NONVIRAL GENE DELIVERY: USING POLYMER AND PEPTIDE TO DEVELOP SAFE AND CELL-SPECIFIC GENE VECTORS ELIZABETH LUO SZE E NATIONAL UNIVERSITY OF SINGAPORE 2004 NONVIRAL GENE DELIVERY: USING POLYMER AND PEPTIDE TO DEVELOP SAFE AND CELL-SPECIFIC GENE VECTORS ELIZABETH LUO SZE E (B Eng.(Hons.), NUS) A THESIS SUBMITED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2004 Acknowledgements I would like to express my deepest gratitude to my supervisor Dr Wang Shu, for his patience and invaluable guidance throughout the course of this work Special thanks goes to Dr Tang Guping, Ms Ma Yue Xia, Dr Wang Xu, and Mr Zeng Jieming for their time on providing me technical support and assistance This project would not have been possible without the generous financial support from Institute of Bioengineering and Nanotechnology and National University of Singapore I am also very grateful to everyone in the lab for his or her support and friendship, without which my stay in the lab would not have been so enjoyable and fulfilling Last but not least, I would like to thank my parents for their love, encouragement and faith in my ability II Publications Zeng, J.-M., Too, H.-P., Ma, Y.-X., Luo, S.E Elizabeth, Wang, S., 2004 A Synthetic Peptide Containing Loop of Nerve Growth Factor Facilitates Gene Delivery to Neurons J Gene Med, in press III Table of Contents ACKNOWLEDGEMENTS II PUBLICATIONS III TABLE OF CONTENTS IV SUMMARY VIII LIST OF FIGURES IX ABBREVIATIONS X INTRODUCTION 1.1 OVERVIEW OF GENE THERAPY 1.2 NAKED DNA 1.3 CATIONIC POLYMER-BASED GENE DELIVERY SYSTEMS 1.4 POLYETHYLENIMINE (PEI) 1.5 MECHANISM OF GENE DELIVERY BY POLYPLEXES 1.5.1 Condensation of DNA 1.5.2 Cellular Uptake 11 1.5.3 Release from the Endosome 12 1.5.4 Nuclear Transport 13 IV 1.5.5 Vector Unpacking 14 1.6 CYTOTOXICITY OF POLYPLEXES 14 1.7 CELL TARGETING 15 1.8 NERVE GROWTH FACTOR (NGF) 18 OBJECTIVES 21 2.1 IMPROVING THE BIOCOMPATIBILITY OF 25KDA PEI 21 2.2 CELL TARGETING USING NERVE GROWTH FACTOR 22 MATERIALS AND METHODS 26 3.1 PEPTIDE DESIGN AND SYNTHESIS 26 3.2 PLASMIDS 26 3.3 POLYMERS 27 3.4 PREPARATION OF GENE VECTOR COMPLEXES 27 3.5 AGAROSE GEL ELECTROPHORESIS 27 3.6 ETHIDIUM BROMIDE DISPLACEMENT ASSAY 28 3.7 CELL CULTURES 28 3.8 ZETA POTENTIAL AND SIZE OF THE COMPLEXES 29 3.9 IN VITRO GENE TRANSFER 29 V 3.10 IN VIVO GENE TRANSFER 30 3.11 CELL VIABILITY ASSAY 31 IMPROVING THE BIOCOMPATIBILITY OF 25KDA PEI 33 4.1 4.1.1 Cytotoxicity of Filtered PEI25k 33 4.1.2 Improving the DNA-Condensation Capability of PEI25k(F) 33 4.1.3 Transfection Efficiency of PEI25k and Various Copolymers In Vitro 35 4.1.4 Biophysical Characterization of PEI25kD and Various Copolymers 36 4.1.5 Cytotoxicity of PEI25k/PEI600 Copolymers 37 4.2 RESULTS 33 DISCUSSION 38 CELL TARGETING USING NERVE GROWTH FACTOR 49 5.1 RESULTS 49 5.1.1 PEI as an Endosome-Disrupting Agent 49 5.1.2 Biophysical Properties of the NL4-10K, PEI600/NL4-10K, 10K and PEI600/10K Gene Vectors 50 5.1.3 NL4-10K-containing Triplexes Mediates In Vitro Gene Transfer in a Dose- dependent Manner 51 VI 5.1.4 Transfection Efficiency Mediated by NL4-10K-containing Triplexes is Dependent on Formulation Order 52 5.1.5 Specificity of NL4-10K-Mediated Gene Delivery 52 5.1.6 Optimizing the Charge Ratios between PEI600, NL4-10K and DNA 54 5.1.7 Biocompatibility of NL4-10K-containing Triplexes 55 5.2 DISCUSSION 56 REFERENCES 74 VII Summary The