WO2020150391A1

WO2020150391A1 - Engineered stem cell constructs and uses thereof

Info

Publication number: WO2020150391A1
Application number: PCT/US2020/013744
Authority: WO
Inventors: Clayton BEARD
Original assignee: Falcon Therapeutics, Inc.
Priority date: 2019-01-15
Filing date: 2020-01-15
Publication date: 2020-07-23

Abstract

Disclosed herein, in certain embodiments, are recombinant polynucleotides which encodes an apoptotic inducing ligand under a first translation initiation signal, and a transdifferentiation factor and an anti-cancer polypeptide under a second translation initiation signal. In some embodiments, also disclosed herein is an engineered stem cell which comprises the recombinant polynucleotide and methods of generating the engineered stem cell. In additional embodiments, disclosed herein are methods of utilizing the engineered stem cell as a therapeutic under a cancer setting.

Description

ENGINEERED STEM CELL CONSTRUCTS AND USES THEREOF

RELATED APPLICATIONS

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

62/792,833 filed on January 15, 2019, which is incorporated herein by reference in its entirety

SUMMARY OF THE DISCLOSURE

[0002] Disclosed herein, in certain embodiments, are recombinant polynucleotides, which include a first anti-cancer therapy segment comprising (a) a first nucleic acid sequence encoding (i) a first translation initiation signal and (ii) an anti-cancer therapeutic, and (b) a second anti cancer therapy segment comprising a second nucleic acid sequence encoding (i) a second translation initiation signal and (ii) an anti-cancer polypeptide, wherein said first translation initiation signal initiates the translation of the first nucleic acid sequence, and wherein said second translation initiation signal initiates the translation of the second nucleic acid sequence. In some embodiments, the second translation initiation signal is an internal ribosome entry site (IRES). In some embodiments, the recombinant polynucleotide further comprises a promoter, wherein the transcription of the first anti-cancer therapy segment and the second anti-cancer therapy segment is mediated by the promoter. In some embodiments, the second nucleic acid sequence encodes a transdifferentiation factor. In some embodiments, the transdifferentiation factor is Sox2. In some embodiments, the secretion of the anti -cancer therapeutic is increased 1.1 -fold compared to the secretion of the anti-cancer polypeptide generated by a recombinant polynucleotide consisting of a transdifferentiation factor, the anti-cancer therapeutic, and the anti-cancer polypeptide under the translational control of a single translation initiation signal. In some embodiments, the anti -cancer therapeutic is TNF-related apoptosis-inducing ligand (TRAIL). In some embodiments, the anti-cancer polypeptide is thymidine kinase (TK). In some embodiments, the recombinant polynucleotide is a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV), or a retrovirus. In some embodiments, the anti -cancer polypeptide is a cytokine. In some embodiments, the cytokine is a protein, peptide, glycoprotein, chemokine, interleukin, tumor necrosis factor (TNF), monocyte chemoattractant protein (MCP), IL-l-like cytokine, gamma chain cytokine, beta chain cytokine, IL-6-like cytokine, IL- 10-like cytokine, interferon, tumor necrosis factor, TGF-beta, macrophage inflammatory protein (MIP), tumor growth factor (TGF), matrix metalloprotease (MMP), or any combination thereof. In some embodiments, the recombinant polynucleotide is a single vector system.

[0003] Disclosed herein, in certain embodiments, are methods of treating cancer in an individual in need thereof, the methods comprising (a) generating a therapeutic cell from a target somatic cell or a target primary cell, comprising: i) introducing into the target somatic cell or the target primary cell the recombinant polynucleotide of claim 1; and ii) contacting the target somatic cell or the target primary cell with one or more reprogramming agents; and b) iii) administering the therapeutic cell to the individual, thereby treating the cancer. In some embodiments, the one or more reprogramming agents are selected from the group consisting of: GSK3 inhibitor, a WT agonist, an ALK4/5/7 inhibitor, an HD AC inhibitor, a p300 activator, a PDE4 inhibitor, an Adenylyl cyclase agonist, a retinoic acid receptor g agonist, a 5-HT3 antagonist, and a metabotropic glutamate (mGlu) receptor agonist. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is pulmonary cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, the breast cancer is triple negative breast cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is lymphoma. In some embodiments, the cancer is pancreatic cancer.

[0004] Disclosed herein, in certain embodiments, are methods of treating cancer in an individual in need thereof, comprising administering a cell comprising the recombinant polynucleotide disclosed herein. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is pulmonary cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, the breast cancer is triple negative breast cancer. In some embodiments, the cancer is melanoma. In some

embodiments, the cancer is leukemia. In some embodiments, the cancer is lymphoma. In some embodiments, the cancer is pancreatic cancer.

[0005] Disclosed herein, in certain embodiments, are methods of generating a therapeutic cell from a target somatic cell or a target primary cell, comprising: a) introducing into the target somatic cell or the target primary cell the recombinant polynucleotide of claim 1; and b) contacting the target somatic cell or the target primary cell with one or more reprogramming agents, wherein the recombinant polynucleotide gives rise, upon transcription, to a factor that contributes to the reprogramming of the target somatic cell or the target primary cell into a therapeutic cell. In some embodiments, the one or more reprogramming agents are selected from the group consisting of: GSK3 inhibitor, a WT agonist, an ALK4/5/7 inhibitor, an HDAC inhibitor, a p300 activator, a PDE4 inhibitor, an Adenylyl cyclase agonist, a retinoic acid receptor g agonist, a 5-HT3 antagonist, and a metabotropic glutamate (mGlu) receptor agonist. In some embodiments, the therapeutic cell is a iTDC. In some embodiments, the therapeutic cell expresses CXCR4.

[0006] Disclosed herein, in certain embodiments, are methods of generating a therapeutic cell from a target somatic cell or a target primary cell, comprising introducing into the target somatic cell or the target primary cell the recombinant polynucleotide of the present disclosure, wherein the recombinant polynucleotide gives rise, upon transcription, to a factor that contributes to the reprogramming of the target somatic cell or the target primary cell into a therapeutic cell. In some embodiments, the therapeutic cell is a iTDC. In some embodiments, the therapeutic cell expresses CXCR4.

[0007] Disclosed herein, in certain embodiments, are vectors, comprising: a) a promoter; b) a first nucleic acid sequence encoding an anti-cancer therapeutic, the first nucleic acid sequence under the transcriptional control of the promoter; c) a second nucleic acid sequence downstream of the first nucleic acid sequence and under the transcriptional control of the promoter, the second nucleic acid sequence encoding an internal ribsome entry site (IRES), an anti -cancer polypeptide and a transdifferentiation factor, the anti-cancer polypeptide and the

transdifferentiation factor under the translational control of the IRES. In some embodiments, the transdifferentiation factor is Sox2. In some embodiments, the anti -cancer therapeutic is TNF- related apoptosis-inducing ligand (TRAIL) or secretable TNF-related apoptosis-inducing ligand (S-TRAIL). In some embodiments, the anti -cancer polypeptide is thymidine kinase (TK) or a herpes simplex virus thymidine kinase (HSV-TK). In some embodiments, the anti -cancer therapeutic is secreted extracellularly. In some embodiments, the secretion of the anti-cancer therapeutic is increased 1.1 fold compared to the secretion of the anti-cancer polypeptide generated by a recombinant polynucleotide consisting of the transdifferentiation factor, the anti cancer therapeutic, the anti-cancer polypeptide under the translational control of a single translation initiation signal. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV), or a retrovirus. In some embodiments, the anti-cancer polypeptide is a cytokine. In some embodiments, the cytokine is a protein, peptide, glycoprotein, chemokine, interleukin, tumor necrosis factor (TNF), monocyte chemoattractant protein (MCP), IL-l-like cytokine, gamma chain cytokine, beta chain cytokine, IL-6-like cytokine, IL- 10-like cytokine, interferon, tumor necrosis factor, TGF-beta, macrophage inflammatory protein (MIP), tumor growth factor (TGF), matrix metalloprotease (MMP), or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which: [0009] FIGS 1A and IB and FIGS 2A and 2B show the results of the viability assays disclosed in Example 1 herein. FIG 1 includes bar graphs depicting the Plate 1 viability results after 24 (FIG 1 A) and 48 (FIG IB) hours, and FIG 2 includes bar graphs depicting the Plate 2 viability results after 24 (FIG 2A) and 48 (FIG 2B) hours. The y-axes of the graphs in FIGS 1 and 2 provide the percent viability of the cancer cell lines (CAOV-3, ES-2 and SKOV-3) exposed to the TRAIL supernatant secreted by cells induced with the vector constructs disclosed in Example 1 herein, which are provided on the X axes.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0008] Disclosed herein, in certain embodiments, are engineered stem cells that are capable of delivering a payload, for example a therapeutic payload or an imaging agent, to a tumor site of interest. Further disclosed herein, in some embodiments, are methods of preparing engineered stem cells ex vivo. Additionally, disclosed herein, in certain embodiments, are methods of treatment utilizing engineered stem cells.

A. Anti-Cancer Polynucleotides Encoding First and Second Translation Initiation Signals

[0009] 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 (iTDCs) 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) for treating the cancer. In many instances, it is desirable to maximize the secretion of and iTDC therapeutic payload in order to maximize cancer cell -killing.

[0010] Disclosed herein, in certain embodiments, are recombinant polynucleotides (e.g., to be used to generate a therapeutic cell or iTDC) comprising, under the control of separate translation initiation signals, (i) a transdifferentiation factor and anti-cancer polypeptide and (ii) an anti -cancer therapeutic. In certain embodiments, the recombinant polynucleotides disclosed herein comprise: a) a first anti -cancer therapy segment comprising a first nucleic acid sequence encoding a first translation initiation signal and an anti -cancer therapeutic; and b) a second anti cancer therapy segment comprising a second nucleic acid sequence encoding a second translation initiation signal and an anti -cancer polypeptide, wherein said first translation initiation signal initiates the translation of the first nucleic acid sequence, and wherein said second translation initiation signal initiates the translation of the second nucleic acid sequence. In some embodiments the recombinant polynucleotides are used to treat breast cancer (e.g., triple negative breast cancer), brain cancer (e.g., glioblastoma), melanoma, ovarian cancer, pancreatic cancer, leukemia, lymphoma or pulmonary cancer. In some embodiments, this construct design allows for increased secretion of the anti -cancer therapeutic, as compared to a construct where the transdifferentiation factor and anti-cancer elements are located on a single peptide that is cleaved when expressed in a cell (e.g., a 2A self-cleaving peptide). In some embodiments, this construct allows for more predictable and safe transduction as compared to a construct where each of the transdifferentiation factor, anti-cancer polypeptide, and anti-cancer therapeutic are delivered to a therapeutic cell by three separate vectors.

i. Constructs

[0011] Disclosed herein, in certain embodiments, are recombinant polynucleotides comprising a first anti-cancer therapy segment and a second anti-cancer therapy segment. In some embodiments, the first anti-cancer therapy segment comprises a polynucleotide encoding a first translation initiation signal. In some embodiments, the first anti-cancer therapy segment comprises a polynucleotide encoding 1 translation initiation signal to 5 translation initiation signals. In some embodiments, the first anti-cancer therapy segment comprises a polynucleotide encoding 1 translation initiation signal to 2 translation initiation signals, 1 translation initiation signal to 3 translation initiation signals, 1 translation initiation signal to 4 translation initiation signals, 1 translation initiation signal to 5 translation initiation signals, 2 translation initiation signals to 3 translation initiation signals, 2 translation initiation signals to 4 translation initiation signals, about 2 translation initiation signals to 5 translation initiation signals, 3 translation initiation signals to 4 translation initiation signals, 3 translation initiation signals to 5 translation initiation signals, or 4 translation initiation signals to 5 translation initiation signals. In some embodiments, the first anti -cancer therapy segment comprises a polynucleotide encoding 1 translation initiation signal, 2 translation initiation signals, 3 translation initiation signals, 4 translation initiation signals, or about 5 translation initiation signals. In some embodiments, the first anti-cancer therapy segment comprises a polynucleotide encoding at least 1 translation initiation signal, at least 2 translation initiation signals, at least 3 translation initiation signals, at least 4 translation initiation signals, or at least 5 translation initiation signals. In some

embodiments, the first anti -cancer therapy segment comprises a polynucleotide encoding at most about 2 translation initiation signals, at most 3 translation initiation signals, at most 4 translation initiation signals, or at most 5 translation initiation signals.

In some embodiments, the first anti-cancer therapy segment comprises a polynucleotide encoding a first translation initiation signal that controls the translation of a first nucleic acid sequence encoding an anti-cancer therapeutic. In some embodiments, the first nucleic acid sequence encodes a pro-apoptotic ligand (or apoptotic-inducing ligand). In some embodiments, the pro- apoptotic ligand utilizes an extrinsic signaling pathway to initiate apoptosis. In some

embodiments, the pro-apoptotic ligand comprises: tumor necrosis factor (TNF)-related apoptosis- inducing ligand (TRAIL) (also known as Apo2 ligand, Apo2L, or TNFSF10), which binds to Death receptor 4 (DR4; also known as TNFRSFIOA, TRAILR1, or AP02) or Death receptor 5 (DR5; also known as TNFRS10B, TRAIL-R2, TRICK2, KILLER, or ZTNFR9); Apo3 ligand (Apo3L) (also known as TNFSF12, TWEAK, or DR3LG), which binds to Death receptor 3 (DR3; also known as TNFRSF12, Apo3, WSL-1, TRAMP, LARD, or DDR3); fatty acid synthetase ligand (FasL) (also known as TNFSF6, Apol, apoptosis antigen ligand 1, CD95L, CD178, or APTILGI), which binds to fatty acid synthetase receptor (FasR) (or TNFRSF6,

APTl, or CD95); or tumor necrosis factor alpha (TNF-a) (also known as TNFA or cachectin), which binds to tumor necrosis factor receptor 1 (TNFR1) (or TNFRSF1 A, p55 TNFR, or CD 120a).

[0012] In some embodiments, the pro-apoptotic ligand is TRAIL. In some embodiments, the pro-apoptotic ligand is a secretable TRAIL (sTRAIL). In some embodiments, the TRAIL protein is a truncated polypeptide comprising a leucine zipper domain, which trimerizes and optionally increases potency. In some embodiments, the truncated TRAIL polypeptide comprises from about residue 95 to about residue 281 of a full-length wild-type TRAIL (e.g., a wild-type human TRAIL). In some embodiments, the truncated TRAIL polypeptide comprises from about residue 95 to about residue 281 of TRAIL of NCBI Ref. No: NP_003801.1, or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence from about residue 95 to about residue 281 of TRAIL of NCBI Ref. No: NP_003801.1.

