nanoRainbow Beads, catalog no. CBS6, Cellarcus Biosciences) and antibody capture beads (vCal nanoCai antibody capture beads, Cellarcus Biosciences) to calibrate flow data. Samples were subjected to a 1,000-fold dilution, stained with vFRed and phycoerythrin (PE)-conjugated antibody corresponding to a cocktail of anti-human monoclonal antibodies against human CD9, CD63, and CD81 (vTag antihuman tetraspanin antibody, catalog no. CBS5-PE, Cellarcus Biosciences), and Atorney Docket No. 15670-0434WO1

[0096] 120 pL measured on the flow cytometer at a flow rate of 60 pL / min for 2 min. Data were analyzed using FCS Express (Dotmatics / De Novo Software) and a standardized layout used to apply gating, compensation, and calibration (Cellarcus Biosciences).

[0097] In vivo wound-healing assay in diabetic mice models: For the wound-healing activity of CDK1 -loaded sEVs in the db / db mice model, hair was removed by shaving and topical treatment with depilatory cream of dorsal skin, a full-thickness 4-mm punch made (catalog no. P450, Acuderm). and the wound site splinted with a silicone ring (catalog no. GBLRD476687, Grace Bio-Labs) by 4-0 nylon suturing (catalog no. MV-662, Med Vet International). We treated 2.0 x io7CDK1 and empty7(mock) sEVs (in a volume of 50 pL PBS per wound) and covered them with 3M Tegaderm dressing film (catalog no. 1622w). The wound site was imaged with iPhone 12 Pro (ISO 125, 26 mm, Oev, Fl.6, l / 60s) and analyzed by ImageJ (1.54i version, NIH). Tissues were harvested for histology7analysis by fixation of skin wound samples in 4% paraformaldehyde into paraffin at the UCSD Tissue Technology7Shared Resource (TTSR) that prepared slides stained with hematoxylin and eosin (H&E). Immunohistochemical (IHC) staining to localize Ki67 (1:50, catalog no. 16667, GeneTex) was performed with an Intellipath Automated IHC Stainer (Biocare) by the TTSR. H&E and IHC images were analyzed using Aperio ImageScope version 12.4.6.5003 software (Leica Biosystems).

[0098] Immunofluorescence: For detecting CDK1 after CDKl-loaded sEVs treatment, immunofluorescence (IF) was imaged as in the following procedure: 1.0 x io5HaCaT cells were seeded onto a 12-well plate (black frame 12-well plate with glass-like polymer bottom, catalog no. P12-1.5P, Cellvis) and incubated in a humidified 5% CO2 incubator at 37°C for 24 h. To synchronize the cell cycle, the cells were starved with culture medium containing 1% FBS and 1 x AA for 24 h. We loaded 2.0 x 105- 108CDKl-loaded sEVs and 2.0 x io7empty (mock) sEVs (in a volume of 100 pL PBS per well) onto each well for 6 h. PBS was treated as a negative control. After EV exposure, EV -treated HaCaT cells were washed twice with cold PBS and fixed by 4% paraformaldehyde for 10 min at 37°C. The cells were permeabilized by 0.15% Triton X-100 for 15 min at room temperature. Non-specific antibody binding was blocked by 2.5% BSA for 1 h at room temperature. Primary antibodies (CDK1, 1 :400, catalog no. 9116, CST, for in vitro study: catalog no. 19532-1-AP. Proteintech, for in vivo study; Ki67-FITC. 1:50. catalog no. 130-130- Atorney Docket No. 15670-0434WO1

[0099] 859, Miltenyi Biotec; vimentin, 1: 100, catalog no. 5741, CST; Keratinl4, 1:200, catalog no. 10143-1-AP, Thermo-Fisher) were incubated overnight at 4°C. Secondary antibodies (4 pg / mL) (anti-mouse IgG, Alexa Fluor 546-linked, catalog no. Al 1030, Thermo Fisher) were incubated for 45 min at room temperature. Nucleus was stained with 1 pg / rnL DAPI for 5 min at room temperature. IFs were imaged with confocal laser scanning microscopy (Model AXR, Nikon). IF images were analyzed by Imaged version 1.54i software.

