Development of new neural stem cell based tumor targeted gene therapy approaches

DEVELOPMENT OF NEW NEURAL STEM CELL-BASED TUMOR-TARGETED GENE THERAPY APPROACHES ZHU DETU NATIONAL UNIVERSITY OF SINGAPORE 2012 DEVELOPMENT OF NEW NEURAL STEM CELL-BASED TUMOR-TARGETED GENE THERAPY APPROACHES ZHU DETU (B. Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE & INSTITUTE OF BIOENGINEERING AND NANOTECHNOLOGY 2012 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ZHU DETU 21 August 2012 I ACKNOWLEDGMENTS I would like to acknowledge all who have helped and inspired me during my study at the National University of Singapore and Institute of Bioengineering and Nanotechnology. I am very grateful to my supervisor, Dr. Wang Shu, Associate Professor, Department of Biological Sciences, National University of Singapore, for his invaluable inspiration and guidance during my PhD study. I would like to dedicate my most sincere gratitude to my parents for their constant encouragement and support. I acknowledge the National University of Singapore, for honoring me with studentship and financial assistance in the form of scholarship. II TABLE OF CONTENTS ACKNOWLEDGMENTS I TABLE OF CONTENTS . III SUMMARY .VI LIST OF PUBLICATIONS .VIII LIST OF TABLES IX LIST OF FIGURES X ABBREVIATIONS XII CHAPTER INTRODUCTION . 1.1 Neural stem cells .2 1.1.1 Tumor tropism .2 1.1.2 Cell source 1.1.3 Genetic engineering .5 1.1.4 Side effects of intravenous injection .6 1.2 Fusogenic membrane glycoproteins .7 1.2.1 Bystander effect .7 1.2.1.1 Cell fusion 1.2.1.2 Antitumor immune response activation 1.2.2 Family members .10 1.2.2.1 GALV.fus 10 III 1.2.2.2 Syncytin-1 11 1.2.2.3 VSV-G 12 1.2.3 Applications in tumor gene therapy 12 1.2.3.1 Enhanced antitumor effect .12 1.2.3.2 Difficulties in large-scale clinical application 14 1.3 CD40-CD40 ligand interaction 14 1.3.2 CD40 expression and function in human cells .15 1.3.2 Direct growth inhibition of cancer .18 1.3.3 Antitumor immune response activation .22 1.4. Purpose 26 CHAPTER SELECTIVE KILLING OF CANCER CELLS BY A NOVEL VSV-G MUTANT THAT PROMOTES LOW PH-DEPENDENT CELL FUSION 28 2.1. Introduction 29 2.2. Materials and Methods 32 2.2.1. Cell culture .32 2.2.2. Mutagenesis, baculovirus preparation and cell transduction .33 2.2.3. Indirect immunofluorescence microscopy .34 2.2.4. Syncytia Formation Assay 35 2.2.5. Cytotoxicity assays .35 2.2.6. Reverse transcriptase polymerase chain reaction .36 2.2.7. Western blot .36 IV 2.2.8. In vitro Boyden chamber cell migration assay .37 2.2.9. Animal studies to evaluate therapeutic efficacy .37 2.2.10. Statistical analysis 38 2.3. Results .40 2.3.1. Whole body biodistribution of intravenously administered NSCs in a mouse 4T1 breast cancer model .40 2.3.2. Mutagenesis of VSV-G 44 2.3.3. pH-responsive Properties of VSV-G(H162R) 47 2.3.4. In vitro bystander effect of VSV-G(H162R) 53 2.3.5. Establishment of VSVG(H162R)-expressing NSCs using baculovirus 57 2.3.6. Metastatic breast cancer therapy using VSVG(H162R)-expressing NSCs .62 2.3.7. Side effects of cancer therapy using VSVG(H162R)-expressing NSC 68 2.4. Discussion 70 CHAPTER SELECTIVE KILLING OF CD40-POSITIVE BREAST CANCER CELLS BY NSC-MEDIATED DELIVERY OF CD40 LIGAND IN A MOUSE MODEL 75 3.1. Introduction 76 3.2. Materials and Methods 79 V 3.2.1. Cell culture .79 3.2.2. Baculovirus preparation and cell transduction .80 3.2.3. Cytokine antibody array 80 3.2.4. Cytotoxicity assays .81 3.2.5. Reverse transcriptase polymerase chain reaction .82 3.2.6. Fluorescent-activated cell sorting analysis 82 3.2.7. Animal studies to evaluate therapeutic efficacy .83 3.2.8. Statistical analysis 83 3.3. Results .85 3.3.1. Establishment of CD40L-expressing iPS-NSCs using a baculovirus 85 2.3.2. CD40L induces cytokine production in CD40-positive 4T1 breast cancer cells .87 2.3.3. In vitro bystander effect of CD40L-expressing iPS-NSCs 92 2.3.4. Metastatic breast cancer therapy using CD40L-expressing NSCs 95 2.3.5. Side effects of cancer therapy using CD40L-expressing NSCs .102 3.4. Discussion 104 CHAPTER CONCLUSION 108 References . 113 VI SUMMARY Neural stem cells (NSCs) have recently emerged as one of the most attractive cellular vehicles for targeted gene delivery to cancers due to their migratory capacity and tumor tropism. However, because NSCs can be chemoattracted to non-target regions, especially after intravenous administration, off-target transgene expression is a concern for the clinical application of NSC-mediated cancer gene therapy. To minimize this side effect, therapeutic transgenes that enable tumor cell-selective killing are needed. In this project, I developed two novel approaches to target tumor cells. The first approach uses vesicular stomatitis virus G glycoprotein (VSV-G) to target tumor acidosis. VSV-G is a viral fusogenic membrane glycoprotein that kills tumor cells via syncytia formation. Tumor acidosis is one of the hallmarks of the tumor microenvironment. Here, I have discovered a novel VSV-G mutant that functions specifically at an acidic tumor extracellular pH, thus enabling VSV-G to selectively kill tumor cells. The second approach uses CD40 ligand (CD40L) to target CD40+ tumor cells. CD40 is a type I TNF receptor that is selectively expressed on a number of epithelial and mesenchymal tumors, such as breast tumors, bladder tumors and lymphomas, but not on most normal, non-proliferating epithelial tissues. VII Expression of CD40L in CD40+ tumors leads to tumor growth inhibition via apoptosis induction and immunity activation. In our studies, both approaches showed significant therapeutic effects in vitro and in vivo. In a mouse model of 4T1 metastatic breast cancer, both VSV-G and CD40L delivered by NSC-based vectors obtained greater therapeutic efficacy and reduced less toxicity to normal tissues than the conventional HSVtk/GCV suicide gene therapy. 