WO2025166262A1 - Auto-loaded vesicular cancer therapy platform derived from induced neural stem cells - Google Patents

Abstract

An extracellular vesicle based therapeutic compound for treating tumors and cancers. The extracellular vesicle based therapeutics use exosomes that are transdifferentiation-derived induced neural stem cells (Exo-iNSCs) that produce an extracellular vesicle having a tumoricidal gene product or an anti-cancer molecule withing the extracellular vesicle. The therapeutic agent is present in a lumen of the extracellular vesicle or is distributed across a membrane surface of the extracellular vesicle. Methods of using the extracellular vesicle based therapeutic compounds are effective in treating tumors and cancers.

Description

AUTO-LOADED VESICULAR CANCER THERAPY PLATFORM DERIVED FROM INDUCED NEURAL STEM CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application Serial No. 63/549,191, filed February' 2, 2024, the disclosure of which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant Numbers NS099368 and TR003715 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present disclosure relates to auto-loaded vesicular cancer therapy platform derived from induced neural stem cells. The present disclosure relates to methods of using the vesicular cancer therapy platform to treat cancers and tumors, including brain cancer.

BACKGROUND

[0004] Malignant brain tumors present diverse and significant therapeutic hurdles, and there is a desperate need for new. creative, approaches to treatment. Standard resection and chemo-radiation treatment lack the ability to eradicate diffuse malignant cells; therefore, recurrence of some brain tumors is almost unavoidable. Engineered tumoricidal neural stem cells (NSCs) have broken new ground as potential therapeutics, demonstrating promise as an alternative cancer treatment approach via targeted migration to brain tumor cells and delivery of a broad selection of therapeutic agents [4], [5], [6], Despite the success of these NSCs, many concerns, ranging from complex manufacturing, enormous production costs, and potentially harmful side effects, remain major limitations of the clinical benefits of NSC therapy [7], [12], [13], [0005] There remains a need for new and improved therapies and therapeutic compounds for treating cancers, including brain tumors, using NSCs and related technologies.

BRIEF SUMMARY

[0006] This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely an example of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise for purposes of example. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary’ or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.

[0007] Provided in one aspect is a therapeutic compound, the therapeutic compound comprising an extracellular vesicle (EV), the EV includes an exosome derived from transdifferentiation (TD)-derived induced neural stem cells (Exo- iNSCs), a therapeutic agent, the therapeutic agent includes a tumoricidal gene product and/or an anti-cancer molecule, where the therapeutic agent is present in a lumen of the EV and/or is distributed across a membrane and/or surface of the EV. The therapeutic compound may also include where the induced neural stem cells are derived from skin fibroblasts. The therapeutic compound may also include where the tumoricidal gene product and/or anti-cancer molecule includes TNF-related apoptosis-inducing ligand (TRAIL). The therapeutic compound may also include where the tumoricidal gene product and/or anti-cancer molecule includes Herpes simplex virus thymidine kinase (TK).

[0008] The therapeutic compound may also include where the tumoricidal gene product and/or anti-cancer molecule includes both TRAIL and TK. The therapeutic compound may also include where the EV includes a concentration of TRAIL present in the lumen, and/or distributed across the membrane and/or surface of the EV at about 100 pg/ug EV to about 500 pg/ug EV, optionally about 250 pg/ug EV to about 400 pg/ug EV, optionally about 100 pg/ug EV to about 250 pg/ug EV, optionally about 250 pg/ug EV to about 500 pg/ug EV, optionally about 300 pg/ug EV to about 350 pg/ug EV.

[0009] The therapeutic compound may also include where the EV has a tumorhoming capacity causing a selective migration to and/or attraction to tumors and/or cancer cells imparted by the induced neural stem cells (iNSCs). The therapeutic compound may also include where the tumoricidal gene product and/or an anti-cancer molecule of the EV has increased tumor-selective accumulation and increased therapeutic activity as compared to free TRAIL and/or free TK. In some embodiments, the increased tumor-selective accumulation of the tumoricidal gene product and/or an anti-cancer molecule is an increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% as compared to free TRAIL and/or free TK, i.e. TRAIL or TK not part of and EV delivery mechanism. In some embodiments, the increased therapeutic activity of the tumoricidal gene product and/or an anti-cancer molecule is an increase of about 5%, about 10%, about 15%, about 20%, about

25%, about 30%, about 35%, about 40%, about 45%. about 50%, about 55%, about 60%, about 65%. about 70%. about 75%, about 80%, about 85%, about

90%, about 95%, or about 100% as compared to free TRAIL and/or free TK, i.e. TRAIL or TK not part of and EV delivery⁷ mechanism. The therapeutic compound may also include where the exosome of the EV generated by TD-iNSC is produced with the therapeutic agent present in a lumen of the EV and/or distributed across the membrane and/or surface of the EV. The therapeutic compound may also include where about 75% or more of the therapeutic agent is distributed across the membrane of the EV, optionally about 50%, about 60%, about 70%. about 80%, about 90%. about 95%, about 99%. or more.

[0010] The therapeutic compound may also include where the therapeutic compound is configured to treat a cancer or tumor, optionally a brain cancer or brain tumor, optionally a glioblastoma. The therapeutic compound may also include where the EV further includes other therapeutic proteins and/or additional tumor-targeting motifs. [0011] Provided in some aspects are therapeutic compositions comprising the therapeutic compound, and an excipient, carrier or buffer, which is configured to be administered to a subject.

[0012] Provided are methods of producing the therapeutic compound, including genetically engineering iNSCs to generate one or more extracellular vesicles (EVs) comprising exosomes loaded with the therapeutic agent, where the therapeutic agent includes a tumoricidal gene product and/or an anti-cancer molecule, where the therapeutic agent is present in a lumen of the EVs and/or is distributed across a membrane and/or surface of the EVs. The method may also include where genetically engineering iNSCs to generate one or more EVs includes transdifferentiating fibroblstats to produce iNSCs followed by transducing iNSCs to produce the tumoricidal gene product and/or an anti-cancer molecule as part of the EVs. The method may also include where the EVs are isolated and formulated into a therapeutic composition. The method may also include where the therapeutic agent is loaded in the one or more EVs at about twice the rate as parent fibroblasts of the one or more EVs. The method may also include comprising generating personalized EVs from iNSCs transdifferentiated from an subject patient's own skin.

[0013] Provided are methods of treating a subject, the methods comprising administering to a subject in need a therapeutic dose of the compounds or compositions. The methods may also include where the subject is a human subject suffering from a cancer, tumor or related condition. The method may also include where the cancer is a brain cancer or brain tumor, optionally a glioblastoma. These and other objects are achieved in whole or in part by the presently disclosed subject matter. Other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, examples, and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The presently disclosed subject matter can be better understood by referring to the following, example figure. The components in the figure are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the presently disclosed subject matter (often schematically). In the figure, like reference numerals designate corresponding parts throughout the different views. A further understanding of the presently disclosed subject matter can be obtained by reference to an embodiment set forth in the illustrations of the accompanying drawing. Although the illustrated embodiment is merely for purposes of example of systems for carrying out the presently disclosed subject matter, both the organization and method of operation of the presently disclosed subject matter, in general, together with further objectives and advantages thereof, may be more easily understood by reference to the drawings and the following description. The drawing is not intended to limit the scope of this presently disclosed subject matter, which is set forth with particularity in the claims as appended or as subsequently amended, but merely to clarify and provide examples of the presently disclosed subject matter.

[0015] FIG. 1 Scheme demonstrating the generation and isolation of Exo-iNSC- TRAIL from iNSCs and the selective cytotoxicity of Exo-iNSC-TRAIL to tumor cells.

[0016] FIG. 2 Isolation, characterization and TRAIL measurement of Exo- iNSC-TRAIL. A, Co-localization of TRAIL and exosomes inside iNSCs. Upper, representative confocal microscopy images of TRAIL-exosome colocalization in a single cell. Lower, representative confocal microscopy images of TRAIL- exosome colocalization in iNSC spheres. Exosomes were stained by CD63 (Red, upper) and TSG101 (Red, lower), TRAIL was stained by TRAIL antibody (green), and nuclei of iNSC spheres were labelled with DAPI (blue). Scale bar represents 10 pm (upper) and 25 pm (lower); B, negative contrast micrograph of Exo-iNSC- TRAIL examined and imaged by transmission electron microscopy (TEM), scale bar 500 nm; C. NTA characterizes the size distribution of Exo-iNSC-TRAIL; D, the measurement of TRAIL expressed on intact Exo-iNSC-TRAIL, intact Exo- NHF1 -TRAIL, Exo-iNSC-TRAIL lysate, and Exo-NHFl -TRAIL lysate by ELISA, analyzed by one-way ANOVA (**P < 0.01); E, flow⁷ cytometry detected the percentage of TRAIL expression on the iNSC Exo-TRAIL surface; F-G, TRAIL expression on Exo-iNSC-TRAIL confirmed by dual-labelling of exosomes for TRAIL and CD63 and quantification/imaging via flow cytometry; H, representative immuno-electron-microscopy images of Exo-iNSC-TRAIL stained with TRAIL primary antibody and 6-nm nanogold secondary antibody ; I, the percentage of the total TRAIL secreted by iNSC-TRAIL cells that is associated with Exo-iNSC-TRAIL (n=6 replicates). All data are shown as mean ± SEM.

[0017] FIG. 3 Toxicity and stability' of TRAIL is higher after associated with surface of Exo-iNSC. A, The scheme to demonstrate post-loading of free TRAIL onto Exo-iNSC; B, Free TRAIL associates with Exo-iNSC after co-incubation and washing (measured by ELISA), analyzed by t-test (**P <0.01); C, postloaded Exo-iNSC-TRAIL displays enhanced cytotoxicity compared to free TRAIL, analyzed by two-way ANOVA (** <0.01 and ***p < 0.001); D, the representative BLI images of U87 viability after treatment with 25 ng/mL TRAIL for 48h, analyzed two-way ANOVA (n = 4-5, ****p < 0.0001);E and F, the thermal stability of the TRAIL in Exo-iNSC-TRAIL after incubation at 4 °C and 37 °C for different time intervals, analyzed by two-way ANOVA (n = 3, ****P < 0.0001). All data are shown as mean ± SEM.

[0018] FIG. 4 The cytotoxicity and apoptotic activity' of Exo-iNSC-TRAIL on brain tumor cell lines. A, the cytotoxicity of Exo-iNSC-TRAIL and free TRAIL on MB231Br, GBM8, and U87 after treatment with increasing concentrations of TRAIL, t = 72h, analyzed by two-way ANOVA (****p< 0.0001); B, the fold change of caspase-3/7, caspase-8, and caspase-9 in MB231Br, GBM8, and U87 after treatment with 5 ng/ml of free TRAIL or Exo-iNSC-TRAIL, t = 24h, analyzed by one-way ANOVA ( *P < 0.05, ** < 0.01, ***/’ < 0.001. and ****/>< 0.0001). All data are shown as mean ± SEM.

