RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

62/754,445 filed on November 1, 2018, which is incorporated herein by reference in its entirety.

SUMMARY

[0002] Disclosed herein, in certain embodiments, are methods of treating ovarian cancer in an individual in need thereof, comprising: administering to the individual an induced tumor-homing drug carrier cell (iTDC) that homes to an ovarian cancer tumor and expresses Sox2 and a therapeutic payload, whereby the cell expresses the therapeutic payload at an ovarian cancer tumor and treats the ovarian cancer. In some embodiments, the iTDC is not an induced pluripotent stem cell (iPSC) or an induced neural stem cell (iNSC). In some embodiments, the therapeutic payload comprises TRAIL. In some embodiments, the therapeutic payload comprises s-TRAIL. In some embodiments, the therapeutic payload comprises thymidine kinase (TK). In some embodiments, the therapeutic payload comprises s-TRAIL and TK. In some embodiments, the method further comprises administering to the individual a therapeutically effective amount of ganciclovir or valganciclovir. In some embodiments, the method further comprises administering an additional therapeutic agent to the individual. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is an alkylating agent, an anti -angiogenic agent, an intercalating agent, a thymidylate synthetase inhibitor, a topoisomerase inhibitor, and/or a PARP inhibitor. In some embodiments, the chemotherapeutic agent is melphalan, bevacizumab, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin hydrochloride, doxorubicin hydrochloride liposome, gemcitabine hydrochloride, topotecan hydrochloride, olaparib, niraparib tosylate monohydrate, niraparib tosylate monohydrate, rucaparib camsylate, paclitaxel, taxol, thiotepa, bleomycin sulfate, etoposide phosphate, and/or vinblastine. In some embodiments, the iTDC is cultured in the presence of a progenitor medium. In some embodiments, the progenitor medium is a neural progenitor medium. In some embodiments, the iTDC is transdifferentiated from a somatic cell autologous to the individual. In some embodiments, the iTDC is produced by a method comprising genetically reprogramming a somatic cell into an induced tumor-homing drug carrier cell. In some embodiments, the somatic cell is isolated from a tissue, a blood sample, a bone marrow sample or a body fluid extracted from the individual. In some embodiments, the somatic cell is a fibroblast. In some embodiments, the fibroblast is a skin fibroblast. In some embodiments, the ovarian cancer is epithelial ovarian cancer. In some embodiments, a tumor growth of the ovarian cancer is reduced or inhibited. In some embodiments, the exogenous Sox2 is encoded by a recombinant nucleic acid. In some embodiments, the recombinant nucleic acid comprises a viral vector.

[0003] Disclosed herein, in certain embodiments, are pharmaceutical compositions, comprising: (a) an isolated and purified induced tumor-homing drug carrier cell (iTDC) expressing (i) exogenous Sox2 and (ii) a therapeutic payload, and (b) a pharmaceutically-acceptable excipient. In some embodiments, the iTDC is not an induced pluripotent stem cell (iPSC) or an induced neural stem cell (iNSC). In some embodiments, the therapeutic agent expressed by the iTDC is TRAIL, s-TRAIL, and/or thymidine kinase (TK). In some embodiments, the pharmaceutical composition comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is not expressed by the iTDC. In some embodiments, the pharmaceutical composition comprises a cryoprotectant. In some embodiments, the exogenous Sox2 is encoded by a recombinant nucleic acid. In some embodiments, the recombinant nucleic acid comprises a viral vector.

[0004] Disclosed herein, in certain embodiments, are induced tumor-homing drug carrier cells (iTDC), comprising (a) an exogenous nucleic acid sequence encoding Sox2, and (ii) an exogenous nucleic acid sequence encoding a therapeutic payload, wherein the iTDC is not a pluripotent stem cell or an induced neural stem cell. In some embodiments, the iTDC is isolated and purified. In some embodiments, the therapeutic agents is TRAIL, s-TRAIL and/or thymidine kinase (TK).

[0005] Disclosed herein, in certain embodiments, are induced tumor-homing drug carrier cells (iTDC) produced by a method comprising transfecting a somatic cell with an exogenous nucleic acid sequence encoding a transdifferentiation factor, and culturing the transfected somatic cell in a progenitor medium, thereby transforming the somatic cell into an induced tumor-homing drug carrier cell, wherein the iTDC is not a pluripotent stem cell or an induced neural stem cell. In some embodiments, the method further comprises transfecting the somatic cells with an exogenous nucleic acid sequence encoding a therapeutic payload. In some embodiments, the therapeutic agents is TRAIL, s-TRAIL and/or thymidine kinase (TK). In some embodiments, the progenitor medium in a neural progenitor culture medium.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1 shows fluorescence images of therapeutic iTDCs or non-therapeutic controls engineered to express green fluorescent protein (GFP) and co-cultured with human ovarian cancer cells expressing mCherry-luciferase. [0007] FIG. 2 shows a summary graph of the data, plotting the surface photon emission (y-axis) of the images therapeutic iTDCs or non-therapeutic controls.

[0008] FIG. 3 shows serial bioluminescence images of mice ovaries in which firefly luciferase ovarian cancer cells were orthotopically implanted, along therapeutic iTDCs or non-therapeutic controls.

[0009] FIG. 4 shows summary graphs generated from the bioluminescent imaging software, plotting the tumor volume in the mice (y axis) over 20 days (x axis).

[0010] FIG. 5 shows white light and fluorescence photomicrographs of human fibroblasts and iTDCs grown as monolayers and neurospheres, or stained with antibodies against nestin (green).

[0011] FIG. 6 shows a summary graph plotting the expression of nestin over time at different days after induction of iTDC generation. Data are shown as means ± SEM (error bars are from three to four independent experiments; n = 3 technical replicates). Scale bars are 200 pm.

“GAPDH” signifies glyceraldehyde-3 -phosphate dehydrogenase.

[0012] FIG. 7 shows immunofluorescence staining images showing iTDC-GFP (green) expression of the transdifferentiation marker nestin (red) and GFAP+ astrocytes and TUJ-1+ neurons after differentiation by mitogen removal (staining shown in red). In contrast, no staining was observed for the pluripotency markers TRA-l-60 or OCT-4. Hoechst staining is shown in blue. Fluorescence images showing only the red (555 nm) secondary antibody channel are shown in the bottom row.

[0013] FIGS. 8A-8D shows RT-PCR analysis charts of nestin, SOX2, nanog, and OCT-4 expression in NHF, and iTDC. Data are shown as means ± SEM (error bars are from three to four independent experiments; n = 3 technical replicates). Scale bars are 200 pm. GAPDH signifies glyceraldehyde-3 -phosphate dehydrogenase.

[0014] FIG. 9 shows time-lapse fluorescent images in which iTDC-mC-FL were seeded 500 pm away from mCherry (mC)-expressing human glioblastoma (GBM) cells and placed in a fluorescence incubator microscope. Time-lapse fluorescence images were captured every 20 min for 22 hours and used to construct movies that revealed the migration of iTDC to GBM in real time. FIG. 9 shows summary images showing migration of iTDC-mC-FL (red) (A) or parental human fibroblasts (B) toward U87-GFP-FL (green) at 0 and 22 hours after plating. FIG 9 also shows single-cell tracings depicting the paths of iTDC-mC-FL or human fibroblast-directed migration toward GBM over 22 hours.

[0015] FIG. 10 shows summary graphs plotting the directionality and Euclidean distance of iTDC or fibroblast migration toward GBM cells determined from the real-time motion analysis. [0016] FIG. 11 shows fluorescence images of the migration of iTDC-mC-FL (red) into U87 spheroids (green) and their penetration toward the core of the tumor spheroid over time in 3D levitation culture systems.

[0017] FIG. 12 are bioluminescent images of iTDC-mC-FL implanted into the frontal lobes of mice taken over 3 weeks. FIG. 12 also shows a summary graph generated from the

bioluminescent imaging software, plotting the tumor volume in the mice (y axis) over 20 days (x axis).

[0018] FIGS. 13 A and 13B show bioluminescence images of iTDC-mC-FL implanted into the frontal lobes of mice over a period of 3 weeks.

[0019] FIG. 14 shows images and summary data of 3D suspension cultures showing the viability of mCherry+ human U87 GBM spheroids (red) mixed with therapeutic iTDC-sTR or control cells at a ratio of 0.5 : 1 or 1 : 1. GBM spheroid viability was determined by BLI 48 hours after treatment. **P = 0.0169, *P = 0.038 by ANOVA.

[0020] FIGS. 15A and 15B show representative BLI and summary data demonstrating the inhibition of solid U87 GBM progression by iTDC -sTR therapy compared to control -treated mice. *P = 0.0044 by repeated-measures ANOVA.

[0021] FIGS. 16 A, 16B, and 16C show representative images demonstrating the expression of cytotoxic, differentiation, and pluripotency markers in iTDC-sTR after therapy. A subset of animals were sacrificed 14 days after therapy; brain sections were stained with antibodies against nestin, TRAIL, GFAP, TUJ-l, OCT-4, or TRA-l-60; and the colocalization between staining (magenta) and GFP+ iTDC -sTR (green) was visualized.

[0022] FIG. 17 shows two different 3D culture models modeling the antitumor effects of iTDC -TK therapy. iTDC-TK (red) were either mixed GFP+ GBM4 patient-derived GBM cells or seeded adjacent to established GBM4 spheroids, and GCV was added to initiate tumor killing. The top panel shows fluorescence images of the mixed therapy, and the bottom shows fluorescence images of the established GBM4 spheroids. Serial fluorescence images showed the time-dependent decrease in GBM4 spheroid volume by iTDC -TK+ GCV therapy. Figure 17 also shows a summary graph demonstrating the reduction in GBM4 spheroid volume over 7 days by iTDC-TK+ GCV therapy either mixed or seeded adjacent to established spheroids.

[0023] FIG. 18A shows bioluminescent images of iTDC-TK therapy that was assessed in vivo by injecting iTDC-TK cells into GBM tumors established 10 days earlier in the brains of mice. FIG. 18B shows Kaplan-Meier survival curves demonstrating the survival of mice bearing GBM tumors treated with iTDC-TK+ GCV therapy or control iTDC. [0024] FIGS. 19A and 19B show representative whole-brain and high-magnification images showing cell nuclei (blue), GBM4 (green), and iTDC-TK (red) distribution 21 days after delivering iTDC-control (I) or iTDC-TK (J) into established GBM tumors. A large GBM tumor was present in the control iTDC-TK animals, and only a small GBM focus was detected in mice treated with iTDC-TK+ GCV.

[0025] FIGS. 20A and 20B show fluorescence imaging of 3D suspension cultures used to assess the migration and antitumor efficacy of sECM-encapsulated iTDC against patient-derived GBM spheroids and summary data.