[0100] Cell proliferation assays: To assess the proliferative effects of CDK1 -loaded sEVs on HaCaT cells, a cell proliferation assay was carried out using Cell Counting Kit-8 (CCK-8, catalog no. CK04, Dojindo). We seeded 1 x 103HaCaT cells into a 96- well plate and incubated in a humidified 5% CO2 incubator at 37°C for 24 h. We treated 2.0 x 106CDK1 and empty (mock) sEVs in each well for 24 h. The cells treated with PBS were used as a control group. After 24 h, a CCK-8 solution containing the water-soluble tetrazolium salt was added to each well, and the plate was incubated for 2 h. Absorbance was measured using a microplate reader at 450 nm. Relative proliferation was calculated as a percentage to the PBS control group.

[0101] Migration assays: We seeded 5 x 103HaCaT cells using a 2-well silicone insert (catalog no. 80209, Ibidi) in a 24-well plate and incubated in a humidified 5% CO2 incubator at 37°C for 24 h. To inhibit cell proliferation, 10 pg / mL mitomycin C (catalog no. M0440, Sigma-Aldrich) was treated to HaCaT cells for 2 h and the media replaced with sEVs (2.0 x 107CDK1- and empty [mock])-containing media for 24 h. The gap between 2-well was imaged over 24 h using a CCD camera (Retiga R6, Teledyne Photometries) on a microscope (1X70. Olympus) to measure cell migration. All images were analyzed using ZEN blue version 3.4.91.00000 (Carl Zeiss Microscopy GmbH).

[0102] Cell-cycle assays: To determine the activity of CDK1 -loaded sEVs in the cell cycle, a cell-cycle assay was carried out using membrane-permeable DNA staining solution (cell-cycle assay solution deep red, catalog no. C548, Dojindo). We seeded 3.0 x io5HaCaT cells into a 6-well plate and incubated the cells in a humidified 5% CO2 incubator at 37°C for 24 h. To synchronize the cell cycle, the cells were starved with culture medium containing 1% FBS and 1 x AA for 24 h. We treated into each well 2.0 x io7CDK1 and empty (mock) sEVs (in a volume of 100 pL PBS per well) Atorney Docket No. 15670-0434WO1 for 3 h with fresh culture medium containing 10% FBS and 1 x AA. PBS was treated as a negative control. After EV exposure, EV -treated HaCaT cells were washed with cold PBS two times and suspended. Cell suspension was washed by PBS twice and resuspended in 500 pL PBS containing 5 pL cell-cycle assay solution deep red. DNA staining was carried out for 15 min at 37°C and protected from light. The stained cells were analyzed by FC (MACSQuant Analyzer 10, Miltenyi-Biotec). The data were analyzed by FlowJo software version 10.8.2 (BD Biosciences).

[0103] In vitro tracking EV uptake: Intracellular EV tracking was carried out by GFP-loaded sEVs. We treated 2.0 * 107GFP-loaded sEVs (in a volume of 100 pL PBS per well) to 1.0 x io5HaCaT cells for 3, 6, 12, and 24 h. After EVs exposure, EVs were discarded and washed by cold PBS twice. To determine a co-localization of GFP-tagged EVs with lysosomes, lysosomes were stained with LysoTracker Red DND-99 (catalog no. L7528, Thermo Fisher) for 30 min at 37°C. The cells were fixed by 4% paraformaldehyde for 10 min at 37°C. IFs were imaged with confocal laser scanning microscopy (Model AXR, Nikon). IF images were analyzed by ImageJ version 1.54i software.

[0104] Signaling pathway assays: We seeded 1.0 x 105HaCaT cells into 12- ell plate (black frame 12-well plate with glass-like polymer bottom, catalog no. P12- 1.5P, Cellvis) and incubated them in a humidified 5% CO2 incubator at 37°C for 24 h. To synchronize the cell cycle, the cells were starved with culture medium containing 1 % FBS and 1 x AA for 24 h. We treated into each cell 2.0 x 107CDK1 and empty (mock) sEVs (in a volume of 100 pL PBS per well) for 1 h (immunoblotting) and 6 h (IF). PBS w as treated as a negative control. After EV exposure, the phosphorylations of Akt, 4E-BP1, and Erk w ere assessed by immunoblotting and IF. For IF, sEV- treated HaCaT cells were washed with cold PBS twice and fixed by 4% paraformaldehyde for 10 min at 37°C. The cells were permeabilized by 0.15% Triton X-100 for 15 min at room temperature. Non-specific antibody binding was blocked by 2.5% BSA for 1 h at room temperature. Primary antibodies (p-Akt [Ser473], 1 :400, catalog no. 4060. CST; p-4E-BPl [Thr37 / 46], 1:400, catalog no. 2855, CST; p-Erk 1 / 2 [Thr202 / 204], 1:400, catalog no. 4370, CST; p27K1?1, 1 :800, catalog no. 3686, CST; pHistone H3 [SerlO], 1:200, catalog no. 9706, CST; acetyl-a-tubulin, 1:800, catalog no. 5335. CST) were incubated overnight at 4°C. Secondary antibodies (4 pg / mL) (anti-rabbit IgG, Alexa Fluor 488 linked, catalog no. Al 1008, Thermo Atorney Docket No. 15670-0434WO1

