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 ...... 104
3 2. DELIVERY SYSTEM DESIGN ...... 106 3. MATERIALS AND METHODS ...... 107 3.1 Plasmid Preparation ...... 107 3.2 Cell Culture ...... 109 3.3 Animal ...... 109 3.4 Gel Electrophoresis ...... 110 3.5 Synthesis of A2E ...... 110 3.6 HPLC Analysis of A2E Samples ...... 111 3.7 Preparation of ECO/pDNA Nanoparticles ...... 111 3.8 Transmission Electron Microscope ...... 112 3.9 Dynamic Light Scattering (DLS) ...... 112 3.10 In Vitro Transfection ...... 113 3.11 In Vivo Subretinal Transfection with ECO/pDNA nanoparticles ...... 113 3.12 Quantitative RT-PCR (qRT-PCR) ...... 114 3.13 Electroretinograms ...... 115 3.14 Histology...... 115 3.15 Statistical Analysis ...... 116 4. RESULTS ...... 117 4.1 Nanoparticle Characterization ...... 117 4.2 Differential Expression of Reporter Genes In Vitro and In Vivo ...... 118 4.3 In Vitro and In Vivo Transfection with ECO/pABCA4 Nanoparticles ...... 121 4.4 Gene Replacement Therapy using ECO/pABCA4 Nanoparticles ...... 124 4.5 Safety of Gene Replacement Therapy using ECO/pABCA4 Nanoparticle ...... 126 5. DISCUSSION ...... 127 CHAPTER Ⅴ. EFFICIENT DELIVERY OF CRISPR/CAS9 VIA PH- SENSITIVE MULTIFUNCTIONAL LIPIDS FOR THE TREATMENT OF AUTOSOMAL DOMINANT RETINAL GENETIC DISEASES ...... 132
1. BACKGROUND ...... 132 2. DELIVERY SYSTEM DESIGN ...... 135 3. MATERIALS AND METHODS ...... 137 3.1 Synthesis of New Carriers ...... 137 3.2 Cell Culture ...... 142 3.3 Particle Formulation and Characterization ...... 142 3.4 Evaluation of pH-Sensitive Membrane Disruption ...... 143 3.5 In vitro Transfection ...... 144 3.6 Cytotoxicity ...... 145 3.7 TEM ...... 145 3.8 qRT-PCR ...... 146 3.9 Western Blot ...... 146 4. RESULTS ...... 147 4.1 Evaluation of GFP-targeting CRISPR/Cas9 System...... 147 4.2 Gel Electrophoresis of Encapsulation and Stability of New Carriers-CRISPR/Cas9 Nanoparticle Formulations...... 151 4.3 Size Evaluation of New Carriers-CRISPR/Cas9 Nanoparticle Formulations...... 152
4 4.4 Zeta Potential Evaluation of New Carriers-CRISPR/Cas9 Nanoparticle Formulations. . 155 4.5 pH-Dependent Hemolytic Activities of New Carriers ...... 157 4.6 In Vitro Transfection of CRISPR/Cas9 System Using New Carriers in NIH3T3-GFP Cells...... 158 5. DISCUSSION ...... 160 CHAPTER Ⅵ. OPTIMIZATION OF PH-SENSITIVE MULTIFUNCTIONAL CATIONIC LIPID GENE DELIVERY SYSTEMS ...... 165
1. TARGETING LIGAND DEVELOPMENT ...... 166 2. ENHANCERS FOR PROLONGED AND EFFICIENT THERAPEUTIC GENE EXPRESSION ...... 170 3. MODIFICATION OF CURRENT ECO BASED CARRIER ...... 171 CHAPTER Ⅶ. OUTLOOK FOR NON-VIRAL GENE THERAPY FOR IRDS ...... 173
1. ADMINISTRATION ROUTES ...... 173 2. GENE THERAPY DEVELOPMENT FOR AUTOSOMAL DOMINANT IRDS .... 174 3. DISEASE AND PATIENT SELECTION ...... 175 4. UNEXPECTED RESULTS ...... 175 5. MANUFACTURE ...... 176 REFERENCES ...... 179
5 List of Tables
Table 1. List of gene mutations associated with IRDs with available animal models...... 23
Table 2. Clinical trials of gene therapies for the treatment of IRDs...... 24
6 List of Figures
Figure 1.1. Structure of the retina: cellular organization within the retina. .. 19 Figure 1.2. Photo transduction and retinoid cycle14...... 21 Figure 1.3. Common administration routes for retinal drug delivery...... 30 Figure 1.4. Chemical structure of commonly used polymer based non-viral gene delivery systems...... 33 Figure 1.5. Schematic showing polymerization of poly(beta-amino ester)s (PBAEs) 76...... 38 Figure 1.6. Chemical structure of lysine peptide gene delivery system (CK30PEG)...... 40 Figure 1.7. Chemical structure of fourth-generation poly(amidoamine) dendrimer (PAMAM-G4).90 ...... 42 Figure 1.8. Chemical structures of common cationic lipid based gene delivery systems...... 43 Figure 1.9. Three main strategies to package genetic materials by non-viral polycations.99 ...... 47 Figure 1.10. pH-sensitive amphiphilic endosomal escape and Reductive cytosolic release of cationic lipid/siRNA nanoparticles. 101 ...... 