[0026] FIGS. 21 A and 21B show representative images and summary data for serial imaging demonstrating the inhibition of tumor recurrence after intracavity iTDC-TK therapy for postoperative minimal GBM8 tumors. FIG. 21C shows Kaplan-Meier survival curves of mice that underwent surgical resection of diffuse patient-derived GBM tumor cells and were treated with control iTDC or iTDC-TK encapsulated in sECM and transplanted into the surgical cavity

DETAILED DESCRIPTION

[0027] While preferred embodiments of the subject matter disclosed herein have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the subject matter disclosed herein. It should be understood that various alternatives to the embodiments of the subject matter disclosed herein may be employed in practicing the subject matter disclosed herein. It is intended that the following claims define the scope of the subject matter disclosed herein and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Definitions

[0028] Throughout this application, various embodiments of this disclosure 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. [0029] The term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example,“about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively,“about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term“about” meaning within an acceptable error range for the particular value should be assumed.

[0030] The terms“subject,”“individual,”“host,”“donor,” and“patient” are used

interchangeably herein to refer to a vertebrate, for example, a mammal. Mammals include, but are not limited to, murine (e.g., mice and rats), simians, humans, farm animals (e.g., livestock and horses), sport animals, and pets (e.g., dogs and cats). Subjects may be of any age, including infant, juvenile, adolescent, adult, and geriatric subjects. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. Designation as a “subject,”“individual,”“host,”“donor,” or“patient” does not necessarily entail supervision of a medical professional.

[0031] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms“a”,“an” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms“including”,“includes”,“having”,“has”,� ��with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term“comprising.”

[0032] As used herein, the term“therapeutically effective amount” refers to an amount of an immunological cell or a pharmaceutical composition described herein that is sufficient and/or effective in achieving a desired therapeutic effect in treating a patient having a pathogenic disease. In some embodiments, a therapeutically effective amount of the iTDC will avoid adverse side effects.

[0033] As used herein, the terms“allogeneic” or“allogenic” means the plurality of iTDCs are obtained from a genetically non-identical donor. For example, allogenic iTDCs are extracted from a donor and returned back to a different, genetically non-identical recipient. [0034] As used herein, the term“autologous” means the plurality of iTDCs are obtained from a genetically identical donor. For example, autologous iTDCs are extracted from a patient and returned back to the same, genetically identical patient.

[0035] As used herein, the terms“transdifferentiation” or“transdifferentiating” refer to a method in which differentiated somatic cells are directly converted to differentiated or multipotent somatic cells of a different lineage without passing through an intermediate pluripotent stem cell (iPSC) stage.

[0036] As used herein, the term“transdifferentiation factor” refers to a protein such as a transcription factor that promotes the direct conversion of one somatic cell type to another.

Examples include, but are not limited to, Oct4, Sox2, Klf4, Myc, Ascll, Brn2, Mytl 1, Olig2, Zicl, or any combinations thereof.

[0037] As used herein, the terms“treat” or“treatment” refer to any type of treatment that imparts a benefit to a patient afflicted with a disease or disorder. Non-limiting examples of the disease or disorder include a cancer, a neurodegenerative disorder, and a neural trauma. Non limiting examples of the benefit imparted to a patient afflicted with a disease or disorder include improvement in the condition of the patient (e.g., in one or more symptoms), a delay in the progression of the disease or disorder, and a delay in an onset or a recurrence of the disease.

[0038] As used herein, the term“transfecting” is the transfer of heterologous genetic material into a cell, often through the use of a vector (e.g., molecule used as a vehicle to carry foreign genetic material into another cell). Methods of transfecting eukaryotic cells are known, and may include, but are not limited to, electroporation, use of cationic liposome-based reagents, nanoparticle-based reagents, polymeric-based reagents, polymeric and liposomal-based reagents, or any combination thereof.

[0039] As used herein, the term“transducing” is the transfer of heterologous genetic material into a cell by means of a virus. Such viral vectors are known and may include, but are not limited to, lentiviral vectors, adenoviral vectors, retroviral vectors, or any combination thereof.

Methods for Making Induced Tumor-Homing Drug Carrier Cell (iTDC)

[0040] The emergence of cellular reprogramming has opened new avenues in cell therapies, but suffers from limitations. For example, the de-differentiation of a fibroblast into an induced pluripotent stem cell and then re-differentiation to a desired therapeutic cell type is a time- consuming process. Further, the efficiency of iPSC generation is low, and significant safety concerns remain regarding the formation of cancerous teratomas by transplanted iPSCs or derivatives. [0041] Neural stem cell generation can be accomplished in 20 days by partially reprogramming human fibroblasts towards iPSCs using the four Yamanaka factors. Neural stem cell generation can also be achieved by expressing Sox2 in fibroblasts, but this strategy requires culturing on specific feeder cells for 40 days to obtain neural stem cell expansion and passaging. However, in some instances, there is a need for alternative methods that can rapidly (e.g., in less than 40 days) provide, for example, engineered iTDCs, for use in cell-based therapies. Tumor-homing iTDCs have been created by transdifferentiation, termed induced tumor-homing drug carrier cells.

[0042] Transdifferentiation is a method in which cells (e.g., somatic cells) are directly converted to differentiated somatic cells of a different lineage without passing through an intermediate iPSC stage. This direct conversion by transdifferentiation obviates the safety concerns associated with the iPSC state and allows faster generation of the desired therapeutic cell type. Induced tumor-homing drug carrier cells are, for example, induced (e.g., derived by

reprogramming) cells which preferentially accumulate at (e.g., home to, migrate to) tumor tissues or tumor cells and which express a therapeutic payload (e.g., thymidine kinase, TRAIL, s-TRAIL).

[0043] Provided herein, according to some embodiments, are methods for producing an induced tumor-homing drug carrier cell (iTDC), comprising: (a) introducing (e.g., by transfecting or transducing) a nucleic acid encoding Sox2 into a somatic cell, whereby said cell expresses Sox2; and (b) transdifferentiating said somatic cell in the presence of a progenitor medium (e.g., a neural progenitor medium), thereby producing an iTDC expressing Sox 2 and capable of homing to a tumor.

Cells

[0044] In some embodiments, the somatic cell is a fibroblast cell (e.g, a skin fibroblast cell). In some embodiments, ddifferentiated somatic cells are collected from any accessible source, such as tissue, bodily fluids (e.g., blood, urine), etc. For example, in some embodiments, skin cells are collected from the border of a surgical incision, e.g., during an accompanying surgical procedure, or using a traditional skin punch as a stand-alone procedure. Skin could be collected from any area, including, but not limited to, collection from the scalp or forearm.

Transdifferentiation

[0045] In some embodiments, transdifferentiation is carried out by exposing the cells to one or more transdifferentiation factors and/or growing the cells in a medium that promotes

transdifferentiation into the desired cell type. [0046] In some embodiments as taught herein, the transdifferentiating is carried out without the use of feeder cells, e.g., in a neural progenitor medium. Feeder cells, as known in the art, are additional cells grown in the same culture dish or container, often as a layer (e.g., a mouse fibroblast layer on the culture dish) to support cell growth.

Transdifferentiation Factors

[0047] In some embodiments, transdifferentiation is single-factor transdifferentiation in that only one transdifferentiation factor is used. Non-limiting examples of factors include Oct4, Sox2, Klf4, Myc, Ascll, Bm2, Mytl 1, Olig2, Zicl, or any combinations thereof.

[0048]“ Sox2” is a member of the Sox family of transcription factors and is expressed in developing cells in the neural tube as well as in proliferating progenitor cells of the central nervous system. In some embodiments, Sox2 is used as the transdifferentiation factor in the methods taught herein. In some embodiments, Sox2 is used to carry out a single-factor transdifferentiation.

[0049]“Nestin” is expressed predominantly in stem cells of the central nervous system, and its expression is typically absent from differentiated central nervous cells.“GFAP” or“glial fibrillary acidic protein,” is an intermediate filament protein expressed by central nervous system cells, including astrocytes.“Tuj-l” or“beta tubulin” is a neural marker.

[0050]“Nanog” and“OCT3/4” are known stem cell markers.

[0051] In some embodiments, the method comprises transducing said somatic cell with a lentiviral vector comprising said nucleic acid encoding Sox2.

Culture Medium

[0052] In some embodiments, somatic cells (for example those expressing Sox2), and/or the iTDCs cells are cultured in a progenitor medium, such as a neural progenitor medium.

“Progenitor medium” as used herein is a medium or media, for example, incorporating supplements, small molecule inhibitors, and growth factors, that promotes the

transdifferentiation of somatic cells into neural stem cells.

[0053] In some embodiments, the neural progenitor medium includes one or more ingredients selected from: a cell culture medium containing growth-promoting factors and/or a nutrient mixture (e.g., DMEM/F12, MEM/D-valine, neurobasal medium etc., including mixtures thereof); media supplements containing hormones, proteins, vitamins and/or amino acids (e.g., N2 supplement, B27 supplement, non-essential amino acids (NEAA), L-glutamine, Glutamax, BSA, insulin, all trans retinoic acid, etc. including mixtures thereof); and optionally small molecule inhibitors (e.g., SB431542 (BMP inhibitor), LDN193189 (TGF-f3 l inhibitor), CHIR99021 (GSK3f3 inhibitor), etc., including mixtures thereof). In some embodiments, ingredients also include one or more of beta-mercaptoethanol, transferrin; sodium selenite; and cAMP. In some embodiments, suitable concentrations of each of these ingredients are known to those of skill in the art and/or are empirically determined. Example concentrations of ingredients is also provided in Example 5 below. In some embodiments, the neural progenitor medium is a premade medium, such as STEMdiff™ Neural Induction Medium (STEM CELL TM

Technologies, Vancouver, British Columbia, Canada).

Payloads

[0054] In some embodiments, the method further comprises transducing the iTDC with a nucleic acid encoding therapeutic payload or a reporter molecule. In some embodiments, iTDCs as taught herein are loaded with (e.g., contain) a therapeutic payload, a reporter molecule and/or a nucleic acid capable of expressing the same. In some embodiments, the therapeutic agent is a protein toxin (e.g., a bacterial endotoxin or exotoxin), an oncolytic virus (e.g., a conditionally replicative oncolytic adenovirus, reovirus, measles virus, herpes simplex virus (e.g., HSV1716), Newcastle disease 15 virus, vaccinia virus, etc.), or a pro-apoptotic agent (e.g., secretable tumor necrosis factor (TNF)-related apoptosis-inducing ligand (S-TRAIL)). TRAIL is a member of the tumor necrosis factor (TNF) cytokine family. In some instances, TRAIL activates rapid apoptosis in ovarian tumor cells. S-TRAIL, a secreted form of TRAIL, in some instances exerts more potent apoptotic effects (e.g., compared to TRAIL) when delivered by the iTDCs.