[0019] FIG. 5 Exo-iNSC-TRAIL accumulates in tumor cells in an organotypic brain slice culture (OBSC) ex vivo model. A, representative confocal images of fluorescently labelled Exo-NHFl -TRAIL and Exo-iNSC-TRAIL in tumorbearing OBSCs with MB231Br cells (red) engrafted atop the OBSCs. Exo-NHFl- TRAIL and Exo-iNSC-TRAIL were labelled by PKH26 (green), MB231Br were transfected with mCherry (red) and nuclei were stained by DAPI (blue), t = 24h, scale bar = 50 pM. B, quantification of Exo-NHFl -TRAIL and Exo-iNSC- TRAIL accumulation on OBSCs with or without tumor foci. Each bar represents the sum of 19 z-stacked images; C, quantification of exosome accumulation along a z-stack of 19 images which shows that Exo-iNSC-TRAIL accumulates more specifically in MB231Br tumor cells than Exo-NHFl -TRAIL cells, t = 24h. [0020] FIG. 6 Ex vivo and orthotopic in vivo tumor killing capabilities of Exo- iNSC-TRAIL. A, representative BLI images of orthotopic MB23 IBr brain tumorbearing mice at different time points after treatment with PBS (negative control), free TRAIL, or Exo-iNSC -TRAIL. At later time points, BLI shows some mice develop spinal tumors; B, tumor fold changes of in vivo MB231Br tumor growth at multiple time points after treatment by PBS, Free TRAL(C) and Exo-iNSC- TRAIL respectively, n = 6-7 mice per group, analyzed by two-way repeated measures ANOVA (*P < 0.05 and **P < 0.01); C, BLI quantification of individual tumor volumes normalized to tumor size at time of treatment; D, Kaplan-Meier survival curve for experiment described in (A)-(C), analyzed by Log-rank (Mantel-Cox) test (**/>< 0.01). All data are shown as mean ± SEM.

[0021] FIG. 7 Measuring Exo-iNSC -TRAIL efficacy in two orthotopic in vivo models of glioblastoma. A, schematic of study design for GBM8 tumor treatment; B. Kaplan-Meier survival curve for mice with a brain intracerebral U87 tumor treated by PBS, Free TRAL(C) and Exo-iNSC-TRAIL respectively, n=6-7 mice per group, analyzed by Log-rank (Mantel-Cox) test (*P < 0.05);C, tumor fold changes of individual tumor volumes normalized to tumor size at day 1 after tumor implantation and treated by PBS, Free TRAL(C) and Exo-iNSC-TRAIL respectively; D, schematic of study design for GBM8 tumor treatments. Kaplan- Meier survival curve for mice with a brain intracerebral GBM8 tumor treated by PBS, Free TRAL(C) and Exo-iNSC-TRAIL respectively, n=7 mice per group, analyzed by Logrank test for trend (**P < 0.01); F, tumor fold changes of individual tumor volumes normalized to tumor size at the time of treatment by PBS, Free TRAL(C) and Exo-iNSC-TRAIL respectively. All data are shown as mean ± SEM.

[0022] FIG. 8 Results of co-culture assay experiments to determine if iNSC- based EV-TK enzyme/prodrug therapy kilss GBM8 cells.

DETAILED DESCRIPTION

[0023] The presently disclosed subject matter now will be described more fully hereinafter, in which some, but not all embodiments of the presently disclosed subject matter are described. Indeed, the presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0024] L Definitions

[0025] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.

[0026] While the following terms are believed to be well understood by one of ordinary' skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0027] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0028] In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

[0029] Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

[0030] Following long-standing patent law convention, the terms “a”, ‘’an”, and ‘‘the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to "a cell" includes a plurality’ of such cells, and so forth. [0031] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0032] As used herein, the term “about,” when referring to a value or to an amount of a composition, dose, sequence identity (e.g., when comparing two or more nucleotide or amino acid sequences), mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

[0033] The term “comprising”, which is synonymous with “including” “containing” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim.

[0034] As used herein, the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0035] As used herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

[0036] With respect to the terms “comprising”, “consisting of’, and “consisting essentially of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.

[0037] As used herein, the term '‘and/or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C. and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

[0038] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1. 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0039] The term “gene” refers broadly to any segment of DNA associated with a biological function. A gene can comprise sequences including but not limited to a coding sequence, a promoter region, a cis-regulatory sequence, a non-expressed DNA segment that is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof. A gene can be obtained by a variety of methods, including cloning from a biological sample, synthesis based on known or predicted sequence information, and recombinant derivation of an existing sequence.

[0040] The term "substantially identical”, as used herein to describe a degree of similarity between nucleotide sequences, peptide sequences and/or amino acid sequences refers to two or more sequences that have in one embodiment at least about least 60%, in another embodiment at least about 70%, in another embodiment at least about 80%, in another embodiment at least about 85%, in another embodiment at least about 90%, in another embodiment at least about 91%, in another embodiment at least about 92%, in another embodiment at least about 93%, in another embodiment at least about 94%, in another embodiment at least about 95%, in another embodiment at least about 96%, in another embodiment at least about 97%, in another embodiment at least about 98%, in another embodiment at least about 99%, in another embodiment about 90% to about 99%, and in another embodiment about 95% to about 99% nucleotide identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.

[0041] As used herein, the term “subject” refers to an individual (e.g., human, animal, or other organism) to be assessed, evaluated, and/or treated by the methods or compositions of the presently disclosed subject matter. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and includes humans. As used herein, the terms “subject” and “patient” are used interchangeably, unless otherwise noted.

[0042] As used herein, the terms “effective amount” and “therapeutically effective amount” are used interchangeably and refer to the amount that provides a therapeutic effect, e.g., an amount of a composition or therapeutic compound that is effective to treat or prevent diseases, cancers, pathological conditions, etc. in a subject.

[0043] As used herein, the term “adjuvant” as used herein refers to an agent which enhances the pharmaceutical effect of another agent.

[0044] A “compound”, as used herein, refers to any ty pe of substance or agent that is commonly considered a chemical, drug, or a candidate for use as a drug, as well as combinations and mixtures of the above. The term compound further encompasses molecules such as peptides and nucleic acids.

[0045] As used herein, a “derivative” of a compound refers to a chemical compound that can be produced from another compound of similar structure in one or more steps, such as in replacement of H by an alkyl, acyl, or amino group. [0046] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. [0047] In contrast, a “disorder’" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0048] The term “modulate”, as used herein, refers to changing the level of an activity, function, or process. The term “modulate” encompasses both inhibiting and stimulating an activity, function, or process.

[0049] As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in an animal. In some embodiments, a pharmaceutically acceptable carrier is pharmaceutically acceptable for use in a human.

[0050] The term “standard”, as used herein, refers to something used for comparison. For example, it can be a known standard agent or compound which is administered or added to a control sample and used for comparing results when measuring said compound in a test sample. Standard can also refer to an “internal standard”, such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured.

[0051] The term “symptom”, as used herein, refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. In contrast, a sign is objective evidence of disease. For example, a bloody nose is a sign. It is evident to the patient, doctor, nurse and other observers.

[0052] As used herein, the term “treating” includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0053] A '‘therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.

[0054] An “exosome” is a nanosized, membrane-bound vesicles that range in size from 30-200 nm [17], They are constituted of many substances, primarily including proteins, lipids, and nucleic acids [18], and can be identified by enrichment of proteins such as tetraspanins (e.g. CD63, CD9, and CD81) and membrane-binding proteins such as TsglOl [19], These tiny sacs can carry proteins, DNA, and RNA from one cell to another. Exosomes can be released by all cells, including cancer cells, and can be found in many body fluids. Exosomes can carry signals between cells, helping them communicate with each other. Exosomes can transport growth factors from stem cells to damaged areas, which can help repair tissue. In therapeutic applications, exosomes can be used as vehicles to carry therapeutic agents and compounds to sites of treatment in the body of a subject.

[0055] An “extracellular vesicle” or “EV” can include an exosome or other cellular derived vesicle can be used as vehicles to carry therapeutic agents and compounds to sites of treatment in the body of a subject. In some aspects EVs and exosomes can be used interchangeably.

[0056] “NSCs” refer to engineered tumoricidal neural stem cells (NSCs) that can be used as therapeutics, including treatment via targeted migration to brain tumor cells and delivery of a broad selection of therapeutic agents.

[0057] “iNSCs” refere to powerful tumor-homing and tumoricidal induced NSC (iNSC) from human skin fibroblasts produced by transdifferentiating fibroblast cells directly into NSCs without a pluripotent intermediate. These iNSCs have been engineered to produce and secrete a number of therapeutic agents which are actively delivered to tumor cells through a robust tumor-homing capacity. This active migration into invasive tumor foci can increase survival by delivering therapeutics into brain tumor regions that conventional surgery, chemotherapy, and radiotherapy routinely miss. [0058] II, Detailed Description

[0059] Transdifferentiation (TD), a somatic cell reprogramming process that eliminates pluripotent intermediates, creates cells that are ideal for personalized anti-cancer therapy. Provided here is the first evidence that extracellular vesicles (EVs) from TD-derived induced neural stem cells (Exo-iNSCs) are an efficacious treatment strategy for brain cancer. It was discovered that genetically engineered iNSCs generated EVs loaded with the tumoricidal gene product TRAIL at nearly twice the rate of their parental fibroblasts, and TRAIL produced by iNSCs was naturally loaded into the lumen of EVs and arrayed across their outer membrane (Exo-iNSC-TRAIL). Uptake studies in ex vivo organotypic brain slice cultures showed that Exo-iNSC-TRAIL selectively accumulates within tumor foci, and coculture assays demonstrated that Exo-iNSC-TRAIL killed metastatic and primary brain cancer cells more effectively than free TRAIL. In an orthotopic mouse model of brain cancer, Exo-iNSC-TRAIL reduced breast-to-brain tumor xenografts by approximately 3000-fold compared to treatment with free TRAIL, with all Exo-iNSC-TRAIL treated animals surviving through 90 days posttreatment. In additional in vivo testing against aggressive U87 and invasive GBM8 glioblastoma tumors, Exo-iNSC-TRAIL also induced a statistically significant increase in survival. This disclosure, including the studies and data described herein, establish a novel, predictably generated, stable, tumor-targeted EV to efficaciously treat multiple forms of brain cancer.