[0105] Fisher; anti-rabbit IgG. Alexa Fluor 546 linked, catalog no. Al 1010, Thermo Fisher; anti-mouse IgG, Alexa Fluor 488 linked, catalog no. Al 1029. Thermo Fisher; antimouse IgG, Alexa Fluor 546 linked, catalog no. Al 1030, Thermo Fisher) were incubated for 45 min at room temperature. Nucleus was stained by 1 pg / mL DAPI for 5 min at room temperature. IFs were imaged with confocal laser scanning microscopy (Model AXR, Nikon). IF images were analyzed by ImageJ version 1.54i.

[0106] Statistical analysis: All statistical analyses were performed with GraphPad Prism 10.0 (GraphPad Software). Data were expressed as the mean (standard deviation [SD]). Differences between different groups were compared by one-way ANOVA and two-way ANOVA with multiple comparisons with statistically significant p values indicated as *p < 0.05; **p < 0.005; *** ? < 0.001; **** / ) < o 0001. All statistical analyses and representative images presented and observed in at least three independent experiments.

[0107] Example 1: Engineering and validation of CDKl-loaded sEVs

[0108] To generate CDKl-loaded sEVs, we generated a fusion protein of CDK1 with an N-terminal myristoylation sequence (myr-CDKl), which we demonstrated efficiently loads sEVs with protein cargoes (FIG. 1A). sEVs were purified from the conditioned media of cells transfected with myr-CDKl or control. Purified EVs were characterized51via vesicle flow cytometry (vFC) (FIG. 7), demonstrating similar sEV concentrations (FIG. IB), sizes (FIG. 1C), and surface expression of the canonical sEV tetraspanin proteins CD9. CD63, and CD81 (FIG. ID) in both myr-CDKl and mock-transfected cell sEVs. Immunoblotting showed an increased expression of CDK1 in CDKl-loaded sEVs compared to mock sEVs, while similar levels of Alix, CD63, and CD81 were observed on both populations of sEVs, and an absence of the endoplasmic reticulum protein Calnexin (FIG. IE). Quantification of CDK1 protein was normalized to Alix (FIG. IF) to establish engineered CDKl-loaded sEVs for further activity testing. As CDK1 is active when inhibitory residues such as Tyrl5 appear hypo-phosphorylated, we confirmed herein the phosphory lation status of CDK1 loaded into sEVs by immunoblot. We observed that overexpression of CDK1 and loading into sEVs did not affect CDK1 phosphorylation, and the amount of pCDKlTyr15in sEVs was similar to that of the wild-type and mock sEVs (FIG. 1G). Atorney Docket No. 15670-0434WO1

[0109] Example 2: Testing the activity of CDKl-loaded sEVs in an animal model of impaired wound healing

[0110] To determine the wound-healing activity of CDKl-loaded sEVs in vivo, we used 12- to 16-week-old leptin receptor knockout mice, an established and well- defined model of impaired wound healing that is the result of delayed epithelialization related to hyperglycemia and obesity', and EVs loaded with human CDK1, which showed >97% homology7to mouse CDK1. To examine the effect of CDK1 in the diabetic wound model immediately after wound formation, purified sEVs were added as a single topical treatment to a splinted full-thickness excisional wound on the dorsum of the mouse, and the wound diameter imaged over a 9-day time course (FIG. 2A). We observed that wounds treated with CDKl-loaded sEVs demonstrated accelerated wound closure kinetics compared to wounds treated with mock sEVs (FIGS. 2B and 2C). Wound treatment with either CDKl-loaded sEVs or mock sEVs increased wound closure compared to PBS treatment. At days 3 and 5, we observed a statistically significant improvement in the pro-reparative activity of CDKl-loaded sEVs vs. mock sEVs. The extent of the wound-healing response was similar between CDK1 and mock sEVs by day 9, suggesting that CDK1 acted in the early phase of tissue repair.