[0055] In some embodiments, the therapeutic payload comprises a pro-inflammatory protein such as an interleukin, cytokine, or antibody.

[0056] In some embodiments, the therapeutic payload comprises an enzyme useful for enzyme/prodrug therapies (e.g., thymidine kinase (e.g., with ganciclovir prodrug),

carboxylesterase (e.g., with CTP-l l), cytosine deaminase, etc.). In some instances, when expressed in cells, thymidine kinase enzymatically cleaves ganciclovir and subsequently transforms the ganciclovir into a cytotoxic agent

[0057] In some embodiments, the therapeutic comprises an RNAi molecule such as miRNA or siRNA.

[0058] In some embodiments, the iTDCs are loaded with a therapeutic payload used for the treatment of cancer. In some embodiments, the therapeutic payload used for the treatment of ovarian cancer is a chemotherapeutic agent, as described elsewhere herein. In some

embodiments, the therapeutic payload used for the treatment of ovarian cancer is a diagnostic therapeutic agent. In some embodiments, the therapeutic payload used for the treatment of ovarian cancer is an imaging agent. In some embodiments, the imaging agent is 2-Deoxy-2- ¹⁸ F- fluoroglucose (FDG), Sodium ¹⁸ F-fluoride (NaF), Anti- 1 -amino-3 - ¹⁸ F-fluorocyclobutane-l- carboxcylic acid ( ¹⁸ F-fluciclovine, FACBC), ^99m Tc-methoxyisobutylisonitrile ( ^99m Tc-sestamibi), 3'-deoxy-3'- ¹⁸ F-fluorothymidine (FLT), l6a- ¹⁸ F-fluoro-l7P-estradiol (FES), 2l- ¹⁸ F-fluoro- l6a, l7a-[(R)-(r-a-furylmethylidene)dioxy]-l9-norpregn-4-ene-3,20 -dione (FFNP), or any combinations thereof.

[0059] In some embodiments, the iTDCs are loaded with nanoparticle/drug conjugates.

[0060] Reporter molecules are known in the art and include, but are not limited to, Green Fluorescent Protein, f3-galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase gene.

[0061] In some embodiments, loading of the iTDCs with a payload is accomplished using art- known methods, such as transfecting the iTDCs with a nucleic acid capable of producing a therapeutic or reporter protein, transducing the iTDCs with a viral vector, lipid-based or polymeric loading of the cells with a therapeutic payload and/or reporter molecule, etc.

Methods of Treating Ovarian Cancer

[0062] Disclosed herein, in certain embodiments, are methods of treating ovarian cancer in an individual in need thereof. In some embodiments, the method comprising administering said iTDC to the subject. In some embodiments, the iTDC is allogeneic with respect to said subject. In some embodiments, the iTDC is syngeneic with respect to said subject. In some

embodiments, the iTDC is autologous with respect to said subject.

[0063] In some embodiments, the ovarian cancer is epithelial ovarian cancer. In some embodiments, the ovarian cancer is metastatic. In some embodiments, the ovarian cancer is non- invasive. In some embodiments, the ovarian cancer is invasive. In some embodiments, the ovarian cancer is a stage I, a stage II, a stage III, or a stage IV ovarian cancer. In some embodiments, the ovarian cancer is an ovarian germ cell tumor. In some embodiments, the ovarian germ cell tumor is a teratoma, a dysgerminoma, an endodermal sinus tumor, a choriocarcinoma, or any combinations thereof. In some embodiments, the ovarian cancer is a sex cord-stromal tumor, an ovarian sarcoma, a Krukenberg tumor, an ovarian cyst that develops into an ovarian cancer, or any combination thereof. In some embodiments, the ovarian cancer is an ovarian stromal tumor. In some embodiments, the ovarian stromal tumor is a granulosa-theca tumor, a Sertoli-Leydig cell tumor, a granulosa cell tumor, a small cell carcinoma of the ovary, a primary peritoneal carcinoma, or any combinations thereof. In some embodiments, the ovarian cancer is an epithelial ovarian cancer, a germ cell tumor, a stromal cell tumor, a steroid cell tumor, or a combination thereof. In some embodiments, the ovarian cancer is a small cell ovarian carcinoma, a neuro-endocrine carcinoma, a squamous cell carcinoma rising within a dermoid cyst, a struma ovarii malignum, a psammocarcinoma, or any combinations thereof. In some embodiments, the epithelial ovarian cancer is a high-grade serous ovarian cancer, a low- grade serous ovarian cancer, a mucinous ovarian cancer, an ovarian endometrioid cancer, a clear cell ovarian cancer, an unclassified ovarian cancer, an undifferentiated ovarian cancer, a Brenner tumor, a borderline tumor, a carcinosarcoma, or any combinations thereof. In some

embodiments, the germ cell tumor is a dysgerminoma, a teratoma, an ovarian yolk sac tumor, a mixed germ cell tumor, an embryonal carcinoma, a polyembryoma, or any combinations thereof. In some embodiments, the stromal cell tumor is an ovarian stromal tumor with sex cord elements, an adult type granulosa cell tumor, a juvenile type granulosa cell tumor, an

androblastoma, a gynandroblastoma, a sex cord tumor with annular tubules, a thecoma, a fibroma, a fibrosarcoma, a sclerosing stromal tumor, a Signet-ring stromal tumor, a microcystic stromal tumor, an ovarian yxo a, or any combinations thereof. In some embodiments, the steroid cell tumor is a stromal luteoma, a Leydig cell tumor, or any combinations thereof. In some embodiments, the iTDCs are obtained by any method described herein. In some embodiments, the iTDC is produced by a method comprising directly genetically

reprogramming a somatic cell into an induced tumor-homing drug carrier cell (iTDC), without an intermediate stem cell phase. In some embodiments, the somatic cell is isolated from a tissue, a blood sample, a bone marrow sample or a body fluid extracted from the individual. In some embodiments, the somatic cell is a fibroblast. In some embodiments, the fibroblast is a skin fibroblast.

[0064] In some embodiments, the iTDCs are transdifferentiated ex vivo before administration to the individual. In some embodiments, the iTDCs are autologous to the individual. In some embodiments, the iTDCs are allogenic.

[0065] In some embodiments, the iTDCs are fresh, i.e., not frozen or previously frozen. In some embodiments, the iTDCs are cryopreserved (frozen). In some embodiments, the iTDCs are frozen and stored for later use (for example to facilitate transport). In some embodiments, the frozen iTDCs are administered to the individual after being thawed.

[0066] In some embodiments, administration of the iTDCs is performed using methods known in the art. For example, in some embodiments, intravenous administration of the cells is performed for the treatment of an ovarian cancer. In some embodiments intraperitoneal administration of the cells is performed for the treatment of an ovarian cancer. In some embodiments, intratumoral administration or intracavity administration is performed after surgical removal of at least a part of an ovarian tumor. [0067] In some embodiments, the cells are encapsulated by a matrix such as a hydrogel matrix (e.g., a synthetic extracellular matrix) and/or seeded onto a scaffold, which is then be administered or implanted, e.g., intratumorally.

Combination Therapies

[0068] Disclosed herein, in certain embodiments, are methods of treating an ovarian cancer, comprising: administering to the individual (a) an isolated and purified iTDC expressing (i) exogenous Sox2 and (ii) a therapeutic payload, and (b) an exogenous therapeutic agent.

[0069] In some embodiments, a method of treating a ovarian cancer in an individual in need thereof, comprises: administering iTDCs produced by any method described herein, and an additional therapeutic agent.

Chemotherapeutics

[0070] In some embodiments, the exogenous therapeutic agent is a chemotherapeutic agent.

In some embodiments, the iTDCs are administered prophylactically in combination with the exogenous chemotherapeutic agent in order to treat an ovarian cancer and/or a tumor.

[0071] In some embodiments, the exogenous chemotherapeutic agent is an alkylating agent, an anti -angiogenic agent, an anthracycline, a cytoskeletal disruptor, an epothilone, a histone deacetylase inhibitor, an intercalating agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a thymidylate synthetase inhibitor, a PARP inhibitor, a kinase inhibitor, a nucleotide analog, a precursor analog, a peptide antibiotic, a platinum-based agent, a retinoid, or a vinca alkaloid. In some embodiments, chemotherapeutic agents include: actinomycin, albumin bound paclitaxel, altretamine, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, liposomal doxorubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, tioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, bevacizumab, doxorubicin hydrochloride, doxorubicin hydrochloride liposome, gemcitabine hydrochloride, topotecan hydrochloride, olaparib, niraparib tosylate monohydrate, niraparib tosylate monohydrate, rucaparib camsylate, taxol, thiotepa, bleomycin sulfate, etoposide phosphate, and/or vinblastine. Pharmaceutical Compositions

[0072] Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising: (a) an isolated iTDC disclosed herein; and (b) a pharmaceutically-acceptable excipient.

[0073] In some embodiments, a pharmaceutical composition includes one population of iTDCs, or more than one, such as two, three, four, five, six or more populations of iTDCs.

[0074] In some embodiments, the components of the pharmaceutical compositions described herein are administered either alone or in combination with pharmaceutically acceptable carriers, excipients, or diluents, in a pharmaceutical composition. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that are used pharmaceutically. Pharmaceutically-acceptable excipients included in the pharmaceutical compositions will have different purposes depending, for example, on the type of iTDCs used and the mode of administration. Non-limiting examples of generally used pharmaceutically- acceptable excipients include, without limitation: saline, buffered saline, dextrose, water-for- injection, glycerol, ethanol, dextran (e.g., low molecular dextran such as Dextran 40),

PlasmaLyte, human serum albumin (HSA), and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives (such as dimethylsulfoxide (DMSO)), tonicity agents, bulking agents, and lubricating agents. The formulations comprising populations of iTDCs are prepared and cultured in the absence of any non-human components, such as animal serum.

[0075] In some embodiments, the pharmaceutical compositions further comprise a

cryoprotectant or a cryopreservative. In some embodiments, the cryoprotectant or the cryopreservative is selected from dimethylsulfoxide (DMSO), formamide, propylene glycol, ethylene glycol, glycerol, trehalose, 2-methyl-2,4-pentanediol, methanol, butanediol, or any combination thereof.

