WO2020198485A1 - Methods and materials for treating cancer - Google Patents
Methods and materials for treating cancer Download PDFInfo
- Publication number
- WO2020198485A1 WO2020198485A1 PCT/US2020/024976 US2020024976W WO2020198485A1 WO 2020198485 A1 WO2020198485 A1 WO 2020198485A1 US 2020024976 W US2020024976 W US 2020024976W WO 2020198485 A1 WO2020198485 A1 WO 2020198485A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- percent
- cells
- cancer
- transcription factors
- mammal
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16041—Use of virus, viral particle or viral elements as a vector
- C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- This document relates to methods and materials for treating a mammal having cancer.
- this document provides methods and materials for converting one or more cancer cells present in a mammal with cancer into non-cancerous cells.
- Cancer is a major public health issue. In the United States alone over 1.7 million new cases were diagnosed in 2019 (National Cancer Institute,“Cancer Stat Facts: Cancer of Any Site,” at“https” colon“seer” dot“cancer” dot“gov/statfacts/html/all.html”.
- Glioblastoma a type of tumor that arises from unchecked proliferation of glial cells, accounts for half of the malignant brain tumor cases and has a 3.6 percent five- year relative survival rate (Ostrom et al, Neuro Oncol .; 17:ivl-iv62 (2015); and Porter et al, Neuroepidemiology .; 36(4):230-239 (2011)).
- Traditional therapies such as
- chemotherapeutics, radiation therapy, and surgery often fail in GBM because of 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)).
- liver cancer or hepatocellular carcinoma (HCC)
- HCC hepatocellular carcinoma
- This document provides methods and materials for treating a mammal having cancer by converting cancer cells within the mammal into non-cancerous cells.
- a transcription factor e.g. , a neuronal transcription factor or a liver transcription factor
- a transcription factor e.g. , a neuronal transcription factor or a liver transcription factor
- nucleic acid designed to express a transcription factor e.g ., a neuronal transcription factor or a liver transcription factor
- delivering nucleic acid designed to express a neuronal transcription factor e.g., nucleic acid designed to express a neurogenic differentiation factor 1 (NeuroDl) polypeptide
- nucleic acid designed to express a neurogenic differentiation factor 1 (NeuroDl) e.g., nucleic acid designed to express a neurogenic differentiation factor 1 (NeuroDl) polypeptide
- neurogenin-2 neurogenin-2 (Neurog2) polypeptide, or nucleic acid designed to express an achaete-scute homolog 1 (Ascii) polypeptide) to human GBM cells
- the converted neurons can express neuron-specific markers, can have functional synaptic networks, and can have active electrophysiological properties.
- the converted neurons also can exhibit downregulated signaling pathways related to cancer progression (e.g, as compared to the GBM cells prior to conversion).
- the in vivo conversion of GBM cells to neurons can reduce cancer cell proliferation and/or can decrease the rate of astrogliosis.
- delivering nucleic acid designed to express a liver transcription factor e.g, delivering nucleic acid designed to express a hepatocyte nuclear factor 4A (HNF4A) polypeptide, nucleic acid designed to express a forkhead box protein (Foxa2) polypeptide, and/or nucleic acid designed to express a GATA binding protein
- GATA4 polypeptide to human liver cancer cells can convert the human liver cancer cells to non-cancerous liver cells (hepatocytes).
- the converted hepatocytes can have decreased proliferation, can have decreased expression of the liver cancer markers alpha fetoprotein (AFP), and/or can express epithelial-specific markers such as the epithelial cell surface molecule E-cadherin.
- AFP alpha fetoprotein
- Having the ability to convert cancer cells into non-cancerous cells within a living mammal using the methods and materials described herein provides clinicians and patients (e.g, cancer patients) with an effective approach to treat cancer.
- the in vivo conversion of cancer cells into non-cancerous cells can be used to control proliferation of cancer cells in the absence of traditional cancer therapy.
- a cancer patient can avoid common side effects caused by traditional cancer therapies.
- one aspect of this document features a method for treating a mammal having a cancer.
- the method comprises (or consists essentially of or consists of) administering nucleic acid encoding one or more transcription factors to cancer cells within the mammal, wherein the one or more transcription factors are expressed by the cancer cells, and wherein the one or more transcription factors convert the cancer cells into non-cancerous cells within the mammal, thereby reducing the number of cancer cells within 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 a neurogenic differentiation factor 1 (NeuroDl) polypeptide, a neurogenin-2 (Neurog2) polypeptide, and an achaete-scute homolog 1 (Ascii) polypeptide.
- the one or more neuronal transcription factors can comprise a NeuroDl polypeptide, a Neurog2 polypeptide, and an Ascii polypeptide.
- the non-cancerous cells can be neurons.
- the neurons can be FoxGl- positive forebrain neurons.
- the cancer can be a liver cancer.
- the liver cancer can be a hepatocellular carcinoma.
- the one or more transcription factors can be liver transcription factors.
- the one or more liver transcription factors can be selected from the group consisting of a hepatocyte nuclear factor 4A (HNF4A) polypeptide, a forkhead box protein (Foxa2) polypeptide, and a GATA binding protein (GATA4) polypeptide.
- the one or more liver transcription factors can comprise a HNF4A polypeptide, a Foxa2 polypeptide, and a GATA4 polypeptide.
- the non-cancerous cells can be hepatocytes.
- the hepatocytes can be hepatocytes that secrete a liver enzyme.
- the liver enzyme can be albumin.
- the nucleic acid encoding the one or more transcription factors can be administered to the cancer cells in the form of a viral vector.
- the viral vector can be a retroviral vector.
- the viral vector can be a lentiviral vector.
- the nucleic acid encoding each of the one or more transcription factors can be operably linked to a promoter sequence.
- the administration of the nucleic acid encoding the one or more transcription factors can comprise a direct injection into a tumor of the mammal.
- the administration of the nucleic acid encoding the one or more transcription factors can comprise an intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intraparenchymal, intratumoral, intranasal, or oral administration.
- the method can comprise, prior to the administering step, identifying the mammal as having the cancer.
- 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 a neurogenic differentiation factor 1 (NeuroDl) polypeptide, a neurogenin-2 (Neurog2) polypeptide, and an achaete-scute homolog 1 (Ascii) polypeptide.
- the one or more neuronal transcription factors can comprise a NeuroDl polypeptide, a Neurog2 polypeptide, and an Ascii polypeptide.
- the non-cancerous cells can be neurons.
- the neurons can be FoxGl -positive forebrain neurons.
- the cancer can be a liver cancer.
- the liver cancer can be a hepatocellular carcinoma.
- the one or more transcription factors can be liver transcription factors.
- the one or more liver transcription factors can be selected from the group consisting of a hepatocyte nuclear factor 4A (HNF4A) polypeptide, a forkhead box protein (Foxa2) polypeptide, and a GATA binding protein (GATA4) polypeptide.
- the one or more liver transcription factors can comprise a HNF4A polypeptide, a Foxa2 polypeptide, and a GATA4 polypeptide.
- the non-cancerous cells can be hepatocytes.
- the hepatocytes can be hepatocytes that secrete a liver enzyme.
- the liver enzyme can be albumin.
- the nucleic acid encoding the one or more transcription factors can be administered to the cancer cells in the form of a viral vector.
- the viral vector can be a retroviral vector.
- the viral vector can be a lentiviral vector.
- transcription factors can be operably linked to a promoter sequence.
- the administration of the nucleic acid encoding the one or more transcription factors can comprise a direct injection into a tumor of the mammal.
- the administration of the nucleic acid encoding the one or more transcription factors can comprise an intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intraparenchymal, intratumoral, intranasal, or oral administration.
- the method can comprise, prior to the administering step, identifying the mammal as having the cancer.
- this document features a composition comprising (or consisting essentially of or consisting of) nucleic acid encoding one or more transcription factors to treat cancer according to a method comprises (or consists essentially of or consists of) administering nucleic acid encoding one or more transcription factors to cancer cells within the mammal, wherein the one or more transcription factors are expressed by the cancer cells, and wherein the one or more transcription factors convert the cancer cells into non-cancerous cells within the mammal, thereby reducing the number of cancer cells within 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 a neurogenic differentiation factor 1 (NeuroDl) polypeptide, a neurogenin-2 (Neurog2) polypeptide, and an achaete-scute homolog 1 (Ascii) polypeptide.
- the one or more neuronal transcription factors can comprise a NeuroDl polypeptide, a Neurog2 polypeptide, and an Ascii polypeptide.
- the non- cancerous cells can be neurons.
- the neurons can be FoxGl -positive forebrain neurons.
- the cancer can be a liver cancer.
- the liver cancer can be a hepatocellular carcinoma.
- the one or more transcription factors can be liver transcription factors.
- the one or more liver transcription factors can be selected from the group consisting of a hepatocyte nuclear factor 4A (HNF4A) polypeptide, a forkhead box protein (Foxa2) polypeptide, and a GATA binding protein (GATA4) polypeptide.
- the one or more liver transcription factors can comprise a HNF4A polypeptide, a Foxa2 polypeptide, and a GATA4 polypeptide.
- the non-cancerous cells can be hepatocytes.
- the hepatocytes can be hepatocytes that secrete a liver enzyme.
- the liver enzyme can be albumin.
- the nucleic acid encoding the one or more transcription factors can be administered to the cancer cells in the form of a viral vector.
- the viral vector can be a retroviral vector.
- the viral vector can be a lentiviral vector.
- the nucleic acid encoding each of the one or more transcription factors can be operably linked to a promoter sequence.
- the administration of the nucleic acid encoding the one or more transcription factors can comprise a direct injection into a tumor of the mammal.
- the administration of the nucleic acid encoding the one or more transcription factors can comprise an
- the method can comprise, prior to the administering step, identifying the mammal as having the cancer.
- 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 a neurogenic differentiation factor 1 (NeuroDl) polypeptide, a neurogenin-2 (Neurog2) polypeptide, and an achaete-scute homolog 1 (Ascii) polypeptide.
- the one or more neuronal transcription factors can comprise a NeuroDl polypeptide, a Neurog2 polypeptide, and an Ascii polypeptide.
- the non-cancerous cells can be neurons.
- the neurons can be FoxGl -positive forebrain neurons.
- the cancer can be a liver cancer.
- the liver cancer can be a hepatocellular carcinoma.
- the one or more transcription factors can be liver transcription factors.
- the one or more liver transcription factors can be selected from the group consisting of a hepatocyte nuclear factor 4A (HNF4A) polypeptide, a forkhead box protein (Foxa2) polypeptide, and a GATA binding protein (GATA4) polypeptide.
- the one or more liver transcription factors can comprise a HNF4A polypeptide, a Foxa2 polypeptide, and a GATA4 polypeptide.
- the non-cancerous cells can be hepatocytes.
- the hepatocytes can be hepatocytes that secrete a liver enzyme.
- the liver enzyme can be albumin.
- the nucleic acid encoding the one or more transcription factors can be administered to the cancer cells in the form of a viral vector.
- the viral vector can be a retroviral vector.
- the viral vector can be a lentiviral vector.
- the nucleic acid encoding each of the one or more transcription factors can be operably linked to a promoter sequence.