[0013] In some embodiments, the TRAIL protein is a fusion protein comprising a truncated TRAIL polypeptide operably linked to a fms-related tyrosine kinase 3 ligand (FLT3LG or FLT3 ligand) secretion sequence. In some embodiments, the FLT3LG secretion sequence comprises the first 182 residue of a wild-type FLT3 ligand. In some embodiments, the FLT3LG secretion sequence comprises the first 182 residue of FLT3 ligand of NCBI Ref. No: NP 001450, or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the first 182 residue of FLT3 ligand of NCBI Ref. No: NP_001450.

[0014] In some embodiments, the TRAIL fusion protein comprises an N-terminal FLT3LG secretion sequence and a C-terminal TRAIL leucine zipper domain. In some embodiments, the N-terminal FLT3LG secretion sequence is directly fused to the C-terminal TRAIL leucine zipper domain. In some embodiments, the N-terminal FLT3LG secretion sequence is indirectly fused to the C-terminal TRAIL leucine zipper domain via a linker. In some embodiments, the linker comprises ASMKQIEDKIEEILSKIYHIENEIARIKKLIGEREEF (SEQ ID NO:5). [0015] In some embodiments, the second anti-cancer therapy segment comprises a polynucleotide encoding a second translation initiation signal. In some embodiments, the second anti-cancer therapy segment comprises a polynucleotide encoding second translation initiation signal, and a third translation initiation signal. In some embodiments, the second anti -cancer therapy segment comprises a polynucleotide encoding 1 translation initiation signal to 5 translation initiation signals. In some embodiments, the second anti -cancer therapy segment comprises a polynucleotide encoding 1 translation initiation signal to 2 translation initiation signals, 1 translation initiation signal to 3 translation initiation signals, 1 translation initiation signal to 4 translation initiation signals, 1 translation initiation signal to 5 translation initiation signals, about 2 translation initiation signals to 3 translation initiation signals, about 2 translation initiation signals to 4 translation initiation signals, about 2 translation initiation signals to 5 translation initiation signals, about 3 translation initiation signals to 4 translation initiation signals, about 3 translation initiation signals to 5 translation initiation signals, or about 4 translation initiation signals to 5 translation initiation signals. In some embodiments, the second anti-cancer therapy segment comprises a polynucleotide encoding 1 translation initiation signal, about 2 translation initiation signals, about 3 translation initiation signals, about 4 translation initiation signals, or about 5 translation initiation signals. In some embodiments, the second anti cancer therapy segment comprises a polynucleotide encoding at least 1 translation initiation signal, about 2 translation initiation signals, about 3 translation initiation signals, or about 4 translation initiation signals. In some embodiments, the second anti -cancer therapy segment comprises a polynucleotide encoding at most about 2 translation initiation signals, about 3 translation initiation signals, about 4 translation initiation signals, or about 5 translation initiation signals.

[0016] In some embodiments, the second anti-cancer therapy segment comprises a polynucleotide encoding a second translation initiation signal that controls the translation of the second nucleic acid encoding a transdifferentiation factor and/or a third nucleic acid sequence encoding an anti-cancer polypeptide. In some embodiments, the second anti-cancer therapy segment comprises a polynucleotide encoding third translation initiation signal that controls the translation of a third nucleic acid encoding a transdifferentiation factor and/or anti-cancer polypeptide.

[0017] In some embodiments, the translation initiation signal is an internal ribosome entry site (“IRES” herein) ,a polynucleotide sequence, which when present in an RNA, promotes direct internal ribosomal entry (e.g., of the 40S ribosomal subunit) upstream of a translation initiation codon (e.g., a codon that initiates translation of mRNA). In some embodiments, an IRES allows for cap-independent translation of a polynucleotide sequence. In some embodiments, an IRES allows for the expression of two or more separate polypeptide constructs from a single polynucleotide sequence. In some embodiments, the first translation initiation signal is an IRES and the second translation initiation signal is an IRES. In some embodiments, the second translation initiation signal is an IRES and the third translation initiation signal is an IRES. In some embodiments, the first translation initiation signal is an IRES, the second translation initiation signal is an IRES, and the third translation initiation signal is an IRES.

[0018] In some embodiments, the translation initiation signal is not an IRES. In some embodiments, the translation initiation signal is a 5’ cap. In some embodiments, the first translation initiation signal is a 5’ cap. In some embodiments, the first translation initiation signal is a 5’ cap, and the second translation initiation is an IRES. In some embodiments, the first translation initiation signal is a 5’ cap, the second translation initiation signal is an IRES, and the third translation initiation signal is an IRES.

[0019] In some embodiments, the recombinant polynucleotide comprises a promoter. In some embodiments, the transcription of the translation initiation signals is mediated (e.g., initiated and directed) by the promoter. In some embodiments, the transcription of the first translation initiation signal and the second translation initiation signal is mediated by the promoter. In some embodiments, the transcription of the first translation initiation signal, the second translation initiation signal, and the third translation initiation signal is mediated by the promoter. In some embodiments, the promoter-first translation initiation signal-second translation initiation signal construct allows for the translation of multiple polypeptide constructs from a single

polynucleotide, mediated by a single promoter.

[0020] In some embodiments, the translation initiation signal of the first anti-cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic. In some embodiments, the translation initiation signal or translation initiation signals of the first anti-cancer therapy segment control the expression of a second nucleic acid encoding a transdifferentiation factor and a third nucleic acid encoding an anti-cancer polypeptide. In some embodiments, the translation initiation signal of the first anti-cancer therapy segment control the expression of a second nucleic acid encoding a transdifferentiation factor. In some embodiments, the translation initiation signal of the first anti-cancer therapy segment control the expression of a third nucleic acid encoding an anti-cancer polypeptide. In some embodiments, the translation initiation signal of the first anti-cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic and the expression of a second nucleic acid encoding a transdifferentiation factor.

[0021] In some embodiments, the translation initiation signal of the second anti-cancer therapy segment control the expression of a second nucleic acid encoding a transdifferentiation factor. In some embodiments, the translation initiation signal of the second anti-cancer therapy segment control the expression of a second nucleic acid encoding a transdifferentiation factor and a third nucleic acid encoding an anti -cancer polypeptide. In some embodiments, the translation initiation signal of the second anti-cancer therapy segment control the expression of a third nucleic acid sequence encoding an anti -cancer polypeptide. In some embodiments, the translation initiation signal of the second anti-cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti -cancer therapeutic.

[0022] In some embodiments, the translation initiation signal of a third anti-cancer therapy segment controls the expression a third nucleic acid encoding an anti-cancer polypeptide. In some embodiments, the translation initiation signal of the third anti-cancer therapy segment control the expression of a second nucleic acid encoding a transdifferentiation factor. In some embodiments, the translation initiation signal of the third anti -cancer therapy segment control the expression of a third nucleic acid encoding an anti-cancer therapeutic.

[0023] In some embodiments, the translation initiation signal that control the expression of the first nucleic acid sequence is or are different from the translation initiation signal that control the expression of the second nucleic acid sequence and/or the third nucleic acid sequence. In some embodiments, the RNA product of the first nucleic acid sequence is translated into a polypeptide construct separate from the RNA product of the second and/or third nucleic acid sequences. In some embodiments, the translation initiation signal that control the expression of the second nucleic acid sequence is or are different from the translation initiation signal that control the expression of the first nucleic acid sequence and/or the third nucleic acid sequence. In some embodiments, the RNA product of the second nucleic acid sequence is translated into a polypeptide construct separate from the RNA product of the first and/or third nucleic acid sequences. In some embodiments, the translation initiation signal that control the expression of the third nucleic acid sequence is or are different from the translation initiation signal that control the expression of the first nucleic acid sequence and/or the third nucleic acid sequence. In some embodiments, the RNA product of the third nucleic acid sequence is translated into a polypeptide construct separate from the RNA product of the first and/or second nucleic acid sequences.

[0024] In some embodiments, the translation initiation signal that control the expression of the second nucleic acid sequence is or are the same from the translation initiation signal that control the expression of the third nucleic acid sequence. In some embodiments, the translation initiation signal that control the expression of the first nucleic acid sequence is or are the same from the translation initiation signal that control the expression of the third nucleic acid sequence. [0025] The transdifferentiation factor is a protein such as a transcription factor that promotes the direct conversion of one somatic cell type to another. Exemplary transdifferentiation factors include, but are not limited to, Oct4, Sox2, Klf4, Myc, Ascii, Bm2, Mytll, 01ig2, or Zicl.

[0026] In some embodiments, the second nucleic acid encodes Sox2. In some embodiments, Sox2 is used as the transdifferentiation factor to reprogram a somatic cell or primary cell into a engineered stem cell. In some embodiments, Sox2 is used to carry out a single-factor

transdifferentiation.

[0027] In some embodiments, the second nucleic acid encodes a full-length Sox2 or a functional fragment thereof. In some embodiments, the Sox2 is a wild-type human Sox2 protein or a variant or isoform thereof. In some embodiments, the functional fragment comprises an N- terminal deletion, a C-terminal deletion, or an internal deletion.

[0028] In some embodiments, the Sox2 sequence comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 90% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 96% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 97% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 98% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 100% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence consists of SEQ ID NO: 2.

[0029] In some embodiments, the third nucleic acid encodes an anti-cancer polypeptide. In some embodiments, the anti -cancer polypeptide is a cytokine. Exemplary cytokines include chemokines such as the CXC, CC, CX3C, and XC subfamilies, e.g., CCL2 (also known as monocyte chemoattractant protein- 1 or MCP-1), CCL3 (also known as macrophage

inflammatory protein la or MIP-la), CCL4 (also known as MPMb), CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, or CXCL13; interferons such as IFN-a, IFN-b, or IFN-g; interleukins such as IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, or IL-21; lymphokines such as Granulocyte-macrophage colony-stimulating factor (GM-CSF); and tumor necrosis factors (TNFs) such as TNF-a.

[0030] In some embodiments, the anti-cancer polypeptide is a gamma chain cytokine, or a cytokine such as IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21 that recognizes the IL-2 receptor gamma. [0031] In some embodiments, the anti-cancer polypeptide is a beta chain cytokine, or a cytokine such as IL-5, IL-3, or GM-CSF that recognizes the common beta chain receptor that is shared among the cytokines.

[0032] In some embodiments, the anti-cancer polypeptide is a tumor growth factor (TGF). In some embodiments, the TGF comprises TGFa or TGFp.

[0033] In some embodiments, the anti-cancer polypeptide is a matrix metalloprotease (MMP) (also known as matrixins). Exemplary MMPs include, but are not limited to, MMP-2, MMP-9, MMP-11, MMP-14, MMP-15, MMP-16, MMP-17, or MMP-19. In some embodiments, the MMP is co-expressed with another anti-cancer protein.

[0034] In some embodiments, the third nucleic acid encodes a thymidine kinase (TK), e.g., a full-length TK or a functionally active fragment thereof. In some embodiments, the TK is a herpes simplex virus thymidine kinase (HSV-TK). In some embodiments, the TK is a HSV1-TK (e.g., a wild-type HSV1-TK).

[0035] In some embodiments, the thymidine kinase is a modified TK. In some embodiments, the modified TK comprises one or more mutations within the catalytic domain, a deletion at a terminus position, or a combination. In some embodiments, the modified TK comprises a mutation at the catalytic domain that decreases or abolishes TK activity but maintains a guanine nucleoside analogue phosphorylating capacity. In some embodiments, the modified TK comprises a mutation at amino acid residue L159, 1160, F161, A167, A168, or L169, or a combination thereof, wherein the residues correspond to positions 159, 160, 161, 167, 168, and 169 of SEQ ID NO: 3. In some embodiments, L159 is mutated to He (I). In some embodiments, 1160 is mutated to Leu (L) or Phe (F). In some embodiments, F161 is mutated to Leu (L), Ala (A), or Val (V). In some embodiments, A167 is mutated to Phe (F). In some embodiments, A168 is mutated Tyr (Y), Phe (F), or Val (V). In some embodiments, L169 is mutated to Asn (N), Tyr (Y), or Met (M).

[0036] In some embodiments, the modified TK comprises mutations LI 591, 1160F, F161L, A168F, and L169M (also known as HSV-TK mutant sr39 or sr39tk), wherein the residues correspond to positions 159, 160, 161, 168, and 169 of SEQ ID NO: 3. In some embodiments, the HSV-TK mutant sr39 further comprises an N-terminal deletion. In some embodiments, the deletion comprises a deletion of about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues from the N- terminus. In some embodiments, the deletion comprises a deletion of about 45 residues from the N-terminus.

[0037] In some embodiments, the HSV-TK mutant sr39 further comprises a modification of the nuclear localization signal, e.g., a mutation at residue 25, 26, 32, or 33, or a combination thereof, in which the residues correspond to positions 25, 26, 32, and 33 of SEQ ID NO: 3. In some embodiments, the residues 25, 26, 32, and 33 are each independently mutated to Ala or Gly.

[0038] In some embodiments, the modified HSV-TK comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises about at least about 90% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises at least about 95% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises at least about 96% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV- TK comprises at least about 97% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises at least about 98% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises at least about 99% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises at least about 100% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK consists of SEQ ID NO: 4.

[0039] In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by a polynucleotide encoding a self-cleaving peptide. Self-cleaving peptide mediates co-translational cleavage of proteins that are upstream and downstream from the cleavage site, allowing the production of both the transdifferentiation factor and the anti-cancer polypeptide under the control of a single promoter. In particular, the self-cleaving sequence causes a translating eukaryotic ribosome to release the growing polypeptide chain that it is synthesizing without dissociating from the mRNA. In some embodiments, the self-cleavage peptide is a 2A peptide, a 2A-like peptide, or a CHYSEL (cis-acting hydrolase element) sequence. Exemplary 2A peptides include, but are not limited to, F2A (foot-and-mouth disease virus): VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 6), E2A (equine rhinitis A virus): QCTNYALLKLAGDVESNPGP (SEQ ID NO: 7), P2A (porcine teschovirus-1 2A):

ATNFSLLKQAGDVEENPGP, and T2A (thosea asigna virus 2A): EGRGSLLTCGDVEENPGP. In some embodiments, a GSG peptide is further added to the N-terminus of the 2A peptide to improve cleavage efficiency. In some embodiments, the 2A-like peptide is a peptide disclosed in U.S. Patent No. 8,975,042. In some embodiments, the CHYSEL sequence is a picornavirus 2A peptide disclosed in de Felipe P (2004) Skipping the co-expression problem: the new 2 A “CHYSEL” technology. Genet Vaccines Ther 2: 13.