[0076] Pharmaceutical compositions comprising: (a) an isolated iTDC; and (b) a

pharmaceutically-acceptable excipient are administered to a subject using modes and techniques known to the skilled artisan. Exemplary modes include, but are not limited to, intraperitoneal (I.P.) injection. Other modes include, without limitation, intravenous, intratumoral, intradermal, subcutaneous (S.C., s.q., sub-Q, Hypo), intramuscular (i.m.), intra-arterial, intramedullary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, intrathecal (spinal fluids), intraduodenal, intramedullary, intraosseous, intrathecal, intravascular, intravitreal, and epidural. In some embodiments, any known device useful for parenteral (e.g., intraperitoneal) injection and/or infusion of the formulations is used to effect such administration.

[0077] In some embodiments, pharmaceutical compositions are formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection are presented in unit dosage form, e.g., in ampoules or in multi -dose containers, with an added preservative. In some embodiments, the compositions take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In some embodiments, the compositions are presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and are stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use. In some embodiments, extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described.

[0078] In some embodiments, pharmaceutical compositions for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compounds which contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which include suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. In some embodiments, aqueous injection suspensions contain substances which increase the viscosity of the suspension, such as sodium

carboxymethyl cellulose, sorbitol, or dextran. In some embodiments, the suspension also contains suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0079] It should be understood that in addition to the ingredients particularly mentioned above, the compounds and compositions described herein include other agents conventional in the art having regard to the type of formulation in question.

Methods of Dosing and Treatment Regimens

[0080] In certain embodiments, the compositions comprising the iTDCs and/or the combination therapies described herein are administered for prophylactic and/or therapeutic treatments of ovarian cancer. In certain therapeutic applications, the compositions are administered to a patient already suffering from ovarian cancer, in an amount sufficient to cure or at least partially arrest at least one of the symptoms of the ovarian cancer. Amounts effective for this use depend on the severity and course of the ovarian cancer, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating physician. Therapeutically effective amounts are optionally determined by methods including, but not limited to, a dose escalation and/or dose ranging clinical trial.

[0081] In some embodiments, the pharmaceutical compositions comprising an iTDC are administered directly at a tumor site in the individual. In some embodiments, the

pharmaceutical compositions comprising an iTDC are administered directly into a tumor, a resection margin, and/or a tumor resected area. In some embodiments, the iTDCs are administered systemically to the individual.

[0082] In some embodiments, administration of the iTDCs is performed using methods known in the art. For example, in some embodiments, intravenous administration of the cells is performed for the treatment of an ovarian cancer. In some embodiments intraperitoneal administration of the cells is performed for the treatment of an ovarian cancer. In some embodiments, intratumoral administration or intracavity administration is performed after surgical removal of at least a part of an ovarian tumor.

[0083] In some embodiments, the cells are encapsulated by a matrix such as a hydrogel matrix (e.g., a synthetic extracellular matrix) and/or seeded onto a scaffold, which is then be administered or implanted, e.g., intratumorally.

[0084] In some embodiments, the iTDCs are encapsulated by a matrix such as a hydrogel matrix (e.g., a synthetic extracellular matrix) and/or seeded onto a scaffold. In some embodiments, the scaffold is biocompatible. In some embodiments, the scaffold is biodegradable and/or bioabsorbable. In some embodiments, the scaffold is sterile. In some embodiments, the scaffold is suitable for intratumoral or intracavity administration after surgical removal of a tumor. In some embodiments, the scaffold is pliable to allow manipulation thereof prior to or during administration to conform the scaffold to the area to which the iTDCs are being delivered. In some embodiments, the scaffold is configured to line the walls of the resection cavity. In some embodiments, the average thickness of the scaffold is in the nanometer, micrometer or millimeter range. In some embodiments, the scaffold includes a polymerized and/or crosslinked material selected from polyanionic polysaccharides (e.g., hyaluronic acid (HA),

carboxymethylcellulose (CMC), carboxymethylamylose (CMA), chondroitin-6-sulfate, dermatin sulfate, dermatin-6-sulfate and combinations thereof), alginic acid, chitin, chitosan, fibrin, dextran, polylactic acid, polyglycolic acid, poly(D-)lactic acid, polyglycoliclactic acid, keratin, laminin, elastin, collagen and other naturally-occurring extracellular matrix proteins, gelatin, polydioxanones, polycaprolactone, and blends and co-polymers thereof. In some embodiments, the scaffold comprises a bioabsorbable gelatin sponge. [0085] In some embodiments, seeding the iTDCs on the scaffold comprises: mixing a polymerizable and/or crosslinkable scaffold material with said induced drug carrier cells to form a mixture of the material and iTDCs, and polymerizing and/or crosslinking said material of said mixture, to thereby form said scaffold comprising iTDCs.

[0086] In some embodiments, the intratumoral or intracavity administration of the scaffold is performed using methods known in the art. In some instances, iTDCs migrate away from the scaffold and towards a cancerous or damaged tissue. In some embodiments, the polymerizing and/or crosslinking are performed in situ during intracavity administration after surgical removal of a brain tumor. In some embodiments, the scaffold is administered to line the walls of a resection cavity of an ovarian tumor. In some embodiments, the scaffold has ridges, channels and/or aligned fibers to promote movement of the drug carrier cells in the direction of the cancer or damaged tissue.

[0087] In prophylactic applications, compositions comprising the iTDC described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition. Such an amount is defined to be a“prophylactically effective amount or dose.” In this use, the precise amounts also depend on the state of health of the patient, the weight of the patient, and the like. When used in patients, effective amounts for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the health status of the patient, response of the patient to the drugs, and the judgment of the treating physician. In one aspect, prophylactic treatments include administering to an individual, who previously experienced at least one symptom of the disease being treated and is currently in remission, a pharmaceutical composition comprising an iTDC described herein, in order to prevent a return of the symptoms of the ovarian cancer.

[0088] In certain embodiments, an iTDC and an additional therapeutic agent described herein are administered at a dose lower than the dose at which either the induced drug carrier cell or the additional therapeutic agent are normally administered as monotherapy agents. In certain embodiments, an iTDC and an additional therapeutic agent described herein are administered at a dose lower than the dose at which either the iTDC or the additional therapeutic agent are normally administered to demonstrate efficacy. In certain embodiments, an iTDC is

administered at a dose lower than the dose at which it is normally administered as a

monotherapy agent, when administered in combination with an additional therapeutic agent described herein. In certain embodiments, an iTDC is administered at a dose lower than the dose at which it is normally administered to demonstrate efficacy, when administered in combination with an additional therapeutic agent described herein. In certain embodiments, an additional therapeutic agent is administered at a dose lower than the dose at which it is normally administered as a monotherapy agent, when administered in combination with an iTDC. In certain embodiments, an additional therapeutic agent is administered at a dose lower than the dose at which it is normally administered to demonstrate efficacy, when administered in combination with an iTDC.

[0089] In certain embodiments, wherein the condition of the patient does not improve, upon the discretion of the doctor, the administration of the iTDC compositions are administered chronically, that is, for an extended period of time, including throughout the duration of the life of the patient in order to ameliorate or otherwise control or limit the symptoms of the patient’s ovarian cancer.

[0090] In certain embodiments wherein a status of a patient does improve, the dose of the pharmaceutical compositions being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a“drug holiday”). In specific embodiments, the length of the drug holiday is between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days. The dose reduction during a drug holiday is, by way of example only, by 10%- 100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[0091] Once improvement of the conditions of the patient has occurred, a maintenance dose is administered if necessary. Subsequently, in specific embodiments, the dosage or the frequency of administration, or both, is reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. In certain embodiments, however, the patient requires intermittent treatment on a long-term basis upon any recurrence of symptoms.

[0092] The amount of a given agent that corresponds to such an amount varies depending upon factors such as the particular compound, disease condition and its severity, the identity (e.g., weight, sex) of the individual in need of treatment, but nevertheless is determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the individual being treated.

[0093] In some embodiments, the pharmaceutical compositions comprising an induced drug carrier cell are administered at a dosage in the range of about 10 ³ to about 10 ¹⁰ iTDC per kg of body weight iTDCs per kg of body weight, including all integer values within those ranges. In one embodiment, the desired dose is conveniently presented in a single dose or in divided doses administered simultaneously or at appropriate intervals, for example as two, three, four or more sub-doses per day. In some embodiments, the desired dose is administered as a single dose or in divided doses within about 72 hours of each other. In some embodiments, the desired dose is administered in divided does within about 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days of each other. In some embodiments, the daily dosage or the amount of active in the dosage form are lower or higher than the ranges indicated herein, based on a number of variables in regard to an individual treatment regime. In various embodiments, the daily and unit dosages are altered depending on a number of variables including, but not limited to, the activity of the compound used, the ovarian cancer to be treated, the mode of administration, the requirements of the individual, the severity of the ovarian cancer being treated, and the judgment of the practitioner.

[0094] In some embodiments, the pharmaceutical compositions comprising an iTDC are administered at a dosage of about 10 x 10 ⁶ cells per kilogram (kg). In some embodiments, the pharmaceutical compositions comprising an iTDC are administered at a dosage of about 12.5 x 10 ⁶ cells/kg. In some embodiments, the pharmaceutical compositions comprising an iTDC are administered at a dosage of about 1,000 cells/kg to about 10,000,000,000 cells/kg. In some embodiments, the pharmaceutical compositions comprising an induced drug carrier cell are administered at a dosage of at least about 1,000 cells/kg. In some embodiments, the

pharmaceutical compositions comprising an induced drug carrier cell are administered at a dosage of at most about 10,000,000,000 cells/kg. In some embodiments, the pharmaceutical compositions comprising induced drug carrier cell are administered at a dosage of about 1,000 cells/kg to about 10,000 cells/kg, about 1,000 cells/kg to about 100,000 cells/kg, about 1,000 cells/kg to about 1,000,000 cells/kg, about 1,000 cells/kg to about 10,000,000 cells/kg, about 1,000 cells/kg to about 100,000,000 cells/kg, about 1,000 cells/kg to about 1,000,000,000 cells/kg, about 1,000 cells/kg to about 10,000,000,000 cells/kg, about 10,000 cells/kg to about 100,000 cells/kg, about 10,000 cells/kg to about 1,000,000 cells/kg, about 10,000 cells/kg to about 10,000,000 cells/kg, about 10,000 cells/kg to about 100,000,000 cells/kg, about 10,000 cells/kg to about 1,000,000,000 cells/kg, about 10,000 cells/kg to about 10,000,000,000 cells/kg, about 100,000 cells/kg to about 1,000,000 cells/kg, about 100,000 cells/kg to about 10,000,000 cells/kg, about 100,000 cells/kg to about 100,000,000 cells/kg, about 100,000 cells/kg to about 1,000,000,000 cells/kg, about 100,000 cells/kg to about 10,000,000,000 cells/kg, about

1,000,000 cells/kg to about 10,000,000 cells/kg, about 1,000,000 cells/kg to about 100,000,000 cells/kg, about 1,000,000 cells/kg to about 1,000,000,000 cells/kg, about 1,000,000 cells/kg to about 10,000,000,000 cells/kg, about 10,000,000 cells/kg to about 100,000,000 cells/kg, about 10,000,000 cells/kg to about 1,000,000,000 cells/kg, about 10,000,000 cells/kg to about 10,000,000,000 cells/kg, about 100,000,000 cells/kg to about 1,000,000,000 cells/kg, about 100,000,000 cells/kg to about 10,000,000,000 cells/kg, or about 1,000,000,000 cells/kg to about 10,000,000,000 cells/kg. In some embodiments, the pharmaceutical compositions comprising induced drug carrier cell are administered at a dosage of about 1,000 cells/kg, about 10,000 cells/kg, about 100,000 cells/kg, about 1,000,000 cells/kg, about 10,000,000 cells/kg, about 100,000,000 cells/kg, about 1,000,000,000 cells/kg, or about 10,000,000,000 cells/kg.