- the administration of the nucleic acid encoding the one or more transcription factors can comprise a direct injection into a tumor of the mammal.
- the administration of the nucleic acid encoding the one or more transcription factors can comprise an intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intraparenchymal, intratumoral, intranasal, or oral administration.
- the method can comprise, prior to the administering step, identifying the mammal as having the cancer.
- Figure 1 Characterization of human glioblastoma cell lines. Representative images of a series of markers (which stained red) to characterize U251 and U118 human
- glioblastoma cells Red boxes (marked with *) indicate high level of immunopositive markers. Scale bars, 50 pm.
- GFAP and S 100b astrocyte markers; Tuj 1 and DCX, immature neuronal markers; Sox2 and Nestin, neural progenitor markers; 01ig2, oligodendrocyte marker; Ki67, cell proliferation marker; EGFR, cancer marker.
- FIG. 2 Confirmation of the overexpression of neural transcription factor Neurog2, NeuroDl or Ascii in human glioblastoma cells.
- A Representative images showing the overexpression of Neurog2, NeuroDl, or Ascii in U251 human glioblastoma cells through immunostaining. Scale bar, 20 pm.
- Figure 3 Rapid induction of neuron-like cells from human glioblastoma cells by Neurog2 and NeuroDl .
- A Immunostaining of immature neuronal markers Doublecortin (DCX, red) and b3 -tubulin (Tuj 1, magenta) in U251 human GBM cells infected by Neurog2- GFP, NeuroDl-GFP or Ascll-GFP retroviruses at 6 dpi. Scale bar, 50 pm.
- B-C Quantitative analyses of neuronal conversion at 6 dpi.
- Neurog2 showed a significant increase in the percentage of DCX + cells (GFP, 0; Neurog2, 12.6% ⁇ 2.0%; NeuroDl, 1.6% ⁇ 0.4%; Ascii, 0; B), and Tuj l + cells (GFP, 0; Neurog2, 46.1% ⁇ 3.2%; NeuroDl, 20.5% ⁇ 4.9%; Ascii, 2.6% ⁇ 0.7%; C), followed by NeuroDl at early stage of viral infection.
- the Ascii conversion efficiency is the lowest among the three neural transcription factors tested. Data are represented as mean ⁇ SEM, and analyzed by one-way ANOVA followed with Dunnett’s test. **, p ⁇ 0.01; ***, p ⁇ 0.001; n > 200 cells from triplicate cultures.
- Figure 4 Single neuronal transcription factor Neurog2, NeuroDl, or Ascii converts human glioblastoma cells into neurons.
- A-B Retroviral expression of Neurog2-GFP, NeuroDl -GFP, or Ascii -GFP in U251 human glioblastoma cells led to a large number of neuronal cells compared with GFP alone (top row).
- Neurog2-, NeuroDl-, or Ascii - converted cells were immunopositive for immature neuronal markers (A; DCX; Tuj l) at 20 days post infection (dpi), and mature neuronal markers (B; MAP2; NeuN) at 30 dpi. Scale bars, 50 pm.
- Neurog2, NeuroDl, or Ascii overexpression in U25 1 cells revealed by real-time qPCR.
- FIG. 5 Induction of neuron-like cells in U118 human glioblastoma cells via combination of Neurog2 overexpression and small molecule treatment.
- A-D U118 cells were converted into neuron-like cells (DCX, which stained in cyan) when Neurog2 was overexpressed together with small molecule treatment (Core: 5 pM DAPT, 1.5 pM
- FIG. 6 Characterization of the converted neurons from human GBM cells.
- A-D Representative images showing the immunostaining of neuronal subtype markers.
- Most of the Neurog2-, NeuroDl-, and Ascii -converted neurons (DCX in A; and MAP2 in B) were immunopositive for hippocampal neuron marker Proxl (A) and forebrain neuron marker FoxGl (B).
- Neurog2-, NeuroDl-, and Ascii -converted neurons (DCX in C) were largely VGluTl+ (C), while some of the Ascii -converted neurons (DCX in D) were also GABA+ (D).
- E-H Quantitative analyses of converted neurons from human GBM cells. Samples were at 20 dpi.
- Figure 8 Comparison with the human astrocyte-converted neurons after infection by Neurog2, NeuroDl, or Ascii.
- A Representative images showing that the majority of human astrocyte-converted neurons induced by Neurog2, NeuroDl or Ascii were immunopositive for hippocampal neuron marker Proxl and forebrain marker FoxGl. Much less Ascii - converted neurons were Ctip2 + . Scale bars, 20 pm.
- B Quantitative analyses of Neurog2-, NeuroDl - and Ascii -converted neurons from human cortical astrocytes (HA1800 cells, ScienCell, San Diego, USA).
- Proxl + /MAP2 + Neurog2, 85.4% ⁇ 3.4%; NeuroDl, 89.2% ⁇ 3.3%; Ascii, 85.0% ⁇ 3.7%.
- FoxGl + /MAP2 + Neurog2, 92.6% ⁇ 3.8%; NeuroDl, 85.1% ⁇ 2.7%; Ascii, 85.7% ⁇ 4.8%.
- Ctip2 + /MAP2 + Neurog2, 46.6% ⁇ 5.1%; NeuroDl, 61.1% ⁇ 2.8%; Ascii, 14.0% ⁇ 5.4%. Samples were at 30 dpi. Data are represented as mean ⁇ SEM. n > 50 cells from triplicate cultures.
- Figure 9 Fate change from glioblastoma cells to neurons induced by Neurog2 overexpression.
- A Downregulation of astrocyte markers vimentin and GFAP in Neurog2- converted neurons (bottom row) compared to the control U251 glioblastoma cells expressing GFP alone (top row). Samples were at 20 dpi.
- B-C Representative images showing the gap junctions (Connexin 43) among U251 GBM cells overexpressing GFP alone (top row) or Neurog2-GFP (bottom row). Quantified data (C) showing a significant reduction of
- D Representative images illustrating a growth cone depicted by GAP43 and phalloidin in U251 cells overexpressing Neurog2 at 6 dpi.
- E-H Distribution and morphological changes of mitochondria (MitoTracker) and the Golgi apparatus (GM130) during neuronal conversion of U251 cells. Quantified data showing changes of MitoTracker intensity (F) and the Golgi apparatus size reflected by GM130 covered area (H) after Neurog2 expression at 30 dpi. n > 150 from triplicate cultures.
- FIG. 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, which stained red) during neuronal conversion of U251 cells.
- B-C Quantified data of ATG5 area (B) and intensity (C) in infected U251 cells at 30 dpi. n > 150 cells from triplicate cultures. Scale bars, 10 pm. Data are represented as mean ⁇ SEM, and analyzed by Student’s /-test. * p ⁇ 0.05; *** p ⁇ 0.001.
- FIG. 12 Functional analyses of human glioblastoma cell-converted neurons.
- B-C Robust synaptic puncta
- A Immunostaining of GSK3P (which stained magenta) in Neurog2-converted neurons (DCX, which stained red) from U251 human GBM cells at 20 dpi. Scale bars, 50 pm.
- B-C Quantitative analyses of GSIOp intensity (B) and neuronal conversion efficiency (C) after inhibiting GSK3p by CHIR99021 (5 pM) or TWS119 (10 pM) for 20 days. Data are represented as mean ⁇ SEM, and analyzed by Student’s /-test n > 3 repeats.
- FIG. 14 Investigation of cancer makers in Neurog2-converted neurons from human glioblastoma cells.
- A-B Immunostaining of IL13Ra2 (which stained red, A) in Neurog2-converted neurons from U251 human glioblastoma cells at 20 dpi. Quantified in panel (B). Scale bar, 50 pm.
- C-D Immunostaining of EGFR (which stained red, C) during neuronal conversion of U251 cells. Samples were collected at 20 dpi. Scale bar, 20 pm. Data are represented as mean ⁇ SEM, and were analyzed by Student’s /-test n > 40 cells from triplicate samples.
- FIG. 15 In vivo neuronal conversion of human glioblastoma cells in a xenograft mouse model.
- A Representative images illustrating the transplanted human U251 GBM cells (mixed with Neurog2-GFP retroviruses) in the brain of Ragl-/- immunodeficient mice at one month post transplantation. Note that U251 cells expressed a high level of vimentin, and Neurog2-GFP infected U251 cells were immunopositive for immature neuronal marker DCX.
- C High magnification images showing that most of the transplanted U251 cells (Vimentin) infected by Neurog2-GFP (bottom row) retroviruses were converted into neurons (DCX) at 1-week post transplantation.
- FIG. 16 Inhibition of cell proliferation and reduction of astrogliosis after in vivo neuronal conversion of glioblastoma cells.
- C-D Reduction of reactive astrocytes (labeled by LCN2) in the Neurog2-infected regions (D), compared to the contralateral GFP-infected regions (C). Samples were at three weeks post transplantation.
- FIG. 17 Resident microglia and blood vessel distribution during in vivo neuronal conversion of glioblastoma cells.
- FIG. Transduction of liver tumor cell line HepG2 by liver transcription factors Foxa2, HNF4A, and GATA4.
- A Transduced cells grown on glass coverslips were fixed with paraformaldehyde and bound with a mixture of chicken anti-GFP plus goat anti-Foxa2 or chicken anti-GFP plus goat anti-HNF4A or chicken anti-GFP plus goat anti-GATA4 antibodies. Secondary antibodies chicken-specific Alexa Fluor 488 and goat-specific Alexa Fluor 594 were used for detection. Fluorescence was visualized with Zeiss LSM800 confocal microscope.
- B Transduced cells grown in 12-well plate were harvested, and cell lysates were processed for SDS-PAGE and analyzed by western blotting with a mouse monoclonal anti-GFP antibody.
- C, D, E Cell lysates were fractionated by SDS-PAGE and processed for immunoblotting with a goat polyclonal anti-Foxa2, goat polyclonal anti- HNF4A or goat polyclonal anti-GATA4 antibody, respectively.
- A Foxa2, HNF4A, GATA4, or GFP transduced HepG2 cells were harvested, and cell lysates were fractionated by SDS-PAGE and analyzed by immunoblotting with a mixture of goat polyclonal anti-Foxa2 and mouse monoclonal anti-HNF4A antibodies. A rabbit GAPDH polyclonal antibody was used as internal loading control.
- B The above cell lysates were fractionated by SDS-PAGE and analyzed by immunoblotting with a mixture of goat polyclonal anti-GATA4 and rabbit polyclonal anti-GAPDH antibodies.
- Figure 20 In vitro and in vivo cell proliferation of Foxa2, GATA4, HNF4A, or GFP transduced cell lines.
- A In vitro proliferation curve of Foxa2, GATA4, HNF4A, or GFP transduced cell lines. Equal amount of Foxa2, GATA4, HNF4A, or GFP transduced cells were seeded in 12-well plate. At different time points, cells were fixed and stained with crystal violet. The stained crystal violet was extracted by acetic acid, and the optical density of each extraction was read by a microplate reader. The volume of optical density represent the cells number grown in each well. Each value represent three individual experiments.