[0111] Immunofluorescent imaging and immunoblotting analysis indicated CDK1 remained significantly elevated for up to 3 days after delivery' by sEV, and declined by day 5 after delivery7(FIGS. 8A-8C). To test the pro-reparative activity of multiple doses of CDK1 sEVs, we administered CDK1 sEVs on days 0 and 3 (double-treated group; CDKl-loaded sEVs vs. mock sEVs). The double-treated CDKl-loaded sEV double-treated mice showed a significant improvement in wound closure in comparison to mice treated with a single dose of CDKl-loaded sEV or mice treated with either one or two doses of mock EVs.

[0112] Analysis of tissue histology7showed increased epithelial thickness and extension of migratory epithelium (epithelial tongue) (FIGS. 2D and 2E), which was associated with increased cell proliferation determined by7Ki67 immunostaining (FIGS. 2F and 2G). These results supported a model that delivery7of CDKl-loaded sEVs enhanced tissue repair and promoted re-epithelialization via increased cell proliferation in vivo. Atorney Docket No. 15670-0434WO1

[0113] Example 3: Testing biochemical activity of CDK1 delivered by sEVs to human keratinocyte

[0114] To test the biochemical activity of CDKl-loaded sEVs on a human keratinocyte cell line (HaCaT), we immunostained sEV -treated cells with an anti- CDK1 antibody to determine the intracellular distribution. We observed that CDK1 protein pay loads were present in the cytosol of cells treated with CDKl-sEVs (FIG. 3A) and distinct from the lysosomal distribution that is often associated with sEV uptake in recipient cells (FIG. 9A). This was consistent with the clathrin- mediated endocytosis and micropinocytosis being the primary mechanism of EV uptake. To further investigate protein uptake and release in recipient cells, we generated EVs that were engineered to express green fluorescent protein (GFP) using the same myristoylation tag approach as the engineering of CDKl-loaded sEVs, and we observed the accumulation of GFP-loaded sEVs over a time course of 6-24 h, with increased uptake by 24 h (FIG. 9B). By quantifying the association of GFP- loaded sEVs with LysoTracker, a lysosome marker, we demonstrated that GFP-loaded sEVs escape into the cytosol within 6 h (FIG. 9C). The release of the engineered sEVs into the cytosol was further investigated by serial dilution showing an optimal dosing of I O7108sEVs / 100 LIL to 105cells (FIGS. 10A-10C). These data demonstrated that GFP-loaded sEVs were taken up by recipient cells, undergo endosomal escape, and had the potential to exert biochemical activity in the cytosol.

[0115] To determine the mechanism for the improvement in wound healing, we assessed the effects of CDKl-loaded sEVs on cell proliferation and cell migration. Cell proliferation was assessed by the increase in the number of cells 24 h after sEV treatment relative to the increase in the number of cells 24 h after treatment with PBS. We observed a 25.6% increase following treatment with CDK1 sEVs compared to mock sEVs (FIGS. 3B and 3C). To determine whether the CDKl-loaded sEVs might regulate migration in addition to proliferation to enhance wound healing, we used a modified scratch assay in which the migration of HaCaT keratinocytes was monitored in the presence or absence of mitomycin C (FIG. 11A). We observed that in the presence of mitomycin C, the treatment of recipient cells with CDKl-loaded sEVs increased migration, even in the absence of proliferation, with accelerated scratch closure (FIGS. 3C and 3D). In the absence of mitomycin C, we observed even more Atorney Docket No. 15670-0434WO1 rapid gap closure in this model (FIGS. 11B and 11C). To determine whether the treatment of cells with CDK1 -loaded sEVs affected the cell cycle, we stained cells to measure DNA content (FIG. 3E) and observed a consistent CDKl-mediated entry into G2 / M phase based on the increase in DNA content (FIG. 3F). These findings supported a model that delivery7of CDKl-loaded sEVs promoted entry in the cell cycle that was associated with increased keratinocyte migration and proliferation.