[0095] In any of the aforementioned aspects are further embodiments in which the effective amount of the pharmaceutical compositions described herein is: (a) systemically administered to the subject; and/or (b) intravenously administered to the subject; and/or (c) administered by injection to the subject; and/or (d) administered non-systemically or locally to the subject.

[0096] In any of the aforementioned aspects are further embodiments comprising single administrations of the effective amount of the pharmaceutical composition, including further embodiments in which (i) the pharmaceutical composition is administered once a day; or (ii) the pharmaceutical composition is administered to the individual multiple times over the span of one day.

[0097] In any of the aforementioned aspects are further embodiments comprising multiple administrations of the effective amount of the pharmaceutical composition, including further embodiments in which (i) the pharmaceutical composition is administered continuously or intermittently: as in a single dose; (ii) the time between multiple administrations is every 6 hours; (iii) the compound is administered to the individual every 8 hours; (iv) the compound is administered to the individual every 12 hours; (v) the compound is administered to the individual every 24 hours; (vi) the compound is administered to the individual every 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days. In further or alternative embodiments, the method comprises a drug holiday, wherein the administration of the compound is temporarily suspended or the dose of the compound being administered is temporarily reduced; at the end of the drug holiday, dosing of the compound is resumed. In one embodiment, the length of the drug holiday varies from 2 days to 1 year.

[0098] In certain instances, it is appropriate to administer at least one pharmaceutical composition described herein, in combination with one or more other therapeutic agents.

[0099] In one embodiment, the therapeutic effectiveness of one of the pharmaceutical compositions described herein is enhanced by administration of an adjuvant (i.e., by itself the adjuvant has minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced). Or, in some embodiments, the benefit experienced by a patient is increased by administering one of the pharmaceutical compositions described herein with another agent (which also includes a therapeutic regimen) that also has therapeutic benefit.

[00100] In one specific embodiment, a pharmaceutical composition described herein, is co- administered with a second therapeutic agent, wherein the pharmaceutical composition described herein, and the second therapeutic agent modulate different aspects of the disease, disorder or condition being treated, thereby providing a greater overall benefit than

administration of either therapeutic agent alone.

[00101] In any case, regardless of the disease, disorder or condition being treated, the overall benefit experienced by the patient is additive of the two therapeutic agents or the patient experiences a synergistic benefit.

[00102] In certain embodiments, different dosages of the pharmaceutical composition disclosed herein are utilized in formulating pharmaceutical composition and/or in treatment regimens when the compounds disclosed herein are administered in combination with one or more additional agent, such as an additional drug, an adjuvant, or the like. Dosages of drugs and other agents for use in combination treatment regimens are optionally determined by means similar to those set forth hereinabove for the actives themselves. Furthermore, the methods of

prevention/treatment described herein encompasses the use of metronomic dosing, i.e., providing more frequent, lower doses in order to minimize toxic side effects. In some embodiments, a combination treatment regimen encompasses treatment regimens in which administration of a pharmaceutical composition described herein, is initiated prior to, during, or after treatment with a second agent described herein, and continues until any time during treatment with the second agent or after termination of treatment with the second agent. It also includes treatments in which a pharmaceutical composition described herein, and the second agent being used in combination are administered simultaneously or at different times and/or at decreasing or increasing intervals during the treatment period. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient.

[00103] It is understood that the dosage regimen to treat, prevent, or ameliorate the condition(s) for which relief is sought, is modified in accordance with a variety of factors (e.g. the disease, disorder or condition from which the individual suffers; the age, weight, sex, diet, and medical condition of the individual). Thus, in some instances, the dosage regimen actually employed varies and, in some embodiments, deviates from the dosage regimens set forth herein.

[00104] For combination therapies described herein, dosages of the co-administered

pharmaceutical compositions vary depending on the type of co-drug employed, on the specific drug employed, on the ovarian cancer being treated and so forth. In additional embodiments, when co-administered with one or more other therapeutic agents, the pharmaceutical

composition provided herein is administered either simultaneously with the one or more other therapeutic agents, or sequentially.

[00105] In some embodiments a co-drug is administered in conjunction with the

pharmaceutical composition. In some embodiments the co-drug is ganciclovir or valganciclovir. In some embodiments, the dosing interval is determined by the bioavailability of the co-drug and its excretion from the body. In some embodiments, the co-drug is administered for at least 5 days, about 10 days to about 3 weeks.

[00106] In combination therapies, the multiple therapeutic agents (one of which is one of the pharmaceutical compositions described herein) are administered in any order or even

simultaneously. If administration is simultaneous, the multiple therapeutic agents are, by way of example only, provided in a single, unified form, or in multiple forms (e.g., as a single pill or as two separate pills).

[00107] The pharmaceutical compositions described herein, or a pharmaceutically acceptable salt thereof, as well as combination therapies, are administered before, during or after the occurrence of ovarian cancer, or a disease or condition associated with ovarian cancer, and the timing of administering the pharmaceutical composition containing a compound varies. Thus, in one embodiment, the pharmaceutical compositions described herein are used as a prophylactic and are administered continuously to individuals with a propensity to develop ovarian cancer in order to prevent the occurrence of ovarian cancer. In another embodiment, the pharmaceutical compositions are administered to an individual during or as soon as possible after the onset of the symptoms. In specific embodiments, a pharmaceutical composition described herein is administered as soon as is practicable after the onset of a ovarian cancer is detected or suspected, and for a length of time necessary for the treatment of the disease. In some embodiments, the length required for treatment varies, and the treatment length is adjusted to suit the specific needs of each individual. For example, in specific embodiments, a compound described herein or a formulation containing the pharmaceutical composition is administered for at least 2 weeks, about 1 month to about 5 years.

EXAMPLES

[00108] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the invention in any fashion. These examples, along with the methods described herein, are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention, as defined by the scope of the claims, will occur to those skilled in the art.

Example 1: In vitro and in vivo experiments confirming the efficacy of iTDCs in treating ovarian cancer

[00109] A combination of bioluminescent and fluorescent imaging and 2-D cell culture techniques were used to investigate the efficacy of iTDC therapy and iTDC-prodrug therapy against human ovarian cancer cells.

Materials

[00110] Cell lines: OVACAR human ovary cancer cells were purchased. Human fibroblast cells were provided by the University of North Carolina (UNC) School of Medicine. Cell lines were grown according to methods known in the art, particularly those described in SD Hingtgen et al ,“A novel molecule integrating therapeutic and diagnostic activities reveals multiple aspects of stem cell-based therapy,” Stem Cells 28, 832-841 (2010), and M Sena-Esteves, et al , “Optimized large-scale production of high titer lentivirus vector pseudotypes,” J. Virol. Methods 122, 131-139 (2004).

[00111] Lentiviral vectors: Reprogramming lentiviral vectors (“LV”) encoding Sox2 were purchased from Addgene (Cambridge, MA, USA). Lentiviral vectors expressing cytotoxic agents and optical reporters were constructed by methods known in the art. The cytotoxic agents were a secreted variant of the proapoptotic molecule TRAIL (“sTR”) and thymidine kinase (“TK”). The reporters were Green Fluorescent Protein (“GFP”), mCherry (“mC”), and Firefly Luciferase (“FL”). The following vectors were constructed: LV-sTR-TK-GFP, LV-GFP, and LV-mC-FL. All lentiviral constructs were packaged as lentiviral vectors in 293T cells using a helper virus-free packaging system.

Experiments and Results

[00112] ITDC Generation: ITDCs were generated following a single-factor Sox2 and feeder-free method. Human fibroblasts were seeded in 6-well plates and transduced with reprogramming vectors in media containing protamine sulfate. Two days after infection, the media was changed to STEMdiff™ Neural Induction Medium (STEMCELL Technologies, Vancouver, Canada) containing doxycycline. Media was changed every 3 days. Neurosphere formation was induced by culturing in low-adherent flasks.

[00113] Co-culture viability assays: The following cells were generated: (1) OVACAR cells expressing mC-FL; (2) iTDC therapeutic cells expressing sTR-TK-GFP; and (3) iTDC control cells expressing sTR-GFP. The therapeutic and control cells were seeded in separate wells. Forty-eight hours later, the OVACAR cells expressing mC-FL were seeded into both the wells containing control cells and the wells containing therapeutic cells. The cells were visualized for fluorescent protein expression by fluorescence microscopy. OVACAR viability was measured by quantitative in vitro bioluminescence imaging. Photon emission was quantified using Livingimage software (PerkinElmer).

[00114] Results: Fluorescence and bioluminescent imaging revealed a significant reduction in the viability of OVACAR cells co-cultured with therapeutic iTDCs. Figure 1 shows fluorescent images of the control treated OVACAR cells (left) and of the therapeutic treated OVACAR cells (right). The OVACAR cells were engineered to express m-Cherry (red) and the iTDC’s were engineered to express GFP (green). These images show that significantly less OVACAR cells survived when treated with therapeutic iTDCs. Bioluminescence imaging was used to quantify the change in volume of the OVACAR cells. The chart in Figure 2 plots the surface photon emission of the control treated OVACAR cells versus the surface photon emission of the therapeutic treated OVACAR cells. The surface photon emission of the control treated

OVACAR cells is significantly higher than that of the therapeutic treated OVACAR cells, reflecting that the therapeutic cells significantly reduced the viability of OVACAR cells as compared to the control.