- FIG 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 plus goat anti-albumin antibodies. Secondary antibodies chicken-specific Alexa Fluor 488 and goat-specific Alexa Fluor 594 were used for detection. Fluorescence was visualized with Zeiss LSM800 confocal microscope as described for Figure 18 A.
- B Albumin expressed in Foxa2, GATA4, HNF4A, or GFP transduced cells were detected by western blot with a goat polyclonal anti-albumin antibody. Rabbit polyclonal anti-GAPDH antibody was used to show internal loading control.
- C Relatively albumin production was calculated as the amount of albumin protein detected in Foxa2, GATA4, or HNF4A transduced cells normalized to the amount of albumin obtained with GFP transduced cells. Results were from three independent experiments.
- D Albumin concentration secreted into culture medium of Foxa2, GATA4, HNF4A, or GFP transduced cells. Albumin produced in Foxa2, GATA4, HNF4A, or GFP transduced cells were secreted into culture medium. The concentration of albumin in culture medium were measured by ELISA according to the ELISA kit instructions. The albumin concentration were obtained by comparison with a standard curve provided in the kit.
- FIG. 22 Liver cancer marker alpha fetoprotein (AFP) expression in Foxa2,
- GATA4, HNF4A, or GFP transduced cells were grown on coverslips, fixed, and stained with a mixture of chicken anti-GFP plus rabbit anti-AFP. A secondary antibodies mixture of chicken-specific Alexa Fluor 488 and rabbit-specific Alexa Fluor 594 was used for detection. Fluorescence was visualized with Zeiss LSM800 confocal microscope as described for Figure 18 A.
- B, Foxa2, GATA4, HNF4A, or GFP transduced cells were lysed and fractionated by SDS-PAGE and probed with a rabbit polyclonal anti-AFP for immunoblotting analysis. A rabbit GAPDH polyclonal antibody was used as internal loading control.
- AFP level was calculated as the amount of AFP detected in Foxa2, GATA4, or HNF4A transduced cells normalized to the amount of AFP obtained with GFP transduced cells. Results were from three independent experiments.
- C AFP levels in tumors formed by GFP, HNF4A, or GATA4 transduced cells. Tumor sections were permeabilized with Triton X-100, followed by incubation with primary antibodies chicken anti-GFP plus rabbit anti-AFP. A secondary antibodies mixture of chicken-specific Alexa Fluor 488 and rabbit-specific Alexa Fluor 594 were used for detection. Fluorescence was visualized with Zeiss LSM800 confocal microscope.
- Xenografted tumors of GATA4, HNF4A, or GFP cell lines were freshly collected and lysed. The lysates were separated by SDS-PAGE gel, and were probed with AFP, GATA4, and GFP antibodies. Actin was shown as internal loading control. Relatively AFP level was calculated as the amount of AFP detected in GATA4 or HNF4A transduced cells normalized to the amount of AFP obtained with GFP transduced cells as described for panel B. Results were from three independent experiments.
- FIG 23 Over-expression of GATA4, Foxa2, or HNF4A leads to an increase of membranous E-cadherin.
- A Foxa2, GATA4, HNF4A, or GFP transduced cells were stained with a mixture of chicken anti-GFP plus rabbit anti-E-cadherin. A secondary antibodies mixture of chicken-specific Alexa Fluor 488 and rabbit-specific Alexa Fluor 594 were used for detection. Fluorescence was visualized with Zeiss LSM800 confocal microscope as described for Figure 18A.
- B Lysed Foxa2, GATA4, HNF4A, or GFP transduced cells were fractionated by SDS-PAGE and probed with a rabbit polyclonal anti-Ecadherin for western blot analysis.
- a rabbit GAPDH polyclonal antibody was used as 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 with GFP transduced cells. Results were from three independent experiments.
- C Immunofluorescent images of E-cadherin showing increased E-cadherin in GATA4 or HNF4A transduced cells formed tumors compared to GFP tumor.
- D Tumor samples of GATA4, HNF4A, or GFP transduced cells were analysed quantitatively by western blot with anti-E-cadherin antibody. E-cadherin expressed more intensely with a measurable 2-fold higher intensity, as shown in GATA4 comparing with GFP expressing tumor cells.
- FIG. 24 Expression and redistribution of beta-catenin in GATA4 overexpressed cell line.
- A GATA4 or GFP transduced cells were stained with rabbit anti-beta-catenin antibody, and immunefluorescent images of beta-catenin were visualized with a microscope. Beta-catenin in GATA4 cells was distributed to cell surface.
- B Western blot analysis of Foxa2, GATA4, HNF4A, or GFP transduced cells with beta-catenin antibody and relative beta-catenin levels detected in Foxa2, GATA4, HNF4A, or GFP transduced cells. Results were from three independent experiments.
- C Immunofluorescent images of beta-catenin showing beta-catenin in GATA4 transduced cells formed tumors compared to GFP tumor.
- FIG. 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 with anti-vimentin antibody revealed vimentin levels decreased in GATA4 transduced cells formed tumors compared to GFP tumor. Decreased vimentin was calculated by comparison of the amount of vimentin detected in GATA4 transduced cells normalized to the amount of obtained with GFP transduced cells.
- Figure 26 Amino acid sequence of a representative NeuroDl polypeptide (SEQ ID NO: 1]
- Figure 27 Amino acid sequence of a representative Neurog2 polypeptide (SEQ ID NO:2).
- This document provides methods and materials for treating a mammal having cancer.
- nucleic acid encoding one or more transcription factors, or one or more transcription factors themselves can be used to treat a mammal having cancer.
- treating a mammal having cancer as described herein can include converting cancer cells within the mammal into non-cancerous cells (e.g ., functional cells or near normal cells) within the mammal.
- treating a mammal having cancer as described herein can have a conversion efficacy of, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
- treating a mammal having cancer as described herein can have a conversion efficacy from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent , from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 50 percent, from 40 to 45 percent, from 40 to 50 percent, from 40 to 55 percent, from 45 to 50 percent, from 45 to 55 percent, from 45 to 60 percent, from 50 to 55 percent, from 50 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 65 percent, from 55 to 70 percent, from 60 to 65 percent, from 60 to 70 percent, from 60 to 75 percent, from 65 to 70 percent, from 65 to 75 percent, from 65 to 80 percent, from 70 to
- treating a mammal having cancer as described herein can be effective to convert cancer cells within the mammal into non-cancerous cells from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent , from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 50 percent, from 40 to 45 percent, from 40 to 50 percent, from 40 to 55 percent, from 45 to 50 percent, from 45 to 55 percent, from 45 to 60 percent, from 50 to 55 percent, from 50 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 65 percent, from 55 to 70 percent, from
- nucleic acid designed to express one or more transcription factors 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).
- a mammal having cancer e.g., a mammal having cancer
- nucleic acid designed to express one or more neuronal agents e.g., a mammal having cancer
- transcription factors can be administered to a mammal (e.g, a human) having brain cancer as described herein to reduce the size of the brain cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
- nucleic acid designed to express one or more liver transcription factors can be administered to a mammal (e.g, a human) having liver cancer as described herein to reduce the size of the liver cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
- nucleic acid designed to express one or more liver transcription factors can be administered to a mammal (e.g, a human) having liver cancer as described herein to reduce the size of the liver cancer from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent , from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent, from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to
- 70 percent from 65 to 75 percent, from 65 to 80 percent, from 70 to 75 percent, from 70 to
- 90 percent from 85 to 95 percent, from 85 to 100 percent, from 90 to 95 percent, from 90 to 100 percent, or from 95 to 100 percent.
- nucleic acid designed to express one or more transcription factors can be administered to a mammal in need thereof (e.g ., a mammal having cancer) to increase the survival rate of the mammal (e.g, increase the five-year relative survival rate of the mammal) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years.
- nucleic acid designed to express one or more transcription factors can be administered to a mammal in need thereof (e.g.
- a mammal having cancer to increase the survival rate of the mammal (e.g, increase the five-year relative survival rate of the mammal) from 1 to 10 years , such as from 1 to 1.5 years, from 1 to 2 years, from 1 to 2.5 years, from 1.5 to 2 years, from 1.5 to
- nucleic acid designed to express one or more neuronal transcription factors can be administered to a mammal (e.g ., a human) having brain cancer as described herein to increase the survival rate of the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
- nucleic acid designed to express one or more neuronal transcription factors can be administered to a mammal (e.g., a human) having brain cancer as described herein to increase the survival rate of the mammal from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent , from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 50 percent, from 40 to 45 percent, from 40 to 50 percent, from 40 to 55 percent, from 45 to 50 percent, from 45 to 55 percent, from 45 to 60 percent, from 50 to 55 percent, from 50 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 65
- nucleic acid designed to express one or more liver transcription factors can be administered to a mammal (e.g, a human) having liver cancer as described herein to increase the survival rate of the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
- nucleic acid designed to express one or more liver transcription factors can be administered to a mammal (e.g, a human) having liver cancer as described herein to increase the survival rate of the mammal from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent , from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 50 percent, from 40 to 45 percent, from 40 to 50 percent, from 40 to 55 percent, from 45 to 50 percent, from 45 to 55 percent, from 45 to 60 percent, from 50 to 55 percent, from 50 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 60 to 65 percent, from 60 to 70 percent, from 60 to 65 percent, from 60 to 70 percent, from
- nucleic acid designed to express one or more transcription factors can be administered to a mammal in need thereof (e.g ., a mammal having cancer) to differentiate cancer cells in the mammal (e.g, to convert cancer cells into terminally differentiated and/or non-dividing cells within the mammal).
- nucleic acid designed to express one or more neuronal transcription factors can be administered to a mammal (e.g, a human) having brain cancer (e.g., a glioma such as GBM) as described herein to differentiate, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99, or more percent of the brain cancer cells (e.g., glioma cells) in the mammal into non-cancerous neurons in the brain of the living mammal (e.g, functional neurons that can be integrated into the brain of the living mammal).
- a mammal e.g, a human having brain cancer (e.g., a glioma such as GBM) as described herein to differentiate, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99, or more percent of the brain cancer cells (e.g., glioma cells) in the mammal into non-cancerous neurons in the brain of
- nucleic acid designed to express one or more neuronal transcription factors can be administered to a mammal (e.g, a human) having brain cancer (e.g., a glioma such as GBM) as described herein to differentiate from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent , from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 50 percent, from 40 to 45 percent, from 40 to 50 percent, from 40 to 55 percent, from 45 to 50 percent, from 45 to 55 percent, from 45 to 60 percent, from 50 to 55 percent, from 50 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 65 percent, from 55 to 70 percent, from
- nucleic acid designed to express one or more liver transcription factors can be administered to a mammal (e.g ., a human) having liver cancer as described herein to differentiate, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99, or more percent of the liver cancer cells in the mammal into non-cancerous hepatocytes in the liver of the living mammal (e.g., functional hepatocytes that can be integrated into the liver of the living mammal).
- a mammal e.g ., a human having liver cancer as described herein to differentiate, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99, or more percent of the liver cancer cells in the mammal into non-cancerous hepatocytes in the liver of the living mammal (e.g., functional hepatocytes that can be integrated into the liver of the living mammal).