49 Figure 2.1. Formation of G4/ECO/pDNA nanoparticles. Hybrid G4/ECO/pDNA nanoparticles are formed following two stepwise electrostatic complexations: plasmid DNA first is condensed by G4 nanoglobules and lipid ECO then is incorporated into the delivery system through electrostatic interactions between the cationic head group of ECO and the negatively charged surface of the G4/pDNA complexes...... 53 Figure 2.2. Agarose gel electrophoresis showing retardation of G4/ECO/pDNA nanoparticles over a range of N/N/P ratios as compared with free pDNA, G4/pDNA nanoparticles, and ECO/pDNA nanoparticles...... 63 Figure 2.3. Size and zeta potential of G4/pDNA and G4/ECO/pDNA nanoparticles over a range of N/P or N/N/P ratios...... 64 Figure 2.4. Morphological characterization of a G4/ECO/pDNA N/N/P ratio 3/3/1 formulation. (a) 2D image from a tapping mode AFM scan. (b) Corresponding 3D image from the same tapping mode AFM scan. (c) TEM image of dry form G4/ECO/pDNA N/N/P ratio 3/3/1 particles. .. 65 Figure 2.5. Viability of ARPE-19 cells incubated without or with 10% serum transfection media. G4/ECO/pDNA nanoparticles were tested over a range of N/N/P ratios with lipofectamine 2000, ECO/pDNA nanoparticle and G4/pDNA nanoparticle serving as controls...... 66 Figure 2.6. (a) Cellular uptake of G4/ECO/Cy3 pDNA nanoparticles cultured with ARPE-19 cells for 4 h. A range of N/P ratios were tested under serum-free and 10% serum culture media. (b) Cellular uptake of G4/ECO/pDNA N/P ratio 3/3/1 particles cultured with ARPE-19 cells for
7 4 h in serum-free media under different inhibitory conditions (cytochalasin D (5 μg · mL-1), nocodazole (20 μM), and 4 °C). (c) Confocal fluorescence image of cytosolic delivery of G4/ECO/pDNA N/P ratio 3/3/1 particles. Late endosomes were stained with LysoTracker Green (green), nuclei were stained with Hoechst 33342 (blue), and DNA plasmid was labeled with Cy3 (red). At 24 h, nanoparticles mostly escape endosomal entrapment, shown by the red signals dispersed throughout the cytoplasm. Green signals represent late endosomes/lysosomes. Co- localization with late endosomes (yellow) appears minor...... 67 Figure 2.7. (a) Confocal microscopic image of GFP expression 48 h post transfection in ARPE-19 cell line under serum-free and 10% serum transfection media. Transfection of G4/ECO/pDNA N/N/P ratios 2/1/1, 2/2/1, 2/3/1, 3/1/1, 3/2/1, and 3/3/1 nanoparticles were evaluated. (b) Flow cytometry of GFP expression 48 h post-transfection of ARPE-19 cell line under serum-free and 10% serum transfection media. G4/ECO/pDNA nanoparticles were tested over a range of N/N/P ratios with lipofectamine 2000, ECO/pDNA particles and G4/pDNA particles used as controls...... 70 Figure 2.8. (a) Ex-vivo transfection of RPE and retinal tissues accomplished with an organotypic culture method. G4/ECO/pDNA N/N/P ratio 3/3/1 nanoparticles were cultured with RPE and retinal tissue from C57/BL6J wild type mice for 8 h. Confocal microscopic images of GFP expression (green) after either 4 or 6 d are shown. (b) In vivo transfection. G4/ECO/pDNA N/N/P ratio 3/3/1 particles were subretinally injected into the eyes of wild type BALB/c mice. Confocal microscopic images of GFP expression (green) in flat mounted retina and the RPE layer are shown 3 and 5 d after the injection. Control groups consisted of mice treated with only DNA plasmid at the same dose. (c) Histology of eye cups from BALB/c mice 7 d post-transfection. GFP antibody and a secondary fluorescent labeling antibody (red) were applied to identify GFP expression...... 72 Figure 3.1. Molecular structure of pH-sensitive multifunctional lipid (1- aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)propionamide] (ECO)...... 78 Figure 3.2. Chemical structure of all-trans-retinylamine targeting ligand. ... 78 Figure 3.3. In Vitro Transfection of ARPE19 Cells with ECO/pDNA Nanoparticles (A and B) Confocal microscopy images (A) and flow cytometry analysis (B) of ARPE-19 cells transfected with ECO/pGFP (N/P = 6) nanoparticles and Lipofectamine 2000/pGFP nanoparticles for 48 hr (**p < 0.005). Each bar represents the mean ± expression level of GFP (n = 3). (C) Confocal fluorescence microscopy images demonstrating intracellular trafficking of ECO/Cy3-pDNA nanoparticles in ARPE-19 cells. Cells were treated with LysoTracker Green (1:2,500 dilution) and Hoechst 33342 (1:10,000 dilution) and then transfected with
