Intrathecal delivery of transgene to promote nerve regeneration

INTRATHECAL DELIVERY OF TRANSGENE TO PROMOTE NERVE REGENERATION SHI LEI NATIONAL UNIVERSITY OF SINGAPORE 2003 Master Thesis Shi Lei INTRATHECAL DELIVERY OF TRANSGENE TO PROMOTE NERVE REGENERATION SHI LEI (B.E., PRC) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF ANATOMY, FACULTY OF MEDICINE AND INSTITUTE OF MATERIALS RESEARCH ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2003 Master Thesis Shi Lei ACKNOWLEDGEMENTS I would like to extend my deepest gratitude to my supervisor Drs Wang Shu, Ng Yee Kong and Xiao Zhicheng for their patient support, guidance and invaluable advice without them none of this work would have been possible Dr Tang Guping deserves special mention for his polymer synthesis and characterization Equal special thanks to Mr Gao Shujun for his patient guidance in animal experiments Also never forget Dr Ma Yuexia for her help in the in vitro experiments Equal thanks are paid to my other colleagues Liu Beihui, Li Ying and Zeng Jieming My sincerest thanks also go to the National University of Singapore for providing me the research scholarship and to Institute of Materials Research and Engineering where A*STAR funds the research work Special thanks also go to our friends, family and others concerned in NUS for their deep concern, kind words and moral support Acknowledgements Shi Lei І PUBLICATIONS International Publications “Repeated Intrathecal Administration of Plasmid DNA Complexed with polyethylenimine for Prolonged Transgene Expression in the Spinal Cord”, Shi L, Tang GP, Gao SJ, Ma YX, Liu BH, Li Y, Zeng JM, Ng YK, Leong KW, Wang S Gene Ther., 2003; 10:1179-88 “Polyethylene Glycol-grafted Polyethylenimine for Improved CNS Gene Transfer: Effects of PEGylation Degrees”, Tang GP, Zeng JM, Gao SJ, Ma YX, Shi L, Li Y, Too H-P and Wang S, Biomaterials, 2003; 24:2351-62 Conference “Intrathecal Delivery of DNA Complexed with Polyethylenimine for Transgene Expression in the Rat Spinal Cord”(Abstract accepted), Wang S, Tang GP, Ma YX, Shi L, Gao SJ, Leong KW, The American Society of Gene Therapy's 5th Annual Meeting, Boston June, 2002 Publications Shi Lei П Page ACKNOWLEDGEMENTS I PUBLICATIONS II TABLE OF CONTENTS III ABBREVIATION VI SUMMARY VIII Chapter One Introduction 1.1 The Most Straight-Forward Way: Naked DNA Gene Delivery 1.2 PEI (Polyethylenimine): Synthesis, Structure and Molecular Species 1.2.1 Formation of DNA/Polycation Nanoparticles 1.2.2 Cellular Mechanisms for Uptake of the PEI/DNA Complexes 1.2.3 Endosomal Escape 1.2.4 Nuclear Entry and Complex Disassembly 1.3 Advantages and Disadvantages of PEI-mediated Gene Delivery 1.4 Repeated Injection Using Non-viral Polymeric Vectors for Prolonged Gene Expression 1.5 Conjugation of PEG to PEI and Its Effect on Gene Expression Levels 10 1.6 Objectives of Present Study 12 Master Thesis Shi Lei III Chapter Two Materials and Methods 2.1 Materials 15 2.2 Synthesis of PEG-PEI 15 2.3 Plasmid 16 2.4 Preparation and Characterization of DNA/Polymer Complexes 16 2.5 In Vitro Gene Transfer 18 2.6 Animals and Injection Procedures 19 2.7 Luciferase Activity Assay 19 2.8 Cell Viability Assay 20 2.9 Histological Examination and Immunostaining 21 2.10 TUNEL Staining 21 Chapter Three 3.1 Results Repeated Administration 3.1.1 Transgene Expression in the Spinal Cord after Intrathecal Delivery of PEI/DNA Complexes 24 3.1.2 Repetitive Injection: Attenuation of Gene Expression Upon Readministration 25 3.1.3 PEI PEGylation Eliminates Attenuation of Gene Expression in Repetitive Administration Master Thesis 27 Shi Lei IV 3.2 Effects of PEGylation Extent upon CNS Gene Transfer 3.2.1 Synthesis and Physicochemical Characterization of PEG-PEI 29 3.2.2 Protection Effects for PEGylated PEI 30 3.2.3 PEI PEGylation Enhanced Gene Delivery In Vitro 30 3.2.4 PEG-PEI Mediated In Vivo Gene Delivery 31 3.3 List of Figures 34 3.4 Table 70 Chapter Four Discussion and Conclusion References 72 81 Master Thesis Shi Lei V ABBREVIATION PEI Polyethylenimine PEG Polyethylene Glycol CNS Central Nervous System CSF Cerebrospinal Fluid NGF Nerve Growth Factor CMV IE Cytomegalovirus Immediate Early TE Tris-EDTA DMEM Dulbecco's Modified Eagle Medium DMSO Dimethyl Sulfoxide TUNEL TdT-mediated dUTP Digoxigenin