- nucleic acid designed to express one or more liver transcription factors can be administered to a mammal (e.g, a human) having liver cancer as described herein to differentiate from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent , from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 50 percent, from 40 to 45 percent, from 40 to 50 percent, from 40 to 55 percent, from 45 to 50 percent, from 45 to 55 percent, from 45 to 60 percent, from 50 to 55 percent, from 50 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 60 to 65 percent, from 60 to 70 percent, from 60 to 75 percent, from 65 to 70 percent
- hepatocytes that can be integrated into the liver of the living mammal.
- nucleic acid designed to express one or more neuronal transcription factors can be administered to a mammal in need thereof (e.g, a mammal having brain cancer) to reduce astrogliosis in the mammal.
- nucleic acid 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 brain cancer as described herein to reduce astrogliosis in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
- nucleic acid designed to express one or more neuronal transcription factors can be administered to a mammal (e.g., a human) having brain cancer as described herein to reduce astrogliosis in the mammal from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent , from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 50 percent, from 40 to 45 percent, from 40 to 50 percent, from 40 to 55 percent, from 45 to 50 percent, from 45 to 55 percent, from 45 to 60 percent, from 50 to 55 percent, from 50 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60
- Any appropriate mammal can be treated as described herein.
- mammals that can have cancer and can be treated as described herein include, without limitation, humans, non human primates (e.g, monkeys), dogs, cats, cows, horses, pigs, rats, mice, rabbits, ferrets, and sheep.
- a human having cancer can be treated as described herein to reduce the number of cancer cells within the human, for example, by 10, 20, 30, 40, 50, 60 ,70, 80, 90, 95, 98, 99, or more percent.
- a human having cancer can be treated as described herein to reduce the number of cancer cells within the human from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent, from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 50 percent, from 40 to 45 percent, from 40 to 50 percent, from 40 to 55 percent, from 45 to 50 percent, from 45 to 55 percent, from 45 to 60 percent, from 50 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 60 to 65 percent, from 60 to 70 percent, from 60 to 75 percent, from 65 to 70 percent, from 65 to 75 percent, from 65 to 80 percent, from 70
- the cancer can be any type of cancer.
- a mammal refers to any organism classified in the class Mammalia.
- a human refers to the species Homo sapiens.
- a cancer can be a blood cancer.
- a cancer can include one or more solid tumors.
- a cancer can be a luminal cancer.
- a cancer can be a carcinoma cancer.
- a cancer can be a sarcoma cancer.
- a cancer can be a myeloma cancer.
- a cancer can be a leukemia cancer.
- a cancer can be a lymphoma cancer. In some cases, a cancer can be a mixed type cancer. In some cases, a cancer can be a primary cancer. In some cases, a cancer can be a secondary cancer. In some cases, a cancer can be a metastatic cancer. In some cases, a cancer can be stage 0 cancer. In some cases, a cancer can be stage I cancer. In some cases, a cancer can be stage II cancer. In some cases, a cancer can be stage IV cancer.
- cancers examples include, without limitation, brain cancers (e.g., gliomas such as GBM), liver cancers (e.g, HCC), breast cancer, prostate cancer, bone cancer, lung cancer, pancreatic cancer, cervical cancer, uterine cancer, gall bladder cancer, bladder cancer, esophageal cancer, skin cancer, kidney cancer, ovary cancer, and leukemia.
- the methods described herein can include identifying a mammal (e.g, a human) as having a cancer.
- Any appropriate method can be used to identify a mammal as having a cancer.
- 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 mammals (e.g, humans) having cancer.
- imaging techniques can be X-ray, computed tomography (CT scan), ultrasound, magnetic resonance imaging (MRI), position emission tomography (PET scan), and sonogram.
- biopsy techniques can be fine needle aspiration biopsy, core needle biopsy, vacuum-assisted biopsy, excisional biopsy, shave biopsy, punch biopsy, endoscopic biopsy, laparoscopic biopsy, and, bone marrow aspiration biopsy.
- a cytology can be fine needle aspiration biopsy, core needle biopsy, vacuum-assisted biopsy, excisional biopsy, shave biopsy, punch biopsy, endoscopic biopsy, laparoscopic biopsy, and, bone marrow aspiration biopsy.
- a cytology can be fine needle aspiration biopsy, core needle biopsy, vacuum-assisted biopsy, excisional biopsy, shave biopsy, punch biopsy, endoscopic biopsy, laparoscopic biopsy, and, bone marrow aspiration biopsy.
- a microscopy technique can be a light microscopy, an electron microscopy, a laser microscopy, and/or an optical microscopy.
- an histological staining technique can be an hematoxylin and eosin (H&E), an alcian blue stain, an aldehyde fuchsin stain, an alkaline phosphatase stain, a bielschowsky stain, a congo red stain, a crystal violet stain, a fontana-masson stain, a giemsa stain, a luna stain, a nissl stain, a periodic acid schiff stain, a red oil o stain, a reticulin stain, a Sudan black b stain, a toluidine blue stain, and/or a van gieson stain.
- a genetic testing technique can be
- one or more appropriate transcription factors for that cancer cell type can be selected for use as described herein.
- transcription factors such as NeuroDl, Neurog2, and/or Ascii can be selected and used to convert brain cancer cells into non-cancerous cells.
- transcription factors such as HNF4A, Foxa2, and/or GATA4 can be selected and used to convert liver cancer cells into non-cancerous cells.
- Other examples of transcription factors that can be selected for particular cancer cell types to convert those particular cancer cells into non-cancerous cells are set forth in Table 1.
- NP_849180.1 HNF4B, Foxal, Foxa2 (AAH11780.1 or ACA06111.1), GATA1, GATA2, GAT A3, and/or GATA4 (AAI43480.1;
- a mammal e.g ., a human having a brain cancer (e.g, a glioma such as GBM) can be treated by administering nucleic acid designed to express one or more neuronal transcription factors within the mammal’s brain (e.g, striatum) in a manner that triggers the brain cancer cells (e.g., glioma cells) to form non-cancerous neurons (e.g, functional, near normal, and/orintegrated neurons) within the mammal’s brain (e.g, striatum).
- neuronal transcription factors include, without limitation, NeuroDl polypeptides, Neurog2 polypeptides, and Ascii polypeptides.
- NeuroDl polypeptides include, without limitation, those polypeptides having the amino acid sequence set forth in GenBank ® accession number NP_002491 (GI number 121114306) or Q13562.3, or SEQ ID NO: l ( Figure 26).
- a NeuroDl polypeptide can be encoded by a nucleic acid sequence as set forth in GenBank ® accession number NM_002500 (GI number 323462174).
- Examples of Neurog2 polypeptides include, without limitation, those polypeptides having the amino acid sequence set forth in GenBank ® accession number NP_076924.1; EAX06278.1; or AAH36847.1, or SEQ ID NO:2 ( Figure 27).
- a Neurog2 polypeptide can be encoded by a nucleic acid sequence as set forth in GenBank ® accession number NM 024019.4.
- Examples of Ascii polypeptides include, without limitation, those polypeptides having the amino acid sequence set forth in GenBank ® accession number NP 004307.2 or SEQ ID NO:3 ( Figure 28).
- An Ascii polypeptide can be encoded by a nucleic acid sequence as set forth in GenBank ® accession number NM 004316.4.
- a mammal e.g ., a human having a liver cancer (e.g ., HCC) can be treated by administering nucleic acid designed to express one or more liver transcription factors within the mammal’s liver in a manner that triggers the liver cancer cells to form non-cancerous hepatocytes (e.g., functional, near-normal, and/or integrated hepatocytes) within the mammal’s liver.
- liver transcription factors include, without limitation, HNF4A polypeptides, Foxa2 polypeptides, and GATA4 polypeptides.
- HNF4A polypeptides include, without limitation, those polypeptides having the amino acid sequence set forth in GenBank® accession number XP_005260464.1; NP_000448.3; NP_001274113.1; NP_001274112.1;
- a HNF4A polypeptide can be encoded by a nucleic acid sequence as set forth in GenBank® accession number NM_178849.3.
- Foxa2 polypeptides include, without limitation, those polypeptides having the amino acid sequence set forth in GenBank® accession number AAH11780.1 or ACA06111.1, or SEQ ID NO:5 ( Figure 30).
- a Foxa2 polypeptide can be encoded by a nucleic acid sequence as set forth in GenBank® accession number NM_021784.5.
- GATA4 polypeptides include, without limitation, those polypeptides having the amino acid sequence set forth in GenBank® accession number AAI43480.1; NP_001295022.1; NP_002043.2; NP_001295023.1; or NP 001361203.1, or and SEQ ID NO:6 ( Figure 31).
- a GATA4 polypeptide can be encoded by a nucleic acid sequence as set forth in GenBank® accession NM_001308093.3.
- nucleic acid designed to express one or more transcription factors can be administered to a mammal using one or more vectors such as viral vectors.
- vectors such as viral vectors.
- separate vectors e.g., one vector for nucleic acid encoding a first transcription factor, and one vector for nucleic acid encoding a second transcription factor
- nucleic acids designed to express a transcription factor are delivered to cells within a living mammal
- a single vector containing both nucleic acid encoding a first transcription factor and nucleic acid encoding a second transcription factor can be used to deliver the nucleic acids to cells.
- Vectors for administering nucleic acid e.g ., nucleic acid designed to express one or more transcription factors
- cells e.g., cells within a living mammal
- nucleic acid e.g., nucleic acid designed to express one or more transcription factors
- a vector can be used to administer nucleic to any appropriate cell.
- a vector can be used to administer nucleic acid encoding a transcription factor to a dividing cell.
- a vector can be used to administer nucleic acid encoding a transcription factor to a non-dividing cell.
- a vector can be used to administer nucleic acid encoding a transcription factor to a cancer cell.
- vectors for administering nucleic acid e.g, nucleic acid designed to express one or more transcription factors
- cells e.g, cells within a living mammal
- nucleic acid e.g, nucleic acid designed to express one or more transcription factors
- vectors for administering nucleic acid e.g, nucleic acid designed to express one or more transcription factors
- cells e.g, cells within a living mammal
- vectors for administering nucleic acid e.g, nucleic acid designed to express one or more transcription factors
- cells e.g, cells within a living mammal
- vectors for administering nucleic acid e.g, nucleic acid designed to express one or more transcription factors
- cells e.g, cells within a living mammal
- a vector for administering nucleic acid e.g, nucleic acid designed to express one or more transcription factors
- administering nucleic acid can be used for stable expression of one or more transcription factors
- the vector can be engineered to integrate nucleic acid designed to express one or more transcription factors into the genome of a cell. In some cases, when vector is
- any appropriate method can be used to integrate that nucleic acid into the genome of a cell.
- gene therapy techniques can be used to integrate nucleic acid designed to express one or more transcription factors into the genome of a cell.
- Vectors for administering nucleic acids 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).
- a vector designed to administer nucleic acid encoding one or more transcription factors to cells can be an appropriate vector including, without limitation, 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.
- 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.
