Methods and materials for treating cancer

Abstract

To provide methods and materials for converting one or more cancer cells present in a mammal having cancer into non-cancerous cells.SOLUTION: The present invention relates to methods and materials for treating a mammal having cancer. For example, methods and materials for converting one or more cancer cells in a mammal into non-cancerous cells by using one or more nucleic acids encoding a transcription factor (for example, a neuronal transcription factor or a hepatic transcription factor) are provided.SELECTED DRAWING: Figure 15

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 823,702, filed March 26, 2019. The disclosure of the prior application is considered part of (and incorporated by reference into) the disclosure of this application.

[0002] This application relates to methods and materials for treating a mammal with cancer. For example, this document provides methods and materials for converting one or more cancer cells present in a mammal with cancer into non-cancerous cells. [Background technology]

[0003] Cancer is a major public health problem. In the United States alone, more than 1.7 million new cases were diagnosed in 2019 (National Cancer Institute, "Cancer Stat Facts: Cancer of Any Site," available at https colon "seer" dot "cancer" dot "gov / statfacts / html / all.html).

[0004] Glioblastoma (GBM), a type of tumor arising from the uncontrolled proliferation of glial cells, accounts for half of all malignant brain tumor cases, with a 5-year relative survival rate of 3.6 percent (Ostrom et al., Neuro Oncol.; 17:iv1-iv62 (2015) and Porter et al., Neuroepidemiology.; 36(4):230-239 (2011)). Conventional therapies, such as chemotherapy, radiation therapy, and surgery, often fail in GBM due to its active cell proliferation, invasive nature, and genomic and epigenetic heterogeneity (Brennan et al., Cell.; 155(2):462 (2013) and McLendon et al., Nature.; 455(7216):1061-1068 (2008)).

[0005] Globally, liver cancer (or hepatocellular carcinoma (HCC)) is the third leading cause of cancer-related deaths and the sixth leading cause of incidence (El-Serag, Gastroenterology.;142:1264-73(2012)). Treatment for liver cancer typically involves surgery and resection, but for patients with advanced or terminal disease, such treatment is often ineffective. Summary of the Invention

[0006] This application provides methods and materials for treating a mammal with cancer by converting cancer cells in the mammal to non-cancerous cells. For example, one or more nucleic acids encoding a transcription factor (e.g., a neuronal transcription factor or a liver transcription factor) can be used to convert one or more cancer cells in the mammal to non-cancerous cells.

[0007] A hallmark of many cancers is the presence of dedifferentiated cancer cells. As described herein, cancer cells can be converted into noncancerous cells (e.g., terminally differentiated, non-dividing cells) in a mammal by delivering a nucleic acid designed to express a transcription factor (e.g., a neuronal transcription factor or a liver transcription factor) to the cells in the mammal. As demonstrated herein, human GBM cells can be converted into noncancerous neurons by delivering a nucleic acid designed to express a neuronal transcription factor (e.g., a nucleic acid designed to express a neurogenic differentiation factor 1 (NeuroD1) polypeptide, a nucleic acid designed to express a neurogenin-2 (Neurog2) polypeptide, or a nucleic acid designed to express an achaete-scute homolog 1 (Ascl1) polypeptide) to the GBM cells. The converted neurons can express neuronal-specific markers, have functional synaptic networks, and have active electrophysiological properties. The converted neurons can also exhibit downregulated signaling pathways related to cancer progression (e.g., compared to the GBM cells prior to conversion). In vivo conversion of GBM cells into neurons can reduce cancer cell proliferation and / or the rate of astrogliosis. Also, as demonstrated herein, human liver cancer cells can be converted into non-cancerous liver cells (hepatocytes) by delivering a nucleic acid designed to express a liver transcription factor (e.g., a nucleic acid designed to express hepatocyte nuclear factor 4A (HNF4A) polypeptide, a nucleic acid designed to express forkhead box protein (Foxa2) polypeptide, and / or a nucleic acid designed to express GATA-binding protein (GATA4) polypeptide) to the cells. The converted hepatocytes can have reduced proliferation, reduced expression of the liver cancer marker alpha-fetoprotein (AFP), and / or express epithelial-specific markers such as the epithelial cell surface molecule E-cadherin.

[0008] The ability to convert cancer cells to non-cancerous cells in living mammals using the methods and materials described herein provides clinicians and patients (e.g., cancer patients) with an effective approach to treating cancer. For example, in vivo conversion of cancer cells to non-cancerous cells can be used to control cancer cell growth in the absence of conventional cancer therapies. In such cases, cancer patients can avoid common side effects caused by conventional cancer therapies.

[0009] In general, one aspect of this document features a method for treating a mammal having cancer. The method includes (or consists essentially of, or consists of) administering to cancer cells in the mammal a nucleic acid encoding one or more transcription factors, wherein the one or more transcription factors are expressed by the cancer cells, and the one or more transcription factors convert the cancer cells in the mammal to non-cancerous cells, thereby reducing the number of cancer cells in the mammal. The mammal can be a human. The cancer can be a glioma. The one or more transcription factors can be one or more neuronal transcription factors. The one or more neuronal transcription factors can be selected from the group consisting of neurogenic differentiation factor 1 (NeuroD1) polypeptide, neurogenin-2 (Neurog2) polypeptide, and achaete-scute homolog 1 (Ascl1) polypeptide. The one or more neuronal transcription factors can include NeuroD1 polypeptide, Neurog2 polypeptide, and Ascl1 polypeptide. The non-cancerous cells can be neuronal cells. The neuronal cells can be FoxG1-positive forebrain neurons. The cancer can be liver cancer. The liver cancer can be hepatocellular carcinoma. The one or more transcription factors may be liver transcription factors. The one or more liver transcription factors may be selected from the group consisting of hepatocyte nuclear factor 4A (HNF4A) polypeptide, forkhead box protein (Foxa2) polypeptide, and GATA-binding protein (GATA4) polypeptide. The one or more liver transcription factors may include HNF4A polypeptide, Foxa2 polypeptide, and GATA4 polypeptide. The non-cancerous cells may be hepatocytes. The hepatocytes may be hepatocytes that secrete liver enzymes. The liver enzymes may be albumin. The nucleic acids encoding the one or more transcription factors may be administered to the cancer cells in the form of a viral vector. The viral vector may be a retroviral vector. The viral vector may be a lentiviral vector. The nucleic acids encoding each of the one or more transcription factors may be operably linked to a promoter sequence. Administration of the nucleic acids encoding the one or more transcription factors may include direct injection into a mammalian tumor. Administration of the nucleic acids encoding the one or more transcription factors may include intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intraparenchymal, intratumoral, intranasal, or oral administration.The method can include, prior to the administering step, identifying the mammal with cancer.

[0010] In another aspect, this document features the use of a composition comprising (or consisting essentially of, or consisting of) a nucleic acid encoding one or more transcription factors to treat cancer according to a method comprising (or consisting essentially of, or consisting of) administering to cancer cells in a mammal a nucleic acid encoding one or more transcription factors, wherein the one or more transcription factors are expressed by the cancer cells, and the one or more transcription factors convert the cancer cells in the mammal to non-cancerous cells, thereby reducing the number of cancer cells in the mammal. The mammal can be a human. The cancer can be a glioma. The one or more transcription factors can be one or more neuronal transcription factors. The one or more neuronal transcription factors can be selected from the group consisting of neurogenic differentiation factor 1 (NeuroD1) polypeptide, neurogenin-2 (Neurog2) polypeptide, and achaete-scute homolog 1 (Ascl1) polypeptide. The one or more neuronal transcription factors can include NeuroD1 polypeptide, Neurog2 polypeptide, and Ascl1 polypeptide. The non-cancerous cells can be neuronal cells. The neuronal cells can be FoxG1-positive forebrain neurons. The cancer may be liver cancer. The liver cancer may be hepatocellular carcinoma. The one or more transcription factors may be liver transcription factors. The one or more liver transcription factors may be selected from the group consisting of hepatocyte nuclear factor 4A (HNF4A) polypeptide, forkhead box protein (Foxa2) polypeptide, and GATA-binding protein (GATA4) polypeptide. The one or more liver transcription factors may include HNF4A polypeptide, Foxa2 polypeptide, and GATA4 polypeptide. The non-cancerous cells may be hepatocytes. The hepatocytes may be hepatocytes that secrete liver enzymes. The liver enzymes may be albumin. The nucleic acids encoding the one or more transcription factors may be administered to the cancer cells in the form of a viral vector. The viral vector may be a retroviral vector. The viral vector may be a lentiviral vector. The nucleic acids encoding each of the one or more transcription factors may be operably linked to a promoter sequence. The administration of the nucleic acids encoding the one or more transcription factors may include direct injection into the mammalian tumor.Administration of the nucleic acid encoding one or more transcription factors can include intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intraparenchymal, intratumoral, intranasal, or oral administration. The method can include identifying the mammal with cancer prior to the administering step.

[0011] In another aspect, this document features a composition comprising (or consisting essentially of, or consisting of) a nucleic acid encoding one or more transcription factors for treating cancer according to a method comprising (or consisting essentially of, or consisting of) administering to cancer cells in a mammal a nucleic acid encoding one or more transcription factors, wherein the one or more transcription factors are expressed by the cancer cells, and the one or more transcription factors convert the cancer cells in the mammal to non-cancerous cells, thereby reducing the number of cancer cells in the mammal. The mammal can be a human. The cancer can be a glioma. The one or more transcription factors can be one or more neuronal transcription factors. The one or more neuronal transcription factors can be selected from the group consisting of neurogenic differentiation factor 1 (NeuroD1) polypeptide, neurogenin-2 (Neurog2) polypeptide, and achaete-scute homolog 1 (Ascl1) polypeptide. The one or more neuronal transcription factors can include NeuroD1 polypeptide, Neurog2 polypeptide, and Ascl1 polypeptide. The non-cancerous cells can be neuronal cells. The neuronal cells can be FoxG1-positive forebrain neurons. The cancer may be liver cancer. The liver cancer may be hepatocellular carcinoma. The one or more transcription factors may be liver transcription factors. The one or more liver transcription factors may be selected from the group consisting of hepatocyte nuclear factor 4A (HNF4A) polypeptide, forkhead box protein (Foxa2) polypeptide, and GATA-binding protein (GATA4) polypeptide. The one or more liver transcription factors may include HNF4A polypeptide, Foxa2 polypeptide, and GATA4 polypeptide. The non-cancerous cells may be hepatocytes. The hepatocytes may be hepatocytes that secrete liver enzymes. The liver enzymes may be albumin. The nucleic acids encoding the one or more transcription factors may be administered to the cancer cells in the form of a viral vector. The viral vector may be a retroviral vector. The viral vector may be a lentiviral vector. The nucleic acids encoding each of the one or more transcription factors may be operably linked to a promoter sequence. The administration of the nucleic acids encoding the one or more transcription factors may include direct injection into the mammalian tumor.Administration of the nucleic acid encoding one or more transcription factors can include intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intraparenchymal, intratumoral, intranasal, or oral administration. The method can include identifying the mammal with cancer prior to the administering step.