Nick End Labelling H&E Hematoxylin and Eosin BSA Bovine serum albumin HIV Human Immunodeficiency Virus RLU Relative Light Units FCS Fetal Calf Serum PBS Phosphate Buffered Saline ELISA Enzyme-Linked Immunosorbent Assay FITC Fluorescein Isothiocyanate OT Object Temperature CT Chamber Temperature RT Room Temperature N/P PEI Nitrogen to DNA phosphate Abbreviation Shi Lei VI MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide CpG Cytosine Linked to a Guanine by a Phosphate Bond CAG Combination of CMV IE enhancer, chicken β-actin promoter and rabbit β-globin polyadenylation signal Abbreviation Shi Lei VII SUMMARY In the current study, it has been demonstrated that PEG-grafted PEI mediated transgene expression without attenuation after repeated intrathecal dosing was administrated, even in those rats injected three times, with a 2-week interval between two continuous injections Meanwhile, extent of PEGylation upon CNS gene transfer was further investigated It showed that an improved in vivo transgene expression was mediated by a lower degree of PEG grafting Though the low degree of PEGylation may be insufficient to enhance the biocompatibility of the polycation, it still helps to reduce the aggregation of polymer DNA complexes, minimize the interaction of the complexes with serum proteins, countering inhibitory effects of serum to gene transfer and consequently enhance the transgene expression A low level of PEGylation, without compromising gene transfer efficiency, may also be useful in polymer conjugation of biologically active molecules, where PEG is often used a spacer One potential application of such a bioconjugation is targeted gene delivery in vivo Summary Shi Lei VIII Meyer et al., 2000; Tan et al., 1999) These studies suggested that lipoplex triggered apoptosis and production of pro-inflammatory cytokines including infereron-γ and tumor necrosis factor-α and unmethylated CpG sequences in plasmid DNA also accounted for the direct immunostimulatory effects Anyway, these two main causes explained the ineffectiveness in the repeat injection (Li et al., 1999; Meyer et al., 2000; Tan et al., 1999) The same mechanism may also apply to PEI-mediated gene delivery system and be accountable for detected apoptotic cell death in the spinal cord after intrathecal administration The CNS is considered as an immune priviledged site and inflammatory and immune cells not function in the CNS as efficiently as in other organs However, CNS ventricles and parenchyma are immunologically separated Delivery of antigens and viruses into CSF elicited immune responses, probably owing to antigenic presentation in the deep cervical lymph nodes (Fabry et al., 1994; Stevenson et al., 1997) Cell death and apoptosis could also be induced by over-expression of gene products (Detrait et al., 2002), but this could not be used to explain what happened in this study, considering the observation that non-coding plasmid pLuc-s complexed with PEI prompted inhibitory effects too The potential problem inherent in physiological conditions when these complexes were injected is much higher ionic strength that may speed up the aggregation of complexes and impair the transfection efficacy (Ogris et al., 1999; Goula et al., 1998) In addition, the larger aggregates showed acute toxicity after intravenous injection and may kill 50% of the animals within 30min further suggesting that PEI/DNA complexes, especially their aggregates or precipitates, may also be accountable for cell death It is noteworthy that apoptosis was found exclusively in meninges The pia mater of spinal meninges builds up Chapter Four the boundary of Discussion and Conclusion the CSF Shi Lei and 74 spinal parenchyma, playing as one of the major barriers in transporting DNA/polymer complexes from the CSF to the spinal cord tissues Consistent with a previous report using intracerebro-ventricular injection of PEI/DNA complexes (Abdallah et al., 1996), it has been also observed in the current study considerable transgene expression in CNS parenchyma after CSF gene delivery, indicating PEI/DNA complexes may penetrate through the pia mater No apparent cell death in the gray and white maters of the spinal cord was observed, further indicating that PEI/DNA entering the