- nucleic acid encoding one or more transcription factors can be delivered to cells using adeno-associated virus vectors (e.g ., an AAV serotype 2 viral vector, an AAV serotype 5 viral vector, an AAV serotype 9 viral vector, or a recombinant AAV serotype viral vector such as an AAV serotype 2/5 viral vector), lentiviral vectors, retroviral vectors, adenoviral vectors, herpes simplex virus vectors, poxvirus vector, oncolytic vector, or non-viral vectors such as nanoparticles that mimic viral vectors.
- adeno-associated virus vectors e.g ., an AAV serotype 2 viral vector, an AAV serotype 5 viral vector, an AAV serotype 9 viral vector, or a recombinant AAV serotype viral vector such as an AAV serotype 2/5 viral vector
- lentiviral vectors e.g ., an AAV serotype 2 viral vector, an AAV
- nucleic acid encoding one or more neuronal transcription factors e.g., nucleic acid encoding a NeuroDl polypeptide, nucleic acid encoding a Neurog2 polypeptide, and/or nucleic acid encoding an Ascii
- nucleic acid encoding one or more liver transcription factors can be delivered to hepatocytes using one or more lentiviral vectors.
- a viral vector can contain regulatory elements operably linked to the nucleic acid encoding a transcription factor.
- regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, or inducible elements that modulate expression (e.g, transcription or translation) of a nucleic acid.
- the choice of element(s) that may be included in a viral vector depends on several factors, including, without limitation, inducibility, targeting, and the level of expression desired.
- a promoter can be included in a viral vector to facilitate transcription of a nucleic acid encoding a transcription factor.
- a promoter can be
- tissue-specific promoters that can be used to drive expression of a neural transcription factor in glial cells (e.g, cancerous glial cells) include, without limitation, GFAP, NG2, 01ig2,
- tissue-specific EFla EFla
- AldhlLl EFla
- CMV ubiquitin promoters
- promoters that can be used to drive expression of a liver transcription factor in hepatocytes include, without limitation, al -antitrypsin, albumin, AFP, CAG, CMV, EFla, and ubiquitin promoters.
- a viral vector can contain a glial-specific promoter operably linked to a nucleic acid encoding a neural transcription factor such that it drives transcription in glial cells (e.g ., cancerous glial cells).
- a viral vector can contain a liver-specific promoter operably linked to a nucleic acid encoding a liver transcription factor such that it drives transcription in hepatocytes (e.g., cancerous hepatocytes).
- Nucleic acid encoding one or more transcription factors can be administered to a mammal using non-viral vectors. Methods of using non-viral vectors for nucleic acid delivery are described elsewhere. See, for example, Gene Therapy Protocols Methods in Molecular Medicine), edited by Jeffrey R. Morgan, Humana Press, Totowa, NJ (2002).
- nucleic acid encoding one or more transcription factors can be administered to a mammal by direct injection of nucleic acid molecules (e.g, plasmids) comprising nucleic acid encoding one or more transcription factors, or by administering nucleic acid molecules complexed with lipids, polymers, or nanospheres.
- a genome editing technique such as CRISPR/Cas9-mediated gene editing can be used to activate endogenous
- Nucleic acid encoding a transcription factor can be produced by techniques including, without limitation, common molecular cloning, polymerase chain reaction (PCR), chemical nucleic acid synthesis techniques, and combinations of such techniques.
- PCR polymerase chain reaction
- RT-PCR can be used with oligonucleotide primers designed to amplify nucleic acid (e.g, genomic DNA or RNA) encoding a transcription factor.
- one or more transcription factors can be administered in addition to or in place of nucleic acid designed to express one or more transcription factors.
- nucleic acid designed to express one or more transcription factors can be administered in addition to or in place of nucleic acid designed to express one or more transcription factors.
- NeuroDl polypeptides, Neurog2 polypeptides, and/or Ascii polypeptides can be administered in addition to or in place of nucleic acid designed to express one or more transcription factors.
- NeuroDl polypeptides, Neurog2 polypeptides, and/or Ascii polypeptides can be administered in addition to or in place of nucleic acid designed to express one or more transcription factors.
- HNF4A polypeptides, Foxa2 polypeptides, and/or GATA4 polypeptides can be administered to a mammal to trigger liver cancer cells within the liver into converting into ( e.g ., to differentiate into) non-cancerous hepatocytes in the liver of the living mammal (e.g ., functional hepatocytes that can be integrated into the liver of the living mammal).
- nucleic acid designed to express one or more transcription factors can be administered to a mammal (e.g., a human) having cancer to treat the mammal.
- 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 that designed viral vector can be administered to a human having brain cancer to treat the mammal.
- a polypeptide having an amino acid sequence with 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: 1 can be used.
- a polypeptide containing the entire amino acid sequence set forth in SEQ ID NO: 1, except that the amino acid sequence contains from one to ten (e.g, ten, one to nine, two to nine, one to eight, two to eight, one to seven, one to six, one to five, one to four, one to three, two, or one) amino acid additions, deletions, substitutions, or combinations thereof, can be used.
- nucleic acid designed to express a polypeptide containing an amino acid sequence with between 90% and 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 can be designed and administered to a mammal (e.g, human) having brain cancer (e.g, a glioma such as GBM) to treat the mammal.
- a mammal e.g, human
- brain cancer e.g, a glioma such as GBM
- a polypeptide having an amino acid sequence with 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:2 can be used.
- a polypeptide containing the entire amino acid sequence set forth in SEQ ID NO:2, except that the amino acid sequence contains from one to ten ( e.g ., ten, one to nine, two to nine, one to eight, two to eight, one to seven, one to six, one to five, one to four, one to three, two, or one) amino acid additions, deletions, substitutions, or combinations thereof, can be used.
- nucleic acid designed to express a polypeptide containing an amino acid sequence with between 90% and 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2 can be designed and administered to a mammal (e.g., human) having brain cancer (e.g, a glioma such as GBM) to treat the mammal.
- a mammal e.g., human
- brain cancer e.g, a glioma such as GBM
- a polypeptide having an amino acid sequence with 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:3 can be used.
- a polypeptide containing the entire amino acid sequence set forth in SEQ ID NO:3, except that the amino acid sequence contains from one to ten (e.g, ten, one to nine, two to nine, one to eight, two to eight, one to seven, one to six, one to five, one to four, one to three, two, or one) amino acid additions, deletions, substitutions, or combinations thereof, can be used.
- nucleic acid designed to express a polypeptide containing an amino acid sequence with between 90% and 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3 can be designed and administered to a mammal (e.g, human) having brain cancer (e.g, a glioma such as GBM) to treat the mammal.
- a mammal e.g, human
- brain cancer e.g, a glioma such as GBM
- nucleic acid designed to express a polypeptide having an amino acid sequence with 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:3 can be designed and administered to a mammal (e.g, human) having brain cancer (e.g, GBM) to treat the mammal.
- a mammal e.g,
- nucleic acid designed to express a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, nucleic acid designed to express a polypeptide having the amino acid sequence set forth in SEQ ID NO:5, and nucleic acid designed to express a polypeptide having the amino acid sequence set forth in SEQ ID NO: 6 can be administered to a mammal (e.g, a human) having liver cancer (e.g, HCC) as described herein to treat the mammal.
- a mammal e.g, a human
- liver cancer e.g, HCC
- 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 that designed viral vector can be administered to a human having liver cancer to treat the mammal.
- a polypeptide having an amino acid sequence with 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:4 can be used.
- a polypeptide containing the entire amino acid sequence set forth in SEQ ID NO:4, except that the amino acid sequence contains from one to ten (e.g, ten, one to nine, two to nine, one to eight, two to eight, one to seven, one to six, one to five, one to four, one to three, two, or one) amino acid additions, deletions, substitutions, or combinations thereof, can be used.
- nucleic acid designed to express a polypeptide containing an amino acid sequence with between 90% and 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 can be designed and administered to a mammal (e.g, human) having liver cancer (e.g, HCC) to treat the mammal.
- a mammal e.g, human
- liver cancer e.g, HCC
- a polypeptide having an amino acid sequence with 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.
- a polypeptide containing the entire amino acid sequence set forth in SEQ ID NO:5, except that the amino acid sequence contains from one to ten (e.g, ten, one to nine, two to nine, one to eight, two to eight, one to seven, one to six, one to five, one to four, one to three, two, or one) amino acid additions, deletions, substitutions, or combinations thereof, can be used.
- nucleic acid designed to express a polypeptide containing an amino acid sequence with between 90% and 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 ., human) having liver cancer (e.g, HCC) to treat the mammal.
- a mammal e.g ., human
- liver cancer e.g, HCC
- a polypeptide having an amino acid sequence with 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:6 can be used.
- a polypeptide containing the entire amino acid sequence set forth in SEQ ID NO: 6, except that the amino acid sequence contains from one to ten (e.g, ten, one to nine, two to nine, one to eight, two to eight, one to seven, one to six, one to five, one to four, one to three, two, or one) amino acid additions, deletions, substitutions, or combinations thereof, can be used.
- nucleic acid designed to express a polypeptide containing an amino acid sequence with between 90% and 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6 can be designed and administered to a mammal (e.g, human) having liver cancer (e.g, HCC) to treat the mammal.
- a mammal e.g, human
- liver cancer e.g, HCC
- nucleic acid designed to express a polypeptide having an amino acid sequence with 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:6 can be designed and administered to a mammal (e.g, human) having liver cancer (e.g, HCC) to treat the mammal.
- a mammal e.g,
- 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, a nucleic acid or amino acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained online at world wide web dot“fr” dot“com/blast” or at world wide web dot“ncbi.nlm.nih” dot“gov”. Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
- BLASTN is used to compare nucleic acid sequences
- BLASTP is used to compare amino acid sequences.
- the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g ., C: ⁇ seql.txt); -j is set to a 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 setting.
- the following command can be used to generate an output file containing a comparison between two sequences: C: ⁇ B12seq -i c: ⁇ seql.txt -j c: ⁇ seq2.txt -p blastn -o c: ⁇ output.txt -q -1 - r 2.
- B12seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g, C: ⁇ seql.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g, C: ⁇ seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g, C: ⁇ output.txt); and all other options are left at their default setting.
- -i is set to a file containing the first amino acid sequence to be compared (e.g, C: ⁇ seql.txt)
- -j is set to a file containing the second amino acid sequence to be compared (e.g, C: ⁇ seq2.txt)
- -p is set to blastp
- -o is set to any desired file name (e.g, C: ⁇ output.txt); and all other options are left at their default setting.
- the following command can be used to generate an output file containing a comparison between two amino acid sequences: C: ⁇ B12seq -i c: ⁇ seql.txt -j c: ⁇ seq2.txt -p blastp -o c: ⁇ output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
- the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.
- the percent sequence identity is determined by dividing the number of matches by the length of the sequence set forth in the identified sequence (e.g, SEQ ID NO: 1), followed by multiplying the resulting value by 100.
- 75.11, 75.12, 75.13, and 75.14 is rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 is rounded up to 75.2. It also is noted that the length value will always be an integer.