[0060] Malignant brain tumors present diverse and significant therapeutic hurdles, and there is a desperate need for new, creative, approaches to treatment[l],[2],[3]. Standard resection and chemo-radiation treatment lack the ability to eradicate diffuse malignant cells; therefore, recurrence of some brain tumors is almost unavoidable. Over the past two decades, engineered tumoricidal neural stem cells (NSCs) have broken new ground as potential therapeutics, demonstrating promise as an alternative cancer treatment approach via targeted migration to brain tumor cells and delivery of a broad selection of therapeutic agents[4],[5],[6]. By trans differentiating fibroblast cells directly into NSCs without a pluripotent intermediate, a powerful tumor-homing and tumoricidal induced NSC (iNSC) from human skin fibroblasts can be produced as disclosed in W02020093003A1 and [7], [8], [9], the methods of which are herein incorporated by reference.

[0061] As disclosed herein, iNSCs can be engineered to produce and secrete a number of therapeutic agents which are actively delivered to tumor cells through a robust tumor-homing capacity. This active migration into invasive tumor foci can increase survival by delivering therapeutics into brain tumor regions that conventional surgery, chemotherapy, and radiotherapy routinely miss. While the iNSCs offer potential for therapeutic agents, improvements must be made to address ongoing concerns and complexities with manufacturing and efficacy. As provided herein, one way to maintain the robust potency of cytotoxic cell therapies while mitigating regulatory, financial, and technical limitations is to isolate and deliver only the secreted therapeutic product.

[0062] Extracellular vesicles (EVs) and exosomes are nanosized, membranebound vesicles that range in size from 30-200 nm. They are constituted of many substances, primarily including proteins, lipids, and nucleic acids, and can be identified by enrichment of proteins such as tetraspanins (e.g. CD63, CD9, and CD81) and membrane-binding proteins such as TsglOl. EVs are produced intracellularly during the process of plasma membrane invagination and multivesicular body (MVB) development and are ultimately secreted via exocytosis from MVBs by fusion with the cellular plasma membrane. EVs are secreted by virtually all types of cells, including NSCs, and are investigated herein as potential alternatives to treatment with cells. EVs can be considered as “miniature surrogates” of their parental cells, because they can partially inherit analogous therapeutic and organotropic properties from their original cells. EVs naturally secreted from stem cells (e.g., NSCs, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs)) have diverse therapeutic properties, including anti -inflammation, immunity modulation, and tissue repair. Furthermore, stem cell-derived EVs can be more easily isolated and preserved, have higher safety and immune tolerance, and present fewer ethical issues when compared to the full cell product. Based on this, the present disclosure includes, for the first time, studies on EVs derived from iNSCs for brain cancer therapy.

[0063] TNF-related apoptosis-inducing ligand (TRAIL), a type-II transmembrane protein of the TNF superfamily, has long held promise as a cancer therapy [31], [32], TRAIL targets the extrinsic apoptotic pathway, triggering caspase-induced apoptosis in malignant cells with high expression of Death Receptor 4 or 5 (DR4/5) while sparing healthy cells [33], While TRAIL has shown strong antitumoral effect in preclinical investigations, clinical trials have shown that TRAIL alone is insufficient to effectively treat patients [34], [35], Without being bound by any particular theory or mechanism of action, this could be due to its poor pharmacokinetics, such as short chemical and biological halflives, insufficient distribution to target areas, inefficiency in stimulating DR4 and DR5 receptors of tumor cells and acquired TRAIL resistance. To help circumvent these constraints, robust TRAIL delivery is required, with a solution provided herein.

[0064] To elaborate, provided herein are therapeutic EVs derived from iNSCs that have been engineered to produce and secrete a therapeutic agent, including a tumoricidal gene product and/or an anti-cancer molecule. One such example therapeutic agent produced and secreted by these therapeutic EVs derived from iNSCs is TRAIL. Disclosed herein for the first time is the finding that a significant proportion of TRAIL is secreted by iNSCs via EVs, which are fully made, loaded, and secreted by the cells themselves. The therapeutic potential of these EVs. termed Exo-iNSC-TRAIL (Fig. 1). isolated from TRAIL-secreting iNSCs in vitro, was evaluated as described herein. Characterization and in vitro/ex vivo in vivo testing of Exo-iNSC-TRAIL as a local brain cancer therapy demonstrates its tumor-selective accumulation and therapeutic superiority to free TRAIL protein in three orthotopic models of glioblastoma and breast cancer brain metastasis.

[0065] Another such example therapeutic agent produced and secreted by these therapeutic EVs derived from iNSCs is the Herpes simplex virus thymidine kinase (TK) gene. Thus, similar to Exo-iNSC-TRAIL, Exo-iNSC-TK is provided herein as an effect cancer and tumor therapeutic.

[0066] In addition to TRAIL and TK. the disclosed Exo-iNSC delivery vehicle can be used with any suitable therapeutic agent comprising a tumoricidal gene product and/or an anti-cancer molecule, and should not be limited to TRAIL and TK. [0067] Likewise, in some aspects EV-based combination therapies are provided. Within a single patient GBM is highly heterogeneous, comprised of morphologically and genetically distinct cell types. Chemotherapy regimens commonly consist of combination strategies to overcome the heterogeneous response of cancer cells. A similar strategy can be employed for EV-based therapy, yet the efficacy of EVs delivering multiple combination therapies or their impact on treatment durability was unknown prior to this disclosure. An example prodrug/enzyme combinations is the drug ganciclovir (GCV) and the Herpes simplex virus thymidine kinase (TK) gene. In this approach, GCV is converted into a toxic metabolite by TK that then induces cell killing in fast dividing tumor cells. This method can in some embodiments be beneficial over other tumoricidal payloads because the dosing and release of the toxic product is precisely controlled by GCV administration. It was reasoned that the non-targeted burstrelease profile of TK is the ideal drug to combine with the highly -targeted anti- GBM mechanism of TRAIL. TRAIL is known to enhance TK-killing of cancer cells. Thus, provided in some aspects are combined TK/TRAIL EV therapies, as well as other combination EV therapies that can achieve tumor killing across a broad range of tumors, cancers and GBMs.

[0068] Thus, provided in one aspect is a therapeutic compound, the therapeutic compound comprising an extracellular vesicle (EV), the EV includes an exosome derived from transdifferentiation (TD)-derived induced neural stem cells (Exo- iNSCs), a therapeutic agent, the therapeutic agent includes a tumoricidal gene product and/or an anti-cancer molecule, where the therapeutic agent is present in a lumen of the EV and/or is distributed across a membrane and/or surface of the EV. The therapeutic compound may also include where the induced neural stem cells are derived from skin fibroblasts. The therapeutic compound may also include where the tumoricidal gene product and/or anti-cancer molecule includes TNF-related apoptosis-inducing ligand (TRAIL). The therapeutic compound may also include where the tumoricidal gene product and/or anti-cancer molecule includes Herpes simplex virus thymidine kinase (TK).

[0069] The therapeutic compound may also include where the tumoricidal gene product and/or anti-cancer molecule includes both TRAIL and TK. The therapeutic compound may also include where the EV includes a concentration of TRAIL present in the lumen, and/or distributed across the membrane and/or surface of the EV at about 100 pg/ug EV to about 500 pg/ug EV, optionally about 250 pg/ug EV to about 400 pg/ug EV, optionally about 100 pg/ug EV to about 250 pg/ug EV, optionally about 250 pg/ug EV to about 500 pg/ug EV, optionally about 300 pg/ug EV to about 350 pg/ug EV.

[0070] The therapeutic compound may also include where the EV has a tumorhoming capacity causing a selective migration to and/or attraction to tumors and/or cancer cells imparted by the induced neural stem cells (iNSCs). The therapeutic compound may also include where the tumoricidal gene product and/or an anti-cancer molecule of the EV has increased tumor-selective accumulation and increased therapeutic activity as compared to free TRAIL and/or free TK. In some embodiments, the increased tumor-selective accumulation of the tumoricidal gene product and/or an anti-cancer molecule is an increase of about 5%, about 10%, about 15%. about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% as compared to free TRAIL and/or free TK, i.e. TRAIL or TK not part of and EV delivery mechanism. In some embodiments, the increased therapeutic activity of the tumoricidal gene product and/or an anti-cancer molecule is an increase of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% as compared to free TRAIL and/or free TK, i.e. TRAIL or TK not part of and EV delivery mechanism. The therapeutic compound may also include where the exosome of the EV generated by TD-iNSC is produced with the therapeutic agent present in a lumen of the EV and/or distributed across the membrane and/or surface of the EV. The therapeutic compound may also include where about 75% or more of the therapeutic agent is distributed across the membrane of the EV, optionally about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or more.

[0071] The therapeutic compound may also include where the therapeutic compound is configured to treat a cancer or tumor, optionally a brain cancer or brain tumor, optionally a glioblastoma. The therapeutic compound may also include where the EV further includes other therapeutic proteins and/or additional tumor-targeting motifs.

[0072] Provided in some aspects are therapeutic compositions comprising the therapeutic compound, and an excipient, carrier or buffer, which is configured to be administered to a subject.

[0073] Provided are methods of producing the therapeutic compound, including genetically engineering iNSCs to generate one or more extracellular vesicles (EVs) comprising exosomes loaded with the therapeutic agent, where the therapeutic agent includes a tumoricidal gene product and/or an anti-cancer molecule, where the therapeutic agent is present in a lumen of the EVs and/or is distributed across a membrane and/or surface of the EVs. The method may also include where genetically engineering iNSCs to generate one or more EVs includes transdifferentiating fibroblstats to produce iNSCs followed by transducing iNSCs to produce the tumoricidal gene product and/or an anti-cancer molecule as part of the EVs. The method may also include where the EVs are isolated and formulated into a therapeutic composition. The method may also include where the therapeutic agent is loaded in the one or more EVs at about twice the rate as parent fibroblasts of the one or more EVs. The method may also include comprising generating personalized EVs from iNSCs transdifferentiated from an subject patient’s own skin.

[0074] Provided are methods of treating a subject, the methods comprising administering to a subject in need a therapeutic dose of the compounds or compositions. The methods may also include where the subject is a human subject suffering from a cancer, tumor or related condition. The method may also include where the cancer is a brain cancer or brain tumor, optionally a glioblastoma.