[0040] In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by a 2A peptide, a 2A-like peptide, or a CHYSEL sequence. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by F2A. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by P2A. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by E2A. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by T2A. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by a 2A-like peptide. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by a CHYSEL sequence.

[0041] In some embodiments, if the second translation initiation signal within the second anti-cancer therapy segment is used as a reference point within the recombinant polynucleotide, the first anti-cancer therapy segment is located upstream of the second translation initiation signal. In some embodiments, the first anti -cancer therapy segment is at least about 10, 20, 30,

40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000 bases, or more upstream of the second translation initiation signal.

[0042] In some embodiments, the first anti-cancer therapy segment is adjacent to the 5’ end of the second translation initiation signal. In such cases, the first anti -cancer therapy segment is less than or about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base from the 5’ end of the second translation initiation signal.

[0043] In some embodiments, the first translation initiation signal is located upstream of the nucleic acid encoding the anti-cancer therapeutic. In such cases when the first anti-cancer therapy segment is located upstream of the second translation initiation signal, the first translation initiation signal is farther away from the second translation initiation signal than the nucleic acid encoding the anti-cancer therapeutic (e.g., the construct order is first translation initiation signal - anti-cancer therapeutics - second translation initiation signal). In other instances, the first translation initiation signal is closer to the second translation initiation signal than the nucleic acid encoding the anti-cancer therapeutic (e.g., the construct order is anti-cancer therapeutics - first translation initiation signal - second translation initiation signal).

[0044] In some embodiments within the second anti-cancer therapy segment, the

transdifferentiation factor is located between the second translation initiation signal and the anti cancer polypeptide. In such cases, the transdifferentiation factor is located about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 bases, or more downstream of the second translation initiation signal.

[0045] In some embodiments, the anti-cancer polypeptide is located between the second translation initiation signal and the transdifferentiation factor. In such cases, the anti -cancer polypeptide is located about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 bases, or more downstream of the second translation initiation signal.

[0046] In some embodiments, the first translation initiation signal and the nucleic acid encoding the anti-cancer therapeutic is upstream of the second translation initiation signal, the transdifferentiation factor, and the anti-cancer polypeptide. In some embodiments, the construct order is: first translation initiation signal - anti-cancer therapeutic - second translation initiation signal-transdifferentiation factor-anti-cancer polypeptide. In some embodiments, the construct order is: first translation initiation signal - anti-cancer therapeutic - second translation initiation signal-anti-cancer polypeptide-transdifferentiation factor.

[0047] In some embodiments, the exemplary construct orders include, but are not limited to,:

(i) Promoter-first translation initiation signal -anti-cancer therapeutic-second translation initiation signal-anti-cancer polypeptide-transdifferentiation factor

(ii) Promoter-first translation initiation signal-anti-cancer therapeutic-second translation initiation signal-transdifferentiation factor-anti-cancer polypeptide

(iii) Promoter-first translation initiation signal -anti-cancer therapeutic-second translation initiation signal-transdifferentiation factor-third translation initiation signal-anti-cancer polypeptide

(iv) Promoter-first translation initiation signal -anti-cancer therapeutic-second translation initiation signal -anti-cancer polypeptide-third translation initiation signal-anti-cancer polypeptide

(v) Promoter-first translation initiation signal -anti-cancer polypeptide-transdifferentiation factor- second translation initiation signal-anti-cancer therapeutic

(vi) Promoter-first translation initiation signal-transdifferentiation factor-anti-cancer polypeptide- second translation initiation signal-anti-cancer therapeutic

(vii) Promoter-first translation initiation signal-transdifferentiation factor-second translation initiation signal -anti-cancer polypeptide-third translation initiation signal-anti-cancer therapeutic (viii) Promoter-first translation initiation signal-transdifferentiation factor-second translation initiation signal- anti -cancer therapeutic -third translation initiation signal-anti-cancer

polypeptide

ii. Vectors

[0048] In some embodiments, a recombinant polynucleotide disclosed above is inserted into a vector. In some embodiments, the vector optionally comprises one or more promoters, enhancers, ribosome binding sites, RNA splice sites, polyadenylation sites, a replication origin, and/or transcriptional terminator sequences.

[0049] Promoters are specific nucleotide sequences in DNAs that allow initiation of transcription using DNAs as templates, and have a consensus sequence in general. In some embodiments, the promoters are constitutive promoters. In other instances, the promoters are inducible promoters. In additional instances, the promoters are specific promoters. In some embodiments, the promoters are eukaryotic promoters, or promoters used in a eukaryotic system.

[0050] Exemplary promoters include, but are not limited to, CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALl-10, TEF1, GDS, ADH1, CaMV35S, Ubi, HI, U6, CaMV35S, SV40, CMV, and HSV TK promoter.

[0051] In some embodiments, the promoter is e.g., CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALl-10, TEF1, GDS, ADH1, CaMV35S, Ubi, HI, U6, CaMV35S, SV40, or HSV TK promoter. In some embodiments, the promoter is CMV. In some embodiments, the promoter is EFla. In some embodiments, the promoter is ubiquitin.

[0052] As disclosed herein, in some embodiments the vector is a bicistronic or polycistronic vector (e.g., having or involving two or more loci responsible for generating a protein) having an internal ribosome entry site (IRES) is for translation initiation in a cap-independent manner. In some embodiments, an IRES is located upstream of the nucleic acids encoding the

transdifferentiation factor and the anti-cancer polypeptide, respectively (e.g., IRES- transdifferentiation factor-anti-cancer polypeptide or IRES-anti-cancer polypeptide- transdifferentiation factor). In some embodiments, the IRES is located at least or about 10, 20,

30, 40, 50, 100, 200, 300, 400, 500 bases, or more upstream of the nucleic acids encoding the transdifferentiation factor and the anti-cancer polypeptide, respectively. In some embodiments, an IRES is located at the 5’ end of the nucleic acids encoding the transdifferentiation factor and the anti -cancer polypeptide, respectively. In some embodiments, an IRES is located between the anti-cancer polypeptide and the anti -cancer therapeutic.

[0053] Enhancers are nucleotide sequences that have the effect of enhancing promoter activity, and in general, often comprise about 100 bp. In some embodiments, enhancers augment transcription regardless of the orientation of their sequence. In some embodiments, enhancers activate transcription from a distance of several kilo base pairs. Furthermore, enhancers are located optionally upstream or downstream of a gene region to be transcribed, and/or located within the gene, to activate the transcription.

[0054] Exemplary enhancers include, but are not limited to, WPRE; CMV enhancers; the R- U5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; the intron sequence between exons 2 and 3 of rabbit b-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981); and the genome region of human growth hormone (J Immunol., Vol. 155(3), p. 1286-95, 1995). [0055] In some embodiments, one or more selectable markers are also present in a vector disclosed herein. In some embodiments, the selectable marker is an antibiotic resistant gene. Exemplary antibiotic resistant genes include, but are not limited to, ampicillin, chloramphenicol, kanamycin, tetracycline, polymyxin B, erythromycin, carbenicillin, streptomycin, spectinomycin, blasticidin S deaminases ( Bsr , BSD), bleomycin-binding protein ( Ble ), Neomycin

phosphotransferase ( neo ), puromycin N-acetyltransferase ( Pac ), zeocin (Sh bla ), and hygromycin B phosphotransferase ( Hph ). In some embodiments, the selectable marker is a eukaryotic antibiotic resistant gene. In some embodiments, the selectable marker is blasticidin S deaminases (Bsr, BSD), bleomycin-binding protein (Ble), Neomycin phosphotransferase (neo), puromycin N- acetyltransferase (Pac), zeocin (Sh bla), or hygromycin B phosphotransferase (Hph).

[0056] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno- associated viral vectors (AAVs), or herpes simplex virus vectors (HSVs). In some embodiments, the retroviral vectors include gamma-retroviral vectors such as vectors derived from the Moloney Murine Keukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Steam cell Virus (MSCV) genome. In some embodiments, the retroviral vectors also include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some embodiments, AAV vectors include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. In some embodiments, viral vector is a chimeric viral vector, comprising viral portions from two or more viruses. In additional embodiments, the viral vector is a recombinant viral vector.

[0057] In some embodiments, the vector is a non-viral vector. In such instances, a physical method or a chemical method is employed for delivery into the somatic cell. Exemplary physical methods include electroporation, gene gun, sonoporation, magnetofection, or hydrodynamic delivery. Exemplary chemical methods include delivery of the recombinant polynucleotide via liposomes such as, cationic lipids or neutral lipids; dendrimers; nanoparticles; or cell -penetrating peptides.

B. Anti-Cancer Polynucleotides Encoding First and Second Promoter

[0058] Disclosed herein, in certain embodiments, are recombinant polynucleotides, comprising: a) a first anti-cancer therapy segment comprising a first promoter and a first nucleic acid sequence encoding an anti-cancer therapeutic, and b) a second anti-cancer therapy segment comprising a second promoter, and a second nucleic acid sequence encoding an anti-cancer polypeptide; wherein the first promoter mediates the transcription of the first nucleic acid sequence encoding the anti -cancer therapeutic, and wherein the second promoter mediates the transcription of the second nucleic acid sequence encoding the anti-cancer polypeptide. In some embodiments the recombinant polynucleotides are used to treat breast cancer (e.g., triple negative breast cancer), brain cancer (e.g., glioblastoma), melanoma, ovarian cancer, pancreatic cancer, leukemia, lymphoma or pulmonary cancer.

i. Constructs

[0059] In some embodiments, the first anti-cancer therapy segment comprises a first promoter and a second promoter. In some embodiments, the first anti -cancer therapy segment comprises a first promoter, a second promoter, and a third promoter. In some embodiments, the first anti-cancer therapy segment comprises a first promoter and a second promoter. In some embodiments, the first anti -cancer therapy segment comprises about 1 promoter to about 5 promoters. In some embodiments, the first anti-cancer therapy segment comprises about 1 promoter to about 2 promoters, about 1 promoter to about 3 promoters, about 1 promoter to about 4 promoters, about 1 promoter to about 5 promoters, about 2 promoters to about 3 promoters, about 2 promoters to about 4 promoters, about 2 promoters to about 5 promoters, about 3 promoters to about 4 promoters, about 3 promoters to about 5 promoters, or about 4 promoters to about 5 promoters. In some embodiments, the first anti -cancer therapy segment comprises about 1 promoter, about 2 promoters, about 3 promoters, about 4 promoters, or about 5 promoters. In some embodiments, the first anti-cancer therapy segment comprises at least about 1 promoter, about 2 promoters, about 3 promoters, or about 4 promoters. In some embodiments, the first anti cancer therapy segment comprises at most about 2 promoters, about 3 promoters, about 4 promoters, or about 5 promoters.

[0060] In some embodiments, the first anti-cancer therapy segment comprises a first promoter that controls the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic. In some embodiments, the first nucleic acid sequence encodes a pro-apoptotic ligand (or apoptotic-inducing ligand). In some embodiments, the pro-apoptotic ligand utilizes an extrinsic signaling pathway to initiate apoptosis. In some embodiments, the pro-apoptotic ligand comprises:

[0061] tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (also known as Apo2 ligand, Apo2L, or TNFSF10), which binds to Death receptor 4 (DR4; also known as TNFRSF10A, TRAILR1, or AP02) or Death receptor 5 (DR5; also known as TNFRS10B, TRAIL-R2, TRICK2, KILLER, or ZTNFR9);

[0062] Apo3 ligand (Apo3L) (also known as TNFSF12, TWEAK, or DR3LG), which binds to Death receptor 3 (DR3; also known as TNFRSF12, Apo3, WSL-1, TRAMP, LARD, or DDR3); [0063] fatty acid synthetase ligand (FasL) (also known as TNFSF6, Apol, apoptosis antigen ligand 1, CD95L, CD178, or APT1LG1), which binds to fatty acid synthetase receptor (FasR) (or TNFRSF6, APT1, or CD95); or

[0064] tumor necrosis factor alpha (TNF-a) (also known as TNFA or cachectin), which binds to tumor necrosis factor receptor 1 (TNFR1) (or TNFRSF1A, p55 TNFR, or CD120a).

[0065] In some embodiments, the pro-apoptotic ligand is TRAIL. In some embodiments, the pro-apoptotic ligand is a secretable TRAIL (sTRAIL). In some embodiments, the TRAIL protein is a truncated polypeptide comprising a leucine zipper domain, which trimerizes and optionally increases potency. In some embodiments, the truncated TRAIL polypeptide comprises from about residue 95 to about residue 281 of a full-length wild-type TRAIL (e.g., a wild-type human TRAIL). In some embodiments, the truncated TRAIL polypeptide comprises from about residue 95 to about residue 281 of TRAIL of NCBI Ref. No: NP_003801.1, or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence from about residue 95 to about residue 281 of TRAIL of NCBI Ref. No: NP_003801.1.

[0066] In some embodiments, the TRAIL protein is a fusion protein comprising a truncated TRAIL polypeptide operably linked to a fms-related tyrosine kinase 3 ligand (FLT3LG or FLT3 ligand) secretion sequence. In some embodiments, the FLT3LG secretion sequence comprises the first 182 residue of a wild-type FLT3 ligand. In some embodiments, the FLT3LG secretion sequence comprises the first 182 residue of FLT3 ligand of NCBI Ref. No: NP 001450, or about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the first 182 residue of FLT3 ligand of NCBI Ref. No: NP_001450.

[0067] In some embodiments, the TRAIL fusion protein comprises an N-terminal FLT3LG secretion sequence and a C-terminal TRAIL leucine zipper domain. In some embodiments, the N-terminal FLT3LG secretion sequence is directly fused to the C-terminal TRAIL leucine zipper domain. In some embodiments, the N-terminal FLT3LG secretion sequence is indirectly fused to the C-terminal TRAIL leucine zipper domain via a linker. In some embodiments, the linker comprises ASMKQIEDKIEEILSKIYHIENEIARIKKLIGEREEF.