- a brain cancer cell e.g., a glioma cell
- a non-cancerous neuron within the brain of a living mammal e.g., a human
- a brain cancer as described herein e.g, by administering nucleic acid encoding one or more neuronal transcription factors such as NeuroDl, Neurog2, and/or Ascii, or one or more neuronal transcription factors themselves
- the converted neuron can be any appropriate type of neuron.
- a converted neuron can be DARPP32-positive.
- a converted neuron can be a FoxGl -positive forebrain neuron.
- a converted neuron can be a functional neuron (e.g, can have functional synaptic networks).
- a functional neuron can be a glutamatergic neuron or a GABAergic neuron.
- a converted neuron can have active electrophysiological properties.
- a converted neuron can be integrated into the brain of a living mammal (e.g, can include axonal projections that extend out of the striatum).
- a converted neuron can exhibit downregulated signaling pathways related to cancer progression (e.g, as compared to the brain cancer cells prior to conversion).
- liver cancer cell When converting a liver cancer cell to a non-cancerous hepatocyte within the liver of a living mammal (e.g, a human) with a liver cancer as described herein (e.g., by administering nucleic acid encoding one or more liver transcription factors (e.g, nucleic acid encoding
- the converted hepatocyte can be any appropriate type of hepatocyte.
- a converted hepatocyte can be a functional hepatocyte (e.g, can produce cholesterol, bile acids, and/or one or more liver enzymes such as albumin).
- a converted hepatocyte can be integrated into the liver of a living mammal (e.g, can form tight junctions and/or adherins junctions with hepatocytes in the liver of a living mammal).
- a converted hepatocyte can have decreased proliferation (e.g, as compared to the liver cancer cells prior to conversion).
- decreased proliferation can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, decreased proliferation can be from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from
- a converted hepatocyte 35 percent, from 25 to 30 percent , from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 50 percent, from 40 to 45 percent, from 40 to 50 percent, from 40 to 55 percent, from 45 to 50 percent, from 45 to 55 percent, from 45 to 60 percent, from 50 to 55 percent, from 50 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 55 to 65 percent, from 55 to 70 percent, from 60 to 65 percent, from 60 to 70 percent, from 60 to 75 percent, from 65 to 70 percent, from 65 to 75 percent, from 65 to 80 percent, from 70 to 75 percent, from 70 to 80 percent, from 70 to 85 percent, from 75 to 80 percent, from 75 to 85 percent, from 75 to 90 percent, from 80 to 85 percent, from 80 to 90 percent, from 80 to 95 percent, from 85 to 90 percent, from 85 to 95 percent, from 85 to 100 percent, from 90 to 95 percent, from 90 to 100 percent, from 90 to 95 percent, from 90 to
- decreased expression of one or more liver cancer markers can be from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent , from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 50 percent, from 40 to 45 percent, from 40 to 50 percent, from 40 to 55 percent, from 45 to 50 percent, from 45 to 55 percent, from 45 to 60 percent, from 50 to 55 percent, from 50 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 60 to 65 percent, from 60 to 70 percent, from 60 to 75 percent, from 65 to 70 percent, from 65 to 75 percent, from 65 to 80 percent, from 70 to 75 percent, from 70 to 80 percent, from 70 to 100 percent, such as from 10 to 15 percent, from 10 to 20
- a liver cancer marker includes, without limitation, AFP.
- a converted hepatocyte can have increased expression of one or more epithelial-specific markers (e.g., as compared to the liver cancer cells prior to conversion).
- increased expression of one or more epithelial-specific markers can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
- increased expression of one or more epithelial-specific markers can be from 10 to 100 percent, such as from 10 to 15 percent, from 10 to 20 percent, from 10 to 25 percent, from 15 to 20 percent, from 15 to 25 percent, from 15 to 30 percent, from 20 to 25 percent, from 20 to 30 percent, from 20 to 35 percent, from 25 to 30 percent , from 25 to 35 percent, from 25 to 40 percent, from 30 to 35 percent, from 30 to 40 percent, from 35 to 45 percent, from 35 to 50 percent, from 40 to 45 percent, from 40 to 50 percent, from 40 to 55 percent, from 45 to 50 percent, from 45 to 55 percent, from 45 to 60 percent, from 50 to 55 percent, from 50 to 60 percent, from 50 to 65 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 60 percent, from 55 to 65 percent, from 55 to 70 percent, from 60 to 65 percent, from 60 to 70 percent, from 60 to 75 percent, from 65 to 70 percent, from 65 to 75 percent, from 65 to 80 percent, from 70 to 75 percent, from 70 to 80 percent, from 70
- Nucleic acid designed to express one or more transcription factors can be administered to a mammal (e.g ., a human) having cancer by any appropriate route.
- administration can be local administration.
- administration can be systemic administration. Examples of routes of administration include, without limitation, intravenous, intramuscular, intrathecal, intracerebral,
- intraparenchymal subcutaneous, oral, intranasal, inhalation, transdermal, parenteral,
- a first round of treatment can include
- administering nucleic acid designed to express one or more transcription factors (or the one or more transcription factors themselves) described herein to a mammal (e.g., a human) by a first route (e.g, intravenously), and a second round of treatment can include administering nucleic acid designed to express one or more transcription factors (or the one or more transcription factors themselves) described herein to a mammal (e.g, a human) by a second route (e.g, intratumorally).
- nucleic acid designed to express one or more transcription factors (or the one or more transcription factors themselves) described herein can be formulated into a composition (e.g, a pharmaceutical composition) for administration to a mammal (e.g, a mammal having, or at risk of having, cancer).
- a composition e.g, a pharmaceutical composition
- nucleic acid 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.
- nucleic acid designed to express one or more transcription factors (or the one or more transcription factors themselves) can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
- composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, wafers, and granules.
- Pharmaceutically acceptable carriers, fillers, and vehicles that may be used in a pharmaceutical composition described herein include, without limitation, 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, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, sodium carboxymethylcellulose, polyacryl
- methods described herein also can include administering to a mammal (e.g., a mammal having cancer) one or more additional agents used to treat a cancer.
- the one or more additional agents used to treat a cancer can include any appropriate cancer treatment.
- a cancer treatment can include surgery and/or radiation therapy.
- a cancer treatment can include administration of a pharmacotherapy such as a chemotherapy, hormone therapy, targeted therapy, and/or cytotoxic therapy.
- a mammal having cancer can be administered nucleic acid designed to express one or more transcription factors (or the one or more transcription factors themselves) described herein and administered one or more additional agents used to treat a cancer.
- nucleic acid designed to express one or more transcription factors (or the one or more transcription factors themselves) described herein and is treated with one or more additional agents used to treat a cancer can be administered at the same time or independently.
- nucleic acid designed to express one or more transcription factors (or the one or more transcription factors themselves) described herein and one or more additional agents used to treat a cancer can be formulated together to form a single composition.
- nucleic acid designed to express one or more transcription factors (or the one or more transcription factors themselves) described herein can be administered first, and the one or more additional agents used to treat a cancer administered second, or vice versa.
- Example 1 Converting human glioblastoma cells into neurons
- GBM is the most prevalent and aggressive adult primary cancer in the central nervous system (CNS). Current standard GBM therapy is surgery, followed by radio- or
- This Example provides an alternative approach for treating GBM through
- transcription factor reprogramming e.g. , Neurog2, NeuroDl, and/or Ascii reprogramming
- GBM cell lines were purchased from Sigma (U251) or ATCC (U118). U251 cells were cultured in GBM culture medium, which included MEM (GIBCO), 0.2% penicillin/streptomycin (GIBCO), 10% FBS (GIBCO), 1 mM Sodium Pyruvate (GIBCO),
- U118 cells were cultured in culture medium including DMEM (GIBCO), 10% FBS, and 1%
- Human astrocytes were purchased from ScienCell (HA1800, San Diego, USA).
- Human astrocytes were cultured in human astrocyte medium, which included 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, Invitogen), and 10 ng/mL epidermal growth factor (EFG, Invitrogen).
- DMEM/F12 DMEM/F12
- FBS fetal bovine serum
- 3.5 mM Glucose Sigma
- penicillin/streptomycin supplemented with B27 (GIBCO), N2 (GIBCO), 10 ng/mL fibroblast growth factor 2 (FGF2, Invitogen), and 10 ng/mL epidermal growth factor (EFG, Invitrogen).
- cells were trypsinized by 0.25% Trypsin (GIBCO) or TrypLE Select (Invitrogen), centrifuged for 5 minutes at 800 rpm, re-suspended and plated in corresponding culture medium with a split ratio around 1 :4. Cells were maintained at 37°C in humidified air with 5% CO2.
- U251 cells were seeded in poly-D-lysine-coated coverslips in 24-well plates at least twelve hours before the virus infection with a density of 10,000 cells per coverslip.
- GFP, Neurog2, NeuroDl, or Ascii retrovirus was added in GBM cells together with 8 pg/mL Polybrene (Santa Cruz Biotechnology). Culture medium was completely replaced by neuronal differentiation medium (NDM) the next day to help with neuronal differentiation and maturation.
- NDM neuronal differentiation medium
- NDM included DMEM/F12 (GIBCO), 0.4% B27 supplement (GIBCO), 0.8% N2 supplement (GIBCO), 0.2% penicillin/streptomycin, 0.5% FBS, Vitamin C (5 pg/mL, Selleck Chemicals), Y27632 (1 pM, Tocris), GDNF (10 ng/mL, Invitrogen), BDNF (10 ng/mL, Invitrogen) and NT3 (10 ng/mL, Invitrogen). Cells were maintained at 37°C in humidified air with 5% CO2.
- U251 cells were infected by retroviruses expressing Neurog2-GFP or GFP alone; the next day, culture medium was completely replaced by neuronal differentiation medium (NDM) with small molecules, or 0.22% DMSO for control.
- NDM neuronal differentiation medium
- the infected glioblastoma cells were treated with 5 pM DAPT, 1.5 pM CHIR99021, 5 pM SB431542, 0.25 pM LDN193189, 1 pM SAG, and 1 pM purmorphamine.
- the small molecule-contained medium was refreshed every 3-4 days. Cells were first treated in small molecules for 12 days and then changed to NDM for desired time periods before immunostaining.
- retroviruses were used to overexpress Neurog2 (CAG: :Neurog2-P2A- eGFP), NeuroDl (CAG::NeuroDl-P2A-eGFP), or Ascii (CAG::Ascll-P2A-eGFP), yielding high infection efficiency in fast-proliferating glioblastoma cells. After twelve-hour incubation with viruses, glioblastoma culture medium was changed to neuronal
- Figure 4B quantified in Figure 4D: Neruog2, 93.2% ⁇ 1.2%, NeuroDl, 91.2% ⁇ 1.1%, Ascii, 62.1% ⁇ 5.9%, MAP2+
- the converted neuronal subtypes were characterized according to the neurotransmitters released, in particular glutamatergic and GABAergic neurons, which are the principal excitatory and inhibitory neurons in the brain, respectively.
- Most Neurog2-, NeuroDl-, and Ascii -converted cells were immunopositive for glutamatergic neuron marker VGluTl (Figure 6C; quantified in Figure 6G: Neurog2, 92.8% ⁇ 0.7%; NeurDl, 86.9% ⁇ 2.7%; Ascii, 80.6% ⁇ 2.1%; VGluTl+/DCX+ cells).
