DEVELOPMENT OF GENE THERAPIES FOR INHERITED RETINAL DEGENERATIONS: A NON-VIRAL APPROACH by DA SUN Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY May 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Da Sun candidate for the degree of Doctor of Philosophy. Thesis Advisor Zheng-Rong Lu Committee Chair Stathis Karathanasis Committee Member Eben Alsberg Committee Member Timothy Kern Committee Member Akiko Maeda Date of Defense March 6, 2017 *We also certify that written approval has been obtained for any proprietary material contained therein 1 Table of Contents TABLE OF CONTENTS ....................................................................................... 2 LIST OF TABLES ................................................................................................. 6 LIST OF FIGURES ............................................................................................... 7 ACKNOWLEDGEMENTS ................................................................................ 13 ABSTRACT.......................................................................................................... 15 CHAPTER I. GENE THERAPY FOR INHERITED RETINAL DEGENERATIONS: A NON-VIRAL APPROACH ........................................ 17 1. THE EYE AND RETINA................................................................................. 18 1.1 Photo transduction and the Retinoid Cycle ....................................................................... 19 2. INHERITED RETINAL DISEASES (IRDS) AND GENE THERAPY ................... 21 2.1 Gene Therapy Development for IRDs .............................................................................. 21 2.2 Clinical Investigations of Gene Therapies for IRDs ......................................................... 24 2.3 Vectors for Gene Delivery to the Retina ........................................................................... 27 2.4 Administration Routes of Gene Therapies for IRDs ......................................................... 30 3. DEVELOPMENT OF NON-VIRAL GENE THERAPIES FOR IRDS ................... 32 3.1 Polymer Based Retinal Gene Therapies ..................................................................... 32 3.2 Cationic lipids ................................................................................................................... 42 4. CHALLENGES OF NON-VIRAL GENE THERAPY FOR IRDS ........................ 44 4.1 Barriers of Retinal Gene Delivery .................................................................................... 44 4.2 Ideal Design of Non-viral Gene Therapy for IRDs ........................................................... 46 CHAPTER Ⅱ. SELF-ASSEMBLY OF A MULTIFUNCTIONAL LIPID WITH CORE–SHELL DENDRIMER DNA NANOPARTICLES ENHANCED EFFICIENT GENE DELIVERY AT LOW CHARGE RATIOS INTO RPE CELLS .............................................................................................. 51 1. BACKGROUND ............................................................................................. 51 2. DELIVERY SYSTEM DESIGN ........................................................................ 52 3. MATERIALS AND METHODS ........................................................................... 54 3.1 Cell Culture ....................................................................................................................... 54 3.2 Animal .............................................................................................................................. 54 3.3 Preparation of G4/ECO/DNA Nanoparticles .................................................................... 54 3.4 Nanoparticle Characterization .......................................................................................... 55 3.5 Atomic Force Microscope ................................................................................................. 55 3.6 Transmission Electron Microscope ................................................................................... 56 3.7 Gel Electrophoresis for Particle Stability .......................................................................... 56 3.8 Cell Viability ..................................................................................................................... 57 3.9 Cellular Uptake ................................................................................................................. 57 3.10 Endosomal Escape .......................................................................................................... 58 3.11 In Vitro Transfection ....................................................................................................... 58 2 3.12 Ex Vivo Retinal Transfection ........................................................................................... 59 3.13 In Vivo Subretinal Transfection With G4/ECO/pDNA Nanoparticles ............................. 60 3.14 Histology......................................................................................................................... 61 3.15 Statistical Analysis .......................................................................................................... 62 4. RESULTS ...................................................................................................... 62 5. DISCUSSION ................................................................................................. 72 CHAPTER Ⅲ. TARGETED MULTIFUNCTIONAL LIPID ECO PLASMID DNA NANOPARTICLES AS EFFICIENT NON-VIRAL GENE THERAPY FOR LEBER’S CONGENITAL AMAUROSIS ................................................ 76 1. BACKGROUND ............................................................................................. 76 2. DELIVERY SYSTEM DESIGN ........................................................................ 77 3. MATERIALS AND METHODS ......................................................................... 79 3.1 Cell Cultures ..................................................................................................................... 79 3.2 Animals ............................................................................................................................. 80 3.3 Synthesis of Ret-PEG-MAL ............................................................................................. 80 3.4 Preparation of ECO/pDNA and Ret-PEG-ECO/pDNA Nanoparticles ............................. 80 3.5 TEM .................................................................................................................................. 81 3.6 DLS ................................................................................................................................... 81 3.7 In Vitro Transfection ......................................................................................................... 82 3.8 Intracellular Uptake .......................................................................................................... 83 3.9 In Vivo Subretinal Transfection with ECO/pDNA and Ret-PEG-ECO/pDNA Nanoparticles .......................................................................................................................... 83 3.10 qRT-PCR ......................................................................................................................... 84 3.11 Electroretinograms .......................................................................................................... 85 3.12 Histology......................................................................................................................... 85 3.13 Statistical Analysis .......................................................................................................... 86 4. RESULTS ...................................................................................................... 86 4.1 In Vitro Transfection with ECO/pDNA Nanoparticles ...................................................... 86 4.2 Preparation of Retinylamine-Targeted ECO/pDNA Nanoparticles ................................... 88 4.3 In Vivo Transfection with Targeted Ret-PEG-ECO/pGFP Nanoparticles in Wild-Type BALB/c Mice.......................................................................................................................... 91 4.4 Gene Replacement Therapy with Ret-PEG-ECO/pRPE65 Nanoparticles in Rpe65-/- Mice ................................................................................................................................................ 92 4.5 Cone Preservation after Gene Replacement Therapy with Ret-PEG-ECO/pRPE65 Nanoparticles in Rpe65-/- mice ................................................................................................ 95 4.6 Therapeutic Effect of Gene Replacement Therapy with Ret-PEG-ECO/pRPE65 Nanoparticles in 3-Month-old Rpe65-/- mice Mice ................................................................. 96 4.7 Safety Assessment of Ret-PEG-ECO/pRPE65 Nanoparticles in BALB/c Mice ............... 97 5. DISCUSSION ................................................................................................. 99 CHAPTER Ⅳ. PH-SENSITIVE MULTIFUNCTIONAL LIPID ECO PLASMID DNA NANOPARTICLES AS EFFICIENT NON-VIRAL GENE THERAPY FOR STARGARDT’S DISEASE ................................................. 104 1. BACKGROUND ..........................................................................................