[0068] In some embodiments, the second anti-cancer therapy segment comprises a first promoter and a second promoter. In some embodiments, the second anti -cancer therapy segment comprises a first promoter, a second promoter, and a third promoter. In some embodiments, the second anti-cancer therapy segment comprises a first promoter and a second promoter. In some embodiments, the second anti -cancer therapy segment comprises about 1 promoter to about 5 promoters. In some embodiments, the second anti-cancer therapy segment comprises about 1 promoter to about 2 promoters, about 1 promoter to about 3 promoters, about 1 promoter to about 4 promoters, about 1 promoter to about 5 promoters, about 2 promoters to about 3 promoters, about 2 promoters to about 4 promoters, about 2 promoters to about 5 promoters, about 3 promoters to about 4 promoters, about 3 promoters to about 5 promoters, or about 4 promoters to about 5 promoters. In some embodiments, the second anti -cancer therapy segment comprises about 1 promoter, about 2 promoters, about 3 promoters, about 4 promoters, or about 5 promoters. In some embodiments, the second anti-cancer therapy segment comprises at least about 1 promoter, about 2 promoters, about 3 promoters, or about 4 promoters. In some embodiments, the second anti -cancer therapy segment comprises at most about 2 promoters, about 3 promoters, about 4 promoters, or about 5 promoters.

[0069] In some embodiments, the second anti-cancer therapy segment comprises a second promoter that controls the expression of a second nucleic acid encoding a transdifferentiation factor and a third nucleic acid sequence encoding an anti -cancer polypeptide.

[0070] In some embodiments, the promoter or promoters of the first anti-cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic and a second nucleic acid encoding a transdifferentiation factor. In some

embodiments, the promoter or promoters of the first anti-cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic and a third nucleic acid encoding an anti-cancer polypeptide. In some embodiments, the promoter or promoters of the first anti-cancer therapy segment control the expression of a second nucleic acid encoding a transdifferentiation factor and a third nucleic acid encoding an anti-cancer polypeptide. In some embodiments, the promoter or promoters of the first anti-cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic, a second nucleic acid encoding a transdifferentiation factor, and a third nucleic acid encoding an anti -cancer polypeptide.

[0071] In some embodiments, the promoter or promoters of the second anti -cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic and a second nucleic acid encoding a transdifferentiation factor. In some

embodiments, the promoter or promoters of the second anti-cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic and a third nucleic acid encoding an anti-cancer polypeptide. In some embodiments, the promoter or promoters of the second anti-cancer therapy segment control the expression of a second nucleic acid encoding a transdifferentiation factor and a third nucleic acid encoding an anti-cancer polypeptide. In some embodiments, the promoter or promoters of the second anti-cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic, a second nucleic acid encoding a transdifferentiation factor, and a third nucleic acid encoding an anti -cancer polypeptide. [0072] In some embodiments, the promoter or promoters of the third anti-cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic and a second nucleic acid encoding a transdifferentiation factor. In some

embodiments, the promoter or promoters of the third anti-cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic and a third nucleic acid encoding an anti-cancer polypeptide. In some embodiments, the promoter or promoters of the third anti -cancer therapy segment control the expression of a second nucleic acid encoding a transdifferentiation factor and a third nucleic acid encoding an anti-cancer polypeptide. In some embodiments, the promoter or promoters of the third anti-cancer therapy segment control the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic, a second nucleic acid encoding a transdifferentiation factor, and a third nucleic acid encoding an anti -cancer polypeptide.

[0073] In some embodiments, the promoter or promoters that control the expression of the first nucleic acid sequence is or are different from the promoter or promoters that control the expression of the second nucleic acid sequence and/or the third nucleic acid sequence. In some embodiments, the promoter or promoters that control the expression of the second nucleic acid sequence is or are different from the promoter or promoters that control the expression of the first nucleic acid sequence and/or the third nucleic acid sequence. In some embodiments, the promoter or promoters that control the expression of the third nucleic acid sequence is or are different from the promoter or promoters that control the expression of the first nucleic acid sequence and/or the third nucleic acid sequence.

[0074] In some embodiments, the promoter or promoters that control the expression of the first nucleic acid sequence is or are the same from the promoter or promoters that control the expression of the second nucleic acid sequence and/or the third nucleic acid sequence. In some embodiments, the promoter or promoters that control the expression of the second nucleic acid sequence is or are the same from the promoter or promoters that control the expression of the first nucleic acid sequence and/or the third nucleic acid sequence. In some embodiments, the promoter or promoters that control the expression of the third nucleic acid sequence is or are the same from the promoter or promoters that control the expression of the first nucleic acid sequence and/or the third nucleic acid sequence.

[0075] The transdifferentiation factor is a protein such as a transcription factor that promotes the direct conversion of one somatic cell type to another. Exemplary transdifferentiation factors include, but are not limited to, Oct4, Sox2, Klf4, Myc, Ascii, Bm2, Mytll, 01ig2, or Zicl.

[0076] In some embodiments, the second nucleic acid encodes Sox2. In some embodiments, Sox2 is used as the transdifferentiation factor to reprogram a somatic cell or primary cell into a engineered stem cell. In some embodiments, Sox2 is used to carry out a single-factor transdifferentiation.

[0077] In some embodiments, the second nucleic acid encodes a full-length Sox2 or a functional fragment thereof. In some embodiments, the Sox2 is a wild-type human Sox2 protein or a variant or isoform thereof. In some embodiments, the functional fragment comprises an N- terminal deletion, a C-terminal deletion, or an internal deletion.

[0078] In some embodiments, the Sox2 sequence comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 90% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 96% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 97% sequence identity to SEQ ID NO:

2. In some embodiments, the Sox2 sequence comprises about 98% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence comprises about 100% sequence identity to SEQ ID NO: 2. In some embodiments, the Sox2 sequence consists of SEQ ID NO: 2.

[0079] In some embodiments, the third nucleic acid encodes an anti-cancer polypeptide. In some embodiments, the anti -cancer polypeptide is a cytokine. Exemplary cytokines include chemokines such as the CXC, CC, CX3C, and XC subfamilies, e.g., CCL2 (also known as monocyte chemoattractant protein- 1 or MCP-1), CCL3 (also known as macrophage

[0080] In some embodiments, the anti-cancer polypeptide is a gamma chain cytokine, or a cytokine such as IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21 that recognizes the IL-2 receptor gamma.

[0081] In some embodiments, the anti-cancer polypeptide is a beta chain cytokine, or a cytokine such as IL-5, IL-3, or GM-CSF that recognizes the common beta chain receptor that is shared among the cytokines.

[0082] In some embodiments, the anti-cancer polypeptide is a tumor growth factor (TGF). In some embodiments, the TGF comprises TGFa or TORb.

[0083] In some embodiments, the anti-cancer polypeptide is a matrix metalloprotease (MMP) (also known as matrixins). Exemplary MMPs include, but are not limited to, MMP-2, MMP-9, MMP-11, MMP-14, MMP-15, MMP-16, MMP-17, or MMP-19. In some embodiments, the MMP is co-expressed with another anti-cancer protein.

[0084] In some embodiments, the third nucleic acid encodes a thymidine kinase (TK), e.g., a full-length TK or a functionally active fragment thereof. In some embodiments, the TK is a herpes simplex virus thymidine kinase (HSV-TK). In some embodiments, the TK is a HSV1-TK (e.g., a wild-type HSV1-TK).

[0085] In some embodiments, the thymidine kinase is a modified TK. In some embodiments, the modified TK comprises one or more mutations within the catalytic domain, a deletion at a terminus position, or a combination. In some embodiments, the modified TK comprises a mutation at the catalytic domain that decreases or abolishes TK activity but maintains a guanine nucleoside analogue phosphorylating capacity. In some embodiments, the modified TK comprises a mutation at amino acid residue L159, 1160, F161, A167, A168, or L169, or a combination thereof, wherein the residues correspond to positions 159, 160, 161, 167, 168, and 169 of SEQ ID NO: 3. In some embodiments, L159 is mutated to He (I). In some embodiments, 1160 is mutated to Leu (L) or Phe (F). In some embodiments, F161 is mutated to Leu (L), Ala (A), or Val (V). In some embodiments, A167 is mutated to Phe (F). In some embodiments, A168 is mutated Tyr (Y), Phe (F), or Val (V). In some embodiments, L169 is mutated to Asn (N), Tyr (Y), or Met (M).

[0086] In some embodiments, the modified TK comprises mutations L159I, I160F, F161L, A168F, and L169M (also known as HSV-TK mutant sr39 or sr39tk), wherein the residues correspond to positions 159, 160, 161, 168, and 169 of SEQ ID NO: 3. In some embodiments, the HSV-TK mutant sr39 further comprises an N-terminal deletion. In some embodiments, the deletion comprises a deletion of about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues from the N- terminus. In some embodiments, the deletion comprises a deletion of about 45 residues from the N-terminus.

[0087] In some embodiments, the HSV-TK mutant sr39 further comprises a modification of the nuclear localization signal, e.g., a mutation at residue 25, 26, 32, or 33, or a combination thereof, in which the residues correspond to positions 25, 26, 32, and 33 of SEQ ID NO: 3. In some embodiments, the residues 25, 26, 32, and 33 are each independently mutated to Ala or Gly.

[0088] In some embodiments, the modified HSV-TK comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises about 90% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises about 95% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises about 96% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises about 97% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises about 98% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises about 99% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK comprises about 100% sequence identity to SEQ ID NO: 4. In some embodiments, the modified HSV-TK consists of SEQ ID NO: 4.

[0089] In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by a polynucleotide encoding a self-cleaving peptide. Self-cleaving peptide mediates co-translational cleavage of proteins that are upstream and downstream from the cleavage site, allowing the production of both the transdifferentiation factor and the anti-cancer polypeptide under the control of a single promoter. In particular, the self-cleaving sequence causes a translating eukaryotic ribosome to release the growing polypeptide chain that it is synthesizing without dissociating from the mRNA. In some embodiments, the self-cleavage peptide is a 2A peptide, a 2A-like peptide, or a CHYSEL (cis-acting hydrolase element) sequence. Exemplary 2A peptides include, but are not limited to, F2A (foot-and-mouth disease virus): VKQTLNFDLLKLAGDVESNPGP, E2A (equine rhinitis A virus):

QCTNYALLKLAGDVESNPGP, P2A (porcine teschovirus-1 2A):

ATNFSLLKQAGDVEENPGP, and T2A (thosea asigna virus 2A): EGRGSLLTCGD VEENPGP . In some embodiments, a GSG peptide is further added to the N-terminus of the 2A peptide to improve cleavage efficiency. In some embodiments, the 2A-like peptide is a peptide described in U.S. Patent No. 8,975,042. In some embodiments, the CHYSEL sequence is a picornavirus 2A peptide described in de Felipe P (2004) Skipping the co-expression problem: the new 2A “CHYSEL” technology. Genet Vaccines Ther 2: 13.

[0090] In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by a 2 A peptide, a 2A-like peptide, or a CHYSEL sequence. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by F2A. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by P2A. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by E2A. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by T2A. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by a 2A-like peptide. In some embodiments, the transdifferentiation factor and the anti-cancer polypeptide are operably linked by a CHYSEL sequence.

[0091] In some embodiments, if the second promoter within the second anti-cancer therapy segment is used as a reference point within the recombinant polynucleotide, the first anti -cancer therapy segment is located upstream of the second promoter. In some embodiments, the first anti cancer therapy segment is about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000 bases, or more upstream of the second promoter.

[0092] In some embodiments, the first anti-cancer therapy segment is adjacent to the 5’ end of the second promoter. In such cases, the first anti-cancer therapy segment is less than or about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base from the 5’ end of the second promoter.

[0093] In some embodiments, the first promoter is located upstream of the nucleic acid encoding the anti-cancer therapeutics. In such cases when the first anti-cancer therapy segment is located upstream of the second promoter, the first promoter is farther away from the second promoter than the nucleic acid encoding the anti-cancer therapeutics (e.g., the construct order is first promoter - anti-cancer therapeutics - second promoter). In other instances, the first promoter is closer to the second promoter than the nucleic acid encoding the anti-cancer therapeutics (e.g., the construct order is anti-cancer therapeutics - first promoter - second promoter).

[0094] In some embodiments within the second anti-cancer therapy segment, the

transdifferentiation factor is located between the second promoter and the anti-cancer polypeptide. In such cases, the transdifferentiation factor is located about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 bases, or more

downstream of the second promoter.

[0095] In some embodiments, the anti-cancer polypeptide is located between the second promoter and the transdifferentiation factor. In such cases, the anti-cancer polypeptide is located about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 bases, or more downstream of the second promoter.

[0096] In some embodiments, the first promoter and the nucleic acid encoding the anti cancer therapeutic is upstream of the second promoter, the transdifferentiation factor, and the anti-cancer polypeptide. In some embodiments, the construct order is first promoter - anti-cancer therapeutic - second promoter-transdifferentiation factor-anti-cancer polypeptide. In some embodiments, the construct order is first promoter - anti-cancer therapeutic - second promoter- anti-cancer polypeptide-transdifferentiation factor.

[0097] In some embodiments, the first anti-cancer therapy segment is located downstream of the second promoter. In some embodiments, the first anti-cancer therapy segment is about 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000 bases, or more downstream of the second promoter and also downstream of the transdifferentiation factor and the anti-cancer polypeptide. [0098] In some embodiments, the exemplary construct orders include:

[0099] second promoter-transdifferentiation factor-anti-cancer polypeptide-first promoter- anti-cancer therapeutic;

[0100] second promoter-anti-cancer polypeptide-transdifferentiation factor-first promoter- anti-cancer therapeutic;

[0101] second promoter-transdifferentiation factor-anti-cancer polypeptide-anti-cancer therapeutic-first promoter; or

[0102] second promoter- anti-cancer polypeptide-transdifferentiation factor- anti-cancer therapeutic-first promoter.

[0103] In some embodiments, the first anti-cancer therapy segment is located downstream of the second promoter and the transdifferentiation factor and the anti-cancer polypeptide are located upstream of the second promoter. In such instances, the first anti-cancer therapy segment is either located downstream of the second promoter or is adjacent to the second promoter. Exemplary construct orders for this instance include:

[0104] transdifferentiation factor-anti-cancer polypeptide-second promoter-first promoter- anti-cancer therapeutic;

[0105] anti-cancer polypeptide-transdifferentiation factor-second promoter-first promoter- anti-cancer therapeutic;

[0106] transdifferentiation factor-anti-cancer polypeptide-second promoter-anti-cancer therapeutic-first promoter; or

[0107] anti-cancer polypeptide-transdifferentiation factor- second promoter-anti-cancer therapeutic-first promoter.