[0012] In another aspect, this document features the use of nucleic acids encoding one or more transcription factors in the manufacture of a medicament for treating cancer, according to a method comprising (or consisting essentially of, or consisting of) administering to cancer cells in a mammal nucleic acids encoding one or more transcription factors, wherein the one or more transcription factors are expressed by the cancer cells, and the one or more transcription factors convert the cancer cells in the mammal to non-cancerous cells, thereby reducing the number of cancer cells in the mammal. The mammal can be a human. The cancer can be a glioma. The one or more transcription factors can be one or more neuronal transcription factors. The one or more neuronal transcription factors can be selected from the group consisting of neurogenic differentiation factor 1 (NeuroD1) polypeptide, neurogenin-2 (Neurog2) polypeptide, and achaete-scute homolog 1 (Ascl1) polypeptide. The one or more neuronal transcription factors can include NeuroD1 polypeptide, Neurog2 polypeptide, and Ascl1 polypeptide. The non-cancerous cells can be neuronal cells. The neuronal cells can be FoxG1-positive forebrain neurons. The cancer can be liver cancer. The liver cancer may be hepatocellular carcinoma. The one or more transcription factors may be liver transcription factors. The one or more liver transcription factors may be selected from the group consisting of hepatocyte nuclear factor 4A (HNF4A) polypeptide, forkhead box protein (Foxa2) polypeptide, and GATA-binding protein (GATA4) polypeptide. The one or more liver transcription factors may include HNF4A polypeptide, Foxa2 polypeptide, and GATA4 polypeptide. The non-cancerous cells may be hepatocytes. The hepatocytes may be hepatocytes that secrete liver enzymes. The liver enzymes may be albumin. The nucleic acids encoding one or more transcription factors may be administered to the cancer cells in the form of a viral vector. The viral vector may be a retroviral vector. The viral vector may be a lentiviral vector. The nucleic acids encoding each of the one or more transcription factors may be operably linked to a promoter sequence. The administration of the nucleic acids encoding one or more transcription factors may include direct injection into the mammalian tumor. Administration of nucleic acids encoding one or more transcription factors can include intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intraparenchymal, intratumoral, intranasal, or oral administration.The method can include, prior to the administering step, identifying the mammal with cancer.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, as exemplified in various art-specific dictionaries. Although the present invention can be practiced using methods and materials similar or equivalent to those described herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0014] The details of one or more aspects of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0015] [Figure 1] Characterization of human glioblastoma cell lines. Representative images of a panel of markers (stained in red) used to characterize U251 and U118 human glioblastoma cells. Red boxes (marked with *) indicate high levels of immunopositive markers. Scale bar = 50 μm. GFAP and S100β are astrocyte markers, Tuj1 and DCX are immature neuron markers, Sox2 and nestin are neural progenitor markers, Olig2 is an oligodendrocyte marker, Ki67 is a cell proliferation marker, and EGFR is a cancer marker. [Figure 2]Confirmation of overexpression of the neural transcription factors Neurog2, NeuroD1, or Ascl1 in human glioblastoma cells. (A) Representative images showing overexpression of Neurog2, NeuroD1, or Ascl1 in U251 human glioblastoma cells via immunostaining. Scale bar: 20 μm. (B) Hierarchical clustering and heatmap of real-time qPCR analysis showing transcriptional changes of different neural transcription factors in U251 GBM cells. Note the significant increase in mRNA levels in U251 GBM cells after viral infection with Neurog2, NeuroD1, or Ascl1. Data were normalized to GFP control virus-infected U251 cells and expressed as mean values. Samples were collected 20 days post-infection (dpi). n = 3 batches of cultures. [Figure 3] Rapid induction of neuron-like cells from human glioblastoma cells by Neurog2 and NeuroD1. A. Immunostaining of immature neuronal markers doublecortin (DCX, red) and β3-tubulin (Tuj1, magenta) in U251 human GBM cells infected with Neurog2-GFP, NeuroD1-GFP, or Ascl1-GFP retroviruses at 6 dpi. Scale bar: 50 μm. B-C. Quantitative analysis of neuronal conversion at 6 dpi. Note that Neurog2 showed a significant increase in the proportion of DCX+ cells (GFP, 0; Neurog2, 12.6% ± 2.0%; NeuroD1, 1.6% ± 0.4%; Ascl1, 0; B) and Tuj1+ cells (GFP, 0; Neurog2, 46.1% ± 3.2%; NeuroD1, 20.5% ± 4.9%; Ascl1, 2.6% ± 0.7%; C), followed by NeuroD1 at the early stage of viral infection. Ascl1 transduction efficiency was the lowest among the three neural transcription factors tested. Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett's test. ** indicates p < 0.01, *** indicates p < 0.001, and n indicates > 200 cells from triplicate cultures. [Figure 4A]The single neuronal transcription factor Neurog2, NeuroD1, or Ascl1 converts human glioblastoma cells into neurons. In A-B, retroviral expression of Neurog2-GFP, NeuroD1-GFP, or Ascl1-GFP in U251 human glioblastoma cells resulted in a higher number of neurons compared to GFP alone (top row). Neurog2-, NeuroD1-, or Ascl1-converted cells were immunopositive for immature neuronal markers (A, DCX, Tuj1) at 20 days post-infection (dpi) and for mature neuronal markers (B, MAP2, NeuN) at 30 dpi. Scale bar is 50 μm. [Figure 4B] The single neuronal transcription factor Neurog2, NeuroD1, or Ascl1 converts human glioblastoma cells into neurons. In A-B, retroviral expression of Neurog2-GFP, NeuroD1-GFP, or Ascl1-GFP in U251 human glioblastoma cells resulted in a higher number of neurons compared to GFP alone (top row). Neurog2-, NeuroD1-, or Ascl1-converted cells were immunopositive for immature neuronal markers (A, DCX, Tuj1) at 20 days post-infection (dpi) and for mature neuronal markers (B, MAP2, NeuN) at 30 dpi. Scale bar is 50 μm. [Figure 4C] The single neuronal transcription factor Neurog2, NeuroD1, or Ascl1 converts human glioblastoma cells into neurons. C-D. Quantitative analysis of conversion efficiency at 20 dpi (C) and 30 dpi (D). **p<0.01, ***p<0.001, one-way ANOVA followed by Dunnett's test. n=200 cells from triplicate cultures. [Figure 4D] The single neuronal transcription factor Neurog2, NeuroD1, or Ascl1 converts human glioblastoma cells into neurons. C-D. Quantitative analysis of conversion efficiency at 20 dpi (C) and 30 dpi (D). **p<0.01, ***p<0.001, one-way ANOVA followed by Dunnett's test. n=200 cells from triplicate cultures. [Figure 4E]Single neuronal transcription factors Neurog2, NeuroD1, or Ascl1 convert human glioblastoma cells into neurons. E. Time course of DCX transcription activation by overexpression of Neurog2, NeuroD1, or Ascl1 in U251 cells as revealed by real-time qPCR. Data were normalized to GFP control and presented as mean ± SEM. n=3 batches. [Figure 5] Induction of neuron-like cells in U118 human glioblastoma cells by the combination of Neurog2 overexpression and small molecule treatment. In A-D, U118 cells were converted to neuron-like cells (DCX stained blue-green) when Neurog2 was overexpressed together with small molecule treatment (core: 5 μM DAPT, 1.5 μM CHIR99021, 5 μM SB431542, 0.25 μM LDN193189). Samples were collected 18 dpi after 12 days of drug treatment. [Figure 6A] Characterization of converted neurons from human GBM cells. A–D are representative images showing immunostaining for neuronal subtype markers. Most Neurog2-, NeuroD1-, and Ascl1-converted neurons (DCX in A and MAP2 in B) were immunopositive for the hippocampal neuronal marker Prox1 (A) and the forebrain neuronal marker FoxG1 (B). Furthermore, Neurog2-, NeuroD1-, and Ascl1-converted neurons (DCX in C) were primarily VGluT1+ (C), whereas some Ascl1-converted neurons (DCX in D) were also GABA+ (D). [Figure 6B] Characterization of converted neurons from human GBM cells. A–D are representative images showing immunostaining for neuronal subtype markers. Most Neurog2-, NeuroD1-, and Ascl1-converted neurons (DCX in A and MAP2 in B) were immunopositive for the hippocampal neuronal marker Prox1 (A) and the forebrain neuronal marker FoxG1 (B). Furthermore, Neurog2-, NeuroD1-, and Ascl1-converted neurons (DCX in C) were primarily VGluT1+ (C), whereas some Ascl1-converted neurons (DCX in D) were also GABA+ (D). [Figure 6C] Characterization of converted neurons from human GBM cells. A–D are representative images showing immunostaining for neuronal subtype markers. Most Neurog2-, NeuroD1-, and Ascl1-converted neurons (DCX in A and MAP2 in B) were immunopositive for the hippocampal neuronal marker Prox1 (A) and the forebrain neuronal marker FoxG1 (B). Furthermore, Neurog2-, NeuroD1-, and Ascl1-converted neurons (DCX in C) were primarily VGluT1+ (C), whereas some Ascl1-converted neurons (DCX in D) were also GABA+ (D). [Figure 6D] Characterization of converted neurons from human GBM cells. A–D are representative images showing immunostaining for neuronal subtype markers. Most Neurog2-, NeuroD1-, and Ascl1-converted neurons (DCX in A and MAP2 in B) were immunopositive for the hippocampal neuronal marker Prox1 (A) and the forebrain neuronal marker FoxG1 (B). Furthermore, Neurog2-, NeuroD1-, and Ascl1-converted neurons (DCX in C) were primarily VGluT1+ (C), whereas some Ascl1-converted neurons (DCX in D) were also GABA+ (D). [Figure 6E] Characterization of converted neurons from human GBM cells. E–H. Quantitative analysis of converted neurons from human GBM cells. Samples were obtained at 20 dpi. Scale bar is 50 μm. Data are expressed as mean ± SEM. n is ≥200 cells from triplicate cultures. [Figure 6F] Characterization of converted neurons from human GBM cells. E–H. Quantitative analysis of converted neurons from human GBM cells. Samples were obtained at 20 dpi. Scale bar is 50 μm. Data are expressed as mean ± SEM. n is ≥200 cells from triplicate cultures. [Figure 6G]Characterization of converted neurons from human GBM cells. E–H. Quantitative analysis of converted neurons from human GBM cells. Samples were obtained at 20 dpi. Scale bar is 50 μm. Data are expressed as mean ± SEM. n is ≥200 cells from triplicate cultures. [Figure 6H] Characterization of converted neurons from human GBM cells. E–H. Quantitative analysis of converted neurons from human GBM cells. Samples were obtained at 20 dpi. Scale bar is 50 μm. Data are expressed as mean ± SEM. n is ≥200 cells from triplicate cultures. [Figure 7] Further characterization of neuronal identity among human GBM cell-converted neurons. A-B are representative images showing immunostaining signals for the cortical neuronal markers Ctip2 (A) or Tbr1 (B) after viral infection with Neurog2, NeuroD1, or Ascl1 among U251 human glioblastoma cells at 20 dpi. Scale bar is 50 µm. [Figure 8]Comparison with human astrocyte-converted neurons after infection with Neurog2, NeuroD1, or Ascl1. (A) The majority of human astrocyte-converted neurons induced by Neurog2, NeuroD1, or Ascl1 were immunopositive for the hippocampal neuronal marker Prox1 and the forebrain marker FoxG1. Far fewer Ascl1-converted neurons were Ctip2+. Scale bar: 20 μm. (B) Quantitative analysis of Neurog2-, NeuroD1-, and Ascl1-converted neurons from human cortical astrocytes (HA1800 cells, ScienCell, San Diego, USA). Prox1+ / MAP2+: Neurog2, 85.4% ± 3.4%; NeuroD1, 89.2% ± 3.3%; Ascl1, 85.0% ± 3.7%. FoxG1+ / MAP2+: Neurog2, 92.6% ± 3.8%; NeuroD1, 85.1% ± 2.7%; Ascl1, 85.7% ± 4.8%. Ctip2+ / MAP2+: Neurog2, 46.6% ± 5.1%; NeuroD1, 61.1% ± 2.8%; Ascl1, 14.0% ± 5.4%. Samples were obtained 30 dpi. Data are expressed as mean ± SEM. n = 50 cells from triplicate cultures. [Figure 9A] Neurog2 overexpression induces neuronal fate change in glioblastoma cells. A, Downregulation of astroglial markers vimentin and GFAP in Neurog2-converted neurons (bottom row) compared with control U251 glioblastoma cells expressing GFP only (top row). Samples were taken at 20 dpi. [Figure 9B] Neurog2 overexpression induces the change in glioblastoma cell fate to neurons. B–C are representative images showing gap junctions (connexin 43) between U251 GBM cells overexpressing GFP alone (top row) or Neurog2-GFP (bottom row). Quantitative data (C) show that connexin 43 intensity was significantly reduced in the Neurog2 group compared to the control GFP group. Samples were taken at 20 dpi. n = 60 or more from triplicate cultures. [Figure 9C]Neurog2 overexpression induces the change in glioblastoma cell fate to neurons. B–C are representative images showing gap junctions (connexin 43) between U251 GBM cells overexpressing GFP alone (top row) or Neurog2-GFP (bottom row). Quantitative data (C) show that connexin 43 intensity was significantly reduced in the Neurog2 group compared to the control GFP group. Samples were taken at 20 dpi. n = 60 or more from triplicate cultures. [Figure 9D] (D) A representative image illustrating the growth cone, indicated by GAP43 and phalloidin, in U251 cells overexpressing Neurog2 at 6 dpi. [Figure 9E] Neurog2 overexpression induces the neuronal fate change of glioblastoma cells. E–H: Distribution and morphological changes of mitochondria (MitoTracker) and Golgi apparatus (GM130) during neuronal conversion of U251 cells. Quantitative data show changes in MitoTracker intensity (F) and Golgi apparatus size (H) as reflected by GM130 coverage area after Neurog2 expression at 30 dpi. n = 150 cells from triplicate cultures. Scale bars are 20 μm for (A), (B), and (D) and 10 μm for (E) and (G). Data are presented as mean ± SEM and analyzed by Student's t-test. * indicates p<0.05, *** indicates p<0.001. [Figure 9F]Neurog2 overexpression induces the neuronal fate change of glioblastoma cells. E–H: Distribution and morphological changes of mitochondria (MitoTracker) and Golgi apparatus (GM130) during neuronal conversion of U251 cells. Quantitative data show changes in MitoTracker intensity (F) and Golgi apparatus size (H) as reflected by GM130 coverage area after Neurog2 expression at 30 dpi. n = 150 cells from triplicate cultures. Scale bars are 20 μm for (A), (B), and (D) and 10 μm for (E) and (G). Data are presented as mean ± SEM and analyzed by Student's t-test. * indicates p<0.05, *** indicates p<0.001. [Figure 9G] Neurog2 overexpression induces the neuronal fate change of glioblastoma cells. E–H: Distribution and morphological changes of mitochondria (MitoTracker) and Golgi apparatus (GM130) during neuronal conversion of U251 cells. Quantitative data show changes in MitoTracker intensity (F) and Golgi apparatus size (H) as reflected by GM130 coverage area after Neurog2 expression at 30 dpi. n = 150 cells from triplicate cultures. Scale bars are 20 μm for (A), (B), and (D) and 10 μm for (E) and (G). Data are presented as mean ± SEM and analyzed by Student's t-test. * indicates p<0.05, *** indicates p<0.001. [Figure 9H] Neurog2 overexpression induces the neuronal fate change of glioblastoma cells. E–H: Distribution and morphological changes of mitochondria (MitoTracker) and Golgi apparatus (GM130) during neuronal conversion of U251 cells. Quantitative data show changes in MitoTracker intensity (F) and Golgi apparatus size (H) as reflected by GM130 coverage area after Neurog2 expression at 30 dpi. n = 150 cells from triplicate cultures. Scale bars are 20 μm for (A), (B), and (D) and 10 μm for (E) and (G). Data are presented as mean ± SEM and analyzed by Student's t-test. * indicates p<0.05, *** indicates p<0.001. [Figure 10A] Neuronal conversion of human glioblastoma cells inhibits proliferation. (A) Representative images examining cell proliferation via BrdU immunostaining in U251 human glioblastoma cells expressing GFP, Neurog2-GFP, NeuroD1-GFP, or Ascl1-GFP. Cell cultures were incubated in 10 mM BrdU for 24 hours and then immunostained at 7 dpi. Scale bar is 50 μm. [Figure 10B] Neuronal conversion of human glioblastoma cells inhibits proliferation. B. Quantitative analysis of proliferating cells (BrdU+ cells / total infected cells) during neuronal conversion of U251 cells. Data were analyzed by one-way ANOVA followed by Dunnett's test. *** indicates p<0.001, and n is ≥200 cells from triplicate cultures. [Figure 10C] Neuronal conversion of human glioblastoma cells inhibits proliferation. C-D. GSK3β expression levels examined by Western blot in U251 GBM cells overexpressing GFP alone or Neurog2-GFP. Data were normalized to the GFP control (D). Samples were collected at 20 dpi. n = 3 batches. [Figure 10D] Neuronal conversion of human glioblastoma cells inhibits proliferation. C-D. GSK3β expression levels examined by Western blot in U251 GBM cells overexpressing GFP alone or Neurog2-GFP. Data were normalized to the GFP control (D). Samples were collected at 20 dpi. n = 3 batches. [Figure 10E] Neuronal conversion of human glioblastoma cells inhibits proliferation. E, GSK3β immunostaining in U251 GBM cells upon GFP or Neurog2-GFP overexpression (GFP) at 20 dpi. Note the significant increase in GSK3β signal in Neurog2-converted neurons. Scale bar is 50 μm. [Figure 10F]Neuronal conversion of human glioblastoma cells inhibits proliferation. F, Quantitative analysis of GSK3β immunostaining intensity during neuronal conversion of U251 cells. Samples were collected at 20 dpi. Data were analyzed by Student's t-test. *** indicates p<0.001, n is 6 batches. Data are expressed as mean ± SEM. [Figure 11] Examination of autophagy / lysosomes during neuronal conversion of human GBM cells. A. Representative images illustrating the distribution and morphological changes of autophagy / lysosomes (ATG5, stained in red) during neuronal conversion of U251 cells. B–C. Quantitative data of ATG5 area (B) and intensity (C) in infected U251 cells at 30 dpi. n is over 150 cells from triplicate cultures. Scale bar is 10 μm. Data are presented as mean ± SEM and analyzed by Student's t-test. * indicates p<0.05, *** indicates p<0.001. [Figure 12] Functional analysis of human glioblastoma cell-converted neurons. In A, robust synaptic puncta (SV2) were detected along dendrites (MAP2, blue-green) in Neurog2-converted neurons derived from U251 human glioblastoma cells. Scale bar: 20 μm. B-C: Representative traces showing Na+ and K+ currents recorded from Neurog2-converted neurons (B), with quantitative analysis shown in (C). In D-E, whole-cell patch-clamp recordings revealed action potentials fired from Neurog2-converted neurons (D). Pie charts show the percentage of cells firing single (dark gray, E), repetitive (light gray, E), or no action potentials (black, E). Samples were taken at 30 dpi. n = ≥ 20 from triplicate cultures. [Figure 13]Effect of GSK-3β inhibition on neuronal conversion of human GBM cells. A. Immunostaining of GSK-3β (stained magenta) in Neurog2-converted neurons (DCX, stained red) derived from U251 human GBM cells at 20 dpi. Scale bar: 50 μm. B-C. Quantitative analysis of GSK-3β intensity (B) and neuronal conversion efficiency (C) after GSK-3β inhibition with CHIR99021 (5 μM) or TWS119 (10 μM) for 20 days. Data are presented as mean ± SEM and analyzed by Student's t-test. n = ≥ 3 replicates. [Figure 14] Investigation of cancer makers in Neurog2-converted neuronal cells derived from human glioblastoma cells. A-B: Immunostaining of IL13Ra2 (stained in red, A) in Neurog2-converted neuronal cells derived from U251 human glioblastoma cells at 20 dpi. Quantification is shown in panel (B). Scale bar is 50 μm. C-D: Immunostaining of EGFR (stained in red, C) during neuronal conversion of U251 cells. Samples were collected at 20 dpi. Scale bar is 20 μm. Data are presented as mean ± SEM and analyzed by Student's t-test. n is ≥40 cells from triplicate cultures. [Figure 15]In vivo neuronal conversion of human glioblastoma cells in a xenograft mouse model. A, Representative image illustrating human U251 GBM cells (mixed with Neurog2-GFP retrovirus) transplanted into the brain of a Rag1- / - immunodeficient mouse one month after transplantation. Note that U251 cells expressed high levels of vimentin, and Neurog2-GFP-infected U251 cells were immunopositive for DCX, an immature neuronal marker. B, Quantitative analysis of conversion efficiency one month after transplantation. Data are presented as mean ± SEM and analyzed by Student's t-test. *** indicates p<0.001, n=3 animals. Note that the in vivo conversion efficiency was also very high (approximately 90%). C, High-magnification image showing that the majority of transplanted U251 cells (vimentin) infected with Neurog2-GFP (bottom) retrovirus were converted to neurons (DCX) one week after transplantation. D-E: Further characterization of Neurog2-converted neurons in vivo, as indicated by the neuronal marker Tuj1 and the hippocampal neuronal marker Prox1, 1 month after transplantation of U251 human glioblastoma cells (labeled vimentin and human nuclei). Scale bars are 200 μm in (A) and 20 μm in (C)-(E). [Figure 16] Inhibition of cell proliferation and reduced astrogliosis after in vivo neuronal conversion of glioblastoma cells. (A-B) Representative images (A) and quantitative analysis (B) of proliferating U251 GBM cells (Ki67+) 7 days after transplantation. Note the significant reduction in cell proliferation in the Neurog2 group. n = 4 animals. (C-D) Reduction of reactive astrocytes (labeled LCN2) in the Neurog2-infected area (D) compared with the contralateral GFP-infected area (C). Samples were taken 3 weeks after transplantation. (E) Quantitative analysis of LCN2 coverage area (infected with Neurog2-GFP or GFP retrovirus) 3 weeks after U251 cell transplantation. n = 5 animals. Scale bar = 50 μm. Data are presented as mean ± SEM and analyzed by Student's t-test. ** = p < 0.01, *** = p < 0.001. [Figure 17]Resident microglia and vascularity during in vivo neuronal conversion of glioblastoma cells. (A-B) Examination of resident microglia (Iba1, red) 3 weeks after U251 GBM cell transplantation with Neurog2 or GFP control virus infection. Quantitative analysis of the mean intensity of Iba1 in panel (B). n = 3 animals. Scale bar = 100 μm. (C-D) Representative images showing vascularity (Ly6c, stained red) 3 weeks after U251 GBM cell transplantation with Neurog2 or GFP control virus infection. Quantitative analysis of Ly6c coverage area in panel (C). Data are presented as mean ± SEM and analyzed by Student's t-test. n = 5 animals. Scale bar = 100 μm. [Figure 18] Transduction of the liver tumor cell line HepG2 with the hepatic transcription factors Foxa2, HNF4A, and GATA4. (A) Transduced cells grown on glass coverslips were fixed with paraformaldehyde and conjugated with a mixture of chicken anti-GFP and goat anti-Foxa2, chicken anti-GFP and goat anti-HNF4A, or chicken anti-GFP and goat anti-GATA4 antibodies. Detection was performed using chicken-specific Alexa Fluor 488 and goat-specific Alexa Fluor 594 secondary antibodies. Fluorescence was visualized using a Zeiss LSM800 confocal microscope. (B) Transduced cells grown in 12-well plates were harvested, and cell lysates were processed for SDS-PAGE and analyzed by Western blot using a mouse monoclonal anti-GFP antibody. In C, D, and E, cell lysates were fractionated by SDS-PAGE and processed for immunoblotting with goat polyclonal anti-Foxa2, goat polyclonal anti-HNF4A, or goat polyclonal anti-GATA4 antibodies, respectively. [Figure 19]Transduction of GATA4 increases endogenous Foxa2 expression levels. (A) Foxa2-, HNF4A-, GATA4-, or GFP-transduced HepG2 cells were harvested, and cell lysates were fractionated by SDS-PAGE and analyzed by immunoblotting using a mixture of goat polyclonal anti-Foxa2 and mouse monoclonal anti-HNF4A antibodies. A rabbit polyclonal GAPDH antibody was used as an internal loading control. (B) The same cell lysates were fractionated by SDS-PAGE and analyzed by immunoblotting using a mixture of goat polyclonal anti-GATA4 and rabbit polyclonal anti-GAPDH antibodies. [Figure 20] Figure 1 shows in vitro and in vivo cell growth of Foxa2-, GATA4-, HNF4A-, or GFP-transduced cell lines. (A) In vitro growth curves of Foxa2-, GATA4-, HNF4A-, or GFP-transduced cell lines. Equal amounts of Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells were seeded into 12-well plates. At different time points, cells were fixed and stained with crystal violet. The stained crystal violet was extracted with acetic acid, and the optical density of each extract was read using a microplate reader. The volume of the optical density represents the number of cells grown in each well. Each value represents three separate experiments. (B) Tumor growth curves of Foxa2-, GATA4-, HNF4A-, or GFP-transduced cell lines. Foxa2-, GATA4-, HNF4A-, or GFP-transduced cell lines were subcutaneously injected into the flanks of nude mice. Tumors were monitored every four days by measuring tumor size using calipers. Tumor volume was calculated by the following formula: V = width x length x length x 0.5. [Figure 21]Expression and secretion of albumin from Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells. (A) Transduced cells grown on glass coverslips were bound with a mixture of chicken anti-GFP and goat anti-albumin antibodies. Detection was performed using chicken-specific Alexa Fluor 488 and goat-specific Alexa Fluor 594 secondary antibodies. Fluorescence was visualized using a Zeiss LSM800 confocal microscope as described for Figure 18A. (B) Albumin expressed in Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells was detected by Western blot using a goat polyclonal anti-albumin antibody. A rabbit polyclonal anti-GAPDH antibody was used as an internal loading control. (C) Relative albumin production was calculated as the amount of albumin detected in Foxa2-, GATA4-, or HNF4A-transduced cells normalized to the amount of albumin obtained in GFP-transduced cells. Results were obtained from three independent experiments. D is the concentration of albumin secreted into the culture medium of Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells. Albumin produced by Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells was secreted into the culture medium. The concentration of albumin in the culture medium was measured by ELISA according to the ELISA kit instructions. Albumin concentrations were obtained by comparison with the standard curve provided with the kit. [Figure 22A] Expression of the liver cancer marker alpha-fetoprotein (AFP) in cells transduced with Foxa2, GATA4, HNF4A, or GFP. In A, Foxa2, GATA4, HNF4A, or GFP transduced cells were grown on coverslips, fixed, and stained with a mixture of chicken anti-GFP and rabbit anti-AFP. Detection was performed using a mixture of chicken-specific Alexa Fluor 488 and rabbit-specific Alexa Fluor 594 secondary antibodies. Fluorescence was visualized using a Zeiss LSM800 confocal microscope as described for Figure 18A. [Figure 22B]Expression of the liver cancer marker alpha-fetoprotein (AFP) in Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells. In B, Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells were lysed and fractionated by SDS-PAGE and probed with rabbit polyclonal anti-AFP for immunoblot analysis. Rabbit GAPDH polyclonal antibody was used as an internal loading control. Relative AFP levels were calculated as the amount of AFP detected in Foxa2-, GATA4-, or HNF4A-transduced cells normalized to the amount of AFP obtained in GFP-transduced cells. Results were derived from three independent experiments. [Figure 22C] Expression of the liver cancer marker alpha-fetoprotein (AFP) in cells transduced with Foxa2, GATA4, HNF4A, or GFP. C: AFP levels in tumors formed by GFP, HNF4A, or GATA4 transduced cells. Tumor sections were permeabilized with Triton X-100 and subsequently incubated with primary antibodies, chicken anti-GFP and rabbit anti-AFP. Detection was performed using a mixture of chicken-specific Alexa Fluor 488 and rabbit-specific Alexa Fluor 594 secondary antibodies. Fluorescence was visualized using a Zeiss LSM800 confocal microscope. [Figure 22D] Expression of the liver cancer marker alpha-fetoprotein (AFP) in Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells. In panel D, xenograft tumors of GATA4-, HNF4A-, or GFP-transduced cell lines were freshly harvested and lysed. Lysates were resolved by SDS-PAGE gel and probed with AFP, GATA4, and GFP antibodies. Actin was shown as an internal loading control. Relative AFP levels were calculated as the amount of AFP detected in GATA4- or HNF4A-transduced cells normalized to the amount of AFP obtained in GFP-transduced cells, as described for panel B. Results were derived from three independent experiments. [Figure 23A]Overexpression of GATA4, Foxa2, or HNF4A leads to increased membranous E-cadherin. In A, Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells were stained with a mixture of chicken anti-GFP and rabbit anti-E-cadherin. Detection was performed using a mixture of chicken-specific Alexa Fluor 488 and rabbit-specific Alexa Fluor 594 secondary antibodies. Fluorescence was visualized using a Zeiss LSM800 confocal microscope as described for Figure 18A. [Figure 23B] Overexpression of GATA4, Foxa2, or HNF4A results in increased membranous E-cadherin. In B, lysed Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells were fractionated by SDS-PAGE and probed with rabbit polyclonal anti-E-cadherin for Western blot analysis. Rabbit GAPDH polyclonal antibody was used as an internal loading control. Relative AFP levels were calculated as the amount of E-cadherin detected in Foxa2-, GATA4-, or HNF4A-transduced cells normalized to the amount of E-cadherin obtained in GFP-transduced cells. Results were derived from three independent experiments. [Figure 23C] Overexpression of GATA4, Foxa2, or HNF4A leads to increased membranous E-cadherin. C, Immunofluorescence images of E-cadherin showing increased E-cadherin in GATA4- or HNF4A-transduced cells formed tumors compared with GFP tumors. [Figure 23D] Overexpression of GATA4, Foxa2, or HNF4A leads to increased membranous E-cadherin. In Figure D, tumor samples from GATA4-, HNF4A-, or GFP-transduced cells were quantitatively analyzed by Western blot using an anti-E-cadherin antibody. E-cadherin was measurably more strongly expressed, with a measurable 2-fold higher intensity, as shown by GATA4, compared with GFP-expressing tumor cells. [Figure 24A]Expression and redistribution of beta-catenin in GATA4-overexpressing cell lines. In A, GATA4- or GFP-transfected cells were stained with rabbit anti-beta-catenin antibody, and immunofluorescence images of beta-catenin were visualized by microscopy. Beta-catenin in GATA4-overexpressing cells was distributed on the cell surface. [Figure 24B] A. Expression and redistribution of beta-catenin in GATA4-overexpressing cell lines. B. Western blot analysis of Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells using a beta-catenin antibody, and the relative beta-catenin levels detected in Foxa2-, GATA4-, HNF4A-, or GFP-transduced cells. Results are from three independent experiments. [Figure 24C] (C) Expression and redistribution of beta-catenin in GATA4-overexpressing cell lines. (D) Immunofluorescence images of beta-catenin showing increased beta-catenin in GATA4-transduced cells formed tumors compared to GFP tumors. [Figure 24D] Expression and redistribution of beta-catenin in GATA4-overexpressing cell lines. In D, tumor samples from GATA4-, HNF4A-, or GFP-transduced cells were analyzed by Western blot using an anti-beta-catenin antibody. The amount of beta-catenin expressed in tumors from GATA4-, HNF4A-, or GFP-transduced cells was calculated. [Figure 25] Vimentin expression in tumors of GATA4-, HNF4A-, and GFP-transduced cells. Western blot analysis of vimentin expressed in tumors of GATA4-, HNF4A-, or GFP-transduced cells using an anti-vimentin antibody revealed reduced vimentin levels in GATA4-transduced cells compared with GFP-transduced tumors. The reduction in vimentin was calculated by comparing the amount of vimentin detected in GATA4-transduced cells normalized to the amount of vimentin obtained in GFP-transduced cells. [Figure 26] 1 is the amino acid sequence of a representative NeuroD1 polypeptide (SEQ ID NO: 1). [Figure 27]1 is the amino acid sequence of a representative Neurog2 polypeptide (SEQ ID NO: 2). [Figure 28] 1 is the amino acid sequence of a representative Ascl1 polypeptide (SEQ ID NO: 3). [Figure 29] 1 is the amino acid sequence of a representative HNF4A polypeptide (SEQ ID NO: 4). [Figure 30] 1 is the amino acid sequence of a representative Foxa2 polypeptide (SEQ ID NO: 5). [Figure 31] 1 is the amino acid sequence of a representative GATA4 polypeptide (Sequence 6). DETAILED DESCRIPTION OF THE INVENTION