[00115] Bioluminescence imaging of iTDC prodrug therapy in vivo: OVACAR cells expressing mC-FL were implanted in the ovaries of 10 mice (2xl0 ⁶ cells/mouse). Three days later, either therapeutic iTDCs expressing TR-TK-GFP (2xl0 ⁶ cells/mouse) or control iTDCs expressing GFP (2xl0 ⁶ cells/mouse) were implanted into the OVACAR implantation site of each mouse. Ganciclovir (“GCV”) was injected into each mouse daily during 20 days at a dose of 100 mg/kg. At 0, 4, 11, and 20 days, mice were given an injection of D-Luciferin and photon emission was determined 1-5 min later using an IVIS Kinetic Optical System (Perkin Elmer) with a 1-5 minute acquisition time. Images were processed and photon emission quantified using Livingimage software (PerkinElmer).

[00116] Results: The in vivo bioluminescent imaging results reflect that iTDCs expressing sTR-TK have a significant therapeutic effect against ovarian cancer. Figure 3 shows

bioluminescent images of two mice taken 0, 4, 11, and 20 days after treatment with either the therapeutic iTDCs (bottom) or with the control (top). The images of the therapeutic treated mouse show a significant reduction in tumor growth by day 4. By day 20, the tumor growth is not visible in the images of therapeutic treated mouse, and has increased significantly in the control treated mouse. [00117] Figure 4 plots the tumor volume (normalized to 1) of the control treated mice (“control”) versus that of the therapeutic-treated mice (“therapy”). On day 0, the tumor volumes of the control treated mice and therapeutic treated mice are equal, but by day 20 the tumor volume of the control-treated mice was nearly eight times greater than that of the therapeutic treated mice. Accordingly, the in vivo bioluminescent imaging results show that the

administration of iTDCs expressing TK-sTR, when administered with GCV, have significant therapeutic effects against ovarian cancer in vivo.

Example 2: Alternative Media for Rapid Trans differentiation of Human Skin Cells

[00118] Transdifferentiation of human skin cells was performed as above in Example 1, but in place of the STEMdiff™ Neural Progenitor Basal Medium was a 1 : 1 mixture ofN-2 medium and B-27 medium as follows. Chemicals were purchased from Gibco® (Invitrogen Corporation, Carlsbad, California), Sigma (Sigma-Aldrich, St. Louis, Missouri) or Selleck Chemicals (Houston, Texas) as indicated.

[00119] N-2 medium: DMEM/F12 (Gibco®), 1 X N2 supplement (Gibco®), 5 pg/ml insulin

(Sigma), 1 mM L-glutamine (Gibco®), 1 mM Glutamax (Gibco®), 100 mM MEM non-essential amino acids (NEAA) (Gibco®), and 15 100 M beta-mercaptoethanol (bME).

[00120] B -27 medium: Neurobasal medium (Gibco®), 1 X B-27 supplement (Gibco®), and 200 mM L-glutamine (Gibco®). To the 1 : 1 mix was added bovine serum albumin (BSA, Sigma) to a :fmal concentration of 5 pg/ml. This medium was supplemented with the following:

SB431542 (Selleck Chemicals) to a final concentration of 10 pM; LDN193189 (Selleck

Chemicals) to a final concentration of 100 nM; all trans retinoic acid to a final concentration of 10 pM (Sigma); and CHIR99021 (Selleck Chemicals) to a final concentration of 3 pM. ETsing this media, nestin+ iNTDs were generated when used with the Sox2 transduction. In some embodiments, the medium included Insulin (25 pg/ml), Transferrin (100 pg/ml), Sodium selenite (30 nM), and/or cAMP (100 ng/ml).

Example 3: Use of iTDCs in Treatment for Ovarian Cancer

[00121] A patient is diagnosed with ovarian cancer (e.g., epithelial ovarian cancer), and surgery is scheduled for removing the tumor soon thereafter (e.g., within 24 days, 4 weeks, or 5 weeks). A skin punch is taken from the patient to obtain skin fibroblast cells. The cells are transdifferentiated as disclosed herein into induced iTDCs and also loaded with a therapeutic agent and/or a reporting molecule. During surgery and after removal of the tumor, the loaded iTDCs are administered into the resection margin and resulting cavity where the tumor had been removed. The iTDCs migrate toward residual ovarian cancer cells and deliver their therapeutic agent and/or reporting molecule payload.

[00122] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Example 4: Use of iTDCs in Treatment for Brain Cancer

[00123] A patient is diagnosed with brain cancer (e.g., glioblastoma), and surgery is scheduled for removing the tumor soon thereafter (e.g., within one, two or three weeks). A skin punch is taken from the patient to obtain skin fibroblast cells. The cells are transdifferentiated as taught herein into induced iTDCs and also loaded with a therapeutic agent and/or a reporting molecule. During surgery and after removal of the tumor, the loaded iTDCs are administered into the resulting cavity where the tumor had been removed. The iTDCs migrate toward residual cancer cells and deliver their therapeutic agent/reporting molecule payload.

[00124] In some embodiments, the ability to rapidly generate iTDCs from human skin enables patient-specific therapies to treat cancer. The efficiency of iTDC generation is significantly higher than other cellular reprogramming strategies, suggesting large numbers of iTDCs could be generated from small amounts of skin. Patient-specific derivation could avoid immune rejection to maximize tumor killing and for treatment durability. Example 5: Rapid Transdifferentiation of Human Skin Cells

[00125] Cell -based drug carriers must be generated quickly in order to treat patients with rapidly progressing cancers. In some embodiments, iTDCs are created in weeks, allowing for reduced turn-around time from patient sample to therapy for aggressive cancers.

[00126] In this study, the potential of TD-derived iTDC therapies was investigated as autologous GBM therapy for human patients. These methods are capable of converting human skin into iTDCs 6-fold faster than previous methods, which is significant because time is a priority for GBM patient therapy. This strategy was used to create the first iTDCs engineered with cytotoxic agents and optical reporters. A combination of real-time molecular imaging, 3-D cell culture, and multiple human GBM xenografts models were used to investigate the fate, tumor-specific homing, and efficacy of iTDC therapy against solid and surgically resected GBM.

Materials and Methods

[00127] Cell lines: U87, GBM8, GBM4, 293T, and human fibroblast cells (CCD-l099Sk, 15 others) were grown as previously described. Lentiviral vectors (LV) encoding hTERT and Sox2 were purchased from Addgene (Cambridge. MA, USA). All cDNA were under control of the tetracycline promoter.

[00128] Human iTDCs were generated following a single-factor Sox2 and feeder-free method. Briefly, 200,000 human fibroblasts were seeded in 6-well plates and transduced with the LV cocktail containing hTERT and Sox2 in media containing protamine sulfate (5 pg/ml, Sigma). Two days after infection, the media was changed to STEMdiff™ Neural Induction Medium (STEMCELL Technologies, Vancouver, Canada) containing doxycycline (1 0 pg/ml, Sigma, St. Louis, MO, USA). Media was changed every 3 days. Neurosphere formation was induced by culturing in low-adherent flasks.

[00129] Lentiviral vectors: In addition to the reprogramming vectors, the following lentiviral vectors were used in this study: LV-GFP-FL, LV-GFP-RLuc, LV-mC-FL, LV-sTR, LV-diTR and LV-mRFP-hRLuc-ttk. GFP-RLuc and GFP-FL were constructed by amplifying the cDNA encoding Renilla luciferase or firefly luciferase using the vectors luciferase-pcDNA3 and pAC-hRluc (Addgene), respectively. The restriction sites were incorporated in the primers, the resulting fragment was digested Bglll and Sail, and ligated in frame in BgllESall digested pEGFP-Cl (Clontech, Mountain View, CA, USA). The GFP-FL or GFP-RLuc fragments were digested with Agel (blunted) and Sail, and ligated into pTK402 (provided by Dr. Tal Kafri, UNC Gene Therapy Center) digested BamHI (blunted) and Xhol to create LV-GFP-FL or LV-GFP- RLuc. Similarly, mCFL was created by amplifying the cDNA encoding firefly luciferase from luciferase-pcDNA3, ligating into BglII/Sall digested mCherry-Cl (Clontech), and ligating the mC-FL fragment into pTK402 LV backbone using blunt/Xhol sites. To create LV-sTR and LV- diTR, the cDNA sequence encoding sTR or diTR was PCR amplified using custom-synthesized oligonucleotide templates (Invitrogen, Carlsbad, CA, USA). The restriction sites were incorporated into the primers, the resulting fragment was digested with BamHI and Xhol, and ligated in-frame into BamHI/Xhol digested pL VX10 plasmid. Both LV -sTR and LV -diTR have 1RES-GFP (internal ribosomal entry sites-green fluorescent protein) elements in the backbone as well as CMV -driven puromycin element. All LV constructs were packaged as LV vectors in 293T cells using a helper virus-free packaging system as described previously. iTDCs and GBM cells were transduced with LVs at varying multiplicity of infection (MOI) by incubating virions in a culture medium containing 5 pg/ml protamine sulfate (Sigma) and cells were visualized for fluorescent protein expression by fluorescence microscopy.

[00130] Cell viability and passage number: To assess the proliferation and passage number of modified and unmodified iTDCs, iTDCs expressing GFP-FL, sTR or unmodified cells were seeded in 96-well plates. Cell viability was assessed 2, 3, 4, 5, 8, and 10 days after seeding using CellTiter-Glo® luminescent cell viability kit (Promega). Maximum passage number was assessed by monitoring the number of times iTDCs, iTDC-sTR, or WT-NTD were subcultured without alterations in morphology, growth rate, or transduction efficiency.

[00131] Immunohistochemistry and in vitro differentiation: To determine the effects of LV modification on iTDC differentiation, iTDCs were transduced with LV-GFP-FL or LV-sTR. Engineered or unmodified cells were fixed, permeabilized, and incubated for lh with anti-nestin Polyclonal antibody (Millipore, MAB353, 1 :500, Billerica, MA, USA). Cells were washed and incubated with the appropriate secondary antibody (Biotium, Hayward, CA, USA) for 1 hr. Cells were then washed, mounted, and imaged using fluorescence confocal 3Q microscopy. For differentiation, engineered or non-transduced iTDCs were cultured for 12 days in N3 media depleted of doxycycline, EGF, and FGF. Cells were then stained with antibodies directed against nestin, glial fibrillary acidic protein (GF AP; Millipore, MAB3405, 1 :250), or Tuj-l (Sigma, T8578, 1 : 1 000) and detected with the appropriate secondary antibody (Biotium). Nuclei were counterstained with Hoechst 33342 and the results analyzed using a FV 1200 laser confocal microscope (Olympus, Center Valley, PA).