spinal parenchyma may not be toxic to glial cells and neurons at these low doses This is also consistent with the previous report that PEI/DNA complexes delivered through a retrograde axonal transport did not inflict toxicity upon brainstem neurons at concentration efficient for DNA transfection (Wang et al., 2001) However, part of the formed aggregates could attach on the surface of the pia/dura mater, leading to death of leptomeningeal cells Further study is needed to identify the underlying mechanism of apoptosis observed in the current study The apoptotic cell death detected in this study was correlated with reduction of transgene expression following a second administration As demonstrated in this study, the attenuation is not due to PEI polymer itself because injection of the polymer alone did not induce any inhibitory effects Neither is the attenuation caused by the gene expression because high dose of naked DNA had no effects and injection of the complexes containing non-encoding plasmid DNA was as inhibitory as those containing functional plasmid DNA It shows that PEI/DNA complexes, rather than separate component alone, are the underlying cause for the attenuation On the other hand, PEI polymer alone is probably capable of inducing apoptosis at higher concentrations, as revealed in the in Chapter Four Discussion and Conclusion Shi Lei 75 vitro experiment of this study In an in vivo study using PEI alone into the CSF, such as the one reported here, opsonization of the highly positively charged polymer might reduce its cytotoxic effects Indeed, PEI polymer alone was less toxic than PEI/DNA complexes in in vitro cell viability assay in the serum-containing medium (Fig 10) Reduced toxicity may also be attributed to relatively inefficient cellular uptake of the branched 25kDa PEI compared to PEI/DNA complexes Meanwhile, naked plasmid DNA alone is very volatile in the body fluid and may degrade quickly DNA degradation may reduce CpG content (Abdallah et al., 1996; Li et al., 1999), thus minimizing CpGtriggered immune response PEG grafting to PEI in this study abolishes apoptosis and inhibitory effects in the repeat administration of DNA/polymer complexes The precise underlying mechanism is largely unknown One of the disadvantages for using polycations, including PEI, is poor solubility of DNA/polymer complexes, especially at higher N/P ratios Precipitates or aggregates of these complexes could be more toxic than their non-aggregated counterparts and polymer alone, as suggested in the finding that the higher the N/P ratio, the stronger the inhibitory effects PEG grafting often can provide improved solubility of macromolecules, reduce aggregation of particulates and minimize their interaction with proteins in the physiological fluid There is evidence from previous studies that PEGylation of PEI reduced surface charge of PEI/DNA particles, increased their dispersion ability at high concentrations, minimized plasma protein binding and erythrocyte aggregation, prolonged blood circulation time and decreased systemic toxicity after intravenous administration (Ogris et al., 1999; Nguyen et al., 2000; Kichler et al., 2002) Some of these effects may have contributed to the improvements in gene Chapter Four Discussion and Conclusion Shi Lei 76 expression upon repeat dosing achieved in the present study Indeed, previous reports that PEGylation of polycations leads to enhanced transfection efficiency (Petersen et al., 2002b; Choi et al., 1998; Lee et al., 2001; Sagara et al., 2002; Ahn et al., 2002) was consistent with the current in vivo study which showed PEGylation improved transfection five fold higher than PEI (data not shown) PEG could modify proteins and peptides, rendering them less immunogenic and antigenic thanks to reduced immune presentation by PEG-conjugated steric hindrance (Francis et al., 1998; Harris et al., 2001; Hinds et al., 2002) Covalent attachment of PEG to the surface of the adenovirus (Chillon et al., 1998; O’Riordan et al., 1999) reduces both humoral and cellular immune responses against viral proteins, attaining substantial gene expression of the vector upon second dosing without compromising the immune system of immunocompetent animals (Croyle et al., 2000) There is still a possibility