GENETIC ENGINEERING AND SURFACE MODIFICATION OF BACULOVIRUS DERIVED VECTORS FOR IMPROVED GENE DELIVERY TO THE CENTRAL NERVOUS SYSTEM YANG YI NATIONAL UNIVERSITY OF SINGAPORE 2006 GENETIC ENGINEERING AND SURFACE MODIFICATION OF BACULOVIRUS DERIVED VECTORS FOR IMPROVED GENE DELIVERY TO THE CENTRAL NERVOUS SYSTEM YANG YI (B Eng.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAMME IN BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE AND INSTITUTE OF BIOENGINEERING AND NANOTECHNOLOGY July 2006 ACKNOWLEDGMENTS First and foremost, I wish to express my appreciation to my supervisor, Dr Wang Shu, Associate Professor, Department of Biological Science, National University of Singapore; Group Leader, Institute of Bioengineering and Nanotechnology, for his continuous support and patient guidance And to my co-supervisors, Dr Feng Si-shen, Associate Professor, Division of Bioengineering, National University of Singapore, for the in-depth discussions and useful suggestions I would also like to acknowledge our exceptional research group at Institute of Bioengineering and Nanotechnology for providing such a fabulous environment for the study Special acknowledgments go to Dr Li Ying, Dr Liu Beihui, and Dr Wu Chunxiao for their assistant in my research project, and to Dr Jurvansuu Jaana for the critical review of the manuscript This thesis is dedicated to my father Yang Zhoumou and my mother Peng Liying, whose love, encouragement and support have always been my greatest impetus I TABLE OF CONTENTS Contents Page Acknowledgments………………………………………………………………….I Table of Contents……………………………………………………………… II Summary………………………………………………………………………… VI List of Publications…………………………………………………… …… VIII List of Tables .IX List of Figures…………………………………………………………………… X Abbreviations… ……………………………………………………………… XII Chapter One: Introduction………………………………………………….1 1.1 General Introduction………………………………………………………….2 1.1.1 Gene Therapy…………………………………………………………… 1.1.2 Non-viral and Viral Gene Delivery Systems……………………………2 1.1.3 PEI and Its Role in Gene Delivery…………………………………… 1.1.4 Viral Gene Delivery to the CNS 1.1.5 Baculovirus Vectors Mediated Gene Delivery……………………… 10 1.2 Purpose of This Study………………………………………………………12 1.3 Specific Objectives………………………………………………………… 13 Chapter Two: Production, Characterization and Purification of Baculovirus Particles…………… 15 2.1 Introduction………………………………………………………………… 16 2.1.1 Baculovirus and the Insect Cell Expression System…………………16 2.1.2 Recombinant Baculovirus Vectors for Gene Delivery… ….… .19 2.1.3 Production and Purification Related Issues……………………… …20 2.1.4 Objectives……………………………………………………………… 20 2.2 Materials and Methods…………………………………………………… 21 2.2.1 Production of Recombinant Baculovirus Particles………………… 21 2.2.2 Viral Stock Amplification……………………………………………… 24 2.2.3 Viral Particles Purification and Concentration……………………… 24 2.2.4 Titering of Viral Stocks………………………………………………… 25 II 2.2.5 Electron Microscopy…………………………………………………… 27 2.2.6 Particle Size and Surface Charge Measurements………………… 27 2.2.7 Cell Lines and Cell Culture…………………………………………… 28 2.2.8 Cell Transduction……………………………………………………… 28 2.2.9 Luciferase Expression Measurement………………………………….28 2.2.10 Flowcytometric Studies………………………………………………….29 2.3 Results…………………………………………………………………………29 2.3.1 The Potential of Baculovirus as a Vector for CNS Gene Delivery In Vitro…………………………………………….29 2.3.2 Studies of Baculovirus Particles Production Procedure…………… 36 2.3.3 Studies of Baculovirus Particles Purification and Concentration Procedures…………………………………………… 41 2.3.4 Studies of Physical Characteristics of Baculovirus Particles……… 45 2.3.5 Baculovirus Particles Interacting with Charged Membrane and a New Purification Method……………………………………… 51 2.4 Discussions………………………………………………………………… 60 Chapter Three: Genetic Engineering of Baculovirus Vectors for Controlled Gene Delivery…………65 3.1 Introduction………………………………………………………………… 66 3.1.1 Genetic Engineering of Baculovirus Vectors for Neuron-targeted Gene Delivery…………………………………………………………….66 3.1.2 Hybrid Promoter with CMV Enhancer………………………………….68 3.1.3 Artificial Transcriptional Factor Boosted Gene Expression………….69 3.1.4 Objectives…………………………………………………………………70 3.2 Improved Neuronal Gene Delivery Achieved by the Adoption of a Hybrid Promoter……………………………………………………… 70 3.2.1 Materials and Methods……………………………………………………70 3.2.1.1 Construction of pGL3-based vectors for promoter strength comparison…………………………………………………70 3.2.1.2 Construction of recombinant baculovirus vectors……………… 71 3.2.1.3 In vitro gene delivery studies……………………………………… 72 3.2.1.4 In vivo gene delivery studies……………………………………… 72 