[0016] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0017] This document provides methods and materials for treating mammals with cancer. For example, nucleic acids encoding one or more transcription factors, or one or more transcription factors themselves, can be used to treat mammals with cancer. In some cases, treating a mammal with cancer as described herein can include converting cancer cells in the mammal into non-cancerous cells (e.g., functional or nearly normal cells) in the mammal. In some cases, treating a mammal with cancer as described herein can have a conversion efficiency of, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more. In some cases, treating a mammal with a cancer described herein may result in a 10 to 100 percent, e.g., 10 to 15 percent, 10 to 20 percent, 10 to 25 percent, 15 to 20 percent, 15 to 25 percent, 15 to 30 percent, 20 to 25 percent, 20 to 30 percent, 20 to 35 percent, 25 to 30 percent, 25 to 35 percent, 25 to 40 percent, 30 to 35 percent, 30 to 40 percent, 35 to 45 percent, 35 to 50 percent, 40 to 45 percent, 40 to 50 percent, 40 to 55 percent, 45 to 50 percent, 45 to 55 percent, 45 to 60 percent, 50 to 55 percent. , 50-60%, 50-65%, 55-60%, 55-65%, 55-70%, 60-65%, 60-70%, 60-75%, 65-70%, 65-75%, 65-80%, 70-75%, 70-80%, 70-85%, 75-80%, 75-85%, 75-90%, 80-85%, 80-90%, 80-95%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% conversion effectiveness.For example, treating a mammal having a cancer as described herein can be effective to convert, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more of the cancer cells in the mammal into non-cancerous cells (e.g., functional or substantially normal cells). In some cases, treating a mammal having a cancer described herein reduces or eliminates 10 to 100 percent, e.g., 10 to 15 percent, 10 to 20 percent, 10 to 25 percent, 15 to 20 percent, 15 to 25 percent, 15 to 30 percent, 20 to 25 percent, 20 to 30 percent, 20 to 35 percent, 25 to 30 percent, 25 to 35 percent, 25 to 40 percent, 30 to 35 percent, 30 to 40 percent, 35 to 45 percent, 35 to 50 percent, 40 to 45 percent, 40 to 50 percent, 40 to 55 percent, 45 to 50 percent, 45 to 55 percent, 45 to 60 percent, 50 to 55 percent, The compound may be 50-60%, 50-65%, 55-60%, 55-65%, 55-70%, 60-65%, 60-70%, 60-75%, 65-70%, 65-75%, 65-80%, 70-75%, 70-80%, 70-85%, 75-80%, 75-85%, 75-90%, 80-85%, 80-90%, 80-95%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100% effective in converting the cells to non-cancerous.

[0018] In some cases, nucleic acids designed to express one or more transcription factors (or one or more transcription factors themselves) can be administered to a mammal in need thereof (e.g., a mammal having cancer) to reduce the size of the cancer in the mammal (e.g., reduce the number of cancer cells in the mammal and / or the volume of one or more tumors in the mammal). For example, nucleic acids designed to express one or more neuronal transcription factors (or one or more neuronal transcription factors themselves) can be administered to a mammal (e.g., a human) having a brain tumor described herein to reduce the size of the brain tumor by, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more. In another example, nucleic acids designed to express one or more liver transcription factors (or one or more liver transcription factors themselves) can be administered to a mammal (e.g., a human) having a liver cancer described herein to reduce the size of the liver tumor by, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more.In some cases, a nucleic acid designed to express one or more liver transcription factors (or one or more liver transcription factors themselves) is administered to a mammal (e.g., a human) having a liver cancer described herein to reduce the size of the liver cancer by 10 to 100 percent, e.g., 10 to 15 percent, 10 to 20 percent, 10 to 25 percent, 15 to 20 percent, 15 to 25 percent, 15 to 30 percent, 20 to 25 percent, 20 to 30 percent, 20 to 35 percent, 25 to 30 percent, 25 to 35 percent, 25 to 40 percent, 30 to 35 percent, 30 to 40 percent, 35 to 45 percent, 35 to 50 percent, 40 to 45 percent, 40 to 50 percent, 40 to 55 percent, 45 to 50 ... It can be reduced by 5 to 55 percent, 45 to 60 percent, 50 to 55 percent, 50 to 60 percent, 50 to 65 percent, 55 to 60 percent, 55 to 65 percent, 55 to 70 percent, 60 to 65 percent, 60 to 70 percent, 60 to 75 percent, 65 to 70 percent, 65 to 75 percent, 65 to 80 percent, 70 to 75 percent, 70 to 80 percent, 70 to 85 percent, 75 to 80 percent, 75 to 85 percent, 75 to 90 percent, 80 to 85 percent, 80 to 90 percent, 80 to 95 percent, 85 to 90 percent, 85 to 95 percent, 85 to 100 percent, 90 to 95 percent, 90 to 100 percent, or 95 to 100 percent.

