SPECIFIC and EFFICIENT in VIVO DELIVERY of DNA and Sirna by POLYETHYLENIMINE and ITS DERIVATIVES
Total Page：16
File Type：pdf, Size：1020Kb
SPECIFIC AND EFFICIENT IN VIVO DELIVERY OF DNA AND siRNA BY POLYETHYLENIMINE AND ITS DERIVATIVES by MASSACHUSETTS INSTITUTE OF TECHNOLOGY JENNIFER A. FORTUNE SEP 2 22010 B.A. Chemistry Wheaton College, 2003 LIBRARIES Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the Degree of ARCHVES Doctor of Philosophy in Biological Chemistry at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY September 2010 @ 2010 Massachusetts Institute of Technology All rights reserved Signature of Author:. Department of Chemistry August 3, 2010 Certified by: Alexander M. Klibanov Novartis ChairProfessor of Chemistry and Bioengineering Thesis Supervisor Accepted by:. Robert W. Field Haslam and Dewey Professor of Chemistry Chairman,Departmental Committee on Graduate Students This Doctoral Thesis has been examined by a committee of the Department of Chemistry as follows: JoAnne Stubbe Novartis Professorof Chemistry and Professor of Biology Thesis Chair Alexander M. Klibanov Novartis Chair Professor of Chemistry and Bioengineering Thesis Supervisor John M.Essigmann William R. and Betsy P. Leitch Professor of Chemistry andiologicalEngineering SPECIFIC AND EFFICIENT IN VIVO DELIVERY OF DNA AND siRNA BY POLYETHYLENIMINE AND ITS DERIVATIVES by JENNIFER A. FORTUNE Submitted to the Department of Chemistry on August 3, 2010 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Chemistry ABSTRACT Linear polyethylenimine (PEI) is the "gold standard" of polycationic gene delivery vectors. However, little focus has been placed on enhancing or understanding the specificity of PEI- mediated gene delivery. Herein we evaluated the effect of chemical modification on the specificity of PEI-mediated nucleic acid delivery. We found that low molecular weight PEI (2 kDa) does not mediate efficient gene expression while high molecular weight (> 87 kDa) leads to toxicity. However, linear PEI of 25 kDa is an efficient gene delivery vector for both DNA and siRNA. Therefore, this PEI was chemically modified to explore the relationship between structure and specificity. First, PEI was covalently attached to a monoclonal anti-angiotensin I-converting enzyme (ACE) antibody (PEI-9B9) and evaluated for its ability to target PEI-9B9 polyplexes following intravenous delivery in a rat. Although mAb 9B9 retains affinity for its substrate ACE, PEI-9B9 does not enhance delivery to its intended target, the lung. Clearance of PEI-9B9 from circulation likely occurs before antibody binding to the surface expressed antigen. Next, we evaluated the ability of hydrophobic modification to modulate specificity of PEI- based gene delivery. Linear PEI was alkylated with variable length hydrocarbon chains at varying percent modification and evaluated for effective and specific gene delivery following intravenous delivery in mice. Modest alkylation (11% modification with ethyl chains to produce N-ethyl-PEI) enhances gene delivery in the lung 26-fold while quadrupling the ratio of gene product expressed in the lung relative to other organs. Interestingly, specificity profiles of the various alkyl chain derivatives vary among the organs examined. Additionally, a topical approach to gene delivery was investigated. Small branched PEI was cross-linked to gold to create PEI-gold nanoparticles (PEI-GNPs). These polycations were complexed with DNA and delivered topically to scratched rabbit cornea. PEI-GNPs effectively transfected corneal endothelium and evoked expression of the plasmid DNA without causing significant immunogenicity or toxicity. Finally, the effect of radiation on biologics was evaluated using a rigorously controlled experimental design with extreme conditions to unequivocally determine if radiofrequency radiation (RFR) has a non-thermal effect on biologics. Neither enzymes nor living cells (both bacterial and mammalian) were affect non-thermally by RFR. Thesis Supervisor: Alexander M. Klibanov Title: Novartis Chair Professor of Chemistry and Bioengineering ACKNOWLEDGMENTS I wish to express my gratitude to my thesis advisor, Alex Klibanov. Thank you for providing an environment where I was granted tremendous independence but also much needed guidance and advice. I take with me many lessons that will help me far beyond science and a surplus of antidotal stories to keep me smiling along the way. The bird is in your hands. I would also like to acknowledge committee members and faculty who played a critical role during my graduate work; JoAnne Stubbe, my thesis chair, John Essigmann, Stuart Licht, Liz Nolan, and Rajiv Mohan. I am exceedingly grateful to both past and present colleagues, especially Ken Hamill, Hector Hernandez, C. Ainsley Davis, Chia H. Wu, Mathew Tantama, Nebojsa Milovic, Alisha Weight, and Alyssa Larson, for helpful discussions and for their friendship. Without you, graduate school would have been a far more frustrating and far less enjoyable place. I am thankful to my parents, Domenic and Karen, my sisters and