III 3.2.1.5 Immunofuluoresence studies……………………………………… 73 3.2.2 Results…………………………………………………………………… 73 3.2.2.1 Neuronal Promoters Display Lower Activities Than Commonly Used Viral Promoters………………………………… 73 3.2.2.2 Improved In Vitro Gene Delivery Mediated by the Hybrid Promoter Baculovirus Vector……………………………… 76 3.2.2.3 Enhanced In Vivo Gene Delivery Mediated by the Hybrid Promoter Baculovirus Vector……………………………… 80 3.2.2.3 Neuronal Specificity of In Vivo Gene Delivery Mediated by the Hybrid Promoter Baculovirus vector……………………… 82 3.2.3 Discussions……………………………………………………………… 85 3.3 Artificial Transcriptional Factor Boosted Neuronal Gene Delivery Driven by PDGF Promoter………………………………………………….86 3.3.1 Materials and Methods……………………………………………………89 3.3.1.1 Construction of recombinant baculovirus vectors…………………89 3.3.1.2 In vitro gene delivery studies……………………………………… 89 3.3.1.3 In vivo gene delivery studies……………………………………… 90 3.3.2 Results…………………………………………………………………… 91 3.3.2.1 In Vitro Studies of the Recombinant Transcriptional Activation….91 3.3.2.2 In Vivo Studies of the Recombinant Transcriptional Activation….96 3.3.3 Discussions………………………………………………………………101 3.4 Summary…………………………………………………………………… 104 Chapter Four: Surface Modification of Baculovirus for Improved In Vivo Gene Delivery…………….… 106 4.1 Introduction………………………………………………………………….107 4.1.1 Susceptibility of Baculovirus to Complement Inactivation………….107 4.1.2 Baculovirus Surface Modification…………………………………… 108 4.1.3 Objectives……………………………………………………………… 108 4.2 Materials and Methods…………………………………………………….109 4.2.1 Preparation of Baculovirus Particles………………………………….109 4.2.2 Preparation of BV-PEI Complex Particles……………………………109 4.2.3 Characterization of BV-PEI Complex Particles………………………110 IV 4.2.4 Serum Complement Inactivation of BV-PEI Vectors……………… 110 4.2.5 Cytotoxicity Assay………………………………………………………110 4.2.6 Electron Microscopy…………………………………………………….111 4.2.7 In Vitro Transduction and Gene Expression Assay…………………111 4.2.8 In Vivo Gene Delivery Studies……………………………………… 111 4.3 Results……………………………………………………………………….112 4.3.1 Baculovirus Particles Interact with PEI Polymers………………… 112 4.3.2 Transduction Capabilities of BV-PEI Vectors……………………… 119 4.3.3 Surface Modification of PEI25k Protect BV Particles against Serum Complement Attack………………………………… 121 4.3.4 In Vitro Studies of BV-PEI25k Vector Mediated Gene Delivery……124 4.3.5 Cytotoxicity of BV-PEI25k Particles………………………………… 129 4.3.6 In Vivo Studies of BV-PEI25k Vector Mediated Gene Delivery……131 4.4 Discussions…………………………………………………………………137 Chapter Five: Conclusion……………………………………………… 140 5.1 Results and Implications………………………………………………….141 5.1.1 Preparation and Characterization of Baculovirus Particles…………141 5.1.2 Genetic Engineering of Baculovirus Vectors for Controlled Gene Expression……………………………………………………… 142 5.1.3 Surface Modifications of Baculovirus Vectors for Improved In Vivo Gene Delivery………………………………………………… 143 5.2 Conclusion………………………………………………………………… 144 References…………………………………………………………………….145 V SUMMARY The studies presented in this thesis focused on development and modification of baculovirus-based vectors in order to achieve improved gene delivery to the Central Nervous System There are many important requirements for an ideal gene delivery system, including feasible vector production procedure, regulated transgene expression, and satisfactory in vivo gene delivery performance Our studies focused on improving all these three aspects First, we studied the gene delivery capability and physical characteristics of baculovirus, as well as several important issues related to baculovirus vector production and purification processes A new baculovirus purification method utilizing charged membrane filters was developed and evaluated Second, through genetic engineering of baculovirus vectors, we achieved targeted gene delivery with enhanced and controlled transgene expression specifically in neuronal cells Two different promoter designs, utilizing a hybrid promoter and a transcriptional activator, were employed to achieve improved transgene expression, both in vitro and in vivo Last, a baculovirus surface modification technique based on electrostatic interactions was developed By incorporating PEI polymers onto the surface of baculovirus particles, we developed a virus-based complex vector which is VI more resistant to the serum inactivation Our studies demonstrated that improved in vivo gene delivery performance of baculovirus-based vectors could be achieved through this surface modification In summary, the information gained from our research, such as development of modified