[0019] In some cases, a nucleic acid designed to express one or more transcription factors (or the one or more transcription factors themselves) can be administered to a mammal in need thereof (e.g., a mammal having cancer) to increase the survival rate of the mammal by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more (e.g., increasing the 5-year relative survival rate of the mammal). In some cases, nucleic acids designed to express one or more transcription factors (or one or more transcription factors themselves) are administered to a mammal in need thereof (e.g., a mammal having cancer) to increase the survival rate of the mammal by 1 to 10 years, e.g., 1 to 1.5 years, 1 to 2 years, 1 to 2.5 years, 1.5 to 2 years, 1.5 to 2.5 years, 1.5 to 3 years, 2 to 2.5 years, 2 to 3 years, 2 to 3.5 years, 2.5 to 3 years, 2.5 to 3.5 years, 2.5 to 4 years, 3 to 3.5 years, 3 to 4 years, 3 to 4.5 years, 3.5 to 4 years, 3.5 to 4.5 years, 3.5 to 5 years, 4 to 4.5 years, 4 to ... ~5 years, 4~5.5 years, 4.5~5 years, 4.5~5.5 years, 4.5~6 years, 5~5.5 years, 5~6 years, 5~6.5 years, 5.5~6 years, 5.5~6.5 years, 5.5~7 years, 6~6.5 years, 6~7 years, 6~7.5 years, 6.5~7 years, 6.5~7.5 years, 6.5~8 years, 7~7.5 years, 7~8 years, 7~8.5 years, 7.5~8 years, 7.5~8.5 years, 7.5~9 years, 8~8.5 years, 8~9 years, 8~9.5 years, 8.5~9 years, 8.5~9.5 years, 8.5~10 years, 9~9.5 years, 9~10 years, or 9.5~10 years, can be increased.

[0020] For example, nucleic acids designed to express one or more neuronal transcription factors (or one or more neuronal transcription factors themselves) can be administered to a mammal (e.g., a human) having a brain tumor described herein to increase the survival rate of the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more. In some cases, administering a nucleic acid designed to express one or more neuronal transcription factors (or one or more neuronal transcription factors themselves) to a mammal (e.g., a human) having a brain tumor described herein increases the survival rate of the mammal by 10 to 100 percent, e.g., 10 to 15 percent, 10 to 20 percent, 10 to 25 percent, 15 to 20 percent, 15 to 25 percent, 15 to 30 percent, 20 to 25 percent, 20 to 30 percent, 20 to 35 percent, 25 to 30 percent, 25 to 35 percent, 25 to 40 percent, 30 to 35 percent, 30 to 40 percent, 35 to 45 percent, 35 to 50 percent, 40 to 45 percent, 40 to 50 percent, 40 to 55 percent, or 45 to 50 percent. The increase can be 45-55 percent, 45-60 percent, 50-55 percent, 50-60 percent, 50-65 percent, 55-60 percent, 55-65 percent, 55-70 percent, 60-65 percent, 60-70 percent, 60-75 percent, 65-70 percent, 65-75 percent, 65-80 percent, 70-75 percent, 70-80 percent, 70-85 percent, 75-80 percent, 75-85 percent, 75-90 percent, 80-85 percent, 80-90 percent, 80-95 percent, 85-90 percent, 85-95 percent, 85-100 percent, 90-95 percent, 90-100 percent, or 95-100 percent. In another example, a nucleic acid designed to express one or more liver transcription factors (or one or more liver transcription factors themselves) can be administered to a mammal (e.g., a human) having a liver cancer described herein to increase the survival rate of the mammal (e.g., a human) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more.In some cases, a nucleic acid designed to express one or more liver transcription factors (or one or more liver transcription factors themselves) is administered to a mammal (e.g., a human) having a liver cancer described herein to increase the survival rate of the mammal by 10 to 100 percent, e.g., 10 to 15 percent, 10 to 20 percent, 10 to 25 percent, 15 to 20 percent, 15 to 25 percent, 15 to 30 percent, 20 to 25 percent, 20 to 30 percent, 20 to 35 percent, 25 to 30 percent, 25 to 35 percent, 25 to 40 percent, 30 to 35 percent, 30 to 40 percent, 35 to 45 percent, 35 to 50 percent, 40 to 45 percent, 40 to 50 percent, 40 to 55 percent, 45 to 50 percent, It can be increased by 45-55 percent, 45-60 percent, 50-55 percent, 50-60 percent, 50-65 percent, 55-60 percent, 55-65 percent, 55-70 percent, 60-65 percent, 60-70 percent, 60-75 percent, 65-70 percent, 65-75 percent, 65-80 percent, 70-75 percent, 70-80 percent, 70-85 percent, 75-80 percent, 75-85 percent, 75-90 percent, 80-85 percent, 80-90 percent, 80-95 percent, 85-90 percent, 85-95 percent, 85-100 percent, 90-95 percent, 90-100 percent, or 95-100 percent.

[0021] In some cases, nucleic acids designed to express one or more transcription factors (or the one or more transcription factors themselves) can be administered to a mammal in need thereof (e.g., a mammal having cancer) to differentiate cancer cells in the mammal (e.g., convert cancer cells into terminally differentiated and / or non-dividing cells in the mammal). For example, nucleic acids designed to express one or more neuronal transcription factors (or the one or more neuronal transcription factors themselves) can be administered to a mammal (e.g., a human) having a brain tumor (e.g., a glioma such as GBM) described herein to, for example, cause 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99 percent or more of the mammal's brain tumor cells (e.g., glioma cells) to differentiate into non-cancerous neurons in the brain of a living mammal (e.g., functional neurons that can integrate into the brain of a living mammal).In some cases, a nucleic acid designed to express one or more neuronal transcription factors (or one or more neuronal transcription factors themselves) can be administered to a mammal (e.g., a human) having a brain tumor (e.g., a glioma such as GBM) described herein to achieve a 10 to 100 percent, e.g., 10 to 15 percent, 10 to 20 percent, 10 to 25 percent, 15 to 20 percent, 15 to 25 percent, 15 to 30 percent, 20 to 25 percent, 20 to 30 percent, 20 to 35 percent, 25 to 30 percent, 25 to 35 percent, 25 to 40 percent, 30 to 35 percent, 30 to 40 percent, 35 to 45 percent, 35 to 50 percent, 40 to 45 percent, 40 to 50 percent, 40 to 55 percent, 45 to 50 percent, or 50 to 55 percent improvement. The percentage can be differentiated into 100 percent, 45-55 percent, 45-60 percent, 50-55 percent, 50-60 percent, 50-65 percent, 55-60 percent, 55-65 percent, 55-70 percent, 60-65 percent, 60-70 percent, 60-75 percent, 65-70 percent, 65-75 percent, 65-80 percent, 70-75 percent, 70-80 percent, 70-85 percent, 75-80 percent, 75-85 percent, 75-90 percent, 80-85 percent, 80-90 percent, 80-95 percent, 85-90 percent, 85-95 percent, 85-100 percent, 90-95 percent, 90-100 percent, or 95-100 percent. For example, a nucleic acid designed to express one or more liver transcription factors (or one or more liver transcription factors themselves) can be administered to a mammal (e.g., a human) having a liver cancer described herein to differentiate, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99 percent or more of the mammal's liver cancer cells into non-cancerous liver cells within the liver of a living mammal (e.g., functional liver cells that can be incorporated into the liver of a living mammal).In some cases, a nucleic acid designed to express one or more liver transcription factors (or one or more liver transcription factors themselves) is administered to a mammal (e.g., a human) having a liver cancer described herein to achieve a proliferation of 10 to 100 percent, e.g., 10 to 15 percent, 10 to 20 percent, 10 to 25 percent, 15 to 20 percent, 15 to 25 percent, 15 to 30 percent, 20 to 25 percent, 20 to 30 percent, 20 to 35 percent, 25 to 30 percent, 25 to 35 percent, 25 to 40 percent, 30 to 35 percent, 30 to 40 percent, 35 to 45 percent, 35 to 50 percent, 40 to 45 percent, 40 to 50 percent, 40 to 55 percent, 45 to 50 percent, 45 to 55 percent, 45 to 60 percent, 50 to 60 percent, 70 to 80 percent, 80 to 90 percent, 90 to 100 percent, 10 to 15 percent, 10 to 20 percent, 10 to 25 percent, 15 to 20 percent, 15 to 25 percent, 15 to 30 percent, 20 to 25 percent, 20 to 30 percent, 20 to 35 percent, 25 to 30 percent, 25 to 35 percent, 25 to 40 percent, 30 to 35 percent, 30 to 40 percent, 35 to 45 percent, 35 to 50 percent, 40 to 45 percent, 40 to 50 percent, 40 to 55 percent, 45 to 50 percent, 45 to 55 percent, 45 to 60 percent, 50 to 100 percent, 1 55 percent, 50-60 percent, 50-65 percent, 55-60 percent, 55-65 percent, 55-70 percent, 60-65 percent, 60-70 percent, 60-75 percent, 65-70 percent, 65-75 percent, 65-80 percent, 70-75 percent, 70-80 percent, 70-85 percent, 75-80 percent, 75-85 percent, 75-90 percent, 80-85 percent, 80-90 percent, 80-95 percent, 85-90 percent, 85-95 percent, 85-100 percent, 90-95 percent, 90-100 percent, or 95-100 percent can be differentiated into non-cancerous hepatocytes within the liver of a living mammal (e.g., functional hepatocytes that can be incorporated into the liver of a living mammal).

[0022] In some cases, nucleic acids designed to express one or more neuronal transcription factors (or the one or more neuronal transcription factors themselves) can be administered to a mammal in need thereof (e.g., a mammal having a brain tumor) to reduce astrogliosis in the mammal. For example, nucleic acids designed to express one or more neuronal transcription factors (or the one or more neuronal transcription factors themselves) can be administered to a mammal (e.g., a human) having a brain tumor described herein to reduce astrogliosis in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more. In some cases, a nucleic acid designed to express one or more neuronal transcription factors (or one or more neuronal transcription factors themselves) is administered to a mammal (e.g., a human) having a brain tumor described herein to reduce astrogliosis in the mammal by 10 to 100 percent, e.g., 10 to 15 percent, 10 to 20 percent, 10 to 25 percent, 15 to 20 percent, 15 to 25 percent, 15 to 30 percent, 20 to 25 percent, 20 to 30 percent, 20 to 35 percent, 25 to 30 percent, 25 to 35 percent, 25 to 40 percent, 30 to 35 percent, 30 to 40 percent, 35 to 45 percent, 35 to 50 percent, 40 to 45 percent, 40 to 50 percent, 40 to 55 percent, 45 to 50 percent, or The reduction can be 100 percent, 45-55 percent, 45-60 percent, 50-55 percent, 50-60 percent, 50-65 percent, 55-60 percent, 55-65 percent, 55-70 percent, 60-65 percent, 60-70 percent, 60-75 percent, 65-70 percent, 65-75 percent, 65-80 percent, 70-75 percent, 70-80 percent, 70-85 percent, 75-80 percent, 75-85 percent, 75-90 percent, 80-85 percent, 80-90 percent, 80-95 percent, 85-90 percent, 85-95 percent, 85-100 percent, 90-95 percent, 90-100 percent, or 95-100 percent.

[0023] Any suitable mammal can be treated as described herein. Examples of mammals that may have cancer and can be treated as described herein include, but are not limited to, humans, non-human primates (e.g., monkeys), dogs, cats, cows, horses, pigs, rats, mice, rabbits, ferrets, and sheep. In some cases, a human with cancer can be treated as described herein to reduce the number of cancer cells in the human by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99, or more. In some cases, a human having cancer is treated as described herein to reduce or eliminate the number of cancer cells in the human by 10-100 percent, e.g., 10-15 percent, 10-20 percent, 10-25 percent, 15-20 percent, 15-25 percent, 15-30 percent, 20-25 percent, 20-30 percent, 20-35 percent, 25-30 percent, 25-35 percent, 25-40 percent, 30-35 percent, 30-40 percent, 35-45 percent, 35-50 percent, 40-45 percent, 40-50 percent, 40-55 percent, 45-50 percent, 45-55 percent, 45-60 percent, 50-55 percent, 55-60 ... It can be reduced by 5 percent, 50-60 percent, 50-65 percent, 55-60 percent, 55-65 percent, 55-70 percent, 60-65 percent, 60-70 percent, 60-75 percent, 65-70 percent, 65-75 percent, 65-80 percent, 70-75 percent, 70-80 percent, 70-85 percent, 75-80 percent, 75-85 percent, 75-90 percent, 80-85 percent, 80-90 percent, 80-95 percent, 85-90 percent, 85-95 percent, 85-100 percent, 90-95 percent, 90-100 percent, or 95-100 percent.

[0024] When treating a mammal (e.g., a human) with cancer as described herein, the cancer may be any type of cancer. As used herein, a mammal refers to any organism classified in the class Mammalia. As used herein, a human refers to the species Homo sapiens. In some cases, the cancer may be a blood cancer. In some cases, the cancer may include one or more solid tumors. In some cases, the cancer may be a luminal cancer. In some cases, the cancer may be a carcinoma. In some cases, the cancer may be a non-epithelial cancer. In some cases, the cancer may be a myeloma cancer. In some cases, the cancer may be a leukemia cancer. In some cases, the cancer may be a lymphoma cancer. In some cases, the cancer may be a mixed cancer. In some cases, the cancer may be a primary cancer. In some cases, the cancer may be a secondary cancer. In some cases, the cancer may be a metastatic cancer. In some cases, the cancer may be a stage 0 cancer. In some cases, the cancer may be a stage I cancer. In some cases, the cancer may be a stage II cancer. In some cases, the cancer may be a stage IV cancer. Examples of cancers that can be treated as described herein include, but are not limited to, brain cancer (e.g., glioma such as GBM), liver cancer (e.g., HCC), breast cancer, prostate cancer, bone cancer, lung cancer, pancreatic cancer, uterine cancer, uterine cancer, gallbladder cancer, bladder cancer, esophageal cancer, skin cancer, kidney cancer, ovarian cancer, and leukemia.

[0025] In some cases, the methods described herein may include identifying a mammal (e.g., a human) as having cancer. Any suitable method can be used to identify a mammal as having cancer. For example, imaging techniques, biopsy techniques, cytology techniques, microscopy techniques, histochemical staining techniques, immunohistochemical staining techniques, flow cytometry techniques, image cytometry techniques, and / or genetic testing techniques can be used to identify a mammal (e.g., a human) as having cancer. In some cases, the imaging technique may be X-ray, computed tomography (CT scan), ultrasound, magnetic resonance imaging (MRI), positron emission tomography (PET scan), and sonogram. In some cases, the biopsy technique may be fine needle aspiration biopsy, core needle biopsy, vacuum-assisted biopsy, excision biopsy, shave biopsy, punch biopsy, endoscopic biopsy, laparoscopic biopsy, and bone marrow aspiration biopsy. In some cases, the cytology technique may be scrape or brush cytology technique. In some cases, the microscopy technique may be light microscopy, electron microscopy, laser microscopy, and / or optical microscopy. In some cases, the tissue staining technique may be hematoxylin and eosin (H&E), Alcian blue stain, aldehyde fuchsin stain, alkaline phosphatase stain, Bielchowsky stain, Congo red stain, crystal violet stain, Fontana-Masson stain, Giemsa stain, Luna stain, Nissl stain, Periodic acid-Schiff stain, Oil Red O stain, reticulin stain, Sudan Black B stain, toluidine blue stain, and / or van Gieson stain. In some cases, the genetic testing technique may be polymerase chain reaction (PCR), gene expression microarray technique, RNA sequencing, and / or DNA sequencing.

[0026] Once a cancer cell type is identified as having a particular type of cancer (e.g., brain cancer, liver cancer, kidney cancer, or lung cancer), one or more transcription factors appropriate for that cancer cell type can be selected for use as described herein. For example, in the case of a brain tumor such as GBM, transcription factors such as NeuroD1, Neurog2, and / or Ascl1 can be selected and used to convert the brain tumor cells into non-cancerous cells. For liver cancer cells, transcription factors such as HNF4A, Foxa2, and / or GATA4 can be selected and used to convert the liver cancer cells into non-cancerous cells. Other examples of transcription factors that can be selected for specific cancer cell types to convert those specific cancer cells into non-cancerous cells are listed in Table 1.