that PEGylation of PEI in the present study might reduce immune stimulatory effects of CpG motifs in plasmid DNA once PEI/DNA complexes were formed, thus affecting cell susceptibility and gene expression profiles in the repetitive injection However, some potential side effect is higher degree of PEGylation can undermine transfection efficiency (Nguyen et al., 2000; Petersen et al., 2002b), probably because of steric hindrance effects of PEG on the interactions of the complexes with the cells (Nguyen et al., 2000) A proper grafting degree may balance the stabilizing benefits and hindrance effects and thereby becomes critical in achieving efficient in vivo gene delivery Out of this consideration, effects of PEG grafting extent on the in vivo gene delivery of PEI were investigated Polymer DNA complexes were delivered into the lumbar subarachnoid space A low degree of grafting, one PEG block per each PEI Chapter Four Discussion and Conclusion Shi Lei 77 polymer, was capable of enhancing transgene expression in the spinal cord and the brain This result is consistent with a recent report (Petersen et al., 2002b) that showed improved in vitro gene expression mediated by PEI after conjugating only one or two PEG blocks per PEI Improvement in transgene expression in the study did not indicate the well-documented effect of PEG in enhancing biocompatibility, since low degree of PEG grafting did not allow cell to survive in the high concentration of polycations The effect of PEG in improving cell viability was only observed when the grafting degree increased up to 2.5 or more blocks of PEG in the study However, as revealed in the SDSPAGE analysis, the low grafting with one PEG block per PEI displayed effective reduction in interaction of polycation DNA complexes with serum proteins It is likely that protein binding to polycation DNA complexes reduce the zeta potential, thus resulting in aggregation of the complexes and/or affecting electrostatic interaction of the complexes with negatively charged cell membrane In the study, it was also observed that the low degree of PEGylation could significantly reduce the interaction between serum protein and polycation DNA complexes in cultured cells In the current study, PEG-PEI (1) was a more qualified candidate than PEI homopolymer in mediating transgene expression As PEG blocks increased per PEI, transfection efficacy decreased Another finding in this study was a higher N/P ratio was necessary for a significant transgene expression, especially for those copolymers with a higher degree of PEGylation For instance, PEG-PEI (14.5) displayed a relatively high transgene expression at N/P ratios ranging from 40 to 70 In the animal experiments, the peak of transgene expression reached by PEG-PEI (1) at the N/P ratio of 30 This is also consistent with the recent report (Petersen et al., 2002b), which showed at N/P ratio of 50 Chapter Four Discussion and Conclusion Shi Lei 78 PEG-PEI complexes showed higher transfection efficacy in the in vitro experiments than that mediated by PEI homopolymer PEG grafting, ever at very low degrees, may considerably reduce the surface charges of the complexes (Petersen et al., 2002b) For binding to negatively charged cell membranes, net positive charge of complexes is required A high N/P ratio may help to keep surface charges of complexes after PEGylation Also, excess positive charges may protect PEI DNA complexes from aggregation under physiological condition (Yang et al., 1990), facilitating cellular uptake of the complexes In summary, the feasibility of PEGylated PEI mediated repetitive administration of DNA/polymer complexes into the spinal cord through lumbar intrathecal injection was achieved A prolonged transgene expression at a significant level persisted for up to weeks after repeated dosing Advantages of this method in spinal cord gene therapy include minimal damage to neural tissues, flexibility in controlling the therapeutic period, and possibility of achieving widespread gene expression It also suggested that a low degree of PEGylation would be capable of altering immune stimulatory effects of CpG motifs in plasmid DNA Further studies are needed to identify whether it is enough to reduce effects of immune inhibitory response