[0108] In some embodiments, the first anti-cancer therapy segment is located downstream and adjacent to the second anti-cancer therapy segment. In such cases, the first anti-cancer therapy segment is located less than or about 9, 8, 7, 6, 5, 4, 3, 2, or 1 base downstream of the first anti-cancer therapy segment.

i. Constructs

[0109] In some embodiments, a recombinant polynucleotide described above is inserted into a vector. As noted herein, the vector optionally comprises one or more promoters, enhancers, ribosome binding sites, RNA splice sites, polyadenylation sites, a replication origin, and/or transcriptional terminator sequences.

[0110] Promoters are specific nucleotide sequences in DNAs that allow initiation of transcription using DNAs as templates, and have a consensus sequence in general. In some embodiments, the promoters are constitutive promoters. In other instances, the promoters are inducible promoters. In additional instances, the promoters are specific promoters. In some embodiments, the promoters are eukaryotic promoters, or promoters used in a eukaryotic system.

[0111] Exemplary promoters include, but are not limited to, CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALl-10, TEF1, GDS, ADH1, CaMV35S, Ubi, HI, U6, CaMV35S, SV40, CMV, and HSV TK promoter.

[0112] In some embodiments, the promoter is e.g., CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALl-10, TEF1, GDS, ADH1, CaMV35S, Ubi, HI, U6, CaMV35S, SV40, or HSV TK promoter. In some embodiments, the promoter is CMV. In some embodiments, the promoter is EFla. In some embodiments, the promoter is ubiquitin.

[0113] In some embodiments, the first promoter is e.g., CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALl-10, TEF1, GDS, ADHl, CaMV35S, Ubi, HI, U6, CaMV35S, SV40, or HSV TK promoter. In some embodiments, the first promoter is CMV. In some embodiments, the first promoter is EFla. In some embodiments, the first promoter is ubiquitin.

[0114] In some embodiments, the second promoter is CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALl-10, TEF1, GDS, ADHl, CaMV35S, Ubi, HI, U6, CaMV35S, SV40, or HSV TK promoter. In some embodiments, the second promoter is EFla. In some embodiments, the first promoter and the second promoter are interchangeable.

[0115] As described herein, in some embodiments the vector is a bicistronic or polycistronic vector (e.g., having or involving two or more loci responsible for generating a protein) having an an internal ribosome entry site (IRES) is for translation initiation in a cap-independent manner. In some embodiments, the IRES is located upstream of the nucleic acids encoding the

transdifferentiation factor and the anti-cancer polypeptide, respectively (e.g., second-promoter- IRES-transdifferentiation factor-anti-cancer polypeptide or second-promoter-IRES-anti-cancer polypeptide-transdifferentiation factor). In some embodiments, the IRES is located at least or about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 bases, or more upstream of the nucleic acids encoding the transdifferentiation factor and the anti-cancer polypeptide, respectively. In some embodiments, the IRES is located at the 5’ end of the nucleic acids encoding the

transdifferentiation factor and the anti-cancer polypeptide, respectively. In some embodiments, the IRES is located between the anti-cancer polypeptide and the anti-cancer therapeutic. In some embodiments, IRES is located upstream of the second promoter and the nucleic acids encoding the encoding the transdifferentiation factor and the anti -cancer polypeptide, respectively (e.g., IRES-second-promoter-transdifferentiation factor-anti-cancer polypeptide). [0116] Enhancers are nucleotide sequences that have the effect of enhancing promoter activity, and in general, often comprise about 100 bp. In some embodiments, enhancers augment transcription regardless of the orientation of their sequence. While enhancers themselves have no promoter activity, In some embodiments, they activate transcription from a distance of several kilo base pairs. Furthermore, enhancers are located optionally upstream or downstream of a gene region to be transcribed, and/or located within the gene, to activate the transcription.

[0117] Exemplary enhancers include, but are not limited to, WPRE; CMV enhancers; the R- U5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; the intron sequence between exons 2 and 3 of rabbit b-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981); and the genome region of human growth hormone (J Immunol., Vol. 155(3), p. 1286-95, 1995).

[0118] In some embodiments, one or more selectable markers are also present in a vector described herein. In some embodiments, the selectable marker is an antibiotic resistant gene. Exemplary antibiotic resistant genes include, but are not limited to, ampicillin, chloramphenicol, kanamycin, tetracycline, polymyxin B, erythromycin, carbenicillin, streptomycin, spectinomycin, blasticidin S deaminases ( Bsr , BSD), bleomycin-binding protein ( Ble ), Neomycin

phosphotransferase ( neo ), puromycin N-acetyltransferase (Pac), zeocin (Sh bid), and hygromycin B phosphotransferase ( Hph ). In some embodiments, the selectable marker is a eukaryotic antibiotic resistant gene. In some embodiments, the selectable marker is blasticidin S deaminases (Bsr, BSD), bleomycin-binding protein (Ble), Neomycin phosphotransferase (neo), puromycin N- acetyltransferase (Pac), zeocin (Sh bla), or hygromycin B phosphotransferase (Hph).

[0119] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno- associated viral vectors (AAVs), or herpes simplex virus vectors (HSVs). In some embodiments, the retroviral vectors include gamma-retroviral vectors such as vectors derived from the Moloney Murine Keukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Steam cell Virus (MSCV) genome. In some embodiments, the retroviral vectors also include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some embodiments, AAV vectors include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. In some embodiments, viral vector is a chimeric viral vector, comprising viral portions from two or more viruses. In additional instances, the viral vector is a recombinant viral vector.

[0120] In some embodiments, the vector is a non-viral vector. In such instances, a physical method or a chemical method is employed for delivery into the somatic cell. Exemplary physical methods include electroporation, gene gun, sonoporation, magnetofection, or hydrodynamic delivery. Exemplary chemical methods include delivery of the recombinant polynucleotide via liposomes such as, cationic lipids or neutral lipids; dendrimers; nanoparticles; or cell-penetrating peptides.

C. Engineered Stem Cells and Methods of Manufacture

[0121] Disclosed herein, in certain embodiments, are engineered stem cells comprising a recombinant polynucleotide comprising, under the control of separate translation initiation signals, (i) a transdifferentiation factor and anti -cancer polypeptide and (ii) an anti -cancer therapeutic. In certain embodiments, the recombinant polynucleotides disclosed herein comprises: a) a first anti-cancer therapy segment comprising a first nucleic acid sequence encoding a first translation initiation signal and an anti -cancer therapeutic; and b) a second anti cancer therapy segment comprising a second nucleic acid sequence encoding a second translation initiation signal and an anti -cancer polypeptide, wherein said first translation initiation signal initiates the translation of the first nucleic acid sequence, and wherein said second translation initiation signal initiates the translation of the second nucleic acid sequence In some

embodiments the recombinant polynucleotides are used to treat breast cancer (e.g., triple negative breast cancer), brain cancer (e.g., glioblastoma), melanoma, ovarian cancer, pancreatic cancer, leukemia, lymphoma or pulmonary cancer. Further disclosed herein, are methods of generating engineered stem cells.

[0122] In some embodiments, the engineered stem cells are derived from a somatic cell or primary cell contacted with a recombinant polynucleotide disclosed supra. In some

embodiments, recombinant polynucleotide disclosed herein is introduced into the somatic or primary cell, and the recombinant polynucleotide gives rise, upon transcription, to a

transdifferentiation factor that contributes to the reprogramming of the targeted somatic cell or targeted primary cell into an engineered stem cell (e.g., a therapeutic cell). In some embodiments, the factor is a transdifferentiation factor, e.g., Oct4, Sox2, Klf4, Myc, Ascii, Brn2, Mytll, 01ig2, or Zicl. In some embodiments, the factor is Sox2. In some embodiments, the engineered stem cells are induced tumor-homing drug carrier cells (iTDC). In some embodiments, the iTDCs are produced by 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. In some embodiments, the iTDC is not a pluripotent stem cell or an induced neural stem cell.

[0001] 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. 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.“Progenitor medium”, as used herein, is a medium or media, for example, incorporating supplements, small molecule inhibitors, and growth factors, that promotes the transdifferentiation (TD) of somatic cells into neural stem cells. In some embodiments, the 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-f31 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 25 below. In some embodiments, the progenitor medium is a premade medium, such as STEMdiff™ Neural Induction Medium (STEM CELL TM

Technologies, Vancouver, British Columbia, Canada).

[0123] In some embodiments, the somatic cell or primary cell is any cell from the body of a subject other than gametocyte, germ cell, or undifferentiated stem cell. In some embodiments, the somatic cell or primary cell comprises a fibroblast cell, a muscle cell, an epithelial cell, or a nerve cell. Epithelial cells include squamous cells, cuboidal cells, and/or columnar cells. Cells of the muscle comprise skeletal muscle, smooth muscle, and/or cardiac muscle.

[0124] In some embodiments, the fibroblast cells are skin fibroblast cells. Skin cells can be collected from a skin punch as a stand-alone procedure or from a surgical incision, e.g., during an accompanying surgery procedure; and can be collected from any area, including, but not limited to, arm (e.g, forearm), leg, or scalp.

[0125] In some embodiments, the somatic cell or primary cell utilized is a fibroblast cell (e.g., a skin fibroblast cell). In some embodiments, a recombinant polynucleotide disclosed herein is introduced into the fibroblast cell (e.g., the skin fibroblast cell), wherein the

recombinant polynucleotide gives rise, upon transcription, to a factor that contributes to the reprogramming of the fibroblast cell (e.g., the skin fibroblast cell) into a therapeutic or engineered stem cell.

[0126] In some embodiments, the somatic cell or primary cell is an autologous cell obtained from the subject to which treatment is to be administered. [0127] In other embodiments, the somatic cell or primary cell is an allogenic cell obtained from a subject which the subject will not receive treatment. In some embodiments, this subject is a healthy subject.

[0128] In some embodiments, an engineered stem cell disclosed herein expresses one or more biomarkers, such as for example, nestin, glial fibrillary acidic protein (or GFAP), Tuj-1 (neuron- specific class III beta-tubulin or bIII tubulin), Nanog, or OCT3/4.

[0129] In some embodiments, the engineered stem cell expresses CXCR4 (e.g., the engineered stem cell is CXCR4 positive).

[0130] In some embodiments, the secretion of the anti-cancer therapeutic (e.g., TRAIL) is enhanced in the engineered stem cell when compared to an equivalent engineered stem cell which comprises a recombinant polynucleotide comprising the anti-cancer therapeutic, the transdifferentiation factor, and the anti-cancer polypeptide under a single translation initiation signal. In some embodiments, the secretion is enhanced by at least about 1.1-fold, 1.5-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 100-fold, or more.

[0131] In some embodiments, the secretion of TRAIL is enhanced in the engineered stem cell when compared to an equivalent engineered stem cell which comprises a recombinant polynucleotide comprising the TRAIL protein, the transdifferentiation factor, and the anti-cancer polypeptide under a single translation initiation signal. In some embodiments, the secretion is enhanced by at least about 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, or more.

[0132] In some embodiments, the engineered stem cell comprises a tumor-homing ability.

C. Methods of Cancer Treatment

[0133] Disclosed herein, in certain embodiments, are methods of treating a cancer in an individual in need thereof. In some embodiments, the method comprises administering to the individual an engineered stem cell (e.g., and iTDC) comprising a recombinant polynucleotide disclosed herein. In certain embodiments, the recombinant polynucleotide comprise: a) a first anti-cancer therapy segment comprising a first nucleic acid sequence encoding a first translation initiation signal and an anti -cancer therapeutic; and b) a second anti -cancer therapy segment comprising a second nucleic acid sequence encoding a second translation initiation signal and an anti-cancer polypeptide, wherein said first translation initiation signal initiates the translation of the first nucleic acid sequence, and wherein said second translation initiation signal initiates the translation of the second nucleic acid sequence In some embodiments the recombinant polynucleotides are used to treat breast cancer (e.g., triple negative breast cancer), brain cancer (e.g., glioblastoma), melanoma, ovarian cancer, pancreatic cancer, leukemia, lymphoma or pulmonary cancer. In some embodiments, the cancer is a solid tumor. In other embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a metastatic cancer, a relapsed cancer, or a refractory cancer.

[0134] In some embodiments, the solid cancer is ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian carcinoma, primary peritoneal carcinoma, or fallopian tube cancer. Epithelial ovarian carcinoma is the most common type of ovarian cancer, comprising about 85% to 90% of all cases. In some embodiments, epithelial ovarian carcinoma is further subtyped into mucinous, endometrioid, clear cell, and undifferentiated. In some embodiments, there are four stages of ovarian cancer. Stage I is characterized by cancerous cells localized within the ovary or fallopian tube (termed Stage IA), cancer that has developed in both ovaries or fallopian tubes but not on their outer surfaces (termed Stage IB), or cancer present in one or both ovaries or fallopian tubes in conjunction with any one of the following: 1) the tissue capsule surrounding the tumor broke during surgery, 2) cancer is on the outer surface of at least one of the ovaries, fallopian tubes, or tissue capsule, 3) cancerous cells were detected in fluid or washings from the abdomen (termed Stage IC) . Stage II is characterized by the presence of cancerous cells in one or both ovaries or fallopian tubes that has invaded the uterus (termed Stage IIA) or cancerous cells that have grown into other nearby pelvic organs (termed Stage IIB).

Stage III is characterized by the presence of cancerous cells in one or both ovaries or fallopian tubes, and in retroperitoneal lymph nodes (termed Stage III A 1 ), or cancerous cells in one or both ovaries or fallopian tubes, in retroperitoneal lymph nodes, and in the lining of the upper abdomen (termed Stage IIIA2), or cancerous cells in one or both ovaries or fallopian tubes and cancer deposits 2 cm or smaller are in the abdomen (termed Stage MB), or cancerous cells in one or both ovaries or fallopian tubes and cancer deposits larger than 2 cm are in the abdomen (termed Stage IIIC). Stage IV is characterized by cancerous cells that have spread to the fluid around the lungs, with no other areas of cancer spread outside the pelvis or peritoneal cavity (termed Stage IV A) or cancerous cells that have spread to the inside of the spleen or liver, lymph nodes, and/or other organs or tissues outside the peritoneal cavity (termed Stage IVB).