[0116] Example 4: Signaling pathway by exosomal CDK1 in cytoplasm

[0117] We next evaluated the effect of CDKl-loaded sEV treatment on known downstream molecular endpoints of CDK1. 4E-BP1 phosphorylation is associated with the phosphatidylinositol 3-kinase (PI3K) / AKT signaling pathway, which integrates both intracellular and extracellular signals to regulate cell metabolism, growth, and proliferation. Because CDK1 regulates 4E-BPlThr202 / Try204through the activation of AKT. we focused on CDKl-mediated phosphorylation changes in AKTSer473and ERKThr37 / 46as key downstream mediators. We observed that treatment of cells with CDKl-loaded sEVs increased the phosphory lation of AKTSer473(2.0- fold; FIGS. 4A and 4B), ERKThr202 / T’,r204(7.4-fold; FIGS. 4C and 4D), and 4E- gp i Thr37 / 46 (g o-fold; FIGS. 4E and 4F) relative to mock EV-treated cells. In addition, activation of AKT and ERK signaling suppresses the p27Kiplthrough the various downstream pathways. After CDKl-loaded sEV treatment, we also identified suppressed p27Kiplexpression by AKT and ERK phosphorylation promoting the cell cycle (FIGS. 12A and 12B). We then examined the effect of CDKl-loaded sEV treatment on changes in the phosphorylation of AKTSe1473and RK11"2'12 1'1204and observed increased phosphorylation in CDKl-loaded sEV -treated groups consistent with the above immunofluorescent studies. A similar increase in 4E- Bp i Thr37 / 46 phosphorylation was also observed (FIGS. 4G-4I), supporting a model that CDKl-loaded sEVs stimulate the phosphorylation of several CDK1 pathway molecular endpoints, and led us to consider the potential effects of CDK1 delivery on chromatin structure.

[0118] Example 5: Delivery of CDKl-loaded sEVs promoted histone phosphorylation

[0119] To determine whether delivery7of CDKl-loaded sEVs affects chromatin remodeling, we focused on monitoring changes in the phosphorylation of histone H3 Atorney Docket No. 15670-0434WO1

[0120] (pHistone H3Ser10), based on it being a target of CDK1 activation through the ERK pathway. Cells were synchronized by serum starvation and sEV -treated cells immunostained with an antibody to pHistone H3Ser10. We observed a 2.8-fold increased pHistone H3Serl° signal in CDK1 EV -treated cells relative to mock EV- treated cells (FIGS. 5A and 5B). To determine whether delivery of CDK1 -loaded sEVs affected progression into specific stages of mitosis, we quantified changes in chromatin structure by enumerating sEV -treated cells in phases of the cell cycle (FIG. SC). We observed that treatment with CDK1 -loaded sEVs increased the overall number of cells entering anaphase, whereas PBS-treated and mock sEV -treated cells remained mainly in metaphase and prophase. The findings showed that CDK1 -loaded sEVs can deliver biochemically active protein pay loads that accelerated wound healing and increased cell proliferation, migration, and cell signaling, which was highlighted by an increased phosphorylation of histone H3 that promoted entry7in anaphase (FIG. 6).

[0121] Example 6: Reactive oxygen species (ROS) sensitive releasable payload of EVs

[0122] Reactive oxygen species (ROS) trigger degradation of oligo-proline. As such, an oligo-proline linker (ROSL) was introduced to a myristylation tag that sorted the payload into extracellular vesicles (EVs) involving ROS sensitive releasable payload of EVs.

[0123] To compare the release and action of Cre recombinase by ROSL in a ROS- rich environment, normal EV, Cre-EV, and ROSL-Cre-EV were tested in ROS- induced LSL-tdTomato transgenic mice. The LSL-tdTomato reporter system of the transgenic mice which was controlled by Cre recombinase, is provided in FIG. 13. In brief, bleomycin was administered on day 1 (DI) by intratracheal administration (i.t.) to induce ROS in the lungs, and EVs were intravenously (i.v) injected into the mice on day 3 (D3) according to the schedule as shown in FIG. 14. Organs were then recovered from the mice on day 10 (DIO) and subjected to immunoblotting compare the enhanced tdTomato expression levels by Cre recombinase release. tdTomato expression in the liver was also compared to organs where ROS was not induced. Comparison of tdTomato expression levels with housekeeping protein (GAPDH) expression showed that mice injected with RCre (ROSL-Cre) EVs showed the highest tdTomato expression level (0.34) in the lungs rich in ROS (FIG. 15). Alpha-smooth Atorney Docket No. 15670-0434WO1 muscle actin (aSMA) expression blot supported the extent of ROS induction (FIG. 15).