[0027] [Table 1]

[0028] As described herein, a mammal (e.g., a human) having a brain tumor (e.g., a glioma such as GBM) can be treated by administering nucleic acids designed to express one or more neuronal transcription factors in the mammal's brain (e.g., striatum) in a manner that induces brain tumor cells (e.g., glioma cells) to form non-cancerous neurons (e.g., functional neurons, substantially normal neurons, and / or integrated neurons) in the mammal's brain (e.g., striatum). Examples of neuronal transcription factors include, but are not limited to, NeuroD1 polypeptide, Neurog2 polypeptide, and Ascl1 polypeptide. Examples of NeuroD1 polypeptides include, but are not limited to, the polypeptide having the amino acid sequence set forth in GenBank® Accession No. NP_002491 (GI No. 121114306) or Q13562.3, or SEQ ID NO: 1 (Figure 26). NeuroD1 polypeptides can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_002500 (GI No. 323462174). Examples of Neurog2 polypeptides include, but are not limited to, the polypeptide having the amino acid sequence set forth in GenBank® Accession No. NP_076924.1, EAX06278.1, or AAH36847.1, or SEQ ID NO: 2 (FIG. 27). Neurog2 polypeptides can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_024019.4. Examples of Ascl1 polypeptides include, but are not limited to, the polypeptide having the amino acid sequence set forth in GenBank® Accession No. NP_004307.2 or SEQ ID NO: 3 (FIG. 28). Ascl1 polypeptides can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_004316.4.

[0029] As described herein, a mammal (e.g., a human) with liver cancer (e.g., HCC) can be treated by administering a nucleic acid designed to express one or more liver transcription factors in the liver of the mammal in a manner that induces liver cancer cells to form non-cancerous liver cells (e.g., functional liver cells, near-normal liver cells, and / or integrated liver cells) in the liver of the mammal. Examples of liver transcription factors include, but are not limited to, HNF4A polypeptide, Foxa2 polypeptide, and GATA4 polypeptide. Examples of HNF4A polypeptides include, but are not limited to, GenBank® Accession Nos. XP_005260464.1, NP_000448.3, NP_001274113.1, NP_001274112.1, NP_001274111.1, NP_001245284.1, NP_001025174.1, NP_787110.2, NP_001025175.1, NP_849181.1, or NP_849180.1, or a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4 (Figure 29). HNF4A polypeptides can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_178849.3. Examples of Foxa2 polypeptides include, but are not limited to, polypeptides having the amino acid sequence set forth in GenBank® Accession No. AAH11780.1 or ACA06111.1, or SEQ ID NO:5 (Figure 30). Foxa2 polypeptides can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_021784.5. Examples of GATA4 polypeptides include, but are not limited to, polypeptides having the amino acid sequence set forth in GenBank® Accession No. AAI43480.1, NP_001295022.1, NP_002043.2, NP_001295023.1, or NP_001361203.1, or SEQ ID NO:6 (Figure 31). GATA4 polypeptides can be encoded by the nucleic acid sequence set forth in GenBank® Accession No. NM_001308093.3.

[0030] Any suitable method can be used to deliver nucleic acids designed to express one or more transcription factors to cells (e.g., cells in a living mammal). For example, nucleic acids encoding transcription factors can be administered to a mammal using one or more vectors, such as viral vectors. In some cases where two or more nucleic acids designed to express transcription factors are delivered to cells in a living mammal, the nucleic acids can be delivered to the cells using separate vectors (e.g., one vector for the nucleic acid encoding the first transcription factor and one vector for the nucleic acid encoding the second transcription factor). In some cases where two or more nucleic acids designed to express transcription factors are delivered to cells in a living mammal, the nucleic acids can be delivered to the cells using a single vector containing both the nucleic acid encoding the first transcription factor and the nucleic acid encoding the second transcription factor.

[0031] A vector for administering a nucleic acid (e.g., a nucleic acid designed to express one or more transcription factors) to a cell (e.g., a cell within a living mammal) can be used to administer the nucleic acid to any suitable cell. In some cases, a vector can be used to administer a nucleic acid encoding a transcription factor to a dividing cell. In some cases, a vector can be used to administer a nucleic acid encoding a transcription factor to a non-dividing cell. In some cases, a vector can be used to administer a nucleic acid encoding a transcription factor to a cancer cell.

[0032] In some cases, vectors for administering nucleic acids (e.g., nucleic acids designed to express one or more transcription factors) to cells (e.g., cells within a living mammal) can be used for transient expression of the transcription factor(s).

[0033] In some cases, a vector for administering a nucleic acid (e.g., a nucleic acid designed to express one or more transcription factors) to a cell (e.g., a cell in a living mammal) can be used for stable expression of the transcription factor(s). When a vector for administering a nucleic acid can be used for stable expression of one or more transcription factors, the vector can be engineered to integrate the nucleic acid designed to express one or more transcription factors into the genome of the cell. In some cases, when a vector is engineered to integrate a nucleic acid into the genome of a cell, any suitable method can be used to integrate the nucleic acid into the genome of the cell. For example, gene therapy techniques can be used to integrate the nucleic acid designed to express one or more transcription factors into the genome of the cell.

[0034] Vectors for administering nucleic acids (e.g., nucleic acids encoding one or more transcription factors) to cells (e.g., cells within a living mammal) can be prepared using standard materials (e.g., packaging cell lines, helper viruses, and vector constructs). See, for example, Gene Therapy Protocols (Methods in Molecular Medicine), edited by Jeffrey R. Morgan, Humana Press, Totowa, NJ (2002), and Viral Vectors for Gene Therapy: Methods and Protocols, edited by Curtis A. Machida, Humana Press, Totowa, NJ (2003). Vectors designed for administering nucleic acids encoding one or more transcription factors to cells (e.g., cells within a living mammal) can be any suitable vector, including, but not limited to, viral vectors such as adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, vaccinia virus, herpes virus, papilloma virus, oncolytic virus, and non-viral vectors such as nanoparticles that mimic viral vectors. In some cases, nucleic acids encoding one or more transcription factors can be delivered to cells using non-viral vectors, such as adeno-associated viral vectors (e.g., recombinant AAV serotype viral vectors, such as AAV serotype 2 viral vectors, AAV serotype 5 viral vectors, AAV serotype 9 viral vectors, or AAV serotype 2 / 5 viral vectors), lentiviral vectors, retroviral vectors, adenoviral vectors, herpes simplex viral vectors, poxvirus vectors, oncolytic vectors, or nanoparticles that mimic viral vectors. For example, nucleic acids encoding one or more neuronal transcription factors (e.g., nucleic acids encoding NeuroD1 polypeptides, Neurog2 polypeptides, and / or Ascl1 polypeptides) can be delivered to glial cells (e.g., cancerous glial cells) using one or more retroviral vectors.For example, nucleic acids encoding one or more hepatic transcription factors (e.g., nucleic acids encoding HNF4A polypeptides, nucleic acids encoding Foxa2 polypeptides, and / or nucleic acids encoding GATA4 polypeptides) can be delivered to hepatocytes using one or more lentiviral vectors.

[0035] In addition to a nucleic acid encoding one or more transcription factors, a viral vector may contain regulatory elements operably linked to the nucleic acid encoding the transcription factor. Such regulatory elements may include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, or inducible elements that regulate the expression (e.g., transcription or translation) of the nucleic acid. The selection of element(s) to be included in the viral vector depends on several factors, including, but not limited to, inducibility, targeting, and the desired expression level. For example, a promoter can be included in the viral vector to promote transcription of the nucleic acid encoding the transcription factor. The promoter can be constitutive or inducible (e.g., in the presence of tetracycline) and can affect the expression of the nucleic acid encoding the polypeptide in a general or tissue-specific manner. Examples of tissue-specific promoters that can be used to drive the expression of neural transcription factors in glial cells (e.g., cancerous glial cells) include, but are not limited to, the GFAP, NG2, Olig2, CAG, EF1a, Aldh1L1, CMV, and ubiquitin promoters. Examples of tissue-specific promoters that can be used to drive expression of liver transcription factors in hepatocytes include, but are not limited to, the alpha 1-antitrypsin, albumin, AFP, CAG, CMV, EF1a, and ubiquitin promoters.

[0036] As used herein, "operably linked" refers to the positioning of regulatory elements within a vector relative to a nucleic acid in a manner that allows or promotes the expression of the encoded polypeptide. For example, a viral vector can contain a neuroglial-specific promoter operably linked to a nucleic acid encoding a neural transcription factor, thereby driving transcription in glial cells (e.g., cancerous glial cells). For example, a viral vector can contain a liver-specific promoter operably linked to a nucleic acid encoding a liver transcription factor, thereby driving transcription in hepatocytes (e.g., cancerous hepatocytes).

[0037] The nucleic acid encoding one or more transcription factors can be administered to mammals using non-viral vectors.The method of using non-viral vectors for nucleic acid delivery has been described elsewhere.For example, see Gene Therapy Protocols (Methods in Molecular Medicine), edited by Jeffrey R. Morgan, Humana Press, Totowa, NJ (2002).For example, the nucleic acid encoding one or more transcription factors can be administered to mammals by directly injecting a nucleic acid molecule (for example, a plasmid) that contains the nucleic acid encoding one or more transcription factors, or by administering the nucleic acid molecule that is complexed with lipid, polymer, or nanosphere.In some cases, genome editing techniques such as CRISPR / Cas9-mediated gene editing can be used to activate endogenous transcription factor expression.

[0038] Nucleic acids encoding transcription factors can be produced by techniques including, but not limited to, conventional molecular cloning, polymerase chain reaction (PCR), chemical nucleic acid synthesis techniques, and combinations of such techniques. For example, PCR or RT-PCR can be used with oligonucleotide primers designed to amplify nucleic acids (e.g., genomic DNA or RNA) encoding the transcription factor.

[0039] In some cases, one or more transcription factors can be administered in addition to or instead of a nucleic acid designed to express one or more transcription factors. For example, NeuroD1 polypeptide, Neurog2 polypeptide, and / or Ascl1 polypeptide can be administered to a mammal to induce the conversion (e.g., differentiation) of brain tumor cells (e.g., glioma cells) in the brain into non-cancerous neurons in the brain of a living mammal (e.g., functional neurons that can be integrated into the brain of a living mammal). In another example, HNF4A polypeptide, Foxa2 polypeptide, and / or GATA4 polypeptide can be administered to a mammal to induce the conversion (e.g., differentiation) of liver cancer cells in the liver into non-cancerous hepatocytes in the liver of a living mammal (e.g., functional hepatocytes that can be integrated into the liver of a living mammal).

[0040] As described herein, nucleic acids designed to express one or more transcription factors (or one or more transcription factors themselves) can be administered to a mammal (e.g., a human) with cancer to treat the mammal. In some cases, a nucleic acid designed to express a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, a nucleic acid designed to express a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, and a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3 (or a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, and / or a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3) can be administered to a mammal (e.g., a human) with a brain tumor (e.g., a glioma such as GBM) as described herein to treat the mammal. For example, a single retroviral vector can be designed to express a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, and a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3, and the designed viral vector can be administered to a human with a brain tumor to treat the mammal.

[0041] In some cases, a polypeptide having an amino acid sequence that is at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) identical to the amino acid sequence set forth in SEQ ID NO: 1 can be used. For example, a polypeptide comprising the entire amino acid sequence set forth in SEQ ID NO: 1 can be used, except that the amino acid sequence contains 1 to 10 (e.g., 10, 1 to 9, 2 to 9, 1 to 8, 2 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1) amino acid additions, deletions, substitutions, or a combination thereof. In some cases, a nucleic acid designed to express a polypeptide comprising an amino acid sequence that is 90% to 99% identical to the amino acid sequence set forth in SEQ ID NO: 1 can be designed and administered to a mammal (e.g., a human) having a brain tumor (e.g., a glioma such as GBM) to treat the mammal.

[0042] In some cases, a polypeptide having an amino acid sequence that is at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) identical to the amino acid sequence set forth in SEQ ID NO: 2 can be used. For example, a polypeptide comprising the entire amino acid sequence set forth in SEQ ID NO: 2 can be used, except that the amino acid sequence contains 1 to 10 (e.g., 10, 1 to 9, 2 to 9, 1 to 8, 2 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1) amino acid additions, deletions, substitutions, or a combination thereof. In some cases, a nucleic acid designed to express a polypeptide comprising an amino acid sequence that is 90% to 99% identical to the amino acid sequence set forth in SEQ ID NO: 2 can be designed and administered to a mammal (e.g., a human) having a brain tumor (e.g., a glioma such as GBM) to treat the mammal.

[0043] In some cases, a polypeptide having an amino acid sequence that is at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) identical to the amino acid sequence set forth in SEQ ID NO: 3 can be used. For example, a polypeptide comprising the entire amino acid sequence set forth in SEQ ID NO: 3 can be used, except that the amino acid sequence contains 1 to 10 (e.g., 10, 1 to 9, 2 to 9, 1 to 8, 2 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1) amino acid additions, deletions, substitutions, or a combination thereof. In some cases, a nucleic acid designed to express a polypeptide comprising an amino acid sequence that is 90% to 99% identical to the amino acid sequence set forth in SEQ ID NO: 3 can be designed and administered to a mammal (e.g., a human) having a brain tumor (e.g., a glioma such as GBM) to treat the mammal.

[0044] In another example, a nucleic acid designed to express a polypeptide having an amino acid sequence that is at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) identical to the amino acid sequence set forth in SEQ ID NO:1, a nucleic acid designed to express a polypeptide having an amino acid sequence that is at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) identical to the amino acid sequence set forth in SEQ ID NO:2, and a nucleic acid designed to express a polypeptide having an amino acid sequence that is at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) identical to the amino acid sequence set forth in SEQ ID NO:3 can be designed and administered to a mammal (e.g., a human) having a mammalian brain tumor (e.g., GBM) to treat the mammal.

[0045] In some cases, a nucleic acid designed to express a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, a nucleic acid designed to express a polypeptide having the amino acid sequence set forth in SEQ ID NO:5, and a nucleic acid designed to express a polypeptide having the amino acid sequence set forth in SEQ ID NO:6 (or a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, a polypeptide having the amino acid sequence set forth in SEQ ID NO:5, and / or a polypeptide having the amino acid sequence set forth in SEQ ID NO:6) can be administered to a mammal (e.g., a human) with liver cancer (e.g., HCC) as described herein to treat the mammal. For example, a single lentiviral vector can be designed to express a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, a polypeptide having the amino acid sequence set forth in SEQ ID NO:5, and a polypeptide having the amino acid sequence set forth in SEQ ID NO:6, and the designed viral vector can be administered to a human with liver cancer to treat the mammal.

[0046] In some cases, a polypeptide having an amino acid sequence that is at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) identical to the amino acid sequence set forth in SEQ ID NO: 4 can be used. For example, a polypeptide comprising the entire amino acid sequence set forth in SEQ ID NO: 4 can be used, except that the amino acid sequence contains 1 to 10 (e.g., 10, 1 to 9, 2 to 9, 1 to 8, 2 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1) amino acid additions, deletions, substitutions, or a combination thereof. In some cases, a nucleic acid designed to express a polypeptide comprising an amino acid sequence that is 90% to 99% identical to the amino acid sequence set forth in SEQ ID NO: 4 can be designed and administered to a mammal (e.g., a human) with liver cancer (e.g., HCC) to treat the mammal.

[0047] In some cases, a polypeptide having an amino acid sequence that has at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 can be used. For example, a polypeptide containing the entire amino acid sequence set forth in SEQ ID NO: 5 can be used, except that the amino acid sequence contains 1 to 10 (e.g., 10, 1 to 9, 2 to 9, 1 to 8, 2 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1) amino acid additions, deletions, substitutions, or a combination thereof. In some cases, a nucleic acid designed to express a polypeptide containing an amino acid sequence that has 90% to 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 can be designed and administered to a mammal (e.g., a human) with liver cancer (e.g., HCC) to treat the mammal.

[0048] In some cases, a polypeptide having an amino acid sequence that is at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) identical to the amino acid sequence set forth in SEQ ID NO: 6 can be used. For example, a polypeptide containing the entire amino acid sequence set forth in SEQ ID NO: 6 can be used, except that the amino acid sequence contains 1 to 10 (e.g., 10, 1 to 9, 2 to 9, 1 to 8, 2 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1) amino acid additions, deletions, substitutions, or a combination thereof. In some cases, a nucleic acid designed to express a polypeptide containing an amino acid sequence that is 90% to 99% identical to the amino acid sequence set forth in SEQ ID NO: 6 can be designed and administered to a mammal (e.g., a human) with liver cancer (e.g., HCC) to treat the mammal.