Examples

[0075] The following examples are included to further illustrate various embodiments of the presently disclosed subject matter. However, those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the presently disclosed subject matter. [0076] Materials and Methods for Examples 1 to 8

[0077] Materials

[0078] TRAIL was purchased from Millipore sigma (CAT No.GF092). TRAIL ELISA kit was purchased from R&D Systems (CAT NO. Invitrogen™ BMS2004). Uranyl Acetate Solution (2%) (CAT NO. 22400-2) were purchased from Electron Micros. Caspase-Gio® 3/7 Assay System (CAT NO. G8090), Caspase-Gio® 8 Assay Systems (CAT NO. G8200), and Caspase-Gio® 9 Assay Systems (CAT NO. G8210), were purchased from Promega. Pierce™ BCA Protein Assay Kit (CAT NO. 23227), RIPA Lysis and Extraction Buffer (CAT NO. 89901), and DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) (CAT NO. D3571) were purchased from Thermo Fisher Scientific. PKH67 Green Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling (CAT NO. MINI67) and anti-GFAP Cy3 conjugate (CAT NO. MAB 3402C3) were purchased from Millipore Sigma. PE anti-human CD253 (Trail) (CAT NO. 308206) was purchased from Biolegend. CD63 Monoclonal Antibody (H5C6). eFluor™ 660, (CAT NO. 50-0639-42) was purchased Thermo Fsher Scientific. Goat anti-mouse IgG H&L (6 nm Gold) (CAT NO. ab39614) and anti-TSGlOl antibody (CAT NO. ab30871) were purchased from Abeam. Alexa Fluor 568 goat anti-rabbit IgG (H+L) secondary antibody (CAT NO. A-11011) was purchased from Invitrogen. Anti-IBAl antibody was purchased from (CAT NO. 019-19741) FUJIFILM Wako Pure Chemical Corporation. Lentivirus expressing Firefly Luciferase-mCherry, puromycin selection and TRAIL-GFP-puro lentivirus nonconcentrated were purchased from Duke Viral Vector Core.

[0079] Cell lines

[0080] Cell lines were received and used as described previously [9]. [8], Briefly, the treatment of triple negative breast cancer (TNBC) brain metastasis cell line MDA-MB-231-Br was obtained through material transfer agreement (MTA) (T. Yoneda). MDA-MB231-Br were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (v/v), 1% penicillin-streptomycin (p/s)(v/v). U87 glioma cells were purchased from the American Type Culture Collection (ATCC) and cultured MEM with 10% FBS and 1% p/s. Human fibroblasts cells (NHF1) were provided by W. Kauffman (The University of North Carolina School of Medicine) and were cultured in DMEM with 10% FBS and 1% p/s. GBM8 glioma neurospheres were a gift from H. Wakimoto (Massachusetts General Hospital) and were cultured in EF medium (neurobasal media, 1% L-glutamine, l x B27 supplement, 0.5x N-2 supplement, 2 pg/mL heparin, 20 ng/mL recombinant human epidermal growth factor, 20 ng/mL recombinant human fibroblast growth factor 2). iNSCs spheres were generated as described previously and maintained in ReNcell NSC Maintenance Media [9], Lentiviral infection of cell lines was completed as described previously [38],

[0081] Isolation and characterization of extracellular vesicles (EVs)

[0082] The iNSCs or NHF1 cells were cultured in EV-depleted ReNcell NSC maintenance and DMEM medium at 37°C respectively, and the conditioned media (CM) was collected after 48 h. The CM was then centrifuged at 1500 rpm for 15 min and 12,000 g for 20 min to get rid of cells and debris. EVs were isolated by ultracentrifugation of the supernatants at 100,000g for 70 min at 4°C. EVs were then washed in PBS at 100,000 g for 70 min at 4°C [39], The isolated EVs were then reconstituted with PBS and stored at 4°C. EV amounts were quantified via protein concentration by a BCA protein assay kit.

[0083] Transmission electron microscope and Immunoelectronmicroscopy

[0084] The morphology of EVs was observed by Transmission Electron Microscope (TEM) via uranyl acetate negative staining [40], To observe the EV surface TRAIL, immuno-electron microscopy was applied to stain and observe the TRAIL. Briefly, EVs were fixed with 4% paraformaldehyde and dropped on carbon-coated copper grids. TRAIL antibodies (1 :5) were co-incubated with fixed EVs at 4°C overnight, and 6 nm gold-conjugated goat anti-mouse IgG secondary antibodies were then used to label the conjugated TRAIL [41], EVs were observed by TEM from Thermo Scientific™ Talos™ F200X.

[0085] ELISA [0086] The level of TRAIL expressed in intact and lysed iNSC-derived EVs was quantified using ELISA following the directions provided by the manufacturer. Lysing buffer was supplemented with a protease inhibitor which did not affect TRAIL stability.

[0087] Flow cytometry for Exo-iNSC-TRAIL

[0088] Approximately 400 pg/ml of isolated Exo-iNSC-TRAIL were stained with CD63 eFluor 660 and TRAIL-PE at a ratio of 1: 100 (v/v) overnight at 4°C. The unbounded antibodies were removed by ultracentrifuge at 100000 g for 70 min and washed one time with PBS. Flow cytometry for Exo-iNSC-TRAIL was performed on a ImageStreamX Mark II flow cytometry.

[0089] Proteomics analysis

[0090] Protein exosome samples (n = 3) were diluted with 100 pL of 8 M urea in 50mM ammonium bicarbonate, pH 7.8. Samples were then reduced with 100 mM DTT at 37 °C for 45 min and alkylated with 15 mM iodoacetamide for 45 min at room temperature. Samples were then diluted to 1 M urea and subjected to digestion with trypsin (Promega, 1:50 w/w) overnight at 37°C. The resulting peptides were acidified to 0.5% trifluoroacetic acid, cleaned using desalting spin columns (Thermo), and eluates were dried via vacuum centrifugation. The resultant peptide samples were quantified by Pierce Fluorometric Peptide Assay, then normalized to 0.4 pg/pL prior to liquid chromatography -tandem mass spectrometry (LC-MS/MS) analysis.

[0091] Samples were analyzed by LC-MS/MS using an Easy nLC 1200 coupled to a QExactive HF mass spectrometer (Thermo Scientific). Samples were injected onto an Easy Spray PepMap C18 column (75 pm id x 25 cm, 2 pm particle size) (Thermo Scientific) and separated over a 120 min method. The gradient for separation consisted of 5-45% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% ACN. The QExactive HF was operated in data- dependent mode (DDA) where the 15 most intense precursors were selected for subsequent fragmentation. Resolution for the precursor scan (m/z 350-1700) was set to 60,000, while MS/MS scans resolution was set to 15,000. The normalized collision energy was set to 27% for HCD. Peptide match was set to preferred, and precursors with unknown charge or a charge state of 1 and > 7 were excluded.

[0092] Raw data were processed using Proteome Discoverer (Thermo Scientific, version 2.5). Data were searched against a reviewed Uniprot human database (containing -20,000 sequences), appended with a common contaminants database, using the Sequest HT search algorithm within Proteome Discoverer. Enzyme specificity was set to trypsin, up to two missed cleavage sites were allowed, carbamidomethylation of Cys was set as fix modification and oxidation of Met was set as variable modification. A precursor mass tolerance of lOppm and fragment mass tolerance of 0.02 Da were used. Label-free quantification (LFQ) using razor + unique peptides was enabled through the Minora node. A 1% peptide false discovery rate (FDR), and a minimum of 2 peptides were used to filter the data. Proteins with > 3 missing LFQ intensities across samples were removed. Further analysis (log2 transformation, normalization, imputation, statistical analysis) was conducted using Argonaut [42], Differentially abundant proteins with a FDR adjusted p-value < 0.01 were analyzed via Reactome Pathway Browser to test for pathway enrichment and pathway topology analysis [43], The false discovery rate was used to correct for multiple hypothesis testing.

[0093] iNSC immunohistochemistry staining

[0094] iNSC-TRAIL were seeded on the laminin coated 15 mm glass cover and cultured for 24h. iNSCs were then fixed with 10% neutral buffered formalin and permeabilized in 0.1% Triton X-100 in phosphate buffered saline (PBS-T) with 1% (w/v) bovine serum albumin (BSA). iNSCs were then incubated with mouse anti human TRAIL-PE conjugate (1 : 1000, v/v), mouse anti human CD63 eFluor 660 conjugate (1: 1000, v/v), and primary rabbit anti human TSG101 (1 : 1000, v/v) antibodies for 2 h at room temperature. For TSG fluorescence labeled secondary antibody, iNSCs were washed with PBS three times and then incubated with Alexa Fluor 568 goat anti-rabbit (1 :1000, v/v) secondary antibodies for 1 h. Then iNSCs were washed with PBS three times and stained with nuclei DAPI stain. [44] After that, iNSCs were mounted on the slides by Pro-Long Gold Antifade Mountant. The slides were observed by Leica SPX8 confocal microscopy. Representative images were analyzed by Leica image software.

[0095] Post-loading of free TRAIL onto Exo-iNSC [0096] Blank Exo-iNSCs were isolated from iNSCs that did not express TRAIL, while free TRAIL was subsequently isolated from iNSC-TRAIL by removing Exo-iNSC-TRAIL via ultracentrifuge. Either 1.5 ng or 3 ng of the isolated free TRAIL (iNSC) was incubated with 1 pg of the blank EVs derived from iNSC overnight, and the EVs were subsequently washed to remove the free TRAIL. The TRAIL associated with Exo-iNSC was measured by TRAIL ELISA kit.

[0097] In vitro studies

[0098] 100 pL of MDA-MB231-Br-mCherry-FLuc cells (5000 cells/well), GBM8-mCherry-FLuc cells (10000 cells/well) and U87-mCherry-FLuc cells (10000 cells/well) were implanted in black 96-well clear-bottom plates. After 24h, the cell media was exchanged by fresh media and different concentrations of free TRAIL and the same amount of TRAIL from Exo-iNSC-TRAIL were added simultaneously. After co-incubation for 72h, cell viability was assessed via bioluminescence (BLI) via AMI optical imaging system. One outlying data point in Fig. 3C was removed using Tukey’s Inter-Quartile Range method.

[0099] EVs uptake by Organotypic Brain Slice Cultures (OBSCs)

[0100] OBSCs were generated as described previously.fi] Briefly, OBSCs were sliced from P8 Sprague-Dawley rat pups. Dissected brains were fixed on a vibratome platform (Leica VT1000S) and immersed in ice-cold brain slice media, also defined previously. [1] Coronal OBSCs were sliced at a thickness of 300 pm. OBSC were then cultured on 6-well matched Millicell culture inserts with 1 mL of brain slice media underneath each insert. For tumor implantation on OBSCs, 1 pL of 20,000 MDA-MB231-Br-mCherry-FLuc cells were added on the center of each OBSC hemisphere at 2 h after OBSC generation. Fresh OBSC medium was changed the day after slicing, and 100 pg/mL PKH26-labeled EVs were added into the OBSC media. After 24 h, the OBSCs were washed with PBS and fixed with 4% paraformaldehyde for immunofluorescent staining.