[0124] In conclusion, these data demonstrated the development of EVs that were capable of environment-specific release (e.g., in a high ROS environment) of injected payloads by introducing an oligo-proline linker.

[0125] Example 7: Formulation of EVs for applications

[0126] Each route of administration for the EVs disclosed herein can have different local or systemic EV retention times. For example, topical administration to the skin resulted in detectable levels of active protein (CDK1) for approximately four days. However, intrapulmonary injection via the intravenous route significantly reduced active protein levels after two days. Furthermore, systemic intravenous injection resulted in decreased active protein levels in the blood within hours, necessitating formulation changes for each injection.

[0127] Hydrogel -based delivery of extracellular vesicles (EVs) offers sustained release at the target site, making it advantageous for various therapeutic applications. Compared to simple saline-based administration, hydrogels significantly enhanced EV retention, local bioavailability, and functional stability-. Cross-linked hydrogel systems can be engineered to respond to environmental cues such as pH or enzymatic activity7, providing controlled and prolonged release profiles. While saline formulations are easy to prepare and may be suitable for acute or systemic delivery, they are rapidly cleared from tissues. In contrast, hydrogel platforms are particularly well-suited for localized and long-term applications such as chronic wound healing, tissue regeneration, and site-specific drug delivery7. In Table 3 below, the observed features of delivering EVs in saline are compared to those observed when delivering the EVs in a hydrogel-based delivery7system.

[0128] TABLE 3: Comparison of EVs Formulations Atorney Docket No. 15670-0434WO1

[0129] References

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[0214] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Atorney Docket No. 15670-0434WO1WHAT IS CLAIMED IS:1 . An engineered extracellular vesicle (EV) comprising: an extracellular vesicle isolated from a biological cell; and a cyclin-dependent kinase (CDK) protein.

2. The engineered EV of claim 1, wherein the EV is engineered to express the CDK protein within the EV.

3. The engineered EV of claim 1 or 2, wherein the EV further comprises a membrane anchor.

4. The engineered EV of claim 1 or 2, wherein the CDK protein is fused to the EV.

5. The engineered EV of claim 1 or 2, wherein the CDK protein is a releasable payload.

6. The engineered EV of any one of claims 1-5, wherein the CDK protein is CDK1.

7. The engineered EV of any one of claims 1-6, wherein the biological cell is a primary mesenchymal stem cell, an embryonic kidney cell, an embryonic fibroblast cell, an alveolar basal epithelial cell, or a monocytic cell or an immortalized cell-line thereof.

8. The engineered EV of claim 7, wherein the biological cell is an embryonic kidney cell.

9. A pharmaceutical composition comprising: any one of the engineered EVs of claims 1-8 and a pharmaceutically acceptable carrier.Attorney Docket No. 15670-0434WO110. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is formulated for local delivery or systemic delivery.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is formulated for topical administration.

12. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is formulated for intravenous delivery.

13. The pharmaceutical composition of any one of claims 9-12, wherein the pharmaceutical composition further comprises a hydrogel.

14. A method of treating a disease in a subject in need thereof, the method comprising: administering to the subject any one of the engineered EVs of claims 1-8 or the pharmaceutical compositions of claims 9-13, thereby treating the disease in the subject.

15. The method of claim 14, wherein the disease is an intractable wound.

16. The method of claim 14, wherein the disease is a skin disease.

17. The method of claim 14, wherein the disease comprises a diabetic wound.

18. The method of any one of claims 14-17, wherein the administering comprises topical administration.

19. The method of any one of claims 14-17, wherein the administering comprises intravenous administration.

20. A method of accelerating tissue regeneration in a subject in need thereof, the method comprising:Atorney Docket No. 15670-0434WO1 administering to the subject any one of the engineered EVs of claims 1-8 or the pharmaceutical compositions of claims 9-13, thereby accelerating tissue regeneration in the subject.

21. The method of claim 20, wherein the disease is an intractable wound.

22. The method of claim 20, wherein the disease is a skin disease.

23. The method of claim 20, wherein the disease comprises a diabetic wound.

24. The method of any one of claims 20-23, wherein the administering comprises topical administration.

25. The method of any one of claims 20-23, wherein the administering comprises intravenous administration.

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