[0049] In another example, a nucleic acid designed to express a polypeptide having an amino acid sequence that is at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) identical to the amino acid sequence set forth in SEQ ID NO:4, a nucleic acid designed to express a polypeptide having an amino acid sequence that is at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) identical to the amino acid sequence set forth in SEQ ID NO:5, and a nucleic acid designed to express a polypeptide having an amino acid sequence that is at least 85% (e.g., 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99.0%) identical to the amino acid sequence set forth in SEQ ID NO:6 can be designed and administered to a mammal (e.g., a human) having mammalian liver cancer (e.g., HCC) to treat the mammal.

[0050] The percent sequence identity between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number (e.g., SEQ ID NO:1 or SEQ ID NO:2) can be determined as follows: First, the nucleic acid or amino acid sequence is compared to the sequence set forth in the particular sequence identification number using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ, including BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ is available online at the world wide web at "fr" dot "com / blast" or at the world wide web at "ncbi.nlm.nih" dot "gov." Instructions for using the Bl2seq program can be found in the readme file provided with BLASTZ. Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to the file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt), -j is set to the file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt), -p is set to blastn, -o is set to any desired file name (e.g., C:\output.txt), -q is set to -1, -r is set to 2, and all other options are left at their default settings.For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\Bl2seq -i c:\seq1.txt-j c:\seq2.txt-p blastn-o c:\output.txt-q-1-r 2. To compare two amino acid sequences, set the Bl2seq options as follows: -i to the file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt), -j to the file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt), -p to blastp, -o to any desired filename (e.g., C:\output.txt), and leave all other options at their default settings. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq-i c:\seq1.txt-j c:\seq2.txt-p blastp-o c:\output.txt. If the two compared sequences share homology, the specified output file will present those homologous regions as aligned sequences. If the two compared sequences do not share homology, the specified output file will not present aligned sequences.

[0051] When aligned, the number of matches is determined by counting the number of positions where the same nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches by the length of the sequence shown in the identified sequence (e.g., SEQ ID NO: 1), and then multiplying the resulting value by 100. For example, an amino acid sequence that has 340 matches when aligned with the sequence shown in SEQ ID NO: 1 is 95.5 percent identical to the sequence shown in SEQ ID NO: 1 (i.e., 340÷356×100=95.5056). Please note that the percent sequence identity value is rounded to two decimal places. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, and 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. Also, please note that the length value is always an integer.

[0052] When brain tumor cells (e.g., glioma cells) are converted into non-cancerous neurons in the brain of a living mammal (e.g., a human) having a brain tumor as described herein (e.g., by administering nucleic acids encoding one or more neuronal transcription factors, such as NeuroD1, Neurog2, and / or Ascl1, or the one or more neuronal transcription factors themselves), the converted neurons can be any suitable type of neuron. In some cases, the converted neurons can be DARPP32-positive. In some cases, the converted neurons can be FoxG1-positive forebrain neurons. In some cases, the converted neurons can be functional neurons (e.g., have functional synaptic networks). For example, the functional neurons can be glutamatergic neurons or GABAergic neurons. In some cases, the converted neurons can have active electrophysiological properties. In some cases, the converted neurons can be integrated into the brain of the living mammal (e.g., include axonal projections extending from the striatum). In some cases, converted neural cells may exhibit downregulated signaling pathways relevant to cancer progression (e.g., compared to pre-converted brain tumor cells).

[0053] When liver cancer cells are converted into non-cancerous hepatocytes in the liver of a living mammal (e.g., a human) having liver cancer as described herein (e.g., by administering nucleic acids encoding one or more liver transcription factors (e.g., nucleic acids encoding HNF4A, Foxa2, and / or GATA4, or one or more liver transcription factors themselves), the converted hepatocytes can be any suitable type of hepatocyte. In some cases, the converted hepatocytes can be functional hepatocytes (e.g., capable of producing one or more liver enzymes, such as cholesterol, bile acids, and / or albumin). In some cases, the converted hepatocytes can be integrated into the liver of the living mammal (e.g., capable of forming tight and / or adherent junctions with hepatocytes in the liver of the living mammal). In some cases, the converted hepatocytes can have reduced proliferation (e.g., compared to the liver cancer cells before conversion). In some cases, the reduction in proliferation can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more.In some cases, the reduction in proliferation is between 10 and 100 percent, e.g., 10 to 15 percent, 10 to 20 percent, 10 to 25 percent, 15 to 20 percent, 15 to 25 percent, 15 to 30 percent, 20 to 25 percent, 20 to 30 percent, 20 to 35 percent, 25 to 30 percent, 25 to 35 percent, 25 to 40 percent, 30 to 35 percent, 30 to 40 percent, 35 to 45 percent, 35 to 50 percent, 40 to 45 percent, 40 to 50 percent, 40 to 55 percent, 45 to 50 percent, 45 to 55 percent, 45 to 60 percent, 50 to 55 percent, 50 to 60 percent, The expression level of the transformed hepatocytes may be decreased, 50-65%, 55-60%, 55-65%, 55-70%, 60-65%, 60-70%, 60-75%, 65-70%, 65-75%, 65-80%, 70-75%, 70-80%, 70-85%, 75-80%, 75-85%, 75-90%, 80-85%, 80-90%, 80-95%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%. In some cases, the transformed hepatocytes may have decreased expression of one or more liver cancer markers (e.g., compared to the liver cancer cells before transformation). In some cases, the decrease in expression of one or more liver cancer markers may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more.In some cases, the decrease in expression of one or more liver cancer cell markers is between 10 and 100 percent, e.g., between 10 and 15 percent, 10 and 20 percent, 10 and 25 percent, 15 and 20 percent, 15 and 25 percent, 15 and 30 percent, 20 and 25 percent, 20 and 30 percent, 20 and 35 percent, 25 and 30 percent, 25 and 35 percent, 25 and 40 percent, 30 and 35 percent, 30 and 40 percent, 35 and 45 percent, 35 and 50 percent, 40 and 45 percent, 40 and 50 percent, 40 and 55 percent, 45 and 50 percent, 45 and 55 percent, 45 and 60 percent, 50 and 55 percent , 50-60%, 50-65%, 55-60%, 55-65%, 55-70%, 60-65%, 60-70%, 60-75%, 65-70%, 65-75%, 65-80%, 70-75%, 70-80%, 70-85%, 75-80%, 75-85%, 75-90%, 80-85%, 80-90%, 80-95%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%. Examples of liver cancer markers include, but are not limited to, AFP. In some cases, the converted hepatocytes may have increased expression of one or more epithelial-specific markers (e.g., compared to the liver cancer cells prior to conversion). In some cases, the increase in expression of one or more epithelial-specific markers may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more.In some cases, the increase in expression of one or more epithelial-specific markers is between 10 and 100 percent, e.g., between 10 and 15 percent, 10 and 20 percent, 10 and 25 percent, 15 and 20 percent, 15 and 25 percent, 15 and 30 percent, 20 and 25 percent, 20 and 30 percent, 20 and 35 percent, 25 and 30 percent, 25 and 35 percent, 25 and 40 percent, 30 and 35 percent, 30 and 40 percent, 35 and 45 percent, 35 and 50 percent, 40 and 45 percent, 40 and 50 percent, 40 and 55 percent, 45 and 50 percent, 45 and 55 percent, 45 and 60 percent, 50 and 55 percent , 50-60%, 50-65%, 55-60%, 55-65%, 55-70%, 60-65%, 60-70%, 60-75%, 65-70%, 65-75%, 65-80%, 70-75%, 70-80%, 70-85%, 75-80%, 75-85%, 75-90%, 80-85%, 80-90%, 80-95%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%. Examples of epithelial-specific markers include, but are not limited to, E-cadherin, claudins, and beta-catenin.

[0054] Nucleic acids designed to express one or more transcription factors (or one or more transcription factors themselves) can be administered to a mammal (e.g., a human) with cancer by any suitable route. In some cases, administration can be local administration. In some cases, administration can be systemic administration. Examples of routes of administration include, but are not limited to, intravenous, intramuscular, intrathecal, intracerebral, intraparenchymal, subcutaneous, oral, intranasal, inhalation, transdermal, parenteral, intratumoral, retroureteral, subcapsular, vaginal, and rectal administration. When multiple treatment rounds are administered, a first treatment round can involve administering nucleic acids designed to express one or more transcription factors described herein (or one or more transcription factors themselves) to the mammal (e.g., a human) by a first route (e.g., intravenous), and a second treatment round can involve administering nucleic acids designed to express one or more transcription factors described herein (or one or more transcription factors themselves) to the mammal (e.g., a human) by a second route (e.g., intratumoral).

[0055] In some cases, nucleic acids designed to express one or more transcription factors described herein (or the one or more transcription factors themselves) can be formulated into compositions (e.g., pharmaceutical compositions) for administration to a mammal (e.g., a mammal having or at risk of having cancer). For example, nucleic acids designed to express one or more transcription factors (or the one or more transcription factors themselves) can be formulated into a pharmaceutically acceptable composition for administration to a mammal having cancer. In some cases, nucleic acids designed to express one or more transcription factors (or the one or more transcription factors themselves) can be formulated with one or more pharmaceutically acceptable carriers (excipients) and / or diluents. Pharmaceutical compositions can be formulated for administration in solid or liquid form, including, but not limited to, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, wafers, and granules. Pharmaceutically acceptable carriers, fillers, and vehicles that may be used in the pharmaceutical compositions described herein include, but are not limited to, saline (e.g., phosphate buffered saline), ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acids, water, salts, or partial glyceride mixtures of electrolytes, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0056] In some cases, the methods described herein may include administering to a mammal (e.g., a mammal having cancer) one or more additional agents used to treat cancer. The one or more additional agents used to treat cancer may include any appropriate cancer therapy. In some cases, the cancer therapy may include surgery and / or radiation therapy. In some cases, the cancer therapy may include administration of drug therapy, such as chemotherapy, hormonal therapy, targeted therapy, and / or cytotoxic therapy. For example, a mammal having cancer may be administered a nucleic acid designed to express one or more transcription factors described herein (or one or more transcription factors themselves) and one or more additional agents used to treat cancer. When a mammal having cancer is treated with a nucleic acid designed to express one or more transcription factors described herein (or one or more transcription factors themselves) and one or more additional agents used to treat cancer, the additional agents used to treat cancer can be administered simultaneously or independently. For example, a nucleic acid designed to express one or more transcription factors described herein (or one or more transcription factors themselves) and one or more additional agents used to treat cancer can be formulated together to form a single composition. In some cases, a nucleic acid designed to express one or more transcription factors described herein (or the one or more transcription factors themselves) can be administered first, and one or more additional agents used to treat cancer can be administered second, or vice versa.

[0057] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0058] Example 1: Conversion of human glioblastoma cells into neural cells GBM is the most common and aggressive adult primary cancer of the central nervous system (CNS). The current standard of care for GBM is surgery followed by radiation therapy or chemotherapy, but due to the heterogeneity and highly invasive nature of GBM, therapeutic advances have been limited.

[0059] This example provides an alternative approach to treating GBM through transcription factor reprogramming (e.g., Neurog2, NeuroD1, and / or Ascl1 reprogramming) of malignant GBM cells into non-proliferating neuronal cells.

[0060] cell culture Human GBM cell lines were purchased from Sigma (U251) or ATCC (U118). U251 cells were cultured in GBM medium containing MEM (GIBCO), 0.2% penicillin / streptomycin (GIBCO), 10% FBS (GIBCO), 1 mM sodium pyruvate (GIBCO), 1% non-essential amino acids (NEAA, GIBCO), and 1x GlutMAX (GIBCO). U118 cells were cultured in medium containing DMEM (GIBCO), 10% FBS, and 1% penicillin / streptomycin.

[0061] Human astrocytes were purchased from ScienCell (HA1800, San Diego, USA) and cultured in human astrocyte medium containing DMEM / F12 (GIBCO), 10% FBS, 3.5 mM glucose (Sigma), and 0.2% penicillin / streptomycin, supplemented with B27 (GIBCO), N2 (GIBCO), 10 ng / mL fibroblast growth factor 2 (FGF2, Invitrogen), and 10 ng / mL epidermal growth factor (EFG, Invitrogen).

[0062] For subculture, cells were trypsinized with 0.25% trypsin (GIBCO) or TrypLE Select (Invitrogen), centrifuged at 800 rpm for 5 min, resuspended, and plated in the corresponding medium at a split ratio of approximately 1:4. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2.

[0063] Reprogramming human GBM cells into neurons U251 cells were seeded onto poly-D-lysine-coated coverslips in 24-well plates at a density of 10,000 cells per coverslip at least 12 h before viral infection. GFP, Neurog2, NeuroD1, or Ascl1 retroviruses were added to GBM cells along with 8 μg / mL polybrene (Santa Cruz Biotechnology). The medium was completely replaced with neuronal differentiation medium (NDM) the following day to support neuronal differentiation and maturation. NDM contained DMEM / F12 (GIBCO), 0.4% B27 supplement (GIBCO), 0.8% N2 supplement (GIBCO), 0.2% penicillin / streptomycin, 0.5% FBS, vitamin C (5 μg / mL, Selleck Chemicals), Y27632 (1 μM, Tocris), GDNF (10 ng / mL, Invitrogen), BDNF (10 ng / mL, Invitrogen), and NT3 (10 ng / mL, Invitrogen). Cells were maintained at 37°C in a humidified atmosphere with 5% CO2.

[0064] Treatment of human glioblastoma cells with small molecules U251 cells were infected with retroviruses expressing Neurog2-GFP or GFP alone. The following day, the culture medium was completely replaced with neuronal differentiation medium (NDM) containing small molecules or 0.22% DMSO for control. Infected glioblastoma cells were treated with 5 μM DAPT, 1.5 μM CHIR99021, 5 μM SB431542, 0.25 μM LDN193189, 1 μM SAG, and 1 μM purmorphamine. The small molecule-containing medium was refreshed every 3–4 days. Cells were first treated with small molecules for 12 days, then switched to NDM for the desired period before immunostaining.

[0065] In vivo neuronal conversion of human glioblastoma cells In vivo neuronal conversion of human glioblastoma cells was performed using Rag1 KO immunodeficient mice (B6.129S7-Rag1tm1Mom / J, The Jackson Laboratory, stock number 002216). 5 ) U251 human glioblastoma cells were transplanted into the striatum of Rag1 KO mouse brains using a stereotaxic instrument (Hamilton). Retroviruses expressing Neurog2-GFP or GFP alone at similar titers were injected intracranially at the same location. Mouse brains were harvested and sliced ​​1, 2, 4, and 8 weeks after injection. Immunostaining of brain slice sections was identical to that of cultured cells.

[0066] Data and statistical analysis Cell counts and fluorescence intensity were performed in a single-blind manner using randomly selected fields of randomly selected photographs and analyzed using Image J software. Data are presented as mean ± SEM. Multiple group comparisons were performed using a two-way analysis of variance followed by Dunnett's test. Two-group comparisons were performed using Student's t-test.