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Hum Gene Ther. 21(6): 683-694.     131 [...]... well-documented oncogenic potential of these genes The ability of human pluripotent stem cells such as human embryonic stem cells (hESCs) to generate virtually any differentiated cell type provides the possibility of using these cells as new sources of human NSCs Self-renewing hESCs are inherently immortal, and their proliferation capacity is preserved during long-term cell culture Hence, they have become... of unlimited amounts of uniform human stem cells (Zhang et al., 2001; Ben-Hur et al., 2004) However, despite their unique potential, the use of hESCs remains ethically controversial, because the process of generating hESC lines involves the destruction of human embryos To circumvent this problem, another type of human pluripotent stem cells, human induced pluripotent stem cells (iPSCs), which are generated... through the reprogramming of adult somatic cells by forced expression of several transcriptional factors, can be used as a new source of NSCs (Takahashi et al., 2007; Yu et al., 2007) Standardization of the large-scale mass production of cell therapeutics is a prerequisite for the widespread application of cell therapy Thus, standardization in generating human iPSC-derived cells offers the potential for...LIST OF PUBLICATIONS Manuscripts Detu Zhu, Lam Dang Hoang and Shu Wang Systemic delivery of fusogenic membrane glycoprotein-expressing neural stem cells to selectively kill tumor cells through low pH-induced cell fusion 2012 (submitted to Molecular Therapy) Detu Zhu, Lam Dang Hoang and Shu Wang Selective killing of CD40-positive breast cancer cells by NSC-mediated delivery of CD40 ligand... The powerful bystander killing effects of FMGs arise from the induction of local syncytia formation, the activation of antitumor immune response and the spread of 7 pro-apoptotic agents via syncytiosomes 1.2.1.1 Cell fusion Overexpression of FMGs in tumor cells leads to massive cell to cell fusion, formation of gigantic, multinucleated syncytia and subsequent cell death in 2-5 days (Higuchi et al.,... delivery vectors for tumor- targeted therapy 1.1.1 Tumor tropism Metastatic tumors are the most aggressive type of neoplasm in humans, characterized by a high infiltrative ability and resistance to conventional therapeutic treatments such as surgical excision, radiotherapy and chemotherapy Currently, gene therapy has emerged as a promising new approach for treating aggressive malignant tumors; however,... cancers (Hoffmann and Wildner, 2006) In addition, FMGs are also intensively explored in other researches as an adjunctive agent for oncolytic virotherapy (Ahmed et al., 2003; Ebert et al., 2004; Grisson et al., 2004), chemotherapy (Hoffmann et al., 2006; Hoffmann et al., 2007) and immunotherapy by cytokine genes (Eslahi et al., 2001; Hoffmann et al., 2007; Hoffmann et al., 2007), allogeneic tumor cell vaccines... transgene expression in hES cells (Zeng et al., 2007) 1.1.4 Side effects of intravenous injection The systemic intravenous administration of NSCs is an attractive option, given that intravenous injection is a minimally invasive procedure, and the injected cells may home in on multiple intracranial tumor foci and solid tumors of a non -neural origin through the circulation However, the majority of intravenously... situation, tumor- targeted therapeutic gene is urgently needed 1.2 Fusogenic membrane glycoproteins Fusogenic membrane glycoproteins (FMGs) are a class of glycoproteins derived from viral envelope genes that can induce cell membrane fusion and cytotoxicity in mammalian cells In the year 2000, a research group found that overexpression of FMGs in tumor cell cultures would induce the formation of gigantic,... chemokines or cytokines throughout the solid tumor, or promote the cross presentation of tumor- associated antigens 1.2.3.2 Difficulties in large-scale clinical application Despite the powerful bystander effects mentioned above, the application of FMG in tumor gene therapy is impeded due to the lack of tumor targeting It has been reported that the generation of high-titer viral vectors encoding the GALV.fus . DEVELOPMENT OF NEW NEURAL STEM CELL- BASED TUMOR- TARGETED GENE THERAPY APPROACHES ZHU DETU NATIONAL UNIVERSITY OF SINGAPORE 2012 1 DEVELOPMENT OF NEW NEURAL STEM. OF NEW NEURAL STEM CELL- BASED TUMOR- TARGETED GENE THERAPY APPROACHES ZHU DETU (B. Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES. HSVtk/GCV suicide gene therapy. These findings are of crucial importance in terms of clinical trials of NSC-mediated cancer gene therapy. This study is the first to deliver tumor- targeted VSV-G