[0135] In some embodiments, the solid tumor is breast cancer. In some embodiments, the breast cancer is triple negative breast cancer. Each year, there are about 2,300 new cases of breast cancer in men and about 230,000 new cases in women. In some embodiments, breast cancer is further classified into invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), ductal carcinoma in situ, lobular carcinoma in situ, infiltrating ductal carcinoma, inflammatory breast cancer, triple-negative breast cancer, paget disease of the nipple, phyllodes tumor, angiosarcoma, adenoid cystic carcinoma, adenocystic carcinoma, low-grade

adenosquamous carcinoma, medullary carcinoma, mucinous carcinoma, colloid carcinoma, papillary carcinoma, tubular carcinoma, metaplastic carcinoma, spindle cell carcinoma, squamous carcinoma, micropapillary carcinoma and mixed carcinoma. IDC is the most common type of breast cancer, comprising about 80% of all cases. In some embodiments, there are four stages of breast cancer. In some embodiments, stage I is characterized by a tumor measuring up to 2 centimeters (cm) localized solely in the breast (termed Stage IA), small groups of cancerous cells found in the lymph nodes, or a tumor in the breast measuring up to 2 cm in addition to small groups of cancerous cells found in the lymph nodes (termed Stage IB). In some embodiments, stage II is characterized by the presence of a tumor larger than 2 millimeters (mm) localized in the lymph nodes (termed Stage IIA), by the presence of a tumor that is between 2 to 5 cm and small groups of cancerous cells localized in the lymph nodes, or a tumor that is larger than 5 cm but has not spread to the lymph nodes (termed Stage IIB). In some embodiments, stage III is characterized by the presence of a tumor larger than 5 cm localized in the lymph nodes (termed Stage IPA), a tumor of any size that has spread to the chest wall and/or skin and has spread to up to 9 axillary lymph nodes or to the lymph nodes near the breastbone (termed Stage MB), or a tumor of any size that has spread to the chest wall and/or skin and has spread to 10 or more axillary lymph nodes or has spread to lymph nodes above or below the collarbone or has spread to axillary lymph nodes or to lymph nodes near the breast bone (termed Stage MC). In some embodiments, stage IV is characterized by cancerous cells that have spread beyond the breast and nearby lymph nodes to other organs of the body.

[0136] In some embodiments, the solid tumor is glioblastoma. Glioblastomas, or

glioblastoma multiforme (GBM), are tumors that arise from astrocytes or the star-shaped cells that make up the“glue-like,” or supportive tissue of the brain. Glioblastoma is fast-growing, is the most common primary tumor of the central nervous system in adults. Glioblastoma is further classified into primary glioblastoma (or de novo glioblastoma) or secondary tumor. In additional cases, glioblastoma is divided into grade I, grade II, grade III and grade IV glioblastoma.

[0137] In some embodiments, the solid tumor is pancreatic cancer. Pancreatic cancer comprises two types, exocrine cancers such as pancreatic adenocarcinoma, acinar cell carcinoma of the pancreas, cystadenocarcinomas, pancreatoblastoma, adenosquamous carcinomas, signet ring cell carcinomas, hepatoid carcinomas, colloid carcinomas, undifferentiated carcinomas, undifferentiated carcinomas with osteoclast-like giant cells, or solid pseudopapillary tumor; and neuroendocrine malignant tumors.

[0138] In some embodiments, the cancer is melanoma. Melanoma is a type of cancer that is developed from melanocytes or pigment-containing cells. In some embodiments, melanoma occurs in the skin, but potentially also occurs in the mouth, intestines, or eye. Exemplary melanoma includes, but is not limited to lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft-tissue melanoma, melanoma with small nevus-like cells, melanoma with features of a Spitz nevus, and uveal melanoma.

[0139] In some embodiments, the solid tumor is pulmonary cancer. In some embodiments, pulmonary cancer is further classified into non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), and lung carcinoid tumor. NSCLC is the most common type of pulmonary cancer, comprising about 85% of all cases. In some embodiments, NSCLC is further subtyped into adenocarcinoma, squamous cell carcinoma (or epidermoid carcinoma), and large cell carcinomas. In some embodiments, there are four stages of NSCLC. Stage I is characterized by cancerous cells localized in the lungs. Stage II is characterized by the presence of cancerous cells in the lung and nearby lymph nodes. Stage III is characterized by the presence of cancerous cells in the lung, and cancer either spreading to the lymph node on the same side of the chest as the original cancer (termed Stage IIIA), or cancer spreading to the lymph node on the opposite side of the chest as the original cancer (termed Stage IIIB). Stage IV is characterized by cancerous cells present in both lungs, in the pleural space surrounding the lungs, or in other parts of the body. In some embodiments, SCLC is subdivided into two stages, limited stage or extensive stage. Limited stage is characterized by the presence of cancerous cells on one side of the chest involving one part of the lung and nearby lymph nodes. Extensive stage is when cancer has spread to other regions of the chest or other parts of the body. Lung carcinoid tumor or lung neuroendocrine tumors, comprises the fewest cases, about less than 5% of all pulmonary cancer cases.

[0140] In some embodiments, an engineered stem cell is administered to a subject for the treatment of a hematologic malignancy. In some embodiments, the hematologic malignancy is a lymphoma (e.g., a Hodgkin’s lymphoma or a non-Hodgkin’s lymphoma). Lymphoma is a cancer that is commonly manifested in the lymph nodes, lymphoid tissue, or lymphoid organs.

[0141] In some embodiments, the hematologic malignancy is a leukemia. Leukemia is a cancer that originates from the bone marrow and/or blood. Exemplary leukemia includes chronic myeloid leukemia (CML) (also known as chronic myelogenous leukemia), acute myeloid leukemia (AML) (also known as acute myelogenous leukemia), acute lymphocytic leukemia (ALL) (also known as acute lymphoblastic leukemia), and chronic lymphocytic leukemia (CLL).

[0142] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises chemotherapeutic agent, immunotherapeutic agent, targeted therapeutic agent, hormone-based therapeutic agent, or a second stem-cell based therapeutic agent. In some embodiments, the additional therapeutic agent is a first-line treatment. In some embodiments, exemplary

therapeutic agents include, but are not limited to, anti -cancer antibodies ( e.g ., HERCEPTIN®), antimetabolites, alkylating agents, topoisomerase inhibitors, microtubule targeting agents, kinase inhibitors, protein synthesis inhibitors, somatostatin analogs, glucocorticoids, aromatose inhibitors, mTOR inhibitors, protein Kinase B (PKB) inhibitors, phosphatidylinositol, 3 -Kinase (PI3K) Inhibitors, cyclin dependent kinase inhibitors, anti-TRAIL molecules, MEK inhibitors, and the like.

[0143] In some embodiments, the additional therapeutic agent comprises a HSV-TK substrate. Exemplary HSV-TK substrates include, but are not limited to, FHBG (9-[4-fluoro-3- (hydroxymethyl)butyl]guanine), FHPG (9-([3-fiuoro- 1 -hydroxy 2 propoxy]meihyl)guaaine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-deoxy-2'-fiuoro-l-P-D- arabinofuranosyi)-5-iodouracil), FEAU (fluoro-5-ethy!-l-beta-D-arabinofuranosyluracil), FMAU (fluoro-5 -methyl- 1- beta-D-arabinofuranosyluracil), FHOMP (6-((l -fluoro-3-hydroxypropan-2- yloxy)methyl)-5-methylpryrimidine-2,4(lH,3H)-dione), ganciclovir, val ganciclovir, acyclovir, valaciviovir, penciclovir, radiolabeled pyrimidine with 4-hydroxy-3- (hydroxymethyl)butyl side chain at N-1 (HHG-5-FEP) or 5-(2-)hydroxyethyl)- and 5-(3- hydroxypropylj-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir and penciclovir-like side chains.

[0144] In some embodiments, the additional therapeutic agents include, but are not limited to flourouracil (5-FU), capecitabine/XELODA, 5-Trifluoromethyl-2'-deoxyuridine, methotrexate sodium, raltitrexed/Tomudex, pemetrexed/Alimta®, cytosine Arabinoside (Cytarabine, Ara- C)/Thioguanine, 6-mercaptopurine (Mercaptopurine, 6-MP), azathioprine/Azasan, 6-thioguanine (6-TG)/Purinethol (TEVA), pentostatin/Nipent, fludarabine phosphate/Fludara®, cladribine (2- CdA, 2-chlorodeoxyadenosine)/Leustatin, floxuridine (5-fluoro-2)/FUDR (Hospira, Inc.), ribonucleotide Reductase Inhibitor (RNR), cyclophosphamide/Cytoxan (BMS), neosar, ifosfamide/Mitoxana, thiotepa, BCNU- 1 ,3-bis(2-chloroethyl)-l-nitosourea, 1 ,-(2-chloroethyl)-3- cyclohexyl-lnitrosourea, methyl CCNU, hexamethylmelamine, busulfan/Myleran, procarbazine HCL/Matulane, dacarbazine (DTIC), chlorambucil/Leukaran®, melphalan/Alkeran, cisplatin (Cisplatinum, CDDP)/Platinol, carboplatin/Paraplatin, oxaliplatin/Eloxitan, bendamustine, carmustine, chloromethine, dacarbazine (DTIC), fotemustine, lomustine, mannosulfan, nedaplatin, nimustine, prednimustine, ranimustine, satraplatin, semustine, streptozocin, temozolomide, treosulfan, triaziquone, triethylene melamine, thioTEPA, triplatin tetranitrate, trofosfamide, uramustine, doxorubicin HCL/Doxil, daunorubicin citrate/Daunoxome®, mitoxantrone HCL/Novantrone, actinomycin D, etoposide/Vepesid, topotecan HCL/Hycamtin, teniposide (VM-26), irinotecan HCL(CPT-1 1), camptosar®, camptothecin, Belotecan, rubitecan, vincristine, vinblastine sulfate, vinorelbine tartrate, vindesine sulphate, paclitaxel/Taxol, docetaxel/Taxotere, nanoparticle paclitaxel, abraxane, ixabepilone, larotaxel, ortataxel, tesetaxel, vinfiunine, and the like.

[0145] In some embodiments the additional therapeutic agents comprise one or more drugs selected from the group consisting of carboplatin(e.^., PARAPLATIN®), Cisplatin (e.g, PLATINOL®, PLATINOL-AQ®), Cyclophosphamide (e.g, CYTOXAN®, NEOSAPv®), Docetaxel ( e.g. , TAXOTERE®), Doxorubicin (e.g, ADRIAMYCIN®), Erlotinib (e.g, TARCEVA®), Etoposide (e.g, VEPESID®), Fluorouracil (e.g, 5-FU®), Gemcitabine (e.g. , GEMZAR®), imatinib mesylate (e.g, GLEEVEC®), Irinotecan (e.g, CAMPTOSAR®), Methotrexate (e.g, FOLEX® , MEXATE®, AMETHOPTERIN®), Paclitaxel (e.g, TAXOL®, ABRAXANE®), Sorafmib (e.g, NEXAVAR®), Sunitinib (e.g, SUTENT®), Topotecan (e.g, HYCAMTIN®), Vinblastine (e.g, VELBAN®), Vincristine (e.g , ONCOVIN®, VINCASAR PFS®). In some embodiments, the additional therapeutic agents comprises one or more drugs selected from the group consisting of retinoic acid, a retinoic acid derivative, doxorubicin, vinblastine, vincristine, cyclophosphamide, ifosfamide, cisplatin, 5 -fluorouracil, a camptothecin derivative, interferon, tamoxifen, and taxol. In some embodiments, the additional therapeutic agent is selected from the group consisting of abraxane, doxorubicin, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestroltamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, zoledronic acid, vinblastine, etc.), an antisense molecule, an siRNA, and the like.

[0146] In some embodiments, the additional therapeutic agent comprises an immune checkpoint modulator. Exemplary checkpoint modulators include:

[0147] PD-L1 modulators such as Genentech’s MPDL3280A (RG7446), Avelumab

(Bavencio) from Merck/Pfizer, durvalumab (Imfinzi) from AstraZeneca, Anti -mouse PD-L1 antibody Clone 10F.9G2 (Cat # BE0101) from BioXcell, anti-PD-Ll monoclonal antibody MDX-1105 (BMS-936559), BMS-935559 and BMS-986192 from Bristol-Meyer’s Squibb, MSB0010718C, mouse anti-PD-Ll Clone 29E.2A3, CX-072 from XytomX Therapeutics, FAZ053 from Novartis Pharmaceuticals, KN035 from 3D Medicine, LY3300054 from Eli Lilly, and AstraZeneca’s MEDI4736;

[0148] PD-L2 modulators such as GlaxoSmithKline’s AMP -224 (Amplimmune), and rHIgM12B7;

[0149] PD-1 modulators such as anti-mouse PD-1 antibody Clone J43 (Cat # BE0033-2) from BioXcell, anti-mouse PD-1 antibody Clone RMPl-14 (Cat # BE0146) from BioXcell, mouse anti-PD-1 antibody Clone EH12, Merck’s MK-3475 anti-mouse PD-1 antibody

(Keytruda, pembrolizumab, lambrolizumab), AnaptysBio’s anti-PD-1 antibody known as ANB011, antibody MDX-1 106 (ONO-4538), Bristol-Myers Squibb’s human IgG4 monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106), AstraZeneca’s AMP-514 and AMP- 224, sintilimab (IBI-308) from Eli Lilly/Innovent Biologies, AGEN 2034 from Agenus, BGB- A317 from BeiGene, Bl-754091 from Boehringer-Ingelheim Pharmaceuticals, CBT-501 (genolimzumab) from CBT Pharmaceuticals, INCSHR1210 from Incyte, JNJ-63723283 from Janssen Research & Development, MED 10680 from Medlmmune, PDR001 from Novartis Pharmaceuticals, PF-06801591 from Pfizer, REGN2810 from Regeneron Pharmaceuticals, and Pidilizumab (CT-011) from CureTech Ltd;

[0150] CTLA-4 modulators such as Bristol Meyers Squibb’s anti-CTLA-4 antibody ipilimumab (also known as Yervoy®, MDX-010, BMS-734016 and MDX-101), anti-CTLA4 antibody clone 9H10 from Millipore, Pfizer’s tremelimumab (CP-675,206, ticilimumab), AGEN 1884 from Agenus, and anti-CTLA4 antibody clone BNI3 from Abeam;

[0151] LAG3 modulators such as anti-Lag-3 antibody clone eBioC9B7W (C9B7W) from eBioscience, anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences, IMP701 and LAG525 from Novartis Pharmaceuticals, IMP321 (ImmuFact) from Immutep, anti-Lag3 antibody BMS- 986016, BMS-986016 from Bristol-Myers Squibb, REGN3767 from Regeneron

Pharmaceuticals, and the LAG-3 chimeric antibody A9H12;

[0152] B7-H3 modulators such as MGA271;

[0153] KIR modulators such as Lirilumab (IPH2101) from Bristol-Myers Squibb;

[0154] CD137 modulators such as urelumab (BMS-663513, Bristol-Myers Squibb), PF-

05082566 (anti-4-lBB, PF-2566, Pfizer), or XmAb-5592 (Xencor);

[0155] PS modulators such as Bavituximab;

[0156] 0X40 modulators such as BMS-986178 from Bristol-Myers Squibb, GSK3174998 from GlaxoSmithKline, INCAGN1949 from Agenus, MEDI0562 from Medlmmune, PF- 04518600 from Pfizer, or RG7888 from Genentech;

[0157] GITR modulators such as GWN323 from Novartis Pharmaceuticals, INCAGN1876 from Agenus, or TRX518 from Leap Therapeutics;

[0158] TIM3 modulators such as MBG453 from Novartis Pharmaceuticals, or TSR-042 from TESARO;

[0159] and modulators such as an antibody or fragments (e.g., a monoclonal antibody, a human, humanized, or chimeric antibody) thereof, RNAi molecules, or small molecules to CD52, CD30, CD20, CD33, CD27, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.