[0101] Immunofluorescence staining of OBSCs

[0102] Fixed OBSCs were first washed by PBS three times. 0.1% Triton X-100 in phosphate buffered saline (PBS-T) was then used to permeabilize the OBSCs for 1 h. OBSCs were then blocked in PBST with 1% BSA for Ih at room temperature. OBSCs were incubated with Cy3 conjugated glial fibrillary acidic protein (GFAP, 1: 1000 v/v), and ionized calcium-binding adapter molecule 1 (IBA1, 1 : 1000 v/v) overnight. After washed by PBS three times, OBSCs were stained with secondary antibody Alexa Fluor 568 goat anti -rabbit IgG (1: 1000 v/v) 1 h. Nuclei were stained by DAPI [45], Finally, OBSCs were mounted on the slides by Pro-Long Gold Antifade liquid mountant and observed by Leica SPX8 confocal microscopy at UNC Neuroscience Microscopy Core.

[0103] In vivo bioluminescence (BLI)

[0104] To monitor the tumor growth of MDA-MB231-Br-mCherry-FLuc cells, GBM8-mCherry-FLuc, and U87- mCherry-FLuc, sequential BLI was conducted by the following method. Each mouse was intraperitoneally injected with 200 pL of 1.5 mg/ml d-luciferin in PBS. 10 min later, BLI was performed on the luciferin- administered mice by an AMI optical imager system, as described previously [9].[11], BLI data were analyzed with Aura software.

[0105] IC-infused therapy studies in TNBC-brain metastasis model

[0106] MDA-MB231-Br-mCherry-FLuc cells (8 x 10⁴) in 3 pL of PBS were implanted in the right hemisphere of the brain in athymic nude mice by stereotactic intracranial (IC) injection, as described previously.fi 1] Four days after tumor implantation, a single dose of 8 ng free TRAIL or an equivalent TRAIL dose of Exo-iNSC-TRAlL in 6 pL of PBS was infused into the tumors. For the control group, 6 pL of PBS was injected in the brain by the same protocol. Serial BLI was completed via the AMI optical imager system to monitor the growth of tumors in mice. When a mouse's body weight decreased by over 20% of its original weight, it was sacrificed, and data on survival was recorded.

[0107] IC-infused therapy studies in Glioblastoma models

[0108] GBM8-mCherry-FLuc cells (1 x 10⁵) and U87-mCherry-FLuc cells (1 x 10⁵) in 3 pL of PBS were implanted in the right hemisphere of the brain in athymic nude mice by stereotactic intracranial (IC) injection as described previously. [11] For the GBM8 model, 8 ng free TRAIL or an equivalent TRAIL dose of Exo-iNSC-TRAIL in 6 pL of PBS was injected IC at day 4, 11 and 25 after tumor implantation. For the U87 model, a single dose of 8 ng free TRAIL or an equivalent TRAIL dose of Exo-iNSC-TRAIL in 6 pL of PBS was infused into the tumors on day 4 after tumor implantation. For the control group, 6 pL of PBS was injected in the brain by the same protocol. Serial BLI was completed via the AMI optical imaging system to monitor the growth of tumors in mice. When a mouse's body weight decreased by over 20% of its original weight, it was sacrificed, and data on survival was recorded.

[0109] Statistical Analysis

[0110] All statistical tests and sample sizes are included in the Figure Legends. All data are shown as mean ± SEM. In all cases, the p values are represented as follows: ****p < 0.0001, ***p < 0.001, ** p < 0.01, *p < 0.05. and not statistically significant when p > 0.05. Mean values between two groups were compared using Student’s t-test or two-way ANOVA. Mean values between three or more groups were compared using one-way ANOVA. Mean values among three groups in longitudinal in vivo studies were compared using two-way repeated measurement ANOVA. All statistical analyses were performed using GraphPad Prism (Version 9.1.0). One outlying data point in Fig 3C was removed using Tukey’s InterQuartile Range method. For all quantifications of immunofluorescence, the samples being compared were processed in parallel and imaged using the same settings and laser power.

[0111] Example 1

[0112] Isolation, characterization and TRAIL measurement of Exo-iNSC- TRAIL

[0113] The present disclosure is based on a second-generation TRAIL- overexpressing iNSC cell type that exhibits improved antitumor properties and significantly increased tumor-homing capability. [9] Since TRAIL protein can be expressed in both membranous and secreted formats, experiments were conducted, as described herein, to determine whether TRAIL was being released from iNSCs not only as free protein, but also associated with iNSC-derived EVs. To investigate this, TRAIL and canonical EVs markers CD63 and TS101 were first intracellularly co-stained in iNSCs (Fig. 2A). Represented fluorescence images reveal the co-localization of TRAIL and EVs within the iNSCs, indicating that TRAIL may be loaded into EVs following EVs secretion. EVs were isolated from conditioned iNSC culture media using a serial centrifugation procedure. [46] The successfully isolated EVs were first confirmed by transmission electron microscopy (TEM) ( Fig. 2B). The represented TEM images show these isolated particles exhibit typical EVs characteristics including a cup-like morphology and a diameter of 50 to 200 nm. Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) confirmed a consistent nanoparticle population with a zeta-potential of - 24 ± 3 and an average size of 138 ± 7 nm (Fig. 2C). The isolated particles were also labeled with CD63 to further validate their EVs property. Flow cytometry of CD63 labeled particles revealed that -80% of isolated particles were CD63 -positive, and these populations maintained a similar size distribution to the unlabeled population, indicating that CD63 -containing EVs had indeed been isolated and termed as Exo-iNSC. Surprisingly, the proteome encapsulated within Exo-iNSC-TRAIL was significantly different than the proteome in EVs derived from pre-transdifferentiated NHF1 fibroblasts (Exo- NHF1 -TRAIL) (p<0.01, n = 3 biological replicates).

[0114] Example 2

[0115] Evaluating iNSC-secreted EVs for functional TRAIL

[0116] Next, experiments were conducted to determine whether the iNSC- secreted EVs contained functional TRAIL. A highly specific commercial ELISA kit was used to investigate the expression of TRAIL in prepared iNSC EVs. Total TRAIL expression in Exo-iNSC-TRAIL was higher than TRAIL expression in EVs generated from pre-transdifferentiated NHF1 fibroblasts (Fig. 2D). Exo- iNSC-TRAIL reproducibly contained nearly double the TRAIL per EV than Exo- NHF1-TRAIL (351±13.3 pg TRAIL/ug EVs vs -187 ± 28.4 pg TRAIL/ug EVs). Interestingly, ELISA quantification of TRAIL on the surface of intact Exo- iNSC-TRAIL (-269.5 ± 1.4 pg TRAIL/ug EVs) was similar to the total EV- associated TRAIL quantified from Exo-iNSC-TRAIL lysate (~351±13.3 pg TRAIL/ug EVs) (Fig. 2D). Lysing buffer was supplemented with a protease inhibitor which did not affect TRAIL stability. This indicates that TRAIL associated with Exo-iNSC-TRAIL was largely bound to the EV surface (-75% of total TRAIL). TRAIL arrayed on the surface of EVs was also validated by image flow cytometry by using the TRAIL-specific and CD63-specific antibodies. Results confirmed that -42% of the isolated vesicles were TRAIL positive and -45% of CD63+ derived EVs expressed with TRAIL (Fig. 2E-G). Moreover, TRAIL expression on the surface of Exo-iNSC-TRAIL was examined by nanogold immunostaining (Fig. 2H). Immuno-electron-microscopy analysis showed vesicles highly positive for surface TRAIL. Using a commercial ELISA, TRAIL expression on Exo-iNSC-TRAIL was compared to the total TRAIL secreted by iNSCs, revealing approximately 26% of the TRAIL produced by iNSCs was associated with Exo-iNSC-TRAIL (Fig. 21) Taken together, these data demonstrate that iNSCs produce and secrete a significant percent of TRAIL within a uniform population of EVs, and that TRAIL was not only loaded into the lumen of the EVs but distributed across the membrane of the particles as well.

[0117] Example 3

[0118] Evaluating secretion and stability of TRAIL within Exo-iNSC- TRAIL

[0119] To determine whether TRAIL is attached to EV surfaces before or after secretion, an experiment was designed to determine whether TRAIL can conjugate to surfaces of EVs after EV isolation (Fig. 3A). Blank Exo-iNSCs were isolated from iNSCs that did not express TRAIL, while free TRAIL was subsequently isolated from iNSC-TRAIL, described as free TRAIL (iNSC). Either 1.5 or 3 ng of the isolated free TRAIL (iNSC) was incubated with 1 pg of the blank EVs overnight, and the EVs were subsequently washed to remove the free TRAIL. ELISA measured a very large amount of TRAIL associated with Exo-iNSC (-1000 pg TRAIL/ pg EVs and -1758 pg TRAIL/ pg EVs respectively, Fig 3B), suggesting that free TRAIL can naturally associate with constituents on the outer membrane of Exo-iNSC. It's worth noting that EVs derived fromnon-TRAIL-expressing iNSCs were negative for TRAIL expression. Interestingly. “post-loaded’^? Exo-iNSC-TRAIL exhibited significantly greater tumor kill against the glioblastoma cell line U87 in vitro when compared to the same dose of free TRAIL (Fig. 3C-D). At a dose of 25 ng/mL, Exo-iNSC-TRAIL induced -85% kill at t = 48 h after treatment compared to just -30% kill by free TRAIL (iNSC). This could be due to the greater stability of TRAIL when associated with EVs (Fig. 3E-F).

[0120] One of the issues preventing the production of recombinant free TRAIL agent is their rapid degradation and clearance when exposed to harsh conditions such as the fluctuating temperature during transportation. Studies have shown that EVs can stabilize TRAIL proteins which could augment their anti-cancer effects. Here it is shown that the stability of TRAIL within Exo-iNSC-TRAIL is significantly enhanced. The stability of the free TRAIL and Exo-iNSC-TRAIL was investigated by monitoring the TRAIL concentration after incubation at 4°C and 37°C for different time intervals (Fig. 3E-F). The results show that incubation of free TRAIL at 4°C and 37°C caused above 90% degradation of the free protein within 1 day. In contrast, the levels of TRAIL in EVs were reduced by only about 10% at 4°C and 37°C after 3 days and about 50% after a week, suggesting the EV thermally stabilizes TRAIL and should allow prolonged activity that could enhance anti-tumor efficacy. (Fig. 3E-F).