brothers, Jamie, Chris, Jess, Jill, and Leif, and my in-laws, Roseann and Chris, for constant support and enjoyable diversions. Thank you for listening to my rants and sharing joy in my successes. And most importantly, to my husband Bill, I can't put into words how integral a part of this thesis process you played. Thank you for making these years memories to be looked upon with a smile. I love you. TABLE OF CONTENTS Abstract 3 Acknowledgements 4 Table of Contents 5 List of Figures 7 List of Tables 8 Abbreviations 9 I. Gene Therapy and Vectors for In Vivo Nucleic Acid Delivery A. Introduction 10 B. References 31 II. Fully Hydrolyzed Linear Polyethylenimine Effects Functional In Vivo Delivery of Plasmid DNA and siRNA A. Introduction 42 B. Results and Discussion 48 C. Materials and Methods 57 D. References 61 III. Specificity of Gene Delivery In Vivo Mediated By Polyethylenimine Conjugated to an Anti-ACE Antibody A. Introduction 65 B. Results and Discussion 66 C. Materials and Methods 74 D. References 79 IV. On the Mechanism of Highly Effictive Gene Transfection In Vivo by Alkylated Polyethylenimine A. Introduction 83 B. Results and Discussion 84 C. Materials and Methods 95 D. References 96 V. Polyethylenimine Mediates Specific In Vivo Gene Delivery Upon Topical Application A. Introduction 100 B. Results and Discussion 101 C. Materials and Methods 109 D. References 113 VI. Radio Frequency Radiation (RFR) Causes No Non-Thermal Damage in Enzymes and Livng Cells A. Introduction 116 B. Results and Discussion 117 C. Materials and Methods 124 D. References 127 Curriculum Vitae 131 LIST OF FIGURES Figure 1.1 DNA transfection of a cell 11 Figure 1.2 Diversity of viruses 16 Figure 1.3 Cationic lipid delivery vectors 20 Figure 1.4 PAMAM dendrimers: structure and characteristics 24 Figure 1.5 Structure of polyethylenimine 28 Figure 2.1 Efficient in vivo gene delivery by linear PEI 45 Figure 2.2 Cytotoxicity of high molecular weight linear PEI 47 Figure 2.3 Schematic of synthesis route for preparation of linear PEI 50 Figure 2.4 Gene delivery by low molecular weight linear PEI in mice 51 Figure 2.5 Biodistribution profile of pDNA delivered by low molecular weight 52 linear PEI in mice Figure 2.6 In vivo siRNA knockdown of caveolin- 1 by linear PEI and its effects 55 Figure 3.1 In vivo gene delivery by linear PEI in rats 67 Figure 3.2 Schematic of synthesis of PEI-9B9 conjugates 69 Figure 3.3 Binding affinity of PEI-conjugated anti-ACE antibody 9B9 70 Figure 3.4 In vivo gene delivery by PEI conjugated to 9B9 71 Figure 3.5 In vivo gene delivery by PEI conjugated to 9B9 at low doses 73 Figure 4.1 Schematic of synthetic route for N-alkylated linear PEI derivatives 85 Figure 4.2 Characterization of N-alkylated linear PEI by buffering capacity and 87 DNA exclusion Figure 4.3 Specificity and efficacy of gene delivery of N-alkylated linear PEI 89 derivatives in mice Figure 4.4 Effect of %modification by N-alkylation on gene delivery in vivo 90 Figure 4.5 Biodistribution profile of N-alkyl PEI derivatives in mice 93 Figure 5.1 Schematic of synthetic route for preparation of PEI-GNPs 102 Figure 5.2 In vivo detection of PEI-GNP/GFP plasmid polyplexes by silver 103 staining and detection of expressed GFP by fluorescence microscopy Figure 5.3 Immunogenicity of topical delivery of PEI-GNP polyplexes in the 105 cornea Figure 5.4 Toxicity of topical delivery of PEI-GNP polyplexes in the cornea 108 Figure 6.1 Effect of RFR on enzymatic activity of p-galactosidase and HRP 120 Figure 6.2 Effect of RFR on bacteria and mammalian cells 122 LIST OF TABLES Table 1.1 Characteristics of viral delivery vectors 17 Table 2.1 Silencing of influenza infection by PEI-mediated siRNA 46 delivery Table 4.1 Biodistribution of plasmid delivered by N-alkylated linear 92 PEI derivatives ABBREVIATIONS Ab-SPDP Antibody conjugated to SPDP ACE angiotensin I-converting enzyme ANOVA analysis of variance s-gal enzyme P-galactosidase BSA bovine serum albumin BCA bicinchoninic acid DAPI 4',6-diamidino-2-phenylindole DNA deoxyribonucleic acid DTT dithiothreitol Epi epithelial scrape GFP green fluorescent protein HCl hydrochloric acid HIV human immunodeficiency virus HRP enzyme horseradish peroxidase kb kilobases (kilonucleotides) mAb 9B9 mouse anti rat ACE monoclonal antibody (mAb) mRNA messenger RNA NMR nuclear magnetic resonance N/P ratio ratio of nitrogen in PEI to phosphate in DNA NP-siRNA influenza nucleopotein siRNA OxPAPC oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine PAMAM polyamidoamine PBS phosphate buffered saline pDNA plasmid DNA PEI polyethylenimine PEI-9B9 PEI-mAb 9B9 conjugates PEI-GNP PEI-gold nanoparticle PEI-SPDP PEI conjugated to SPDP PEOZ poly(2-ethyl-2-oxazoline) PLL poly-(L)-lysine RFID radiofrequency identification RFR radiofrequency radiation RFID radiofrequency identification RLU relative light unit RNA ribonucleic acid RNAi RNA interference SAR specific absorption rate SCID severe combined immunodeficiency siRNA short interfering RNA TUNEL terminal deoxyribonucleotidyl transferase dUTP nick end labeling VILI ventilator induced lung injury I.