- the Neurog2-induced conversion process was investigated.
- the astrocyte marker GFAP and the epithelial-mesenchymal transition (EMT) marker vimentin were both highly expressed in human U251 cells. After Neurog2 overexpression for 20 days, both GFAP and vimentin were downregulated compared with control ( Figure 9A). This further confirmed the fate change from glioblastoma cells to neurons.
- EMT epithelial-mesenchymal transition
- the gap junction marker Connexin 43 was downregulated in U251 glioblastoma cells with Neurog2 overexpression (Figure 9B; quantified Connexin 43 intensity in Figure 9C: Neurog2, 19.4 ⁇ 0.7 a.u.; GFP control, 11.6 ⁇ 0.8 a.u.; at 20 days post infection), consistent with the fact that neurons have less gap junctions compared with glial cells.
- Figure 9D A typical axonal growth cone structure was found in some of the Neurog2-converted neurons ( Figure 9D), with fmgerlike filopodia labeled by filamentous actin (F-actin) probe Phalloidin and growth cone marker GAP43 ( Figure 9D).
- Neurog2-converted cells The capability of the Neurog2-converted cells to form synapses was investigated by performing immunostaining for synaptic vesicle marker SV2. Intensive synaptic puncta were detected along MAP2-labeled dendrites in the Neurog2-converted neurons from human GBM cells at 30 days post infection (Figure 12A). Patch-clamp recordings showed significant sodium and potassium currents in the converted cells at 30 days post infection ( Figure 12B and 12C). The majority of Neurog2-converted cells fired single action potential (14 out of 23), with a subset of the converted neurons (8 out of 23) firing multiple action potentials ( Figure 12D and 12E).
- Neurons are terminally differentiated non-proliferating cells. Therefore, neuronal transdifferentiation may be a promising strategy to control cancer cell proliferation.
- Cell proliferation was examined at the early stage of conversion. U251 cells were incubated with 10 mM BrdU for 24 hours to trace the proliferative cells before fixation and staining at 7 days post viral infection (Figure 10A). Quantification of the percentage of BrdU positive cells showed that the proliferation of Neurog2- and NeuroDl -infected cells decreased significantly compared with GFP control (Figure 10B: GFP, 64.8% ⁇ 4.1%; Neurog2, 11.9% ⁇ 2.9%; NeuroDl, 24.5% ⁇ 2.4%).
- Transplanted U251 human GBM cells were identified by vimentin ( Figure 15 A) or human nuclei staining ( Figure 15D).
- Neurog2 overexpression ( Figure 15 A) in the transplanted human glioblastoma cells led to an efficient neuronal conversion, indicated by immature neuronal marker DCX ( Figure 15A-C, quantified in 15B: Neurog2, 92.8% ⁇ 1.2%, DCX+ neurons/total infected cells at 3 weeks post transplantation).
- Other neuronal makers such as Tuj 1 and Proxl were also detected in the Neurog2-converted cells at one-month post transplantation ( Figure 15D and 15E).
- Neurog2 as a representative reprogramming factor, efficiently reprograms human glioblastoma cells into neuron-like cells in vivo in a xenograft mouse model. Moreover, 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 neurons both in vitro and in vivo , leading to an alternative therapeutic approach to treat cancer (e.g., brain tumors).
- cancer cells e.g., GBM cells
- Example 2 Conversion of liver cancer cells into non-cancerous hepatocytes
- liver transcription factors e.g ., GATA4, Foxa2, and/or HNF4A
- GATA4, Foxa2, and/or HNF4A can be used to mediate tumor cell reprogramming and convert tumor cells into normal-like cells, establishing a novel strategy for the treatment of liver cancer or other type of cancers.
- 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 My coSolutionsTM (AKRON) to detect Mycoplasma contamination.
- AKRON My coSolutionsTM
- Chicken polyclonal or mouse monoclonal antibodies specific to GFP was purchased from Abeam. 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 from Santa Cruz and rabbit GAPDH polyclonal antibody was from Abeam. Rabbit monoclonal antibodies specific to AFP protein and E-cadherin, and mouse monoclonal antibodies specific to HNF4A protein were purchased from Abeam. Rabbit anti- B-catenin polyclonal antibody and goat anti-vimentin polyclonal antibody were obtained from Abeam 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, IRDye 800 Donkey anti-Goat secondary antibodies were purchased from LI-COR.
- mice at 4-5 weeks old Male immunodeficient athymic nude mice at 4-5 weeks old were obtained from Charles River.
- the Agel/Ecorl fragment of GATA4 (or Foxa2 or HNF4A) -P2A-GFP was cloned into the 3nd generation lentivirus vector, pLJMl (Addgene), replacing the existing green fluorescent protein (GFP) sequence.
- the resultant vector plasmids were used to generate the lentiviruses. Lentiviruses were produced by using PEI transfection method.
- plasmid psPAX2 (Addgene). The virus-containing medium was harvested 72 hours after transfection, filtered to remove cells or cell debris, and concentrated by ultracentrifugation. Viruses titers were determined by infection of HEK293T cells, and GFP positive cells were counted for calculating transducing units per milliliter (TU/mL).
- PFA paraformaldehyde
- GATA4, Foxa2, HNF4A, or GFP transduced liver tumor HepG2 cell lines were grown to 80% confluence, counted, and suspended in PBS. Each mouse was subcutaneously injected with 1.0 x 10 6 tumor cells into the right flank. Animals were inspected and tumor growth was monitored every 3 to 4 days throughout the experiment. Tumors were measured with a sliding caliper, and tumor volume was calculated using the following formula: 0.5 x ah 2 (a, major axis; b, minor axis). Mice were euthanized, and tumors were dissected and incubated in 4% PFA at 4°C. Tumors were sliced and analyzed by immunefluorescent staining.
- HepG2 cells were plated in 6 cm culture dish at density of 5 x 10 5 cells and incubate for overnight to allow cells attach. HepG2 cells were infected with lentiviruses at an MOI of 1 in 2 mL of fresh DMEM supplemented with 2% FBS. Cultures were incubated at 37°C for 2 days.
- PVDF polyvinylidene difluoride
- Tumor sections were permeabilized in PBS with 0.3% Triton X-100 for 1 hour, followed by incubation in blocking solution with 0.3% Triton X-100 in PBS for 1 hour. Tumor sections were incubated with primary antibodies mixed in blocking solution overnight at 4°C. After washing away unbound primary antibodies with PBS, tumor sections were incubated with a mixture of Alexa Fluor 488- or Alexa Fluor 594- secondary antibodies (Jackson ImmunoReaseach) for 1 hour at room temperature, unbound secondary antibodies were washed away with PBS, and nuclei were stained with DAPF Tumor sections were visualized with a confocal microscope (Zeiss LSM800).
- GATA4, Foxa2, HNF4A, or GFP transduced liver tumor HepG2 cell lines were seeded in 12-well plate, incubated for six hours to allow cells attached to plate. The culture medium was replaced with serum free DMEM, and incubation was continued for 16 hours. Culture medium from each cell line was collected, and the albumin amount was measured with the Human Serum Albumin ELISA Kit (Molecular Innovations) according to the kit instructions.
- liver tumor cell line HepG2 with liver transcription factors Foxa2, HNF4A, and GATA4
- pLJMl lentiviral vectors carrying Foxa2-P2A-GFP, HNF4A-P2A-GFP, GATA4- P2A-GFP, or GFP were used to infected HepG2 cells. Forty-eight hours later, puromycin was added into the medium to eliminate uninfected cells. Puromycin resistant cells were routinely propagated, and transcription factors or GFP expression in cells were evaluated by immunostaining or western blot.
- HepG2-Foxa2 Foxa2-P2A-GFP transduced
- HepG2-HNF4A HNF4A- P2A-GFP transduced
- HepG2-GATA4 GATA4-P2A-GFP transduced
- HepG2-GFP GFP transduced
- GATA4, Foxa2, HNF4A, or GFP transduced cell lines were cultured in 12-well dish. At 6-, 24-, 48-, and 72-hour time points, cells from each cell line were fixed with 4% PFA and stained with 0.1% crystal violet. Stained crystal violet was extracted by 10% acetic acid, and relative growth rates of GATA4, Foxa2, HNF4A, or GFP transduced cell lines were compared by spectrophotometric measurement. As shown in Figure 20A, cell lines transduced with GATA4, Foxa2, or HNF4A exhibited reduced growth rates compared with the GFP transduced control cell line. Furthermore, Foxa2 and GATA4 mediated cell lines achieved much lower cell growth rate.
- GATA4, Foxa2, HNF4A, or GFP transduced cell lines were subcutaneously xenografted into a nude mouse model. Nude mice were randomly assigned into 4 groups with 6 mice/group, and 1 x 10 6 cells transduced with GATA4, Foxa2, HNF4A, or GFP were implanted into the flank of nude mice. After 4 days, GFP and HNF4A transduced cell lines started to form tumors. The Foxa2 transduced cell line did not form any visible tumor. The GATA4 transduced cell line showed small tumor growth at later time points. The results revealed that Foxa2 or GATA4 in reprogrammed HepG2 cells decreased cell proliferation (Figure 20B).
- AFP expressed in GATA4, Foxa2, HNF4A, or GFP transduced cell lines localized in the cytosol ( Figure 22A) as shown by immunostaining. In the GATA4 and the Foxa2 transduced cell lines, AFP expression was reduced. By western blot examination, the AFP expression amount in the GATA4 cell line was reduced more 60 percent as compared with GFP transduced cell line ( Figure 22B).
- HepG2 tumors were developed in nude mice with subcutaneously xenografted GATA4, HNF4A, or GFP cell lines, and tumor samples were collected. The Foxa2 transduced cell line lost the ability of tumor formation.
- GATA4, HNF4A, or GFP cell lines were fixed in PFA, cut, and analyzed by Immunofluorescence. As shown in Figure 22C, AFP produced in the GATA4 cell line was decreased, and AFP produced in the HNF4A cell line was mildly reduced.
- the fresh HepG2 tumor samples developed with GATA4, HNF4A, or GFP cell lines were also subjected to western blot analysis. GATA4 was overexpressed in the tumors formed from the GATA4 cell line, and AFP expression level was reduced (Figure 22D). Decreased AFP expression was observed both the in vivo and in vitro tests using the GATA4 cell line.
- 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 antibody.
- the immunofluorescence analysis indicated that E-cadherin was expressed more intensely in Foxa2, HNF4A, and GATA4 transduced cells when compared to GFP expressing cells.
- E-cadherin localized at the cell membrane ( Figure 23 A).
- To examine whether GATA4 expression rescues E- cadherin expression cell lines that express GATA4, Foxa2, HNF4A, and GFP were harvested, lysed, and analyzed by western blot. E-cadherin was rescued by more than 2-fold in GATA4 expression line ( Figure 23B), reflecting the results obtained by
- E-cadherin level elevation may be used as an indicator of functional improvement for reprogrammed tumor cells.
- transduced cells also showed increased beta-catenin expression.