[0121] Example 4

[0122] Evaluating the proapoptotic potential of Exo-iNSC-TRAIL

[0123] The proapoptotic potential of Exo-iNSC-TRAIL was next investigated in vitro against the breast cancer brain metastasis cell line MB231Br, the glioma cell line U87, and primary patient-derived stem-like GBM8 cells. According to previous reports, MB231Br were susceptible to TRAIL and had an IC50 of approximately 2 ng/mL, whereas U87 and GBM8 cells were more resistant to TRAIL-induced apotosis[47] [48]. As shown in Fig. 4A, MB231Br shows high sensitivity' to both Exo-iNSC-TRAIL and free TRAIL, with an IC50 of about 3 ng/mL at 72 h. U87 and GBM8 cells were more resistant to free TRAIL, with GBM8 neurospheres showing no apoptosis after being treated with various concentrations of TRAIL for 72 h. Exo-iNSC-TRAIL was more effective than free TRAIL at inducing apoptosis in all three cell lines. Against U87 cells, Exo- iNSC-TRAIL induced an IC50 of 9 ng/mL. which is 5-fold lower than that of free TRAIL. Against GBM8 cells, Exo-iNSC-TRAIL induced nearly 50% killing at 100 ng/mL (Fig. 4A). These data suggest that the cytotoxic potency of TRAIL is increased when loaded on Exo-iNSC-TRAIL.

[0124] Soluble or transmembrane TRAIL can initiate the extrinsic apoptotic pathway by binding to DR4/ DR5 receptors overexpressed on many cancer cells, triggering a proteolytic cascade of caspase activation that subsequently induces apoptosis[49]’[50]. To confirm that the reduction in cell viability in response to Exo-iNSC-TRAIL is mediated by apoptosis, the activation of caspase-3/7, 8 and 9 was detected MB231Br, GBM8, and U87 cell lines were treated with a low dose of TRAIL (5 ng/mL) for 24h. Very low-to-no caspase 3/7,8 and 9 activations were observed in all cells treated with free TRAIL. In contrast, around a threefold increase in caspase 3/7 activity, and around 1.4-fold increases in caspase 8 and 9 activities were observed in all 3 cell lines after treatment with Exo-iNSC-TRAIL (Fig. 4B). These results show that the increased potency of Exo-iNSC-TRAIL compared to free TRAIL is due to greater upregulation of apoptotic signaling rapidly after treatment initiation.

[0125] Example s

[0126] Evaluating the tumor-specific accumulation of Exo-iNSC-TRAIL

[0127] Tumor-tropic targeting of iNSC-derived EVs would be an important advantage to their potential as drug delivery' vehicles. Exo-iNSC-TRAIL are derived from second-generation iNSCs that exhibit significantly increased tumorhoming capability; therefore, it was hypothesized that Exo-iNSC-TRAIL may themselves have an ability to selectively target tumor cells. Using previously validated living ex vivo organotypic brain slice cultures (OBSCs),[l] [ll]’[51] it was found that EVs robustly and selectively accumulate in engrafted MB231Br tumor foci while sparing normal brain tissue (Fig. 5). In six-well plates, OBSCs were cultured atop transwell inserts with 0.4-pm pore size and EVs were diluted in media underneath the inserts, where they were allowed to passively diffuse into OBSCs for 24h. Quantitative imaging of living OBSCs showed that iNSC- derived EVs did not accumulate at significant levels in normal brain or within activated microglia or astrocytes. In contrast, nearly 20-fold greater levels of EVs were detected in OBSCs that had been engrafted with brain tumor foci. Throughout z-stacks of 19 10 pun-thick confocal images of OBSCs harboring MB231Br tumors, both Exo-iNSC-TRAIL and Exo-NHFl -TRAIL showed significant accumulation (Fig. 5A). Interestingly, while the absolute difference in EV signal between these groups was only a 1.4-fold increase in Exo-iNSC-TRAIL compared with that of Exo-NHFl -TRAIL (Fig. 5B), there was a significantly greater co-localization of Exo-iNSC-TRAIL within the tumor cells themselves compared to Exo-NHFl -TRAIL. An analysis of Exo/Tumor signal in each region along the z-axis revealed an about 4-fold increase in tumor-specific accumulation of Exo-iNSC-TRAIL over Exo-NHFl -TRAIL (Fig. 5C). Without being bound by any particular theory or mechanism of action, there are several possible mechanisms for this phenomenon, including (1) that Exo-iNSC-TRAIL has tropism for tumor cells, possibly via an abundance of surface-conjugated TRAIL acting as a tumor-selective targeting ligand and leading to preferential uptake over other cell types; or (2) that other, yet uncharacterized, proteins upregulated in iNSCs are transferred to Exo-iNSC-TRAIL and drive their tumor-specific accumulation.

[0128] Example 6

[0129] Evaluating the killing potential of Exo-iNSC-TRAIL against tumors

[0130] After showing that Exo-iNSC-TRAIL were able to accumulate in the tumor foci on living brain tissue, experiments were conducted to explore the tumor killing potential of Exo-iNSC-TRAIL against breast-to-brain metastasis tumors in an orthotopic xenograft model of MB23 IBr. MB231Br cells expressing firefly luciferase were stereotactically implanted into the brain parenchyma of nude mice according to a previously validated protocol. [9] Four days after tumor implantation, a single dose of phosphate-buffered saline, 6 ng free TRAIL, or an equal amount of TRAIL in Exo-iNSC-TRAIL was directly injected into the tumor. Longitudinal bioluminescence imaging (Fig. 6A) revealed robust, sustained tumor killing by Exo-iNSC-TRAIL (Fig. 6B). In contrast, free TRAIL initially induced modest tumor growth suppression, but tumors quickly grew and many mice developed spinal tumors in addition to the primary brain mass. By 32 days after treatment, there was a statistically significant difference in brain tumor burden among animals given PBS (-971.5-fold tumor growth), those given free TRAIL (-1160-fold tumor growth), and Exo-iNSC-TRAIL (-2.7-fold tumor growth) (Fig. 6B). While a majority of untreated animals and animals given free TRAIL had succumbed to tumor burden by 70 days after treatment, all animals who received Exo-iNSC-TRAIL showed robust and sustained tumor growth suppression (Fig. 6C) and survived over 90 days after treatment (Fig. 6D).

[0131] Example 7

[0132] Evaluating the killing potential of Exo-iNSC-TRAIL against GBM tumors

[0133] The robust tumor killing effect of Exo-iNSC-TRAIL against MB231Br in vivo prompted the design and completion of tests of the efficacy in two additional GBM tumor models. Against the rapidly growing U87 line, a single dose of Exo-iNSC-TRAIL induced a statistically significant increase in survival compared to free TRAIL (p < 0.05) (Fig. 7A-7C). Against the invasive GBM8 glioblastoma line, three doses of Exo-iNSC-TRAIL also led to a statistically significant survival benefit compared to the untreated control (p < 0.01) and a trend toward significance when compared against free TRAIL (p < 0.07) (Fig. 7D-7F).

[0134] Example 8

[0135] Evaluating the killing potential of Exo-iNSC-TK against GBM tumors

[0136] iNSC producer cells engineered with a hyperactive TK mutant has been developed. This variant contained 6 amino acid mutations in the wild-ty pe TK gene that confers 10-100x more effective killing of cancer cells than wild-type TK. Using co-culture assays, it was found that iNSC-based EV-TK, or Exo-iNSC- TK, enzyme/prodrug therapy killed GBM8 cells (FIG. 8). Similar to Exo-iNSC- TRAIL, Exo-iNSC-TK is an effective anti-caner or anti-tumor therapeutic. [0137] Discussion of Examples 1 to 8

[0138] Disclosed here, for the first time, are EVs derived from engineered iNSCs as effective drug carriers for pro-apoptotic agents, including TRAIL and TK, to improve its therapeutic efficacy in the treatment of brain cancers. As reported herein, iNSCs auto-load EVs with a significant proportion of the tumorocidal agent, e.g. TRAIL or TK, produced by those iNSCs, both within the lumen of the EVs as well as arrayed within the outer membrane. Exo-iNSC- TRAIL and Exo-iNSC-TK therapy significantly upregulated tumor apoptotic pathways and killing capacity against brain cancer cells in vitro, displayed selective accumulation in tumor cells ex vivo, and significantly increased survival in three orthotopic mouse models of human TNBC brain metastasis and GBM.

[0139] As shown herein, EVs can be an effective drug carrier to deliver anticancer products with poor pharmacokinetics. Initial goals of these experiments were to load high levels of soluble TRAIL into the lumen of the EVs to decrease clearance, increase stability⁷, and improve therapeutic efficacy. Surprisingly, and as shown herein, when the amount of TRAIL in EVs was quantified by ELISA, it was found that TRAIL was also detectable on the surfaces of intact EVs; in fact, this surface-bound TRAIL comprised about 75% of the total EV-associated TRAIL. The immunoelectronmicroscopy and flow cytometry data both confirm that TRAIL was presented on the outer membrane of iNSC-derived EVs. Since the transfected TRAIL structure is soluble format and without the transmembrane domain of the natural TRAIL, the exact mechanism of EV membrane binding is not clear. Without being bound by any particular theory or mechanism of action, one possibility⁷ is that the Fas ligand (CD95L/ApolL), which can be found both on the cell surface membrane and as a soluble protein and has a similar structure to TRAIL (aka Apo2L), can bind directly to fibronectin. Fibronectin is also found on EV surfaces, suggesting that this could be a membrane protein TRAIL can bind to in Exo-iNSC-TRAIL. Furthermore, membrane-bound Fas ligand shows increased cytotoxicity compared to soluble Fas [56], As disclosed herein, a similar phenomenon was observed: even in vitro, where no clearance occurs, Exo- iNSC-TRAIL is much more potent than free TRAIL (Fig 3C). This could be due to an enhanced multivalent interaction of TRAIL molecules on the surface of Exo- iNSC-TRAIL with DR4/5 on tumor cells, which could enhance DR4/5 clustering and in turn the tumor killing cascade. Exo-iNSC-TRAIL potency could be even further aided by the increased TRAIL stability afforded by association with the EV membrane (Fig 3E-G).

[0140] The observed tumor tropism of iNSC-derived EVs is another important aspect of their potential as drug delivery vehicles. This advantage is likely due to abundant surface-bound tumorocidal agent, e.g. TRAIL or TK, acting as a targeting ligand to cells with high DR4/5. Transdifferentiation ofNHFl cells into iNSCs imparts complex tumor-homing properties to this unique neural stem cell population, and while EVs are unable to “home” like living cells can, it is feasible that some relevant proteins could be transferred from iNSCs to Exo-iNSC-TRAIL which increase tumor tropism relative to Exo-NHFl -TRAIL.

[0141] To summarize, auto-loaded EVs derived from iNSCs are a promising and efficient treatment for brain cancer, particularly when used as local injection. Exo-iNSC-TRAIL and Exo-iNSC-TK can be easily produced, carries high levels of therapeutics, and improves anti-tumor efficacy compared to free TRAIL or free TK. In some instances, these therapeutic compounds, namely Exo-iNSC-TRAIL and Exo-iNSC-TK, or other Exo-iNSC therapeutics, can be use in combination.