[0067] Efficient neuronal conversion of human GBM cells by a single neuronal transcription factor, Neurog2, NeuroD1, or Ascl1 In this study, two different human GBM cell lines (U251, Sigma; U118, ATCC) were used (Figure 1). To determine whether neuronal transcription factors could convert human malignant glioblastoma cells into neurons, the transcription factors NeuroD1, Neurog2, and Ascl1 were tested. Considering that AAV does not infect cultured glial cells or glioblastoma cells with high efficiency, retroviruses were used to overexpress Neurog2 (CAG::Neurog2-P2A-eGFP), NeuroD1 (CAG::NeuroD1-P2A-eGFP), or Ascl1 (CAG::Ascl1-P2A-eGFP) to achieve high infection efficiency in fast-growing glioblastoma cells. After 12 hours of incubation with the virus, the glioblastoma culture medium was changed to neuronal differentiation medium to aid in neuronal maturation. Overexpression of Neurog2, NeuroD1, or Ascl1 was confirmed by immunohistochemistry (IHC) (Figure 2A) and real-time quantitative PCR (RT-qPCR) (Figure 2B). A few days after transduction, U251 glioblastoma cells began to adopt a neuronal morphology after expressing neuronal transcription factors (Figure 2A), whereas control cells expressing only GFP did not (Figure 2A, top). A series of pan-neuronal markers was examined for the potential for neuronal conversion from human glioblastoma cells. Immature neuronal markers DCX and Tuj1 were detected as early as 6 days after viral infection (Figure 3A-C). By 30 days after infection, both mature neuronal markers MAP2 and NeuN were detected (Figure 4B). Conversion efficiency was high for all three factors, especially Neurog2 and NeuroD1 (Figure 4A, quantification shown in Figure 4C: Neurog2, 98.2% ± 0.3%, NeuroD1, 88.7% ± 5.2%, Ascl1, 24.6% ± 4.0%, DCX+ neurons / total infected cells 20 days after infection; Figure 4B, quantification shown in Figure 4D: Neurog2, 93.2% ± 1.2%, NeuroD1, 91.2% ± 1.1%, Ascl1, 62.1% ± 5.9%, MAP2+ neurons / total infected cells 30 days after infection). Neuronal conversion was also confirmed by RT-qPCR, which detected transcriptional activation of DCX after overexpression of Neurog2, NeuroD1, or Ascl1 (Figure 4E).To test whether neuronal conversion was limited to U251 cells, we investigated another human GBM cell line, U118, following a similar protocol. Although the conversion efficiency was low, we found that neuronal conversion could be achieved in U118 cells through the combination of neuronal transcription factors and small molecules (e.g., DAPT, CHIR99021, SB431542, and LDN193189) (Figure 5A-D). After 18 days of Neurog2-GFP virus infection with 12 days of small molecule treatment, some U118 cells began to express the neuronal marker DCX (Figure 5D). However, small molecule treatment alone or Neurog2 alone did not convert U118 cells into neuronal-like cells (Figure 5B and 5C).

[0068] Characterization of converted neuronal cells from human glioblastoma cells We characterized neurons converted from U251 human glioblastoma cells with neuronal markers expressed in different brain regions. We found that the majority of converted cells were immunopositive for the hippocampal granule neuron marker Prox1 (Figure 6A; quantification in Figure 6E: Neurog2, 90.4% ± 1.9%; NeuroD1, 89.9% ± 1.2%; Ascl1, 83.0% ± 1.4%; Prox1+ / DCX+ cells) and the forebrain marker FoxG1 (Figure 6B; quantification in Figure 6F: Neurog2, 99.2% ± 0.8%; NeuroD1, 87.9% ± 4.8%; Ascl1, 81.3% ± 3.6%; FoxG1+ / MAP2+ cells). A small number of converted neurons from GBM cells expressed the cortical neuron markers Ctip2 or Tbr1 (Figures 7A and 7B). These results suggest that the intrinsic imprinting of human glioblastoma cells may differ from that of astrocytes, potentially affecting the outcome of cell conversion. Control comparisons were performed using neurons converted from human astrocytes (HA1800, ScienCell, San Diego, USA). The majority of Neurog2-, NeuroD1-, or Ascl1-converted neurons from human astrocytes were positive for FoxG1 and Prox1, and a significant proportion were immunopositive for Ctip2 (Figures 8A and 8B). Thus, neurons converted from GBM cells shared some common characteristics with neurons converted from astrocytes but differed in specific neuronal subtypes.

[0069] We next characterized the converted neuronal subtypes according to the neurotransmitters they released, specifically glutamatergic and GABAergic neurons, the primary excitatory and inhibitory neurons in the brain, respectively. Most Neurog2, NeuroD1, and Ascl1 converted cells were immunopositive for the glutamatergic neuronal marker VGluT1 (Figure 6C; quantification shown in Figure 6G: Neurog2, 92.8% ± 0.7%; NeuroD1, 86.9% ± 2.7%; Ascl1, 80.6% ± 2.1%; VGluT1+ / DCX+ cells). The majority of Neurog2 and NeuroD1 converted cells were immunonegative for GABA (Figure 6D; quantification shown in Figure 6H: Neurog2, 11.1% ± 3.8%; NeuroD1, 8.6% ± 2.5%; GABA+ / DCX+ cells). Approximately half of the Ascl1-converted cells were GABA-positive neurons ( Fig. 6<em>D ; quantification in Fig. 6<em>H : Ascl1, 49.3% ± 6.4%, GABA+ / DCX+ cells), reflecting differences between different neuronal conversion factors.

[0070] In summary, the majority of Neurog2-, NeuroD1-, or Ascl1-converted neurons from U251 GBM cells were forebrain glutamatergic neurons, whereas Ascl1-converted neurons exhibited a propensity to generate GABAergic neurons. These results suggest that the endogenous GBM cell lineage and ectopically expressed transcription factors significantly influence the converted neuronal subtypes.

[0071] Neurog2 overexpression induces a change in the fate of glioblastoma cells into neuronal cells We investigated the Neurog2-induced transformation process. Both the astrocyte marker GFAP and the epithelial-mesenchymal transition (EMT) marker vimentin were highly expressed in human U251 cells. After 20 days of Neurog2 overexpression, both GFAP and vimentin were downregulated compared to controls (Figure 9A). This further confirmed the fate change of glioblastoma cells to neurons. In addition, the gap junction marker connexin 43 was downregulated in U251 glioblastoma cells with Neurog2 overexpression (Figure 9B; quantification of connexin 43 intensity is shown in Figure 9C: Neurog2, 19.4 ± 0.7 au; GFP control, 11.6 ± 0.8 au; 20 days after infection), consistent with the fact that neurons have fewer gap junctions than glial cells. Typical axonal growth cone structures were found in some of the Neurog2-converted neurons (Figure 9D), with interdigitating filopodia labeled by the filamentous actin (F-actin) probe phalloidin and the growth cone marker GAP43 (Figure 9D).

[0072] We investigated intracellular changes during Neurog2-induced neuronal conversion of U251 glioblastoma cells. Mitochondria and the Golgi apparatus exhibited distinct distribution patterns in Neurog2-converted neurons compared with control GBM cells, as demonstrated by Mitotracker labeling assays (Figure 10E-F) and immunostaining for the Golgi apparatus marker GM130 (Figure 10G-H). Mitochondria are known to be located in areas of high energy demand. In control U251 cells, mitochondria were distributed in the cytoplasm without obvious polarization. In Neurog2-converted neurons, mitochondria were found in both the soma and neurites, with a polarized distribution pattern in the soma (Figure 10E). In addition, the average mitochondrial intensity in converted neurons increased compared with controls 30 days after infection, which may reflect both structural and metabolic changes (Figure 10F). The distribution of the Golgi apparatus was also distinct between Neurog2-converted neurons and control GBM cells (Figure 10G). The area of ​​the Golgi apparatus was much smaller in Neurog2-converted cells compared to controls (Figure 10H), suggesting possible changes in protein trafficking during the neuronal conversion process. Meanwhile, autophagic activity (as indicated by immunostaining for the autophagy regulator ATG5) was found to be comparable between Neurog2-converted and control cells (Figure 11A-C), suggesting that protein degradation was not significantly affected by the conversion process.

[0073] In all respects, the distinct cellular and subcellular patterns between the converted neurons and the control glioblastoma cells further demonstrated the change in fate of human glioblastoma cells to neurons.

[0074] Functional analysis of human glioblastoma cell-converted neuronal cells The ability of Neurog2-converted cells to form synapses was investigated by immunostaining for the synaptic vesicle marker SV2. Thirty days after infection, we detected concentrated synaptic puncta along dendrites labeled with MAP2 in Neurog2-converted neurons derived from human GBM cells (Fig. 12A). Patch-clamp recordings demonstrated prominent sodium and potassium currents in the converted cells 30 days after infection (Figs. 12B and 12C). The majority of Neurog2-converted cells fired single action potentials (14 of 23), while a subset of converted neurons (8 of 23) fired multiple action potentials (Figs. 12D and 12E). However, no spontaneous synaptic events were recorded in the Neurog2-converted cells 30 days after infection, suggesting that the converted neurons are still immature or that surrounding glioma cells may exert an inhibitory effect on synaptic release. In summary, these results demonstrate that human GBM cells can be reprogrammed into partially functional neuron-like cells by neuronal transcription factors.

[0075] Neuronal transcription factors inhibit GBM cell proliferation Neurons are terminally differentiated, non-proliferating cells. Therefore, neuronal transdifferentiation may be a promising strategy for controlling cancer cell proliferation. Cell proliferation was examined at the early stage of transformation. U251 cells were incubated with 10 mM BrdU for 24 hours, and proliferating cells were traced 7 days after viral infection before fixation and staining (Figure 10A). Quantification of the percentage of BrdU-positive cells showed that proliferation of Neurog2- and NeuroD1-infected cells was significantly reduced compared to GFP controls (Figure 10B: GFP, 64.8% ± 4.1%; Neurog2, 11.9% ± 2.9%; NeuroD1, 24.5% ± 2.4%). Proliferation of Ascl1-converted cells remained active 7 days after infection (Figure 10A; quantification in Figure 10B: Ascl1, 54.6% ± 1.2%), likely due to the slow action of Ascl1 in GBM cells (Figures 4A-D, 3A-C). The proliferation rate of GBM cells was significantly reduced with overexpression of Neurog2 or NeuroD1, consistent with the rapid conversion rate of Neurog2 and NeuroD1 after infection of GBM cells. These results suggest that, in addition to neuronal conversion, ectopic expression of neuronal transcription factors may also be a promising method for controlling GBM cell proliferation, which is a hallmark of GBM and a major target for GBM treatment.

[0076] We also tested whether neuronal conversion induces changes in any of the signaling pathways or biomarkers associated with glioblastoma progression. Western blot analysis revealed that the expression level of total GSK3β was upregulated 20 days after Neurog2 virus infection compared with control U251 cells (Figure 10C; quantified GSK3β intensity fold change was 3.0 ± 0.5 in Neurog2 cells, Figure 10D). This was further confirmed using immunostaining analysis (Figure 10E-F). These results demonstrate that GSK3β is involved in the neuronal conversion of U251 glioblastoma cells. We treated Neurog2-infected U251 GBM cells with the GSK3β antagonists CHIR99021 (5 μM) or TWS119 (10 μM) and found that inhibition of GSK3β reduced the neuronal conversion efficiency of U251 cells (Figure 13A-C). In addition, two glioma markers, EGFR and IL13Ra2 39-45, were found to have stable expression after neuronal conversion 20 days after Neurog2 infection (Figure 14A-D), suggesting that GBM cell-converted neurons may still retain certain imprints of cancer cells, at least for a certain period after conversion.

[0077] In vivo neuronal conversion of human glioblastoma cells using a xenograft mouse model To confirm that the in vitro cell culture results also apply to the in vivo environment inside the brain, the transformation ability of human glioblastoma cells was tested in vivo in the mouse brain. To mitigate complications from immune rejection, human U251 GBM cells (5 × 10 5 U251 cells) were intracranially transplanted into the striatum of both sides of Rag1- / - immunodeficient mice (Figure 15A). The same volume (2 μL) and titer (2 × 10 5Neurog2-GFP or control GFP retroviruses with a total concentration of 100 pfu / mL were injected into each side of the striatum along with the transplanted GBM cells. Transplanted U251 human GBM cells were identified by vimentin (Figure 15A) or human nuclei staining (Figure 15D). Overexpression of Neurog2 in transplanted human glioblastoma cells (Figure 15A) resulted in efficient neuronal conversion, as indicated by the immature neuronal marker DCX (Figures 15A-C, quantification in Figure 15B: Neurog2, 92.8% ± 1.2%, DCX+ neurons / total infected cells 3 weeks after transplantation). Other neuronal markers, such as Tuj1 and Prox1, were also detected in Neurog2-converted cells 1 month after transplantation (Figures 15D and 15E). Consistent with the in vitro conversion results, the proliferation rate of transplanted glioma cells was significantly reduced in those infected with Neurog2-GFP virus compared to the GFP control (Figures 16A and 16B). Although many LCN2-positive reactive astrocytes were observed in the brain regions transplanted with GBM cells, the number of reactive astrocytes was significantly reduced in the transplanted areas with Neurog2 overexpression compared to the control side (Figure 16C-E). In addition, other possible local environmental factors, such as vascular or resident microglia distribution, were also examined in this in vivo model. We found that neither was significantly affected by Neurog2-induced neuronal conversion (Figure 17A-D).

[0078] In summary, Neurog2, as a representative reprogramming factor, effectively reprograms human glioblastoma cells into neuronal-like cells in vivo in xenograft mouse models.Furthermore, this reprogramming approach significantly inhibits the proliferation of glioma cells and reduces reactive astrogliosis.Together, these results demonstrate that cancer cells (e.g., GBM cells) can be reprogrammed into different subtypes of neural cells both in vitro and in vivo, which may provide an alternative therapeutic approach for treating cancer (e.g., brain tumors).

[0079] Example 2: Conversion of liver cancer cells to non-cancerous liver cells This example demonstrates that liver transcription factors (e.g., GATA4, Foxa2, and / or HNF4A) can be used to mediate tumor cell reprogramming, converting tumor cells into normal-like cells, and establishing a novel strategy for the treatment of liver cancer or other types of cancer.

[0080] cell line Human liver cancer cell lines HepG2 and HEK293T (obtained from ATCC) were maintained in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. All cell lines were routinely treated with MycoSolutions™ (AKRON) to detect mycoplasma contamination.

[0081] antibody Chicken polyclonal or mouse monoclonal antibodies specific for GFP were purchased from Abcam. Goat polyclonal antibodies against GATA4, Foxa2, and HNF4A proteins were obtained from R&D Systems. Mouse beta-actin monoclonal antibody and goat albumin polyclonal antibody were obtained from Santa Cruz, and rabbit GAPDH polyclonal antibody was obtained from Abcam. Rabbit monoclonal antibodies specific for AFP protein and E-cadherin, and mouse monoclonal antibody specific for HNF4A protein were purchased from Abcam. Rabbit anti-B-catenin polyclonal antibody and goat anti-vimentin polyclonal antibody were obtained from Abcam and R&D Systems, respectively. IRDye 680 Donkey anti-Mouse, IRDye 680 Donkey anti-Rabbit, IRDye 680 Donkey anti-Goat, IRDye 800 Donkey anti-Mouse, IRDye 800 Donkey anti-Rabbit, and IRDye 800 Donkey anti-Goat secondary antibodies were purchased from LI-COR.

[0082] animal Four- to five-week-old male immunodeficient athymic nude mice were obtained from Charles River.

[0083] Generation of lentiviral expression plasmids and viruses The AgeI / EcorI fragment of GATA4 (or Foxa2 or HNF4A)-P2A-GFP was cloned into the third-generation lentiviral vector pLJM1 (Addgene), replacing the existing green fluorescent protein (GFP) sequence. The resulting vector plasmid was used to generate lentivirus. Lentivirus was generated using the PEI transfection method. Briefly, 80% confluent 293T cells grown on 15 cm culture dishes were transfected with 12 μg of the lentiviral vector encoding GATA4 (or Foxa2 or HNF4A)-P2A-GFP, 2.4 μg of the envelope plasmid pMD2.G (Addgene) encoding the VSV glycoprotein G, and 12 μg of the packaging plasmid psPAX2 (Addgene). The virus-containing medium was harvested 72 hours posttransfection, filtered to remove cells or cell debris, and concentrated by ultracentrifugation. Viral titers were determined by infection of HEK293T cells, and GFP-positive cells were counted for calculation of transducing units per milliliter (TU / mL).

[0084] Cell proliferation assay Cell proliferation assays of GATA4-, Foxa2-, HNF4A-, or GFP-transduced cells were initiated with 15,000 cells per 12-well plate. Cells were counted at 6, 24, 48, and 72 hours. At each time point, cells were washed once with phosphate-buffered saline (PBS), and 4% paraformaldehyde (PFA) was added to each well for 15 minutes. Cells were then stained with 0.1% crystal violet for 20 minutes. The stained crystal violet was extracted with 10% acetic acid and transferred to a 96-well plate for optical density measurement at 590 nm using a microplate reader (Bio-Rad Laboratories, CA).