[00132] Three-dimensional tissue culture. Three-dimensional levitation cell cultures were performed using the Bio- Assembler Kit (Nano3D Biosciences, Houston, TX). Confluent 6 well plates with GBM or iTDC were treated with a magnetic nanoparticle assembly (8 mΐ cm ² of cell culture surface area or 50 mΐ cm ¹ medium, NanoShuttle (NS), Nano3D Biosciences) for overnight incubation to allow for cell binding to the nanoparticles. NS was fabricated by mixing iron oxide and gold nanoparticles cross-linked with poly- 1 -lysine to promote cellular uptake. Treated GBM and iTDC were then detached with trypsin, resuspended and mixed at different ratios (1 : 1 and 1 :0.5) in an ultra-low attachment 6 well plate with 2 ml of medium. A magnetic driver of 6 neodymium magnets with field strength of 50 G designed for 6-well plates and a plastic lid insert were placed atop the well plate to levitate the cells to the air-liquid interface. Media containing 4 pg/ml GCV was added to the co-culture of GBM with iTDC expressing ttk. Fluorescence images where taken over time to track the cell viability of both populations (previously labeled with different fluorescence). For BLI of 3D cell culture, 100 mΐ/well of Flue substrate stock reagent was added to the media and imaged using an IVIS Kinetic Optical System (PerkinElmer) with a 5 minute acquisition time. Images were processed and photon emission quantified using Livingimage software (PerkinElmer).

[00133] Real-time imaging and motion analysis: Migration was assessed in novel 2- dimensional and 3-dimensional culture systems.

[00134] 2-dimensional migration: iTDCs expressing RFP were seeded in micro-culture inserts in glass bottom microwell dishes (MatTek, Ashland, MA, EISA) using 2-chamber cell 25 culture inserts (ibidi, Verona, WI, EISA). U87 glioma cells expressing GFP were plated into the adjacent well (0.5mm separation) or the well was left empty. 24 hrs after plating, cells were placed in a VivaView live cell imaging system (Olympus) and allowed to equilibrate. The insert was removed and cells were imaged at 10X magnification every 20 minutes for 36 hours in 6 locations per well (to monitor sufficient cell numbers) in three independent experiments. NIH Image was then used to generate movies and determine both the migrational velocity, total distance migrated, and the directionality of migration.

[00135] 3-dimensional migration: iTDC migration to GBM spheroids was assessed in 3-D culture systems by creating iTDC and GBM spheroids using levitation culture as described above. iTDC and GBM spheroids were co-cultured in levitation systems. Real-time imaging was performed to visualize the penetration of GBM spheroids by iTDCs in suspension.

[00136] Co-culture viability assays: mNTD expressing sTR or control GFP-RL (5xl0 ³ ) were seeded in 96 well plates. 24 hrs later, Ei87-mC-FL, LNl8-mC-FL, or GBM8-mC-FL human GBM cells (5xl0 ³ ) were seeded into the wells and GBM cell viability was measured 24 hrs later by quantitative in vitro bioluminescence imaging. GBM cells were also assessed at 18 hrs for caspase-3 l7 activity with a caged, caspase 3 l7-activatable DEVD-aminoluciferin (Caspase-Glo 317, Promega, Madison, WI, EISA). [00137] iTDC survival and fate in vivo: To determine the survival of iTDCs in vivo, iTDC expressing mCherry-FL (7.5xl0 ⁶ cells/mouse) were suspended in PBS and implanted stereotactically into the right frontal lobe of mice (n=7). iTDC survival was determined by serial bioluminescence imaging performed for 20 days. To determine the fate of iTDCs at a cellular resolution, animals were sacrificed 21 days post-implantation, brains extracted sectioned. Tissue sections were stained with antibodies against nestin, GFAP, Tuj-l, Oct-4, and TRA-160, and visualized using a secondary antibody labeled with CF™488.

[00138] Co-culture viability assays: 3-D levitation culture was used in three separate in vitro cytotoxicity studies. iTDCs expressing 2 different cytotoxic agents were used to treat 1 established GBM cell line (U87) and 2 patient-derived GBM lines (GBM4, GBM8). 1) To determine the cytotoxicty of TRAIL therapy, iTDC-sTR or iTDC-mCherry spheroids were co cultured in suspension with U87-GFP-FLuc spheroids at a iNTD:GBM ratio of 1 :2 or 1 : 1. GBM spheroid viability was determined 48 hrs later by FLuc imaging. 2) To determine the

cytotoxicity of pro-drug enzyme therapy for patient-derived GBMs, iTDC-TK spheroids were co-cultured in suspension with patient-derived GBM4-GFP-FLuc spheroids or mixed with GBM cells prior to sphere formation. Spheroids were cultured with or without ganciclovir (GCV) and GBM spheroid viability was determine 0, 2, 4, or 7 days after addition of the pro-drug by FLuc imaging. 3) To determine the cytotoxicity of sECM-encapsulated iNTD pro-drug/enzyme therapy, iTDC-TK were encapsulated in sECM and placed in levitation cultured with patient- derived GBM8-GFP-FLuc spheroids. Viability was determine by FLuc imaging.

[00139] Anti-GBM Efficacy of iTDC Therapy In Vivo: Three different xenograft studies were performed to assess the anti-GBM effects of iTDC therapy. iTDC-sTR and iTDC-TK therapy was tested against solid (U87), diffused patient-derived (GBM8), and surgically resected patient-derived (GBM4) xenograft models.

[00140] 1) To determine the therapeutic efficacy of iTDC-TRAIL against solid human U87 tumors, a combination of iTDC-TRAIL or iNTD-GFP-RLuc (7.5xl0 ⁵ cells/mouse) were stereotactically implanted into the right frontal lobe of mice (n=7) together with U87-mC-FL cells (lxlO ⁶ cells/mouse). Therapeutic response was then determined by following tumor volumes with FL bioluminescence imaging as described previously. Briefly, mice were given an intraperitoneal injection of D-Luciferin (4.5 mg/mouse in 150 mΐ of saline) and photon emission was determined 5 minutes later using an IVIS Kinetic Optical System (PerkinElmer) with a 5 minute acquisition time. Images were processed and photon emission quantified using

Livingimage software (PerkinElmer). Additionally, mice were followed for survival over time. [00141] 2) To investigate the efficacy of iTDC prodrug/enzyme therapy against invasive patient-derived GBM, mice were stereotactically implanted in the right frontal lobe with GBM8 cells expressing mC-FL (l.5xl0 ⁵ cells/mouse). Three days later, iTDC-TK (n=7, 7.5xl0 ⁵ cells/mouse) or iTDC-mRFP-hRLuc (n=7, 7.5xl0 ⁵ cells/mouse) were implanted into the tumor implantation site. GCV was injected i.p. daily during two weeks at a dose of 100 mg/kg.

Changes in tumor volume were assessed by FLuc imaging as described above and mice were followed for survival over time.

[00142] 3) To determine the efficacy of iTDC therapy against post-surgical minimal GBM, image-guided GBM resection in mice was performed according to our previously reported strategy. Patient-derived GBM8-GFP-FLuc were harvested at 80% confluency and implanted stereotactically (5xl0 ⁵ cells) in the right frontal lobe: 2 mm lateral to the bregma and 0.5 mm from the dura. Following immobilization on a stereotactic frame, mice were placed under an Olympus MVX-10 microscope. Intraoperative microscopic white light, GFP, and RFP images were captured throughout the procedure using with a Hamamatsu ORCA 03 G CCD (high resolution) camera and software (Olympus). A midline incision was made in the skin above the skull exposing the cranium of the mouse. The intracranial xenograft was identified using GFP fluorescence. A small portion of the skull covering the tumor was surgically removed using a bone drill and forceps and the overlying dura was gently peeled back from the cortical surface to expose the tumor. Under GFP fluorescence, the GBM8-GFPFL tumor was surgically excised using a combination of surgical dissection and aspiration, and images of GFP were continuously captured to assess accuracy of GFP -guided surgical resection. Following tumor removal, the resulting resection cavity was copiously irrigated and the skin closed with 7-0 Vicryl suture. No procedure-related mortality was observed. All experimental protocols were approved by the Animal Care and Use Committees at The University of North Carolina at Chapel Hill and care of the mice was in accordance with the standards set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, USD A regulations, and the American Veterinary Medical Association. Following surgical resection, iTDC-TK or iTDC-mC-FL (5xl0 ⁵ cells) were encapsulated in hyaluronic sECM hydro gels (Sigma) and transplanted into the post-operative GBM cavity. GBM recurrence was visualized by FLuc imaging as described above and mice were followed for survival.

[00143] Tissue processing: Immediately after the last imaging session, mice were sacrificed, perfused with formalin, and brains extracted. The tissue was immediately immersed in formalin. 30 pm coronal sections were generated using a vibrating microtome (Fisher Waltham, MA, USA). For nestin, GFAP, and Tuj-l staining, sections were incubated for 1 hr in a blocking solution (0.3% BSA, 8% goat serum, and 0.3% Triton X-100) at room temperature, followed by incubation at 4 °C overnight with the following primary antibodies diluted in blocking solution: (1) anti-human nestin (Millipore), (2) anti GFAP (Millipore), (3) anti TRAIL (ProSci, Poway, CA) and (4) anti-Tuj-l (Sigma). Sections were washed three times with PBS, incubated in the appropriate secondary antibody, and visualized using a confocal microscope (Olympus).

Results

[00144] The Rapid Transdifferentiation of Human Fibroblasts into iTDCs. The rapid and efficient generation of iTDC therapies is essential for treating patients with aggressive cancer.

As a new strategy, human fibroblasts were transduced with Sox2 and performed iTDC generation without feeder cells. Then, diagnostic iTDCs expressing optical reporters or therapeutic iTDCs expressing different cytotoxic agents were generated. First was evaluated the kinetics of generating iTDCs using the feeder-free/Sox2 strategy. Human fibroblasts were transduced with Sox2 and cultured in NTD-inducing media. Changes in cell morphology were observed within 48 hrs of activating Sox2 expression. Additionally, wide-spread nestin expression was detected and the iTDCs could form neurosphere formation. Figure 5 shows white light and fluorescence photomicrographs of the human fibroblasts and iTDCs grown as monolayers and neurospheres or stained with antibodies against nestin (green). Figure 6 is a summary graph showing the expression of nestin over time at different days after induction of iTDC generation. Quantification showed nestin expression in iTDCs remained constant from day 2 through day 10. Figure 7 shows images of the immunofluorescence staining. When induced to differentiate, the iTDCs expressed the astrocyte marker GF AP and the neural marker Tuj-l. Staining revealed the cells did not express the pluripotency makers TRA-160 or OCT4. These findings were confirmed by RT-PCR analysis. Figures 8A - 8D show the RT-PCR analysis of nestin, SOX2, nanog, and OCT-4 expression in NHF, iTDC, and h-iPSC. The iTDCs showed high level of nestin expression that was absent in parental fibroblasts or human iPSC (h- iPSC). Sox2 expression was high in both iTDCs and h-iPSCs because Sox2 overexpression was used to generate both cell lines. Unlike h-iPSCs, iTDCs did not express high levels of the pluripotency markers Nanog or OCT3/4. Together, these data demonstrate the ability to create multi-potent iTDCs within 48 hrs using single-factor Sox2 expression.