upon gene expression Recently, many therapeutic genes have been delivered to protect against neurodegenerative disease (Bowers et al., 1997; Raymon et al., 1997), instead of therapeutic proteins For example, GDNF and BDNF were delivered to nigrostriatal system to protect the DA neurons in the Parkinson’s disease animal model (Bohn, 1999) Strategies were also employed to enhance regeneration in the spinal cord injury by transfecting neurotrophic factors genes into the spinal cord or subarachnoid space (Bohn, Chapter Four Discussion and Conclusion Shi Lei 79 1999; Blesch et al., 1999; Schnell et al., 1994; Tuszynski et al., 1996) The advantage of the gene therapy method is that it can provide a localized source of trophic factors at the site of the cell soma to stimulate neuronal survival and possibly axon growth (Ye et al., 1997; Blesch et al., 2002) At this point, PEGlyation PEI may be one of the promising candidates to provide a localized source of trophic factors at the site of the cell soma to stimulate neuronal survival and possibly axon growth Chapter Four Discussion and Conclusion Shi Lei 80 References [1] Abdallah B, Hassan A, Benoist C, Goula D, Behr JP, Demeneix BA A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine Hum Gene Ther 1996; 7:1947-54 [2] Ahn CH, Chae SY, Bae YH, Kim SW Biodegradable polyethylenimine for plasmid DNA delivery J Control Release 2002; 80:273-282 [3] Blesch A, Uy HS, Grill RJ, Cheng JG, Patterson PH, Tuszynski MH LIF augments corticospinal axon growth and neurotrophin expression after adult CNS injury J Neurosci 1999; 19:3556-3566 [4] Blesch A, Lu P, Tuszynski MH Neurotrophic factors, gene therapy, and neural stem cells for spinal cord repair Brain Res Bull 2002; 57:833-838 [5] Bohn MC A commentary on glial cell line-derived neurotrophic factor From a glial secreted molecule to gene therapy Biochem Pharmacol 1999; 57:135-142 [6] Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine Proc Natl Acad Sci USA 1995; 92:7297-7302 [7] Boussif O, Zanta MA, Behr JP Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold Gene Ther 1996; 3:1074-80 [8] Bowers WJ, Howard DF, Federoff H Gene therapeutic strategies for neuroprotection: implications for Parkinson’s disease Exp Neurol 1997; 144:58-68 [9] Budker V, Budker T, Zhang G, Subbotin V, Loomis A, Wolff JA Hypothesis: naked plasmid DNA is taken up by cells in vivo by a receptor-mediated process J Gene Med 2000; 2:76-88 References Shi Lei 81 [10] Chillon M, Lee JH, Fasbender A, Welsh MJ Adenovirus complexed with polyethylene glycol and cationic lipid is shielded from neutralizing antibodies in vitro Gene Ther 1998; 5:995-1002 [11] Choi YH, Liu F, Kim JS, Choi YK, Park JS, Kim SW Polyethylene glycol-grafted poly-L-lysine as polymeric gene carrier J Control Release 1998; 54:39-48 [12] Choi JH, Choi JS, Suh H, Park JS Effects of poly(ethylene glycol) grafting on polyethlenimine as a gene transfer vector in vitro Bull Korean Chem Soc 2001; 22: 46-52 [13] Costantini LC, Bakowska JC, Breakefield XO, Isacson O Gene therapy in the CNS Gene Ther 2000; 7:93-109 [14] Cotten M, Wagner E, Birnstiel ML Receptor-mediated transport of DNA into eukaryotic cells Methods Enzymol 1993; 217:618-44 [15] Croyle MA, Chirmule N, Zhang Y, Wilson JM "Stealth" adenoviruses blunt cellmediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung J Virol 2000; 75:4792-801 [16] Detrait ER, Bowers WJ, Halterman MW, Giuliano RE, Bennice L, Federoff HJ, Richfield EK Reporter gene transfer induces apoptosis in primary cortical neurons Mol Ther 2002; 5:723-730 [17] Fabry Z, Raine CS, Hart MN Nervous tissue as an immune compartment: the dialect of the immune response in the CNS Immunol Today 1994; 15:218-224 [18] Francis GE, Fisher D, Delgado C, Malik F, Gardiner A, Neale D.PEGylation of cytokines and other therapeutic proteins and peptides: the importance of biological optimization of coupling techniques Int J Hematol 1998; 68:1-18 References Shi Lei 82 [19] Godbey WT, Wu KK, Mikos AG Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle J Biomed Mater Res 1999a; 45:268-75 [20] Godbey WT, Wu KK, Hirasaki GJ, Mikos AG Improved packing of poly(ethylenimine)/DNA complexes increases transfection efficiency Gene Ther 1999b; 6:1380-8 [21] Godbey WT, Wu