- Tumor samples 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 as compared to GFP tumors. Vimentin expression levels in GATA4 tumor were quantified, and the reduction of vimentin in GATA4 tumors was more than 2-fold when compared to GFP tumors.
- MET mesenchymal to epithelial transformation
- EMT epithelial to mesenchymal transition
- cancer cells e.g., liver cancer cells
- normal-like cells e.g., normal-like liver cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/439,733 US20220152224A1 (en) | 2019-03-26 | 2020-03-26 | Methods and materials for treating cancer |
CN202080030149.8A CN113710285A (en) | 2019-03-26 | 2020-03-26 | Methods and materials for treating cancer |
CA3134656A CA3134656A1 (en) | 2019-03-26 | 2020-03-26 | Methods and materials for treating cancer |
EP20777797.0A EP3946469A4 (en) | 2019-03-26 | 2020-03-26 | Methods and materials for treating cancer |
JP2021557370A JP2022527277A (en) | 2019-03-26 | 2020-03-26 | Methods and Materials for Treating Cancer |
AU2020248012A AU2020248012A1 (en) | 2019-03-26 | 2020-03-26 | Methods and materials for treating cancer |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201962823702P | 2019-03-26 | 2019-03-26 | |
US62/823,702 | 2019-03-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020198485A1 true WO2020198485A1 (en) | 2020-10-01 |
Family
ID=72609115
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2020/024976 WO2020198485A1 (en) | 2019-03-26 | 2020-03-26 | Methods and materials for treating cancer |
Country Status (7)
Country | Link |
---|---|
US (1) | US20220152224A1 (en) |
EP (1) | EP3946469A4 (en) |
JP (1) | JP2022527277A (en) |
CN (1) | CN113710285A (en) |
AU (1) | AU2020248012A1 (en) |
CA (1) | CA3134656A1 (en) |
WO (1) | WO2020198485A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10973930B2 (en) | 2016-02-18 | 2021-04-13 | The Penn State Research Foundation | Generating GABAergic neurons in brains |
US20220098617A1 (en) * | 2020-09-29 | 2022-03-31 | NeuExcell Therapeutics Inc. | Ascl1 vector |
EP4045094A4 (en) * | 2019-10-16 | 2024-02-21 | Univ Pittsburgh Commonwealth Sys Higher Education | Compositions and methods for treating liver disease |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2007277341A1 (en) * | 2006-07-19 | 2008-01-31 | University Of Florida Research Foundation, Inc. | Compositions for reprogramming a cell and uses therefor |
WO2009136168A1 (en) * | 2008-05-09 | 2009-11-12 | The University Court Of The University Of Glasgow | Materials and methods relating to cell based therapies |
WO2009143578A1 (en) * | 2008-05-28 | 2009-12-03 | The Council Of The Queensland Institute Of Medical Research | Cancer drug target and methods of diagnosis and therapy |
KR20200057108A (en) * | 2013-10-25 | 2020-05-25 | 웨인 스테이트 유니버시티 | Methods, systems and compositions relating to cell conversion via protein-induced in-vivo cell reprogramming |
CN104830906B (en) * | 2014-02-12 | 2018-09-04 | 北京维通达生物技术有限公司 | A method of reprogramming obtains functional people's liver parenchymal cell |
JP2017514514A (en) * | 2014-03-21 | 2017-06-08 | エージェンシー フォー サイエンス,テクノロジー アンド リサーチ | Fusion genes in cancer |
EP3250609A4 (en) * | 2015-01-26 | 2018-07-11 | The University of Chicago | Il13ra alpha 2 binding agents and use thereof in cancer treatment |
CN109069544B (en) * | 2016-02-18 | 2023-05-09 | 宾州研究基金会 | Intra-brain generation of GABAergic neurons |
-
2020
- 2020-03-26 AU AU2020248012A patent/AU2020248012A1/en active Pending
- 2020-03-26 JP JP2021557370A patent/JP2022527277A/en active Pending
- 2020-03-26 US US17/439,733 patent/US20220152224A1/en active Pending
- 2020-03-26 EP EP20777797.0A patent/EP3946469A4/en active Pending
- 2020-03-26 CN CN202080030149.8A patent/CN113710285A/en active Pending
- 2020-03-26 WO PCT/US2020/024976 patent/WO2020198485A1/en unknown
- 2020-03-26 CA CA3134656A patent/CA3134656A1/en active Pending
Non-Patent Citations (5)
Title |
---|
MÉLANIE LAMBERT, JAMBON SAMY, DEPAUW SABINE, DAVID-CORDONNIER MARIE-HÉLÈNE: "Targeting Transcription Factors for Cancer Treatment", MOLECULES, vol. 23, no. 6, 1479, 19 June 2018 (2018-06-19), pages 1 - 51, XP055744317, ISSN: 1420-3049, DOI: 10.3390/molecules23061479 * |
PIERRE-OLIVIER GUICHET, BIECHE IVAN, TEIGELL MARISA, SERGUERA CHÉ, ROTHHUT BERNARD, RIGAU VALÉRIE, SCAMPS FRÉDÉRIQUE, RIPOLL CHANT: "Cell Death and Neuronal Differentiation of Glioblastoma Stem-Like Cells Induced by Neurogenic Transcription Factors", GLIA, vol. 61, no. 2, 9 October 2012 (2012-10-09), US, pages 225 - 239, XP055744225, ISSN: 0894-1491, DOI: 10.1002/glia.22429 * |
UAN-FENG ZHAO, ZHAO QIU, HU HUI, LIAO JIA-ZHI, LIN JU-SHENG, XIA CHAO, CHANG YING, LIU JING, GUO AN-YUAN, HE XING-XING: "The ASH1-miR-375-YWHAZ Signaling Axis Regulates Tumor Properties in Hepatocellular Carcinoma", MOLECULAR THERAPY NUCLEIC ACIDS, vol. 11, 25 April 2018 (2018-04-25), US, pages 538 - 553, XP055744231, ISSN: 2162-2531, DOI: 10.1016/j.omtn.2018.04.007 * |
XING-XING HE, KUANG SHU-ZHEN, LIAO JIA-ZHI, XU CHUAN-RUI, CHANG YING, WU YU-LIANG, GONG JING, TIAN DE-AN, GUO AN-YUAN, LIN JU-SHEN: "The regulation of microRNA expression by DNA methylation in hepatocellular carcinoma", MOLECULAR BIOSYSTEMS, vol. 11, no. 2, 18 November 2014 (2014-11-18), pages 532 - 539, XP055744313, ISSN: 1742-206X, DOI: 10.1039/C4MB00563E * |
YASUO TAKASHIMA, HORISAWA KENICHI, UDONO MIYAKO, OHKAWA YASUYUKI, SUZUKI ATSUSHI: "Prolonged inhibition of hepatocellular carcinoma cell proliferation by combinatorial expression of defined transcription factors", CANCER SCIENCE, vol. 109, no. 11, 16 September 2018 (2018-09-16), pages 3543 - 3553, XP055744305, ISSN: 1347-9032, DOI: 10.1111/cas.13798 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10973930B2 (en) | 2016-02-18 | 2021-04-13 | The Penn State Research Foundation | Generating GABAergic neurons in brains |
EP4045094A4 (en) * | 2019-10-16 | 2024-02-21 | Univ Pittsburgh Commonwealth Sys Higher Education | Compositions and methods for treating liver disease |
US20220098617A1 (en) * | 2020-09-29 | 2022-03-31 | NeuExcell Therapeutics Inc. | Ascl1 vector |
Also Published As
Publication number | Publication date |
---|---|
US20220152224A1 (en) | 2022-05-19 |
CN113710285A (en) | 2021-11-26 |
EP3946469A4 (en) | 2022-12-28 |
CA3134656A1 (en) | 2020-10-01 |
AU2020248012A1 (en) | 2021-10-07 |
EP3946469A1 (en) | 2022-02-09 |
JP2022527277A (en) | 2022-06-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220152224A1 (en) | Methods and materials for treating cancer | |
Neal et al. | Prokineticin‐2 promotes chemotaxis and alternative A2 reactivity of astrocytes | |
Yao et al. | Wnt regulates proliferation and neurogenic potential of Müller glial cells via a Lin28/let-7 miRNA-dependent pathway in adult mammalian retinas | |
Espuny-Camacho et al. | Hallmarks of Alzheimer’s disease in stem-cell-derived human neurons transplanted into mouse brain | |
Luo et al. | Enhanced transcriptional activity and mitochondrial localization of STAT3 co-induce axon regrowth in the adult central nervous system | |
Nakatani et al. | Ascl1/Mash1 promotes brain oligodendrogenesis during myelination and remyelination | |
Jakovcevski et al. | Olig transcription factors are expressed in oligodendrocyte and neuronal cells in human fetal CNS | |
López-Juárez et al. | Thyroid hormone signaling acts as a neurogenic switch by repressing Sox2 in the adult neural stem cell niche | |
Neddens et al. | Conserved interneuron-specific ErbB4 expression in frontal cortex of rodents, monkeys, and humans: implications for schizophrenia | |
Wang et al. | Silencing of circular RNA HIPK2 in neural stem cells enhances functional recovery following ischaemic stroke | |
Fernando et al. | Optimal myelin elongation relies on YAP activation by axonal growth and inhibition by Crb3/Hippo pathway | |
Li et al. | Caveolin-1 plays a crucial role in inhibiting neuronal differentiation of neural stem/progenitor cells via VEGF signaling-dependent pathway | |
Neman et al. | Co-evolution of breast-to-brain metastasis and neural progenitor cells | |
Qiao et al. | Nap1l1 controls embryonic neural progenitor cell proliferation and differentiation in the developing brain | |
Zhou et al. | Hippocampal TERT regulates spatial memory formation through modulation of neural development | |
Karakatsani et al. | Neuronal LRP4 regulates synapse formation in the developing CNS | |
Scordel et al. | Borna disease virus phosphoprotein impairs the developmental program controlling neurogenesis and reduces human GABAergic neurogenesis | |
Ma et al. | Over-expression of cyclin D1 promotes NSCs proliferation and induces the differentiation into astrocytes via Jak-STAT3 pathways | |
Massart et al. | Developmental and adult expression patterns of the G‐protein‐coupled receptor GPR88 in the rat: Establishment of a dual nuclear–cytoplasmic localization | |
Jiang et al. | Histamine H2 receptor negatively regulates oligodendrocyte differentiation in neonatal hypoxic-ischemic white matter injury | |
Wang et al. | Transcription factor-based gene therapy to treat glioblastoma through direct neuronal conversion | |
Oh et al. | In vivo bioluminescence reporter gene imaging for the activation of neuronal differentiation induced by the neuronal activator neurogenin 1 (Ngn1) in neuronal precursor cells | |
He et al. | A novel CCK receptor GPR173 mediates potentiation of GABAergic inhibition | |
Ferent et al. | Investigation of the proteolipid protein promoter activity during demyelination and repair | |
Xu et al. | Upregulation of SYF2 is associated with neuronal apoptosis caused by reactive astrogliosis to neuroinflammation |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20777797 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 3134656 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2021557370 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2020248012 Country of ref document: AU Date of ref document: 20200326 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2020777797 Country of ref document: EP Effective date: 20211026 |