primary requirements for a clinically effective vehicle for human gene therapy are efficient gene transfer and safety PEI25kDa has been reported to be one of the most efficient non-viral gene vectors However, the main obstacle for its use in gene therapy is the cytotoxicity associated with it In this study, we found that the cytotoxicity is attributed to free PEI present in the gene vector solution, and they must be removed from the solution in order to reduce the toxicity level Moreover, this study also showed the existence of gaps between molecules on the outer layer of PEI-DNA complexes, and it is possible to improve the transfection efficiencies through better packing of the plasmid DNA by inserting smaller PEI molecules of the appropriate size and charge One of the major approaches toward cell-specific gene delivery is to target vehicle binding to a cell-specific receptor through receptor-mediated endocytosis In this study, we present a non-viral gene transfer vector for targeted gene delivery into TrkA-positive cells This gene transfer vehicle consists of the hairpin motif of loop linked to 10 lysine residues (NL4-10K) for targeting and nucleic acid binding purposes, respectively, and a low molecular weight polyethylenime, PEI600 for endosomal escape upon cellular uptake This gene vector is capable of mediating gene delivery into TrkA-expressing cells only, and the transfection efficiency is dependent on the formulation order of the triplexes as well as the N/P ratios between the plasmid DNA, PEI600 and NL4-10K VIII List of Figures FIG 1-1: CATIONIC POLYMERS MOST FREQUENTLY USED FOR NUCLEIC ACID DELIVERY FIG 1-2: SCHEMATIC REPRESENTATION OF DNA UPTAKE BY MAMMALIAN CELLS FIG 1-3: SCHEMATIC DIAGRAM OF RECEPTOR-MEDIATED ENDOCYTOSIS 16 FIG 4-1: CYTOTOXICITY OF PEI25K AND PEI25K/PEI600 COPOLYMERS IN NIH3T3.E25 CELL LINES 42 FIG 4-2: ELECTROPHORETIC MOBILITY OF PLASMID DNA THROUGH A 0.7% AGAROSE GEL WAS REDUCED BY (A) PEI25KD(O), (B) PEI25KD(F), (C) PEI25KD(F) 99, (D) PEI25KD(F) 97 AND PEI25KD(F) 96 44 FIG 4-3: ETHIDIUM BROMIDE DISPLACEMENT ASSAY OF PEI25K(O), PEI25K(F), PEI25K(F) 99, AND PEI25K(F) 97 45 FIG 4-4: EFFECT OF TRANSFECTION EFFICIENCIES MEDIATED BY PEI25K(F) AND PEI25K(F)/PEI600 COPOLYMERS IN NIH3T3.E25 CELLS 46 FIG 5-1: DNA RETARDATION BY (A) NL4-10K, (B) PEI600 AND NL4-10K, (C) 10K AND (D) PEI600 AND 10K IN AGAROSE GEL UNDER ELECTROPHORESIS 64 FIG 5-2: DOSE-DEPENDENT RESPONSE OF TRANSGENE EXPRESSION MEDIATED BY NL4-10K 67 FIG 5-3: EFFECTS OF THE FORMULATION ORDER OF VECTOR TRIPLEXES ON THE GENE DELIVERY EFFICIENCY 68 FIG 5-4: SPECIFICITY OF NL4-10K MEDIATED GENE DELIVERY 70 FIG 5-5: OPTIMIZING THE TRANSFECTION EFFICIENCY MEDIATED BY NL4-10K AND PEI600CONTAINING TRIPLEXES 72 FIG 5-6: CYTOTOXICITY OF NL4-10K-CONTAINING TRIPLEXES IN NIH3T3.E25 CELLS 73 IX Chapter 5: Cell Targeting Using Nerve Growth Factor (A) 10 10 10 10 10 1000 N/P ratio 71 Chapter 5: Cell Targeting Using Nerve Growth Factor (B) 10 10 10 10 10 1000 N/P ratio Fig 5-5: Optimizing the transfection efficiency mediated by NL4-10K and PEI600containing triplexes (A) Triplexes were prepared with PEI600 first complexes with 0.5 µg of plasmid DNA/well at indicated charge ratios NL4-10K was added after 30 incubation at fixed N/P ratio of (B) Triplexes were prepared with PEI600/DNA ratio of 1/1, and NL4-10K were added afterward at various N/P ratios *P