KK, Mikos AG Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery Proc Natl Acad Sci USA 1999c; 96:5177-81 [22] Godbey WT, Wu KK, Mikos AG Poly(ethylenimine) and its role in gene delivery J Control Release 1999d ; 60:149-60 [23] Godbey WT, Barry MA, Saggau P, Wu KK, Mikos AG Poly(ethylenimine)mediated transfection: a new paradigm for gene delivery J Biomed Mater Res 2000; 51:321-8 [24] Godbey WT, Wu KK, Mikos AG Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability Biomaterials 2001; 22:471-80 [25] Goula D, Remy JS, Erbacher P, Wasowicz M, Levi G, Abdallah B, Demeneix BA Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system Gene Ther 1998; 5:712-717 [26] Harris JM, Martin NE, Modi M Pegylation: a novel process for modifying pharmacokinetics Clin Pharmacokinet 2001; 40:539-51 [27] Harris JM, Zalipsky S (Eds.), Poly(ethylene glycol): Chemistry and Biological Applications ACS Book:Washington, DC, 1997 References Shi Lei 83 [28] Hinds KD, Kim SW Effects of PEG conjugation on insulin properties Adv Drug Deliv Rev 2002; 54:505-30 [29] Imaoka T, Date I, Ohmoto T, Nagatsu T Significant behavioral recovery in Parkinson's disease model by direct intracerebral gene transfer using continuous injection of a plasmid DNA-liposome complex Hum Gene Ther 1998; 9:1093-102 [30] Jackson CA et al Repetitive intrathecal injections of poliovirus replicons result in gene expression in neurons of the central nervous system without pathogenesis Hum Gene Ther 2001; 12:1827-41 [31] Jenkins RG et al An integrin-targeted non-viral vector for pulmonary gene therapy Gene Ther 2000; 7:393-400 [32] Kichler A, Chillon M, Leborgne C, Danos O, Frisch B Intranasal gene delivery with a polyethylenimine-PEG conjugate J Control Release 2002; 81:379-88 [33] Lachmann RH, Efstathiou S Utilization of the herpes simplex virus type latencyassociated regulatory region to drive stable reporter gene expression in the nervous system J Virol 1997; 71:3197-207 [34] Lee M, et al Repression of GAD Autoantigen Expression in Pancreas -Cells by Delivery of Antisense Plasmid/PEG-g-PLL Complex Mol Ther 2001; 4:339-346 [35] Lemkine GF, Mantero S, Migne C, Raji A, Goula D, Normandie P, Levi G, Demeneix BA Preferential transfection of adult mouse neural stem cells and their immediate progeny in vivo with polyethylenimine Mol Cell Neurosci 2002; 19:16574 [36] Li S et al Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors Am J Physiol 1999; 276(5 Pt 1):L796-804 References Shi Lei 84 [37] Liu F, Song Y, Liu D Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA Gene Ther 1999; 6:1258-66 [38] Mannes AJ, Caudle RM, O’Connell BC, Iadarola MJ Adenoviral gene transfer to spinal cord neurons: intrathecal vs intraprenchymal administration Brain Res 1998; 793:1-6 [39] Martino G, Furlan R, Comi G, Adorini L The ependymal route to the CNS: an emerging gene therapy approach for MS Trends Immunol 2001; 22:483-490 [40] Meuli-Simmen C et al Gene expression along the cerebral-spinal axis after regional gene delivery Hum Gene Ther 1999; 10:2689-2700 [41] Meyer O, Schughart K, Pavirani A, Kolbe HV Multiple systemic expression of human interferon-beta in mice can be achieved upon repeated administration of optimized pcTG90-lipoplex Gene Ther 2000; 7:1606-11 [42] Mislick KA, Baldeschwieler JD Evidence for the role of proteoglycans in cationmediated gene transfer Proc Natl Acad Sci USA 1996; 93:12349-54 [43] Nguyen H-K, Lemieux P, Vinogradov SV, Gebhart CL, Guérin N, Paradis G, Bronich TK, Alakhov VY, Kabanov AV Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents Gene Ther 2000; 7:126-138 [44] Ogris M, Steinlein P, Kursa M, Mechtler K, Kircheis R, Wagner E The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells Gene Ther 1998; 5:1425-33 [45] Ogris M, Brunner S, Schuller S, Kircheis R, Wagner E PEGylated DNA/transferrinPEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery Gene Ther 1999; 6:595-605 References Shi Lei 85 [46] Oh YK, Kim JP, Yoon H, Kim JM, Yang JS, Kim CK Prolonged organ retention and safety of plasmid DNA administered in polyethylenimine complexes Gene Ther 2001; 8:1587-92 [47] O’Leary MT, Charlton HE A model for long-term transgene expression in spinal cord regeneration studies Gene Ther 1999; 6:1351-1359 [48] O'Riordan CR et al PEGylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo Hum Gene Ther 1999; 10:1349-58 [49] Petersen H, Fechner PM, Martin AL, Kunath K, Stolnik S, Roberts CJ, Fischer D, Davies MC, Kissel T Polyethylenimine-graft-poly(ethylene glycol) copolymers: Influence of copolymer clock structure on DNA complexation and biological activities as gene delivery system Bioconjug Chem 2002a; 13:845-854 [50] Petersen H, Petra M Fechner, Dagmar Fischer, and Thomas Kissel Synthesis, characterization, and biocompatibility of polyethylenimine-graft poly(ethylene glycol) block copolymers Macromoleculars 2002b; 35:6867-6874 [51] Pollard H, Toumaniantz G, Amos JL, Avet-Loiseau H, Guihard G, Behr JP, Escande D Ca2+-sensitive cytosolic nucleases prevent efficient delivery to the nucleus of injected plasmids J Gene Med 2001; 3:153-64 [52] Ram Z, Walbridge S, Oshiro EM, Viola JJ, Chiang Y, Mueller SN, Blaese RM, Oldfield EH Intrathecal gene therapy for malignant leptomeningeal neoplasia Cancer Res 1994; 54:2141-2145 [53] Raymon H, Thode S, Gage FH Application of ex vivo gene therapy in the treatment of Parkinson disease Exp Neurol 1997; 144:82-91 References Shi Lei 86 [54] Rizzuto G, Cappelletti M, Maione D, Savino R, Lazzaro D, Costa P, Mathiesen I, Cortese R, Ciliberto G, Laufer R, La Monica N, Fattori E Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation Proc Natl Acad Sci USA 1999; 96:6417-22 [55] Rols MP, Delteil C, Golzio M, Dumond P, Cros S, Teissie J In vivo electrically mediated protein and gene transfer in murine melanoma Nat Biotechnol 1998; 16:168-71 [56] Sagara K, Kim SW A new synthesis of galactose-poly(ethylene glycol)polyethylenimine for gene delivery to hepatocytes J Control Release 2002; 79:271281 [57]Schnell L, Schreider R, Kolbeck R, Barde YA, Schwab ME Neurotrophin-3 enhances sprouting of corticospinal tract during development and after adult spinal cord lesion Nature 1994; 367:170-173 [58]Stevenson PG, Hawke S, Sloan DJ, Bangham RM The immunogenicity of intracerabral virus infection depends on anatomical site J Virol 1997; 71:145-151 [59]Suh J, Paik H J and Hwang B K Ionization of Poly(ethylenimine) and Poly(allylamine) at Various pH's Bioorg Chem 1994; 22:318-327 [60]Tan Y, Li S, Pitt BR, Huang L The inhibitory role of CpG immunostimulatory motifs in cationic lipid vector-mediated transgene expression in vivo Hum Gene Ther 1999; 10:2153-61 [61]Tang MX, Szoka FC The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes Gene Ther 1997; 4:823-32 References Shi Lei 87 [62] Tuszynski, MH, Gabriel K, Gage FH, Suhr S, Meyer S, Rosetti A Nerve growth factor delivery by gene transfer induces differential outgrowth of sensory, motor , and noradrenergic neurites after adult spinal cord injury Exp Neurol 1996; 137:157173 [63]Uyechi LS, Gagne L, Thurston G, Szoka FC Jr Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components.Gene Ther 2001; 8:828-36 [64]Wolff JA Naked DNA transport and expression in mammalian cells Neuromuscul Disord 1997; 7:314-8 [65]Wang S, Ma N, Gao SJ, Yu H, Leong KW Transgene expression in the brain stem effected by intramuscular injection of polyethylenimine/DNA complexes Mol Ther 2001; 3:658-64 [66]Yamamura J, Kageyama S, Uwano T, Kurokawa M, Imakita M, Shiraki K Longterm gene expression in the anterior horn motor neurons after intramuscular inoculation of a live herpes simplex virus vector Gene Ther 2000; 7:934-941 [67]Yang Y, Li Q, Ertl HC, Wilson JM Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses J Virol 1995; 69:2004-15 [68]Yang NS, Burkholder J, Roberts B, Martinell B, McCabe D In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment Proc Natl Acad Sci USA 1990; 87:9568-72 [69]Ye JH, Houle JD Treatment of the chronically injured spinal cord with neurotrophic factors can promote axonal regeneration from supraspinal neurons Exp Neurol 1997; 143:70-81 References Shi Lei 88 [...]