[0142] Likewise, the present disclosure supports the ability to engineer iNSCs to generate EVs loaded with other therapeutic proteins and/or additional tumortargeting motifs, as well as generating personalized EVs from iNSCs transdifferentiated from an individual patient’s own skin.

[0143] References:

[0144] [1] B. Mann, X. Zhang, N. Bell, A. Adefolaju, M. Thang, R. Dasari, K. Kanchi. A. Valdivia. Y. Yang, A. Buckley, V. Lettry. C. Quinsey, Y. Rauf, D. Kram, N. Cassidy, C. Vaziri, D.L. Corcoran, S. Rego, Y. Jiang, L.M. Graves, D. Dunn, S. Floyd, A. Baldwin, S. Hingtgen, A.B. Satterlee, A living ex vivo platform for functional, personalized brain cancer diagnosis, Cell Rep. Med. 4 (2023) 101042.

[0145] [2] K. Aidape, K.M. Brindle, L. Chesler, R. Chopra, A. Gajjar, M.R. Gilbert, N. Gottardo, D.H. Gutmann, D. Hargrave, E.C. Holland, Challenges to curing primary brain tumours, Nat. Rev. Clin. Oncol. 16 (2019) 509-520. [0146] [3] Y. Xiong, C. He, X. Lin, K. Cheng, F. He, J. Zhao, M. Yang, H. Gao, F. He, X. Zhang, Black phosphorus nanosheets inhibit glioblastoma cell migration and invasion through modulation of WNT/p-catenin and NOTCH signaling pathways, Chem. Eng. J. (2024) 148614.

[0147] [4] J.R. Bago. K.T. Sheets, S.D. Hingtgen, Neural stem cell therapy for cancer, Methods 99 (2016) 37-43.

[0148] [5] S. Benedetti, B. Pirola, B. Polio. L. Magrassi, M.G. Bruzzone, D. Rigamonti, R. Galli, S. Selleri, F. Di Meco, C. De Fraja, Gene therapy of experimental brain tumors using neural progenitor cells, Nat. Med. 6 (2000) 447-450.

[0149] [6] D.W. Stuckey, K. Shah, Stem cell-based therapies for cancer treatment: separating hope from hype, Nat. Rev. Cancer 14 (2014) 683-691.

[0150] [7] J.R. Bago. A. Alfonso-Pecchio, O. Okolie, R. Dumitru, A. Rinkenbaugh, A.S. Baldwin, C.R. Miller, S.T. Magness, S.D. Hingtgen, Therapeutically engineered induced neural stem cells are tumour-homing and inhibit progression of glioblastoma, Nat. Commun. 7 (2016) 10593.

[0151] [8] J.R. Bago. O. Okolie, R. Dumitru. M.G. Ewend, J.S. Parker. R.V. Werff, T.M. Underhill, R.S. Schmid, C.R. Miller, S.D. Hingtgen, Tumorhoming cytotoxic human induced neural stem cells for cancer therapy, Sci. Transl. Med. 9 (2017) eaah6510.

[0152] [9] W. Jiang, Y. Yang, A.R. Mercer-Smith, A. Valdivia, J.R. Bago, A.S. Woodell, A. A. Buckley, M.H. Marand, L. Qian, C.K. Anders, S.D. Hingtgen, Development of next-generation tumor-homing induced neural stem cells to enhance treatment of metastatic cancers. Sci. Adv. 7 (2021) eabfl526.

[0153] [10] A.S. Woodell, E. Landoni, A. Valdivia, A. Buckley, E.A. Ogunnaike, G. Dotti, S.D. Hingtgen, Utilizing induced neural stem cell-based deliver}’ of a cytokine cocktail to enhance chimeric antigen receptor-modified T- cell therapy for brain cancer, Bioeng. Transl. Med. (2023) el 0538.

[0154] [11] A.B. Satterlee, D.E. Dunn, D C. Lo, S. Khagi, S. Hingtgen, Tumoricidal stem cell therapy enables killing in novel hybrid models of heterogeneous glioblastoma, Neuro Oncol. 21 (2019) 1552-1564. [0155] [12] K. Shah, Mesenchymal stem cells engineered for cancer therapy, Adv. Drug Deliv. Rev. 64 (2012) 739-748.

[0156] [13] W. Zakrzewski, M. Dobrzynski, M. Szymonowicz, Z. Rybak, Stem cells: past, present, and future, Stem Cell Res. Ther. 10 (2019) 1-22.

[0157] [ 14] C. Han, X. Sun, L. Liu, H. Jiang, Y. Shen, X. Xu, J. Li, G. Zhang, J. Huang, Z. Lin, Exosomes and their therapeutic potentials of stem cells, Stem Cells Int., 2016 (2016).

[0158] [ 15] D. Jafari, S. Shajari, R. Jafari, N. Mardi, H. Gomari, F. Ganji, M. Forouzandeh Moghadam, A. Samadikuchaksaraei, Designer exosomes: a new platform for biotechnology therapeutics, BioDrugs 34 (2020) 567-586.

[0159] [16] C. Liu, C. Su, Design strategies and application progress of therapeutic exosomes, Theranostics 9 (2019) 1015.

[0160] [17] L. Ding, X. Yang. Z. Gao, C.Y. Effah. X. Zhang, Y. Wu. L. Qu. A Holistic Review of the State-of-the-Art Microfluidics for Exosome Separation: An Overview of the Current Status, Existing Obstacles, and Future Outlook, Small 17 (2021) 2007174.

[0161] [18] B.S. Batista, W.S. Eng, K.T. Pilobello, K.D. Hendricks-Munoz, L.K. Mahal, Identification of a conserved glycan signature for microvesicles, J. Proteome Res. 10 (2011) 4624-4633.

[0162] [19] S. Mathivanan, R.J. Simpson, ExoCarta: A compendium of exosomal proteins and RNA, Proteomics 9 (2009) 4997-5000.

[0163] [20] E.R. Abels, X.O. Breakefield, Introduction to extracellular vesicles: biogenesis, RNA cargo selection, content, release, and uptake, Cell Mol. Neurobiol. 36 (2016) 301-312.

[0164] [21] R. Kalluri. V.S. LeBleu, The biolog}’, function, and biomedical applications of exosomes, Science 367 (2020) eaau6977.

[0165] [22] K. Zhang, K. Cheng, Stem cell-derived exosome versus stem cell therapy, Nat. Rev. Bioeng. (2023) 1-2.

[0166] [23] F. Tan, X. Li, Z. Wang, J. Li, K. Shahzad, J. Zheng, Clinical applications of stem cell-derived exosomes, Signal Transduct. Target. Ther. 9 (2024) 17. [0167] [24] F.J. Vizoso, N. Eiro, S. Cid, J. Schneider, R. Perez-Fernandez, Mesenchymal stem cell secretome: toward cell-free therapeutic strategies in regenerative medicine, Int. J. Mol. Sci. 18 (2017) 1852.

[0168] [25] Y. Wang, Y. Huo, C. Zhao, H. Liu, Y. Shao, C. Zhu, L. An, X. Chen. Z. Chen. Engineered exosomes with enhanced stability and delivery’ efficiency for glioblastoma therapy, J. Controlled Release, 368 (2024) 170-183.

[0169] [26] K. Ren, Exosomes in perspective: a potential surrogate for stem cell therapy, Odontology 107 (2019) 271-284.

[0170] [27] S. Hastuti, R. Idroes, I. Imran, Y. Ramli, A.H. Abas, T.E. Tallei, hUMSC vs. hUMSC-Exosome: Which One Is Better for Epilepsy?, Pharmaceuticals 15 (2022) 1247.

[0171] [28] A.D. Vogel, R. Upadhya, A.K. Shetty, Neural stem cell derived extracellular vesicles: attributes and prospects for treating neurodegenerative disorders, EBioMedicine 38 (2018) 273-282.

[0172] [29] C. Qian, Y. Wang, Y. Ji, D. Chen, C. Wang, G. Zhang, Y. Wang, Neural stem cell-derived exosomes transfer miR-124-3p into cells to inhibit glioma growth by targeting FLOT2, Int. J. Oncol. 61 (2022) 1-12.

[0173] [30] K. Aboody, J. Najbauer, M. Danks, Stem and progenitor cell- mediated tumor selective gene therapy. Gene Ther. 15 (2008) 739-752.

[0174] [31] T. Horiuchi, H. Mitoma, S.-i. Harashima, H. Tsukamoto, T.

Shimoda, Transmembrane TNF-a: structure, function and interaction with anti- TNF agents, Rheumatology 49 (2010) 1215-1228.

[0175] [32] R.J. Armitage, Tumor necrosis factor receptor superfamily members and their ligands, Curr. Opin. Immunol. 6 (1994) 407-413.

[0176] [33] P. Nair, M. Lu, S. Petersen. A. Ashkenazi. Apoptosis initiation through the cell-extrinsic pathway, Methods in enzymology, Elsevier 2014, pp. 99-128.

[0177] [34] S. Von Karstedt, A. Montinaro, H. Walczak, Exploring the TRAILs less travelled: TRAIL in cancer biology and therapy, Nat. Rev. Cancer. 17 (2017) 352-366.

[0178] [35] B. Thapa, K. Remant. H. Uludag, TRAIL therapy and prospective developments for cancer treatment, J. Controlled Release 326 (2020) 335-349. [0179] [36] H. Liu, D. Su, J. Zhang, S. Ge, Y. Li, F. Wang, M. Gravel, A. Roulston. Q. Song, W. Xu, Improvement of pharmacokinetic profile of TRAIL via trimer-tag enhances its antitumor activity in vivo, Sci. Rep. 7 (2017) 8953.

[0180] [37] M. Snajdauf. K. Havlova, J. Vachtenheim Jr, A. Ozaniak, R. Lischke, J. Bartunkova, D. Smrz, Z. Strizova, The TRAIL in the treatment of human cancer: an update on clinical trials, Front. Mol. Biosci. 8 (2021) 628332.

[0181] [38] A. Buckley, S.B. Hagler, V. Lettry, J.R. Bago, S.M. Maingi, S. Khagi, M.G. Ewend. C.R. Miller. S.D. Hingtgen, Generation and profiling of tumor-homing induced neural stem cells from the skin of cancer patients. Mol. Ther. 28 (2020) 1614-1627.

[0182] [39] J. El-Saghir, F. Nassar, N. Tawil, M. El-Sabban, ATL-derived exosomes modulate mesenchymal stem cells: potential role in leukemia progression, Retrovirology 13 (2016) 1-13.

[0183] [40] X. Zhang, X. Zhu. Y. Lu. l-Asparaginaseln situ encapsulated into zwitterionic nanocapsules with a prolonged half-Life, ACS Appl. Polym. Mater. 4 (2022) 2757-2766.