[0160] In some embodiments, the additional therapeutic agent comprises a cytokine therapy. Exemplary cytokine drugs include interferon gamma 1-b (Actimmune) from Horizon Pharma; IL-2 based recombinant fusion protein (ALKS 4230) from Alkermes; ALT-801 or ALT-803 from Altor BioScience; AM0010 from ARMO Biosciences; APN301 from Apeiron Biologies; CDX- 301/CDX-1401 from Celldex; cergutuzumab amunaleukin (RG7813) or RG7461 from Genentch; CYT-6091 from Cytimmune Sciences; DI-Leul6-IL2 from Alopexx Oncology; GEN-1 from Celsion; heterodimeric IL-15 from Admune Therpeutics; HL143 from HanAll Biopharma;

IGN002 from ImmunGene; ImmunoPulse IL-12 from OncoSec Medical; IRX-2 from IRX Therapeutics; M9241 (NHS-IL12) from EMD Serono; MDNA55 from Medicenna Therapeutics; NGR-hTNF from MolMed; or rSIFN-co from Sichuan Huiyang Life Science.

[0161] In some embodiments, the additional therapeutic agent comprises an adoptive cell therapy. Exemplary adoptive cell therapies include AFP TCR, MAGE-A10 TCR, or NY-ESO- TCR from Adaptimmune; ACTR087/rituximab from Unum Therapeutics; anti-BCMA CAR-T cell therapy, anti-CD19“armored” CAR-T cell therapy, JCAR014, JCAR018, JCAR020, JCAR023, JCAR024, or JTCR016 from Juno Therapeutics; JCAR017 from Celgene/Juno Therapeutics; anti -CD 19 CAR-T cell therapy from Intrexon; anti -CD 19 CAR-T cell therapy, axicabtagene ciloleucel, KITE-718, KITE-439, or NY-ESO-1 T-cell receptor therapy from Kite Pharma; anti-CEA CAR-T therapy from Sorrento Therapeutics; anti-PSMA CAR-T cell therapy from TNK Therapeutics/Sorrento Therapeutics; ATA520 from Atara Biotherapeutics; AU101 and AU105 from Aurora BioPharma; baltaleucel-T (CMD-003) from Cell Medica; bb2121 from bluebird bio; BPX-501, BPX-601, or BPX-701 from Bellicum Pharmaceuticals; BSK01 from Kiromic; IMCgplOO from Immunocore; JTX-2011 from Jounce Therapeutics; LN-144 or LN- 145 from Lion Biotechnologies; MB-101 or MB-102 from Mustang Bio; NKR-2 from Celyad; PNK-007 from Celgene; tisagenlecleucel-T from Novartis Pharmaceuticals; or TT12 from Tessa Therapeutics.

[0162] In some embodiments, the additional therapeutic agent comprises a proteasome inhibitor. Exemplary proteasome inhibitors include bortezomib, carfilzomib, delanzomib, ixazomib, marizomib, oprozomib, or derivatives or analogs thereof.

[0163] In some embodiments, the additional therapeutic agent comprises an HD AC inhibitor. Exemplary HDAC inhibitors include ACY-1215 (Rocilinostat), Apicidin, CI-994 (Tacedinaline), CR-2408, entinostat (SNDX-275 or MS-275), ITF2357 (Gavinostat), KD5170, JNJ-26481585, LBH589 (Panobinostat), NVP-LAQ824 (Dacinostat), PXDIOI (Belinostat), romidepsin, phenyl butyrate (S-HD AC-42, AR-42), RAS2410 (Resminostat), sodium butyrate, suberoylanilide bis- hydroxamic acid (SBHA), trichostatin-A (TSA), tubacin, valproic acid (VP A), or vorinostat (SAHA).

[0164] In some embodiments, the engineered stem cell is administered to the subject prior to administering the additional therapeutic agent. [0165] In other instances, the engineered stem cell is administered to the subject in conjunction with the additional therapeutic agent.

[0166] In additional instances, the engineered stem cell is administered to the subject after administering the additional therapeutic agent.

[0167] In further instances, the subject undergoes radiation treatment, and the engineered stem cell is administered to the subject before, during, or after radiation treatment.

[0168] In still further instances, the subject undergoes surgery, and the engineered stem cell is administered to the subject either before or after surgery.

D. Methods of Target Site Visualization

[0169] Disclosed herein, in certain embodiments, are methods of imaging an engineered stem cell at a target site of interest. In some embodiments, the methods comprise (a) introducing into a somatic or primary cell a recombinant polynucleotide comprising i) a first anti-cancer therapy segment comprising a first nucleic acid sequence encoding a first translation initiation signal and an anti -cancer therapeutic; ii) a second anti-cancer therapy segment comprising a second nucleic acid sequence encoding a second translation initiation signal and an anti-cancer polypeptide, and iii) a reporter gene; and (b) imaging the target site of interest.

[0170] In some embodiments, the methods of imaging a therapeutic cell at a target site of interest comprise the use of an imaging modality. In some embodiments, the imaging modality is positron emission tomography (PET). In some embodiments, the imaging modality is magnetic resonance imaging (MRI), ultrasound, X-ray imaging, computer tomography (CT), nuclear medicine, elastography, photoacoustic imaging, echocardiography, functional near-infrared spectroscopy, magnetic particle imaging, or any combination thereof. In some embodiments, the imaging modality uses a volume rendering technique to produce a three-dimensional (3D) image. In some embodiments, the imaging modality is fluorescence microscopy, confocal microscopy, bright field microscopy, oblique illumination microscopy, dark field microscopy, dispersion staining microscopy, phase contrast microscopy, differential interference contrast microscopy, electron microscopy, scanning probe microscopy, ultraviolet microscopy, infrared microscopy, digital holographic microscopy, digital pathology microscopy, laser microscopy, photoacoustic microscopy, or any combinations thereof.

[00182] In some embodiments, the first and second cells comprise a label. In some

embodiments, the imaging modality detects the label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is encoded by the reporter gene. In some embodiments the reporter gene encodes a fluorescent marker or label. In some embodiments, the first recombinant polynucleotide and the second recombinant polynucleotide encode a nucleic acid sequence encoding the fluorescent label. Non-limiting examples of fluorescent labels include green fluorescent protein, tdTomato, E2-Crimson, mCherry, red fluorescent protein, cyan fluorescent protein, or any combination thereof. In some embodiments, the label is a magnetic resonance imaging (MRI) contrast agent or a positron emission tomography (PET) contrast agent.

[00183] In some embodiments, thymidine kinase is used as a label. In some embodiments, thymidine kinase is used as an in vivo label. In some embodiments, thymidine kinase is able to be detected via PET. Thymidine kinase (TK) is amenable to detection via PET. In some embodiments, thymidine kinase is used as a PET tracer. In some embodiments, thymidine kinase is the label. In some embodiments, TK phosphorylates ganciclovir. In some embodiments, phosphorylated ganciclovir is the label. In some embodiments, phosphorylated TK accumulates inside cells, thereby enabling detection via PET. In some embodiments, the uptake of TK is regulated by thymidine kinase 1, and it is therefore taken up preferentially by rapidly

proliferating tumor cells. The fluorine isotope 18 is a positron emitter that is used in positron emission tomography (PET). The fluorine- 18 radiolabeled fluorothymidine F-18 is therefore useful for PET imaging of active tumor proliferation, and compares favorably with the more commonly used marker fludeoxyglucose (18F). In some embodiments, the label is

fluorothymidine F-18.

[0171] In some embodiments, the target is site is a tumor. In some embodiments, the target site is a solid tumor. In some embodiments, the target is site is a cancer cell. In some

embodiments, the target is site is an inflamed tissue or an area of inflammation in a tissue. In some embodiments, the target is site is an atherosclerotic plaque. In some embodiments, the target is site is a bone, cartilage, or tendon. In some embodiments, the target is site is a tumor resection area. In some embodiments, the target is site is a blood vessel.

[0172] In some embodiments, the first anti-cancer therapy segment comprises a first promoter that controls the expression of a first nucleic acid sequence encoding an anti-cancer therapeutic. In some embodiments, the second anti-cancer therapy segment comprises a second promoter that controls the expression of a second nucleic acid encoding a transdifferentiation factor and a third nucleic acid sequence encoding an anti -cancer polypeptide. In some embodiments, the reporter gene is under control of a third promoter. In some embodiments, the reporter gene is part of the second anti-cancer segment and is under control of the second promoter.

[0173] In some embodiments, the reporter gene encodes a fluorescent protein. Exemplary fluorescent proteins include, but are not limited to: [0174] Green fluorescent protein family members such as: green fluorescent protein (GFP), enhanced GFP (EGFP), Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, or mNeonGreen;

[0175] Blue fluorescent protein family members such as: TagBFP, mTagBFP2, Azurite, EBFP2, mKalamal, Sirius, Sapphire, or T-Sapphire;

[0176] Cyan fluorescent protein family members such as: enhanced cyan fluorescent protein (ECFP), Cerulean, SCFP3 A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, or mTFPl;

[0177] Yellow fluorescent protein family members such as: enhanced yellow fluorescent protein (EYFP), Citrine, Venus, SYFP2, or TagYFP;

[0178] Orange fluorescent protein family members such as: monomeric Kusabira-Orange, ihKOk;, mK02, mOrange, or mOrange2;

[0179] Red fluorescent protein family members such as: mRaspberry, mCherry,

mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, or mRuby2;

[0180] Far-Red fluorescent protein family members such as: mPlum, HcRed-Tandem, mKate2, mNeptune, or NirFP;

[0181] Near-IR protein family members such as: TagRFP657, IFP1.4, or iRFP;

[0182] Long Stokes Shift protein family members such as: mKeima Red, LSS-mKatel, LSS- mKate2, or mBeRFP;

[0183] Photoactivatible protein family members such as: PA-GFP, PAmCherryl, or

PATagRFP;

[0184] Photoconvertible protein family members such as: Kaede (green), Kaede (red), KikGRl (green), KikGRl (red), PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), or PSmOrange; and

[0185] Photoswitchable protein family members such as: Dronpa.

[0186] In some embodiments, the recombinant polynucleotide comprises a nucleic acid encoding a thymidine kinase. Thymidine kinase is a phosphotransferase that can phosphorylate a radiolabeled TK substrate, enabling imaging studies. In particular, TK phosphorylates its substrate into a monophosphate form. Next, cellular enzymes metabolize the monophosphate substrate into its triphosphate form, which is then incorporated into nascent DNA.

[0187] Exemplary HSV-TK substrates include, but are not limited to, FHBG (9-[4-fluoro-3- (hydroxymethyl)butyl]guanine), FHPG (9-([3-fiuoro- 1 -hydroxy 2 propoxy]meihyl)guaaine), FGCV (fluorogancielovir), FPCV (fluoropencielovir), FIA!J (l-(2'-deoxy-2'-fiuoro-l-P-D- arabinofuranosyl)-5-iodouracil), FEAU (fIuoro-5-ethyl-l-beta-D-arabinofuranosyluracil), FMAU (fluoro-5 -methyl- 1- beta-D-arabinofuranosy!uracil), FHOMP (6-((l -fluoro-3-hydroxypropan-2- y[oxy)methyl)-5-methylpryrimidine-2,4(lH,3H)-dione), ganciclovir, valgancielovir, acyclovir, valaciviovir, penciclovir, radiolabeled pyrimidine with 4-hydroxy-3- (hydroxymethyl)butyl side chain at N-1 (HHG-5-FEP) or 5-(2-)hydroxyethyl)- and 5 -(3- hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir and penciclovir-like side chains.

[0188] In some embodiments, the radioactive tracer includes ^l8F, ⁶⁴Cu, ^99mTe, C, ^l4C, ¹²⁴I, ¹²³I, ¹³¹I, ¹⁵0, ¹³N and/or ⁸²RbCl.

E. Kits/Article of Manufacture

[0189] Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more methods disclosed herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method disclosed herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

[0190] The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

[0191] For example, the container(s) include a recombinant polynucleotide disclosed herein, or engineered stem cells disclosed herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods disclosed herein.

[0192] A kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0193] In one embodiment, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods disclosed herein.

II. Certain Terminology

[0194] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms“a,”“an” and“the” include plural referents unless the context clearly dictates otherwise. In this application, the use of“or” means“and/or” unless stated otherwise. Furthermore, use of the term“including” as well as other forms, such as“include”, “includes,” and“included,” is not limiting.

[0195] Although various features of the disclosure may be disclosed in the context of a single embodiment, the features may also be provided separately or in any suitable combination.

Conversely, although the disclosure may be disclosed herein in the context of separate embodiments for clarity, the disclosure may also be implemented in a single embodiment.

[0196] Reference in the specification to“some embodiments”,“an embodiment”,“one embodiment” or“other embodiments” means that a particular feature, structure, or characteristic disclosed in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the disclosures.

[0197] As used herein, ranges and amounts can be expressed as“about” a particular value or range. About also includes the exact amount. Hence“about 5 pL” means“about 5 pL” and also “5 pL.” Generally, the term“about” includes an amount that would be expected to be within experimental error.

[0198] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter disclosed.