... interactions of the polycation with negatively charged phosphate groups of DNA With regard to the protonation versus pH profile of PEI, at physiological pH only one fifth to sixth amino nitrogens are protonated (Suh et al., 1994) Electrostatic interactions of nucleic acid and PEI will slightly change the protonation profile of PEI, but still only one second to third of nitrogens will be protonated at... Repeated Administration 3.1.1 Transgene Expression in the Spinal Cord After Intrathecal Delivery of PEI/DNA Complexes First, transgene expression with intrathecal delivery of naked DNA was investigated Different amounts of naked plasmid pCAGLuc, ranging from 0 to 40µg, induced dosedependent transgene expression in the spinal cord The peak of gene expression was seen when 20µg of DNA was used (Fig.1a) When... solution) was mixed with 96µg of pREluc and diluted to a final volume of 6ml 50µl of the DNA-ethidium bromide solution was added into each well of a 96-well-microplate, followed by adding various concentrations of PEI (25kDa), PEG-PEI (1.3), PEG-PEI (2.5) and PEG-PEI (14.5) to obtain a final volume of 100µl per well After mixing for 5min, 100µl of distilled-water was added to each well The fluorescene... spinal cord gene delivery is concerned, lumbar intrathecal injection to the CSF is a less invasive route which makes multiple administrations feasible Lumbar puncture due to the relatively wide subarachnoid space at the site and a decent measure of mobility of nerves within the CSF hence imposes little damage to the spinal cord This route of delivery results in relatively prevalent transgene expression... the transgene expression was observed For instance, DNA/PEI complexes containing 4µg of DNA (at charge ratio of 15) yielded transgene expression 40-fold higher than equal amount of naked DNA When comparing maximum levels, gene expression induced by PEI/DNA complexes (4µg of DNA at N/P ratio of 15) could be near 5-fold of that induced by 20µg of naked DNA (Fig 1b) The efficiency of PEI-mediated transgene. .. are still unknown Indeed, one of the major barriers for efficient non-viral gene delivery is nuclear transport The trafficking of the complexes through cytoplasm and the nucleus is poorly understood but has been shown to be inefficient in polycation condensed DNA However, recent studies that used microinjection of DNA complexes into the cytoplasm or exogenous administration of PEI/DNA complexes indicated... The final stage in transgene expression includes the disassembly of the complexes (Godbey et al., 1999d) This enables transcription apparatus to access DNA efficiently Reasonably, premature degradation of DNA and hence low efficacy of gene delivery will follow if disassembly of the complexes comes too early On the other hand, a belated or insufficient assembly will not allow efficient transgene expression... processes such as absorptive endocytosis, pinocytosis and phagocytosis At N/P ratios of 2-3, complete condensation of DNA seems to happen with formation of neutral particles However, the problem with the complex neutrality is the possibility of particle aggregation Small-sized particles are usually formed only at higher polycation/DNA ratios, i.e N/P ratios, giving rise to complexes with a strong positive... degree of the PEGylation would affect the efficacy of gene delivery In the previous study, it has been shown that PEGylation could attain pronged transgene expression via repeated intrathecal administration Then the optimization of PEGylation degree, if any would have provided further clues for repeated dosing In this current study, it showed that a lower degree of PEGylation to PEI improved transgene. .. combining the intrathecal route with PEI mediated gene transfer, may lead to establishment of new non-viral gene delivery in the central nervous system On the other hand, due to some potential toxicity inherent in this polymer found in this study, various amounts of PEG have been conjugated to PEI and optimal conditions have been figured out Anyway, this strategy not only makes it possible to achieve .. .INTRATHECAL DELIVERY OF TRANSGENE TO PROMOTE NERVE REGENERATION SHI LEI (B.E., PRC) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF ANATOMY, FACULTY OF MEDICINE... Transgene Expression in the Spinal Cord After Intrathecal Delivery of PEI/DNA Complexes First, transgene expression with intrathecal delivery of naked DNA was investigated Different amounts of. .. of the polycation with negatively charged phosphate groups of DNA With regard to the protonation versus pH profile of PEI, at physiological pH only one fifth to sixth amino nitrogens are protonated