[0184] [41] L. Rivoltini, C. Chiodoni. P. Squarcina, M. Tortoreto, A. Villa, B. Vergani, M. Biirdek, L. Botti, I. Arioli, A. Cova, G. Mauri, E. Vergani, B.

Bianchi, P. Della Mina, L. Cantone, V. Bollati, N. Zaffaroni, A.M. Gianni, M.P. Colombo, V. Huber, TNF-Related Apoptosis-Inducing Ligand (TRAIL)-Armed Exosomes Deliver Proapoptotic Signals to Tumor Site, Clin. Cancer Res. 22 (2016) 3499-3512.

[0185] [42] D.R. Brademan, I. J. Miller, N.W. Kwiecien, D.J. Pagliarini, M.S. Westphall, J. J. Coon, E. Shishkova, Argonaut: A web platform for collaborative multi-omic data visualization and exploration. Patterns 1 (2020).

[0186] [43] B. Jassal, L. Matthews, G. Viteri, C. Gong, P. Lorente, A. Fabregat, K. Sidiropoulos, J. Cook. M. Gillespie, R. Haw, The reactome pathway knowledgebase, Nucleic Acids Res. 48 (2020) D498-D503.

[0187] [44] X. Zhang, D. Xu, X. Jin, G. Liu, S. Liang, H. Wang, W. Chen, X. Zhu, Y. Lu, Nanocapsules of therapeutic proteins with enhanced stability and long blood circulation for hyperuricemia management, J. Controlled Release. 255 (2017) 54-61. [0188] [45] J. Zhang. E.S. Park, H.-J. Park, R. Yan, M. Grudniewska, X.

Zhang, S. Oh, X. Yang, J. Baum, M.M. Mouradian, Apoptosis signal regulating kinase 1 deletion mitigates a-synuclein pre-formed fibril propagation in mice, NeurobioL Aging 85 (2020) 49-57.

[0189] [46] P. Li. M. Kaslan, S.H. Lee, J. Yao. Z. Gao, Progress in exosome isolation techniques, Theranostics 7 (2017) 789.

[0190] [47] K. Shah, Y. Tang, X. Breakefield, R. Weissleder, Real-time imaging of TRAIL-induced apoptosis of glioma tumors in vivo, Oncogene 22 (2003) 6865-6872.

[0191] [48] C.E. Badr, T. Wurdinger, J. Nilsson, J.M. Niers, M. Whalen, A. Degterev, B.A. Tannous, Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor-related apoptosis-inducing ligand and induces an alternative cell death pathway, Neuro oncol. 13 (2011) 1213-1224.

[0192] [49] C.E. Badr, T. Wurdinger, J. Nilsson. J.M. Niers, M. Whalen. A. Degterev, B.A. Tannous, Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor-related apoptosis-inducing ligand and induces an alternative cell death pathway, Neuro Oncol. 13 (2011) 1213-1224.

[0193] [50] H. LeBlanc, A. Ashkenazi, Apo2L/TRAIL and its death and decoy receptors, Cell Death Differ. 10 (2003) 66-75.

[0194] [51] A.B. Satterlee. D.E. Dunn, A. Valdivia, D. Malawsky, A. Buckley, T. Gershon, S. Floyd, S. Hingtgen, Spatiotemporal analysis of induced neural stem cell therapy to overcome advanced glioblastoma recurrence, Mol. Ther. Oncolytics 26 (2022) 49-62.

[0195] [52] A. Montinaro, H. Walczak, Harnessing TRAIL-induced cell death for cancer therapy: a long walk with thrilling discoveries, Cell Death Differ. 30 (2023) 237-249.

[0196] [53] S.K. Kelley, L A. Harris, D. Xie, L. DeForge, K. Totpal, J.

Bussiere, J. A. Fox, Preclinical studies to predict the disposition of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety, J. Pharmacol. Exp. Ther. 299 (2001) 31-38. [0197] [54] J. Lemke, S. von Karstedt, J. Zinngrebe, H. Walczak, Geting TRAIL back on track for cancer therapy. Cell Death Differ. 21 (2014) 1350- 1364.

[0198] [55] D. Chanda, E. Otoupalova, K.P. Hough, M.L. Locy, K. Bernard, J.S. Deshane, R.D. Sanderson, J. A. Mobley, V.J. ThannickaL Fibronectin on the surface of extracellular vesicles mediates fibroblast invasion, Am. J. Respir. Cell Mol. Biol. 60 (2019) 279-288.

[0199] [56] K. Aoki, M. Kurooka. J. -J. Chen, J. Petryniak, E.G. NabeL G.J. Nabel, Extracellular matrix interacts with soluble CD95L: retention and enhancement of cytotoxicity, Nat. Immunol. 2 (2001) 333-337.

[0200] [57] Z. Yuan, K.K. Kolluri, K.H. Gowers, S.M. Janes, TRAIL delivery by MSC-derived extracellular vesicles is an effective anticancer therapy, J. Extracell. Vesicles 6 (2017) 1265291.

[0201] [58] M.a.J. Martinez-Lorenzo. A. AneL S. Gamen. I. Monleon, P. Lasierra, L. Larrad, A.s. Pineiro, M.a.A. Alava, J. Naval, Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles, J. Immun. 163 (1999) 1274-1281.

[0202] [59] H. Liu, D. Su, J. Zhang, S. Ge, Y. Li, F. Wang, M. Gravel, A. Roulston, Q. Song, W. Xu, J.G. Liang, G. Shore, X. Wang, P. Liang, Improvement of Pharmacokinetic Profile of TRAIL via Trimer-Tag Enhances its Antitumor Activity’ in vivo, Sci. Rep. 7 (2017) 8953.

[0203] [60] D. De Miguel, A. Gallego-Lleyda, J.M. Ayuso, D. Pejenaute- Ochoa, V. Jarauta, I. Marzo, L.J. Fernandez, I. Ochoa, B. Conde, A. Anel, L. Martinez-Lostao, High-order TRAIL oligomer formation in TRAIL-coated lipid nanoparticles enhances DR5 cross-linking and increases antitumour effect against colon cancer. Cancer Lett. 383 (2016) 250-260.

[0204] [61] Z. Yuan, K.K. Kolluri, E.K. Sage, K.H. Gowers, S.M. Janes. Mesenchymal stromal cell delivery of full-length tumor necrosis factor-related apoptosis-inducing ligand is superior to soluble type for cancer therapy, Cytotherapy. 17 (2015) 885-896. [0205] [62] J. Teng, S. Hejazi. C.E. Badr, B.A. Tannous, Systemic anticancer neural stem cells in combination with a cardiac glycoside for glioblastoma therapy, Stem Cells. 32 (2014) 2021-2032. [0206] It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMS What is claimed is:

1. A therapeutic compound, the therapeutic compound comprising: an extracellular vesicle (EV), the EV comprising an exosome derived from transdifferentiation (TD)-derived induced neural stem cells (Exo-iNSCs); a therapeutic agent, the therapeutic agent comprising a tumoricidal gene product and/or an anti-cancer molecule, wherein the therapeutic agent is present in a lumen of the EV and/or is distributed across a membrane and/or surface of the EV.

2. The therapeutic compound of claim 1, wherein the induced neural stem cells are derived from skin fibroblasts.

3. The therapeutic compound of claim 1, wherein the tumoricidal gene product and/or anti-cancer molecule comprises TNF-related apoptosis-inducing ligand (TRAIL).

4. The therapeutic compound of claim 1, wherein the tumoricidal gene product and/or anti-cancer molecule comprises Herpes simplex virus thymidine kinase (TK).

5. The therapeutic compound of claim 3 or 4, wherein the tumoricidal gene product and/or anti-cancer molecule comprises both TRAIL and TK.

6. The therapeutic compound of any one of claims 1 to 5. wherein the EV has a tumor-homing capacity causing a selective migration to and/or attraction to tumors and/or cancer cells imparted by the induced neural stem cells (iNSCs).

7. The therapeutic compound of any one of claims 1 to 6. wherein the tumoricidal gene product and/or an anti-cancer molecule of the EV has increased tumor-selective accumulation and increased therapeutic activity as compared to free TRAIL and/or free TK.

8. The therapeutic compound any one of claims 1 to 7, wherein the exosome of the EV generated by TD-iNSC is produced with the therapeutic agent present in a lumen of the EV and/or distributed across the membrane and/or surface of the EV.

9. The therapeutic compound of any one of claims 3, 5 or 7, wherein the EV comprises a concentration of TRAIL present in the lumen, and/or distributed across the membrane and/or surface of the EV at about 100 pg/ug EV to about 500 pg/ug EV, optionally about 250 pg/ug EV to about 400 pg/ug EV.

10. The therapeutic compound of any one of claims 1-9, wherein about 75% or more of the therapeutic agent is distributed across the membrane of the EV.

11. The therapeutic compound of any one of claims 1-10, wherein the therapeutic compound is configured to treat a cancer or tumor, optionally a brain cancer or brain tumor, optionally a glioblastoma.

12. The therapeutic compound of any one of claims 1-11, wherein the EV further comprises other therapeutic proteins and/or additional tumor-targeting motifs.

13. A therapeutic composition comprising the therapeutic compound of any one of claims 1-12 and a excipient, carrier or buffer, and configured to be administered to a subject.

14. A method of producing the therapeutic compound of any one of claims 1-12, the method comprising genetically engineering iNSCs to generate one or more extracellular vesicles (EVs) comprising exosomes loaded with the therapeutic agent, wherein the therapeutic agent comprises a tumoricidal gene product and/or an anti-cancer molecule, wherein the therapeutic agent is present in a lumen of the EVs and/or is distributed across a membrane and/or surface of the EVs.

15. The method of claim 14, wherein genetically engineering iNSCs to generate one or more EVs comprises transdifferentiating fibroblstats to produce iNSCs followed by transducing iNSCs to produce the tumoricidal gene product and/or an anti -cancer molecule as part of the EVs.

16. The method of claim 14 or 15, wherein the EVs are isolated and formulated into a therapeutic composition.

17. The method of claim 14, wherein the therapeutic agent is loaded in the one or more EVs at about twice the rate as parent fibroblasts of the one or more EVs.

18. The method of any one of claims 14-17, comprising generating personalized EVs from iNSCs transdifferentiated from an subject patient’s own skin.

19. A method of treating a subject, the method comprising administering to a subject in need a therapeutic dose of the compound of any one of claims 1-12 or composition of claim 13.

20. The method of claim 19, wherein the subject is a human subject suffering from a cancer, tumor or related condition.

21. The method of claim 20, wherein the cancer is a brain cancer or brain tumor, optionally a glioblastoma.