[0199] As used herein, the terms“individual(s)”,“subject(s)” and“patient(s)” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker).

III. EXAMPLES

[0200] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

A. EXAMPLE 1: Viability Assay of Cancer Cells Transfected with Vectors Having First and Second Translation Initiation Signals

[0201] A viability assay (an indirect measure of TRAIL secretion) was conducted to compare fibroblasts transfected with vectors comprising the first/second translation initiation signal construct when compared to fibroblasts transfected with vectors comprising TRAIL, TK, and Sox2 under the control of a single translation initiation signal (referred to herein as the“Triple Fusion Vector”). Also compared were fibroblasts transfected with a vector containing only TRAIL and an optical reporter (referred to herein as the“positive control”). Supernatants from the fibroblasts transfected with the first and second translation initiation signal constructs induced more cell killing when compared to the supernatants collected from the fibroblasts transfected with the Triple Fusion vector. Thus, it can be concluded that the first/second translation initiation signal construct enhances TRAIL secretion of transfected fibroblasts when compared to a construct having TRAIL, TK, and a transdifferentiation factor under the control of a single translation initiation signal.

[0202] Four vectors - 347887, 347888, 347889, and 347890 - comprising the first/second translation initiation signal construct disclosed herein, a vector encoding TRAIL, GFP, and TK under a single promoter and single translation initiation signal (the“Triple Fusion Fusion”), and a vector encoding only TRAIL and an optical reporter (referred to herein as the“Positive

Control”) were used in this experiment.

[0203] Cryopreserved fibroblast cells were thawed and cultured in a lOmL DMEM solution comprising 10% NZ FBS. The fibroblast cells were next transfected with the vector constructs disclosed above (347887, 347888, 347889, 347890, the Triple Fusion Vector, the Positive Control) in four six -well plates. Plates Pl.l and P1.2 were used for a 24 hour timepoint and Plates P2.1 and P2.2 were used for a 48 hour timepoint. The remaining plates were used for biological duplicates.

[0204] Twenty-four hours after transfection, the first batch of supernatants was harvested, aliquoted and frozen at -80°C. At the same time, the cell culture medium on the second plate was exchanged for fresh medium. 48 hours after transfection, the second batch of supernatants was harvested, aliquoted and frozen at -80°C. At the same timepoints, conditioned medium was collected from the un-transfected HEK293T cells for control supernatants.

[0205] The ovarian cancer cell lines CAOV-3, ES-2 and SKOV-3 were seeded into 2 opaque- walled 96 well plates at 20,000 cells/well and returned to the incubator for re-attachment. The next day, the supernatants were thawed on ice and pre-warmed in the incubator to 37°C for 10 minutes. The culture medium was removed from the cells and the supernatants added. Sixteen hours after supernatant addition, cell viability was assessed by CellTiter Glo assay. Attached as Figure 1, is the CellTiter Glo Readout analysis for Plate 1. Attached as Figure 2, is the CellTiter Glo Readout analysis for Plate 2.

[0206] For the CAOV-3 and SKOV-3 cell lines, supernatants from the positive control induced the highest amount of cell killing, particularly for the supernatants collected 48 horns after transfection. Supernatants from the fibroblast cells transfected with the Triple Fusion vector induced very little killing for both cell lines and both timepoints. All of the 347887, 347888, 347889, and 347890 vectors performed similarly well, with two performing slightly better in the 48 hour timepoint: 347889 and 347890. Thus, it can be concluded that the 347887, 347888, 347889, and 347890 vectors enhance TRAIL secretion in transfected fibroblasts when compared to the Triple Fusion Vector.

[0207] For the ES-2 cell line, the 20,000 cells per well seeding density was too high for the ES-2 cell line, as it grows very fast. The cells were already completely confluent at the time of supernatant addition. Therefore, the reduced sensitivity of the cells to the TRAIL-containing supernatant is likely due to the too high density. It is also likely that the assay readout was over saturated for the ES-2 cell line as a result.

B. EXAMPLE 2: Exemplary 2D Culture Protocols for Engineered Stem Cells

[0208] Cryopreserved fibroblast cells were thawed and cultured in a lOmL DMEM solution comprising 10% NZ FBS on a tissue culture plate at 37°C for 3 days or until cells were 80% confluent. Next, the fibroblast cells were counted and then passaged so that lxlOE6 cells were passaged to each 100mm tissue culture plate. Post-seeding, the plates were transduced with a solution comprising a lentiviral vector comprising the recombinant polynucleotide disclosed herein.

[0209] At 24 h post-seeding, the plates were transduced with a solution comprising a lentiviral vector comprising the recombinant polynucleotide, at an MOI (multiplicity of infection) of 25. The plates were then incubated at 37°C for about 24 hours.

[0210] After about 24 hours post transduction, the cell media was replaced with fresh cGMP- like NIM (cGMP-like neural induction medium). This time point was marked as DO of conversion. On D2 and D4, the cell media was replaced with fresh cGMP-like NIM,

respectively. On D5, the cells were dissociated from the plates utilizing a TrypLE Express solution, counted, and cell numbers and viability were recorded. Upon viability assessment, the harvested cells (now engineered stem cells) were stored at -20°C until further use.

[0211] The cGMP-like NIM was prepared in a 50 mL volume and includes 48 mL CTS Neurobasal -A, 500pL CTS Glutamax-I (IX), 500pL CTS N2-Supplement (IX), 1 mL CTS B27 without vitamin A (IX), and EGF and bFGF each at a final concentration of 20 ng/mL. Sequences

[0212] The following Table exemplifies sequences disclosed above.

[0213] While preferred embodiments of the present disclosure have been shown and disclosed 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 disclosure. It should be understood that various alternatives to the embodiments of the disclosure disclosed herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A recombinant polynucleotide, comprising:

a) a first anti-cancer therapy segment comprising a first nucleic acid sequence encoding

(i) a first translation initiation signal and (ii) an anti-cancer therapeutic; and b) a second anti-cancer therapy segment comprising a second nucleic acid sequence

encoding (i) a second translation initiation signal and (ii) an anti-cancer polypeptide,

wherein said first translation initiation signal initiates the translation of the first nucleic acid sequence, and wherein said second translation initiation signal initiates the translation of the second nucleic acid sequence.

2. The recombinant polynucleotide of claim 1, wherein the second translation initiation signal is an internal ribosome entry site (IRES).

3. The recombinant polynucleotide of claim 1, further comprising a promoter, wherein the transcription of the first anti-cancer therapy segment and the second anti-cancer therapy segment is mediated by the single promoter.

4. The recombinant polynucleotide of claim 1, wherein the second nucleic acid sequence encodes a transdifferentiation factor.

5. The recombinant polynucleotide of claim 4, wherein the transdifferentiation factor is Sox2.

6. The recombinant polynucleotide of claim 1, wherein the anti-cancer therapeutic is

secreted extracellularly.

7. The recombinant polynucleotide of claim 1, wherein the secretion of the anti-cancer therapeutic is increased 1.1 fold compared to the secretion of the anti-cancer polypeptide generated by a recombinant polynucleotide consisting of the transdifferentiation factor, the anti -cancer therapeutic, the anti -cancer polypeptide under the translational control of a single translation initiation signal.

8. The recombinant polynucleotide of claim 1, wherein the anti-cancer therapeutic is TNF- related apoptosis-inducing ligand (TRAIL) or secretable TNF-related apoptosis-inducing ligand (S -TRAIL).

9. The recombinant polynucleotide of claims 1, wherein the anti -cancer polypeptide is

thymidine kinase (TK) or a herpes simplex virus thymidine kinase (HSV-TK).

10. The recombinant polynucleotide of claim 1, wherein the recombinant polynucleotide is a vector.

11. The recombinant polynucleotide of claim 10, wherein the vector is a viral vector.

12. The recombinant polynucleotide of claim 11, wherein the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV), or a retrovirus.

13. The recombinant polynucleotide of claim 1, wherein the anti-cancer polypeptide is a

cytokine.

14. The recombinant polynucleotide of claim 11, wherein the cytokine is a protein, peptide, glycoprotein, chemokine, interleukin, tumor necrosis factor (TNF), monocyte

chemoattractant protein (MCP), IL-l-like cytokine, gamma chain cytokine, beta chain cytokine, IL-6-like cytokine, IL-10-like cytokine, interferon, tumor necrosis factor, TGF- beta, macrophage inflammatory protein (MIP), tumor growth factor (TGF), matrix metalloprotease (MMP), or any combination thereof.

15. The recombinant polynucleotide of claim 1, wherein the recombinant polynucleotide is a single vector system.

16. The recombinant polynucleotide of claim 1, wherein the first nucleic acid sequence is 5’ from the second nucleic acid sequence.

17. The recombinant polynucleotide of claim 1, wherein the first nucleic acid sequence is 3’ from the second nucleic acid sequence.

18. A method of treating cancer in an individual in need thereof comprising, (a) generating an induced tumor from a target somatic cell or a target primary cell, comprising: ii) introducing into the target somatic cell or the target primary cell the recombinant polynucleotide of claim 1 ; and ii) contacting the target somatic cell or the target primary cell with one or more reprogramming agents; and b) administering the therapeutic cell to the individual, thereby treating the cancer.

19. The method of claim 18, wherein the one or more reprogramming agents are selected from the group consisting of: GSK3 inhibitor, a WT agonist, an ALK4/5/7 inhibitor, an HD AC inhibitor, a p300 activator, a PDE4 inhibitor, an Adenylyl cyclase agonist, a retinoic acid receptor g agonist, a 5-HT3 antagonist, and a metabotropic glutamate (mGlu) receptor agonist.

20. The method of claim 19, wherein the cancer is ovarian cancer.

21. The method of claim 19, wherein the cancer is pulmonary cancer.

22. The method of claim 19, wherein the cancer is breast cancer.

23. The method of claim 19, wherein the cancer is glioblastoma.

24. The method of claim 20, wherein the breast cancer is triple negative breast cancer.

25. The method of claim 19, wherein the cancer is melanoma.

26. The method of claim 19, wherein the cancer is leukemia.

27. The method of claim 19, wherein the cancer is lymphoma.

28. The method of claim 19, wherein the cancer is pancreatic cancer.

29. A method of treating cancer in an individual in need thereof, comprising administering a cell comprising the recombinant polynucleotide of claim 1.

30. The method of claim 29, wherein the cancer is ovarian cancer.

31. The method of claim 29, wherein the cancer is pulmonary cancer.

32. The method of claim 29, wherein the cancer is breast cancer.

33. The method of claim 29, wherein the cancer is glioblastoma.

34. The method of claim 29, wherein the breast cancer is triple negative breast cancer.

35. The method of claim 29, wherein the cancer is melanoma.

36. The method of claim 29, wherein the cancer is leukemia.

37. The method of claim 29, wherein the cancer is lymphoma.

38. The method of claim 29, wherein the cancer is pancreatic cancer.

39. A method of generating a therapeutic cell from a target somatic cell or a target primary cell, comprising: a) introducing into the target somatic cell or the target primary cell the recombinant polynucleotide of claim 1; and b) contacting the target somatic cell or the target primary cell with one or more reprogramming agents, wherein the recombinant polynucleotide gives rise, upon transcription, to a factor that contributes to the

reprogramming of the target somatic cell or the target primary cell into a therapeutic cell.

40. The method of claim 39, wherein the one or more reprogramming agents are selected from the group consisting of: GSK3 inhibitor, a WT agonist, an ALK4/5/7 inhibitor, an HD AC inhibitor, a p300 activator, a PDE4 inhibitor, an Adenylyl cyclase agonist, a retinoic acid receptor g agonist, a 5-HT3 antagonist, and a metabotropic glutamate (mGlu) receptor agonist.

41. The method of claim 40, wherein the therapeutic cell is an induced tumor homing cell (iTDC).

42. The method of claim 40, wherein the therapeutic cell expresses CXCR4.

43. A method of generating a therapeutic cell from a target somatic cell or a target primary cell, comprising introducing into the target somatic cell or the target primary cell the recombinant polynucleotide of claim 2, wherein the recombinant polynucleotide gives rise, upon transcription, to a factor that contributes to the reprogramming of the target somatic cell or the target primary cell into a therapeutic cell.

44. The method of claim 43, wherein the therapeutic cell is an iTDC.

45. The method of claim 43, wherein the therapeutic cell expresses CXCR4.

46. A vector, comprising:

a) a promoter; b) a first nucleic acid sequence encoding an anti-cancer therapeutic, the first nucleic acid sequence under the transcriptional control of the promoter;

c) a second nucleic acid sequence downstream of the first nucleic acid sequence and under the transcriptional control of the first promoter, the second nucleic acid sequence encoding an internal ribsome entry site (IRES), an anti-cancer polypeptide and a transdifferentiation factor, the anti-cancer polypeptide and the transdifferentiation factor under the translational control of the IRES.

47. The vector of claim 46, wherein the transdifferentiation factor is Sox2.

48. The vector of claim 46, wherein the anti-cancer therapeutic is TNF-related apoptosis- inducing ligand (TRAIL) or secretable TNF-related apoptosis-inducing ligand (S- TRAIL).

49. The vector of claims 46, wherein the anti-cancer polypeptide is thymidine kinase (TK) or a herpes simplex virus thymidine kinase (HSV-TK).

50. The vector of claim 46, wherein the anti -cancer therapeutic is secreted extracellularly.

51. The vector of claim 46, wherein the secretion of the anti-cancer therapeutic is increased 1.1 fold compared to the secretion of the anti-cancer polypeptide generated by a recombinant polynucleotide consisting of the transdifferentiation factor, the anti-cancer therapeutic, the anti -cancer polypeptide under the translational control of a single translation initiation signal.

52. The vector of claim 46, wherein the vector is a viral vector.

53. The vector of claim 46, wherein the viral vector is a lentiviral vector, an adenoviral

vector, an adeno-associated virus (AAV), or a retrovirus.

54. The vector of claim 46, wherein the anti -cancer polypeptide is a cytokine.

55. The vector of claim 54, wherein the cytokine is a protein, peptide, glycoprotein,

chemokine, interleukin, tumor necrosis factor (TNF), monocyte chemoattractant protein (MCP), IL-l-like cytokine, gamma chain cytokine, beta chain cytokine, IL-6-like cytokine, IL-10-like cytokine, interferon, tumor necrosis factor, TGF-beta, macrophage inflammatory protein (MIP), tumor growth factor (TGF), matrix metalloprotease (MMP), or any combination thereof.