vectors and evaluation of pertinent methods, should contribute to the development of Central Nervous System gene therapies as well as to the progression of various virus- and gene delivery-related studies VII LIST OF PUBLICATIONS Publications: Y Li, Y Yang, S Wang Neuronal gene transfer by baculovirus-derived vectors accommodating a neurone-specific promoter Experimental Physiology, v 90, p 39-44, 2005 CY Wang, F Li, Y Yang, HY Guo, CX Wu, S Wang Recombinant baculovirus containing the diphtheria toxin a gene for malignant glioma therapy Cancer Research, 1:66 (11) 5798-5806, 2006 BH Liu, Y Yang, JFR Paton, F Li, J Boulaire, S Kasparov and S Wang GAL4-NFkappaB Fusion Protein Augments Transgene Expression from Neuronal Promoters in the Rat Brain Molecular Therapy, in press, 2006 Manuscript: Y Yang, CX Wu, S Wang Surface Modification of Baculovirus for Improved In Vivo Gene Delivery 2006 The studies presented in this thesis are based on the research work in the above publications and manuscript VIII materials; therefore, these vectors could be used in areas such as neuroscience related studies, neuron degenerative disease studies and CNS gene therapy applications Although in this part, we only focused on the studies of neuron-targeted vectors, we successfully demonstrated the feasibility of using genetic engineering to control the transgene expression regulation at the transcriptional level Moreover, similar strategies and designs would be applicable in the development of other kind of customized baculovirus vectors For example, when other target-specific genetic components are utilized, it should be possible to target the baculovirus vectors to other types of cells and tissues 5.1.3 Surface Modifications of Baculovirus Vectors for Improved In Vivo Gene Delivery As shown in Chapter 4, PEI25k polymers were successfully incorporated onto the surface of baculovirus particles through electrostatic interactions Our studies demonstrated that surface modification can provide protection to retain the morphology and activity of baculovirus vectors against serum complement inactivation, and hence, improve the in vivo performance of baculovirus-based vectors Our results showed that after intravenous administration, the modified BV-PEI25k complex vectors efficiently delivered reporter genes to different organs and tissues, as well as pre-inoculated tumors This surface modification overcomes the vulnerability of baculoviruses due to serum inactivation; and therefore, it makes baculovirus-based gene delivery and gene therapy possible through systemic administration of the vectors Moreover, compare to biological modifications (Grabherr et al., 143 2001;Ojala et al., 2001;Raty et al., 2004), the surface modification method developed in our studies would be more flexible, easier to operate and less time consuming Further more, this part of investigations may also provide useful information for future studies on interactions between viral vectors and non-viral components Nevertheless, in this part of studies, we mainly focused on the investigations of the effects of PEI25k; and the reason is that our primary objective was to develop a flexible, non-biological surface modification method to enhance the viral gene delivery at transductional level However, in future studies, further modifications based on similar concept should be possible For example, transduction facilitating elements or targeting peptides could be utilized to improve and direct the viral transductions, and non-immunogenic polymers could be employed to reduce the immunological risk of virus mediated gene delivery Various functional components may even be incorporated simultaneously to modify and functionalize baculovirus-based vectors, thus fulfilling the requirements of ideal vectors for gene therapy applications 5.2 Conclusion The research work presented in this thesis investigated the preparation, characterization, genetic engineering, and surface modification processes of baculovirus-based gene 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DERIVED VECTORS FOR IMPROVED GENE DELIVERY TO THE CENTRAL NERVOUS SYSTEM YANG YI (B Eng.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR... Chapter Three: Genetic Engineering of Baculovirus Vectors for Controlled Gene Delivery? ??………65 3.1 Introduction………………………………………………………………… 66 3.1.1 Genetic Engineering of Baculovirus Vectors for Neuron-targeted... SUMMARY The studies presented in this thesis focused on development and modification of baculovirus- based vectors in order to achieve improved gene delivery to the Central Nervous System There