[00145] iTDCs Migrate Selectively to CRM The ability to home to solid and invasive GBM deposits is one of the most beneficial characteristics of induced tumor homing cell based cancer therapies. To investigate the tumor-tropic nature of iTDCs, we used real-time motion analysis of iTDCs co-cultured with human GBM cells. For reference, iTDC migration was compared to the parental human fibroblasts from which they were derived. It was found that iTDCs rapidly migrated towards the co-cultured GBM cells, covering the 500 pm gap in 22 hrs. Figure 9 shows summary images showing migration of iTDC -mC-FL (red) or parental human fibroblasts toward U87-GFP-FL (green) at 0 and 22 hours after plating. Figure 9 also shows single-cell tracings depicting the paths of iTDC -mC-FL or human fibroblast-directed migration toward GBM over 22 hours. The dashed line indicates the site of GBM seeding. Figure 10 shows summary graphs of the directionality and Euclidean distance of iTDC or fibroblast migration toward GBM cells determined from the real-time motion analysis.

[00146] Single cell migratory path analysis showed that the presence of GBM cells induced iTDC to selectively migrate towards the co-cultured GBM cells. In contrast, human fibroblasts demonstrated very little migration. Single cell migration analysis of human fibroblasts confirmed the random migratory patterns with very little displacement towards the co-cultured GBM cells. The directionality of the migration of iTDC was analyzed by calculating the ratio of Euclidian distance to overall accumulated distance, with perfect single direction movement yielding a ratio of 1.0 and perfectly non-directional movement yielding a ratio of 0.0. LTsing this analysis, we calculated an average directionality ratio that was significantly higher for iTDCs (0.65) than human fibroblasts (0.28). Further analysis of single cell migration patterns demonstrated significantly increased average Euclidian distance migrated by iTDC (340 pm) as compared to human fibroblasts (200 pm). The average cell velocity by iTDC was lower as 30 compared to human fibroblasts (0.4 vs 0.62). Lastly, we performed 3-D migration assays to mimic the in vivo migration of iTDCs into GBM foci. mCherry+ iTDC spheroids were co- cultured with GFP+ GBM4 spheroids and both cell types were levitated using magnetic force. We discovered that the iTDCs began penetrating the GBM4 spheroids within hours of seeding. The iTDC spheroids continued to penetrate the GBM4 spheroids, extensively co-localizing within 8 days. Together, these observations support the conclusion that iTDCs possess tumoritropic properties and home to GBM cells. Figure 11 shows fluorescence images of the migration of iTDCs-mC-FL (red) into U87 spheroids (green) and their penetration toward the core of the tumor spheroid over time in the 3D levitation culture systems. The top panel shows images of the mixed therapy, and the bottom panel shows images of the established GBM4 spheroids.

[00147] iTDC Persistence and In Vivo Fate. We next utilized the engineered iTDCs to investigate the survival and fate of these cells in vivo in the brain. A previous study of in vitro proliferation after engineering of iTDC with GFPFL and rnCFL showed no significant differences with non-engineered iTDCs. For in vivo study, iTDCs engineered with mCFL was stereotactically implanted in the brain of mice and real-time non-invasive imaging was used to monitor cell survival over time. Capturing images periodically, we found that iTDCs survive more than 20 days post implantation. Figure 12 shows serial bioluminescent images taken over the course of the 20 days. Post-mortem IHC revealed that approximately half of iTDC-mCFL expressed the NTD marker nestin and the other half were positive for the neuronal marker Tuj-l. No astrocyte marker GF AP was observed. Additional IHC, images of which are shown in

Figures 13A and 13B, verified the transplanted iTDCs did not express the pluripotency markers Oct-4 and TDR-160.

[00148] Efficacious Treatment of Malignant and Invasive GBM using Tumoricidal iNTDs. To investigate the therapeutic efficacy of iTDC-based GBM treatment, we first engineered h- iNTDs to express a secreted variant of the pro-apoptotic molecule TNFa-related apoptosis- inducing ligand (TRAIL; diTR) in frame with Gaussia luciferase and upstream of an IRES-GFP element (iNTD-diTR). Anti -cancer effects of TRAIL when delivered from engineered cell carriers were established previously; therefore it is the ideal tumoricidal molecule for

characterizing new iTDC delivery vehicles. Robust expression of the GFP reporter was detected following transduction of the iTDCs. We observed that iTDC-diTR efficiently formed neurospheres when cultured in suspension, and displayed proliferative capacity and passage numbers equivalent to unmodified cells. Nestin expression and differentiation capacity were the same as observed in previous engineered and not engineered iTDC, suggesting that modification of iTDCs with TRAIL does not interfere with their properties as stem cells.

[00149] To evaluate the anti-GBM efficacy of engineered iTDCs, iTDC-diTR or control iNTD-GFPRL were co-cultured at different ratios with human GBM cells expressing mCherry and firefly luciferase (mC-FL). In order to mimic the in vivo characteristics, GBM and iTDC were mixed and cultured in three-dimensional levitation system for 48 hours. The fluorescent and bioluminescent images, Figure 14, revealed a significant reduction in the viability of GBMs co-cultured with iTDC-sTR. This reduction was significantly greater if a higher iTDGGBM ratio was used.

[00150] iTDC Secretion of a Pro-Apoptotic Agent Reduces Solid GBM To test the in vivo efficacy of iTDC-sTR based therapy, we determined the effects of iTDC-sTR treatment on solitary human GBMs. Human U87 GBM cell expressing mC-FL were implanted intracranially with iNTD-sTR or 'control iNTD-GFP and tumor volumes were followed using serial bioluminescence imaging. Figures 15A and 15B show representative bioluminescent images and summary data. We found that iTDC-sTR treatment induced a statistically significant reduction in tumor growth by day 3 and decreased GBM volumes 50-fold by day 24. In addition, iTDC-sTR-treated animals survived more than 51 days, while control animal succumbed to GBM growth in only 25 days. IHC examination of mouse brains showed a robust expression of TRAIL by the iTDC-sTR after two weeks. Figures 16A, 16B, and 16C shows representative images demonstrating the expression of cytotoxic, differentiation, and pluripotency markers in iTDCTE -sTR after therapy. The iTDC-sTR in the GBM were positive for the expression of the Nestin 15 and Tuj-l, and negative for GFAP and pluripotency markers Oct-4 and TRD-160.

[00151] Efficacious Treatment of Malignant and Invasive GBM CD 133+ using Tumoricidal iNTDs. To determine the efficacy of iTDC prodrug/enzyme therapy for patient-derived CD133+ human GEM-initiating cells, we co-cultured GBM4 cells expressing GFP and firefly luciferase (GBM4-GFPFL) with iTDC expressing a trifunctional chimeric reporter including Rluc, RFP and thymidine kinase (TK) activities, to generate iTDC-TK. The thymidine kinase encoded by herpes simplex virus (HSV-TK) was used in the first cell suicide gene therapy proof of principle and still is one of the most widely used systems in clinical and experimental applications.

GBM4-GFPFL and iTDC-TK were co-cultured in three-dimensional levitation system in two different models. Figure 17 shows the fluorescent images taken of both models and the corresponding summary data. The first model (top) the two cell types were mixed and cell survival monitored over time by fluorescence. The second model (bottom), the two cell types were cultured side by side to mimic the treatment of an established GBM. Cell survival was monitored over time by fluorescence. In both cases, a significant reduction of the GBM survival was observed over time, being more significant in the mixed model. We next determined the efficacy of iTDC-TK therapy in vivo on established BM4 by implanting GBM4-GFPFL cancer cells into the parenchyma of mice. Three days later, iTDC-TK or control cells were

administered directly into the established tumors. Figures 18A and 18B show the serial bioluminescence data and corresponding summary data. Serial bioluminescence imaging showed that iTDC-TK treatment attenuated the progression of GBM4 tumors, reducing tumor burden by 9-fold compared to control 28 days after injection. iTDC-TK therapy also led to a significant extension in survival as iTDC-TK treated animals survived an average of 67 days compared to only 37 days in control -treated mice. Post-mortem IHC, the images of which are shown in Figures 19A and 19B, verified the significant reduction in tumor volumes by iTDC- TK injection. Figures 19A and 19B show whole-brain and high-magnification images showing cell nuclei (blue), GBM4 (green), and iTDCTE -TK (red) distribution 21 days after delivering iTDCTE -control (I) or iTDCTE -TK (J) into established GBM4 tumors. A large GBM4 tumor was present in the control iTDCTE -TK animals, and only a small GBM4 focus was detected in mice treated with iTDCTE -TK+ GCV. Together, these results show that iTDC-TK therapy has significant therapeutic effects against malignant and invasive GBM and markedly prolongs the survival of tumor-bearing mice.

[00152] Intracavity iTDC-TK Therapy inhibits surgically resected GBM recurrence. Surgical resection is part of the clinical standard of care for GBM patients. We previously discovered that encapsulation of stem cells is required for intracavity therapy to effectively suppress GBM recurrence. To determine the efficacy of iTDC therapy encapsulated in synthetic extracellular matrices (sECM), we co-cultured GBM-8 GFPFL (patient-derived CD133+ human GEM- initiating cell) with iTDC-TK embedded in ELLA hydrogels. Figures 20A and 20B show the fluorescent images of the cultures and the summary data. We found that mCherry+ iTDCs migrated from the sECM matrix and populated GFP+ GBM8 spheroids within 3 days.

Additionally, sECM/iTDC-TK therapy dramatically reduced the viability of GBM8 spheroids in 3 days.

[00153] To mimic iTDC therapy for surgically resected human GBM patients, we tested h- iNTD-TK therapy against highly diffuse patient-derived GBM8 cells in a mouse model of GBM resection. iTDC-TK embedded in HLA were transplanted into the surgical resection cavity following GBM debulking. Figures 21A and 21B show the resulting serial bioluminescence images and summary data. Serial bioluminescence imaging showed that iTDC-TK therapy attenuated the recurrence of GBM8 tumors, reducing tumor burden by 350% compared to control 14 days after implantation. iTDC-TK therapy also led to a significant extension in survival as iTDC-TK treated animals survived an average of 59 days compared to 46 days in control -treated mice.