[0085] Mouse tumor model Before implantation into mice, GATA4, Foxa2, HNF4A, or GFP-transduced liver tumor HepG2 cell lines were grown to 80% confluence, counted, and suspended in PBS. Each mouse was implanted with 1.0 × 10 6 Tumor cells were injected subcutaneously. Animals were examined and tumor growth was monitored every 3-4 days throughout the experiment. Tumors were measured with calipers and tumor volume was calculated using the formula: 0.5 × ab 2 (a is the long axis, b is the short axis). Mice were euthanized, and tumors were dissected and incubated in 4% PFA at 4°C. Tumors were sliced ​​and analyzed by immunofluorescence staining.

[0086] Lentiviral transduction of liver cancer cells HepG2 For lentiviral transduction, human liver cancer HepG2 cells were cultured at 5 × 10 5 Cells were plated onto 6 cm culture dishes at a density of 1000 x 1000 and incubated overnight to allow cells to attach. HepG2 cells were infected with lentivirus at an MOI of 1 in 2 mL of fresh DMEM supplemented with 2% FBS. Cultures were incubated at 37°C for 2 days.

[0087] The culture medium was replaced with DMEM containing 10% FBS and 2 μg / mL puromycin. Puromycin-resistant cells were maintained in DMEM containing 10% FBS and 2 μg / mL puromycin.

[0088] Western blot analysis Cultured cells resuspended in 1x PBS were mixed with an equal volume of 2x NuPAGE LDS Sample Buffer (Invitrogen). Fresh tumor samples were mixed with 5 volumes of 1x RIPA buffer (Invitrogen) and homogenized for 45 seconds at maximum speed using a BEAD RUPTOR homogenizer (OMNI International, Inc.). An equal volume of 2x NuPAGE LDS Sample Buffer was added to the lysed tumor samples.

[0089] Protein samples were separated on a 10% polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Amersham, Piscataway, NJ). The membrane was blocked in 5% nonfat dry milk and incubated with primary antibodies followed by appropriate secondary antibodies. Protein band detection was performed using a LI-COR ODYSSEY CLx scanner. Protein bands were quantified using LI-COR Image Studio Ver. 3.1 software, and the relative amounts of each protein were obtained according to the software instructions.

[0090] Immunofluorescence staining and microscopy Cell cultures grown on coverslips were fixed with 4% paraformaldehyde (PFA) in PBS for 10 minutes. The PFA was washed away with PBS, and the cells were incubated for 30 minutes in a blocking solution containing 2.5% NDS (Normal Donkey Serum), 2.5% NGS (Normal Goat Serum), and 0.1% Triton X-100 in PBS. The cells were then incubated overnight with primary antibodies mixed in the blocking solution. The cells were washed three times with PBS and incubated for 1 hour with a mixture of Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies (Jackson ImmunoResearch). Unbound secondary antibodies were washed away with PBS, and nuclei were stained with DAPI. The cells were visualized using a confocal microscope (Zeiss LSM800).

[0091] Tumor sections were permeabilized in 0.3% Triton X-100 in PBS for 1 hour, followed by incubation in a blocking solution containing 0.3% Triton X-100 in PBS for 1 hour. The tumor sections were incubated overnight at 4°C with the primary antibodies mixed in the blocking solution. After washing away unbound primary antibodies with PBS, the tumor sections were incubated with a mixture of Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies (Jackson ImmunoResearch) for 1 hour at room temperature. Unbound secondary antibodies were washed away with PBS, and nuclei were stained with DAPI. The tumor sections were visualized using a confocal microscope (Zeiss LSM800).

[0092] ELISA assay GATA4, Foxa2, HNF4A, or GFP-transduced liver tumor HepG2 cell lines were seeded into 12-well plates and incubated for 6 hours to allow cells to adhere to the plate. The culture medium was replaced with serum-free DMEM, and incubation continued for 16 hours. Culture medium from each cell line was collected, and albumin levels were measured using a Human Serum Albumin ELISA Kit (Molecular Innovations) according to the kit's instructions.

[0093] Transduction of the liver tumor cell line HepG2 with hepatic transcription factors Foxa2, HNF4A, and GATA4 HepG2 cells were infected with pLJM1 lentiviral vectors carrying Foxa2-P2A-GFP, HNF4A-P2A-GFP, GATA4-P2A-GFP, or GFP. After 48 hours, puromycin was added to the medium to eliminate uninfected cells. Puromycin-resistant cells were grown as usual, and intracellular transcription factor or GFP expression was assessed by immunostaining or Western blot. To examine the expression and localization of Foxa2, HNF4A, GATA4, and GFP, HepG2-Foxa2 (Foxa2-P2A-GFP transduced), HepG2-HNF4A (HNF4A-P2A-GFP transduced), HepG2-GATA4 (GATA4-P2A-GFP transduced), or HepG2-GFP (GFP transduced) cell lines were subjected to fluorescence microscopy. Foxa2, HNF4A, GATA4, and GFP were all highly expressed in each cell line. The transcription factors Foxa2, HNF4A, and GATA4 were localized in the nucleus, while GFP was distributed throughout the cell body (Figure 18A). All cells in each transduced line expressed the transduced vector. Expression of the transcription factors Foxa2, HNF4A, and GATA4 was further verified by Western blot (Figures 18B, C, D, and E).

[0094] GATA4 increases endogenous Foxa2 polypeptide levels HepG2 cells were individually transduced with the hepatic transcription factors Foxa2, HNF4A, and GATA4. Western blot analysis showed that GATA4 overexpression increased endogenous Foxa2 expression, whereas HNF4A overexpression decreased Foxa2 expression (Figure 19A). Endogenous GATA4 expression was not affected by overexpression of either HNF4A or Foxa2 (Figure 19B).

[0095] Proliferation in reprogrammed HepG2 cells To investigate the functional relevance of hepatic transcription factor expression for the proliferation of liver tumor cells, HepG2, GATA4, Foxa2, HNF4A, or GFP-transduced cell lines were cultured in 12-well dishes. At 6, 24, 48, and 72 hours, cells from each cell line were fixed with 4% PFA and stained with 0.1% crystal violet. The stained crystal violet was extracted with 10% acetic acid, and the relative proliferation rates of the GATA4, Foxa2, HNF4A, or GFP-transduced cell lines were compared spectrophotometrically. As shown in Figure 20A, cell lines transduced with GATA4, Foxa2, or HNF4A exhibited reduced proliferation rates compared to the control cell line transduced with GFP. Furthermore, Foxa2- and GATA4-mediated cell lines achieved much lower cell proliferation rates.

[0096] To examine the in vivo proliferation rate, GATA4-, Foxa2-, HNF4A-, or GFP-transduced cell lines were xenografted subcutaneously into nude mice. Nude mice were randomly assigned to four groups (six mice per group) and 1 × 10 GATA4-, Foxa2-, HNF4A-, or GFP-transduced cell lines were implanted subcutaneously into nude mice. 6 The cells were implanted into the flanks of nude mice. After 4 days, the GFP- and HNF4A-transduced cell lines began to form tumors. The Foxa2-transduced cell line did not form any visible tumors. The GATA4-transduced cell line showed small tumor growth at later time points. The results revealed that Foxa2 or GATA4 in reprogrammed HepG2 cells reduced cell proliferation (Figure 20B).

[0097] Function of reprogrammed HepG2 cells Foxa2-, GATA4-, HNF4A-, or GFP-transduced HepG2 cells were stained with anti-albumin antibodies to examine albumin production in the cell lines. As shown in Figure 21A, both Foxa2- and GATA4-transduced cell lines produced increased albumin compared with HNF4A- and GFP-transduced cell lines examined by immunostaining. This result was confirmed by Western blot analysis, as shown in Figure 21B. The increase in albumin in Foxa2- and GATA4-transduced cell lines was more than two-fold (Figure 21C). Albumin in Foxa2- and GATA4-transduced cell lines could be secreted outside the cells. Compared with the GFP-transduced cell line, albumin secretion from Foxa2- or GATA4-transduced cell lines was more than four-fold increased as detected by ELISA (Figure 21D). Thus, transduction and overexpression of Foxa2 or GATA4 promoted the display of physiological characteristics of normal liver cells by the transduced cell lines.

[0098] Liver cancer markers in reprogrammed HepG2 cells As shown by immunostaining, AFP expressed in GATA4-, Foxa2-, HNF4A-, or GFP-transduced cell lines was localized in the cytoplasm (Figure 22A). AFP expression was reduced in GATA4- and Foxa2-transduced cell lines. Western blot analysis revealed that AFP expression in the GATA4 cell line was reduced by 60% compared to the GFP-transduced cell line (Figure 22B). For in vivo studies, HepG2 tumors were established in nude mice subcutaneously xenografted with GATA4-, HNF4A-, or GFP-transduced cell lines, and tumor samples were collected. The Foxa2-transduced cell line lost tumorigenicity. Tumors formed in GATA4-, HNF4A-, or GFP-transduced cell lines were fixed in PFA, sectioned, and analyzed by immunofluorescence. As shown in Figure 22C, AFP production in the GATA4 cell line was reduced, while AFP production in the HNF4A cell line was only slightly reduced. Fresh HepG2 tumor samples developed with GATA4, HNF4A, or GFP cell lines were also subjected to Western blot analysis. GATA4 was overexpressed in tumors formed from the GATA4 cell line, and AFP expression levels were reduced (Figure 22D). A decrease in AFP expression was observed in both in vivo and in vitro studies using the GATA4 cell line.

[0099] Hepatocyte markers in reprogrammed HepG2 cells E-cadherin expression was observed in GATA4-, Foxa2-, HNF4A-, and GFP-transduced cell lines in vitro and in vivo. GATA4-, Foxa2-, HNF4A-, and GFP-transduced cell lines were grown on coverslips and stained with anti-E-cadherin antibodies. Immunofluorescence analysis showed that E-cadherin was more strongly expressed in Foxa2-, HNF4A-, and GATA4-transduced cells than in GFP-expressing cells. E-cadherin was localized to the plasma membrane (Figure 23A). To examine whether GATA4 expression rescues E-cadherin expression, cell lines expressing GATA4, Foxa2-, HNF4A-, and GFP were harvested, lysed, and analyzed by Western blot. E-cadherin was rescued more than twofold in the GATA4-expressing lines (Figure 23B), mirroring the results obtained by immunofluorescence analysis and Western blot (Figure 23A). In vivo experiments were performed to determine E-cadherin expression levels in GATA4-, HNF4A-, and GFP-overexpressing tumors. As shown in Figures 23C and 23D, tumors with GATA4 overexpression had more than twofold higher levels of E-cadherin when examined by either immunofluorescence analysis or Western blot. Although E-cadherin expression was increased in the HNF4A-overexpressing cell lines, tumor growth rates were not reduced compared with the GFP control cell lines. These data indicate that elevated E-cadherin levels can be used as an indicator of improved function of reprogrammed tumor cells.

[0100] These findings indicate that the transcription factor GATA4 promoted E-cadherin expression. To examine whether GATA4 overexpression affected beta-catenin, we performed immunofluorescence analysis on both GATA4- and GFP-transfected cell lines. GATA4-transfected cell lines showed increased beta-catenin expression compared with GFP-overexpressing cells (Figure 24A). These results also showed that beta-catenin in GFP cells was distributed in the perinuclear region with a slight preference for nuclear distribution, whereas beta-catenin in GATA4-transfected cell lines was distributed on the cell surface. Western blot analysis was used to analyze beta-catenin expression in GATA4-transfected cell lines. GATA4-transfected cell lines showed increased beta-catenin expression (Figure 24B). HNF4A-transfected cells also showed increased beta-catenin expression.

[0101] When tumor samples derived from tumors formed in vivo from GATA4- or GFP-transduced cell lines were stained for beta-catenin, immunofluorescence analysis showed that beta-catenin expression was higher in GATA4 tumors than in GFP tumors. Due to the small, crowded cytoplasmic space of cells in the tumors, differences in beta-catenin distribution between these two tumor types could not be determined (Figure 24C). The increased amount of beta-catenin in GATA4 tumors was threefold higher than in GFP tumors, as assessed by Western blot (Figure 24D).

[0102] Tumor samples derived from tumors formed in vivo from GATA4, HNF4A, or GFP transduced cell lines were also analyzed for vimentin by Western blot. As shown in Figure 25, vimentin expression was reduced in GATA4-overexpressing tumors compared to GFP tumors. When vimentin expression levels in GATA4 tumors were quantified, the reduction in vimentin in GATA4 tumors was more than two-fold compared to GFP tumors.

[0103] These findings indicate that the fate of HCC tumor cells was altered through the overexpression of transcription factors. The altered mechanism may be associated with mesenchymal-to-epithelial transition (MET), the reverse process of epithelial-to-mesenchymal transition (EMT). Taken together, these results suggest that reprogramming cancer cells (e.g., liver cancer cells) into normal-like cells (e.g., normal-like liver cells) both in vitro and in vivo may provide an alternative therapeutic approach for treating cancer (e.g., liver cancer).

[0104] Other Aspects While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is illustrative of, but not intended to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for treating a mammal having cancer, the method comprising administering to cancer cells in the mammal a nucleic acid encoding one or more transcription factors, wherein the one or more transcription factors are expressed by the cancer cells, and the one or more transcription factors convert the cancer cells into non-cancerous cells in the mammal, thereby reducing the number of cancer cells in the mammal.

2. The method of claim 1 , wherein the mammal is a human.

3. 3. The method of claim 1 or 2, wherein the cancer is glioma.

4. The method of claim 3 , wherein the one or more transcription factors are one or more neuronal transcription factors.

5. 5. The method of claim 4, wherein the one or more neuronal transcription factors are selected from the group consisting of neurogenic differentiation factor 1 (NeuroD1) polypeptide, neurogenin-2 (Neurog2) polypeptide, and achaete-scute homolog 1 (Ascl1) polypeptide.

6. The method of claim 4 or 5, wherein the one or more neuronal transcription factors comprise a NeuroD1 polypeptide, a Neurog2 polypeptide, and an Ascl1 polypeptide.

7. The method of any one of claims 3 to 6, wherein the non-cancerous cell is a neuronal cell.

8. The method of claim 7, wherein the neuronal cells are FoxG1-positive forebrain neuronal cells.

9. The method of claim 1 or 2, wherein the cancer is liver cancer.

10. The method of claim 9, wherein the liver cancer is hepatocellular carcinoma.

11. 11. The method of claim 9 or 10, wherein the one or more transcription factors are liver transcription factors.

12. 11. The method of claim 10, wherein the one or more liver transcription factors are selected from the group consisting of hepatocyte nuclear factor 4A (HNF4A) polypeptide, forkhead box protein (Foxa2) polypeptide, and GATA binding protein (GATA4) polypeptide.

13. 13. The method of claim 11 or 12, wherein the one or more hepatic transcription factors comprise an HNF4A polypeptide, a Foxa2 polypeptide, and a GATA4 polypeptide.

14. 14. The method of any one of claims 9 to 13, wherein the non-cancerous cells are hepatocytes.

15. 15. The method of claim 14, wherein the hepatocytes are hepatocytes that secrete liver enzymes.

16. 16. The method of claim 15, wherein the liver enzyme is albumin.

17. 17. The method of any one of claims 1 to 16, wherein the nucleic acids encoding the one or more transcription factors are administered to the cancer cells in the form of a viral vector.

18. 18. The method of claim 17, wherein the viral vector is a retroviral vector.

19. 18. The method of claim 17, wherein the viral vector is a lentiviral vector.

20. 20. The method of any one of claims 1 to 19, wherein the nucleic acid encoding each of the one or more transcription factors is operably linked to a promoter sequence.

21. 21. The method of any one of claims 1 to 20, wherein the administration of the nucleic acid encoding the one or more transcription factors comprises direct injection into a tumor of the mammal.

22. 21. The method of any one of claims 1 to 20, wherein the administration of the nucleic acids encoding the one or more transcription factors comprises intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intraparenchymal, intratumoral, intranasal, or oral administration.

23. 23. The method of any one of claims 1 to 22, wherein the method comprises identifying the mammal as having the cancer prior to the administering step.

24. 24. Use of a composition comprising nucleic acids encoding one or more transcription factors for treating cancer according to the method of any one of claims 1 to 23.

25. 24. A composition comprising nucleic acids encoding one or more transcription factors for treating cancer according to the method of any one of claims 1 to 23.

26. 24. Use of nucleic acids encoding one or more transcription factors in the manufacture of a medicament for treating cancer according to the method of any one of claims 1 to 23.

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