8 ECO/Cy3-labeled nanoparticles at N/P = 6. After 1, 4, and 24 h of transfection, cells were fixed and imaged. Green, endosomes; blue, nuclei; red, Cy3-labeled pDNA. Arrows denote the ECO/Cy3-pDNA nanoparticles and Cy3-pDNA. Scale bars, 20 μm...... 88 Figure 3.4. Preparation of the Targeting Ligand and Characterization of Ret- PEG-ECO/pDNA Nanoparticles. (A) Synthesis route. (B) MALDI-TOF mass spectrum of Ret-PEG-MAL. (C) TEM images of Ret-PEG- ECO/pDNA nanoparticles. (D) Size distribution of Ret-PEG-ECO/pDNA nanoparticles measured by DLS. (E) Sizes and zeta potentials of Ret- PEG-ECO/pDNA nanoparticles. Each bar represents the mean ± size of particles and each dot represents the mean ± zeta potential of particles (n = 3)...... 91 Figure 3.5. In Vivo Gene Transfection with Targeted Ret-PEG-ECO/pGFP Nanoparticles in Wild-Type BALB/c Mice. Mice (1 month old) were subretinally injected with ECO/pGFP or Ret-PEG-ECO/pGFP nanoparticles. RPE flat mounts were obtained 3 days post-transfection. (A) Fluorescence microscopic images showing enhanced GFP expression with Ret-PEG-ECO/pGFP nanoparticles in the RPE 3 days post-injection. (B) Confocal fluorescence microscopic images revealing GFP expression specifically in the RPE with anti-ZO-1 antibody staining (white). The tight junction protein ZO-1 represents the borders of the RPE cells. .... 92 Figure 3.6. Gene Replacement Therapy with Ret-PEG-ECO/pRPE65 Nanoparticles in Rpe65-/- mice. Mice were subretinally injected with Ret- PEG-ECO/pRPE65 nanoparticles or Ret-PEG-ECO. (A) Relative RPE65 mRNA levels in treated (pRPE65-injected) versus control groups 15 days after treatment. Each bar represents the mean ± mRNA expression level (n = 3). (B) Representative scotopic and photopic electroretinograms acquired from Rpe65-/- mice under the light intensity of 1.6 log cd × s/m2 7 days after treatment. (C–F) Amplitudes of scotopic a-waves (C), photopic a-waves (D), scotopic b-waves (E), and photopic b-waves (F) of treated and control Rpe65-/- mice 3, 7, 30, and 120 days post-injection (**p < 0.005). Each bar represents the mean ± wave amplitude (n ≥ 3)...... 94 Figure 3.7. Cone Preservation after Gene Replacement Therapy with Ret- PEG-ECO/pRPE65 Nanoparticles in Rpe65-/- mice 120 Days after Treatment. (A–C) Peanut agglutinin (green) was used to stain cone photoreceptors. Nuclei were stained with DAPI (blue)...... 96 Figure 3.8. Therapeutic Effect of Gene Replacement Therapy with Ret-PEG- ECO/pRPE65 Nanoparticles in 3-Month-Old Rpe65-/- Mice. Shown are ERG amplitudes of major response waveforms. (A–D) Scotopic a-waves (A), photopic a-waves (B), scotopic b-waves (C), and photopic b-waves (D) in the treatment and control groups of Rpe65-/- mice. Each bar represents the mean ± wave amplitude (n ≥ 3)...... 97 Figure 3.9. Safety Assessment of Ret-PEG-ECO/pRPE65 Nanoparticles in 1-
9 Month-Old BALB/c Mice. (A) Representative ERG traces of scotopic waveforms in the PEG-ECO/pRPE65-treated group and untreated mice 30 days post-injection. (B–E) ERG amplitudes of scotopic a-waves (B), photopic a-waves (C), scotopic b-waves (D), and photopic b-waves (E) in treated and control animals. Each bar represents the mean ± wave amplitude (n ≥ 3)...... 99 Figure 3.10. Targeting Mechanism of All-trans-Retinylamine-Modified ECO Nanoparticles. When injected into the subretinal space, all-trans- retinylamine-modified ECO nanoparticles will bind to IRBP in the interphotoreceptor matrix. IRBP binding helps to retain the nanoparticles in the space and transports the nanoparticles to the target cells in the RPE. Following cellular uptake by endocytosis, the nanoparticles escape from the endosomal compartment and release the RPE65 plasmid DNA via the PERC mechanism. Finally, the RPE65 gene is expressed by the RPE cell, where it slows cone cell degeneration and preserves visual function. 101 Figure 4.1. Map of therapeutic ABCA4 plasmid with photoreceptor specific bovine rhodopsin promoter (RHO)...... 107 Figure 4.2. Characterization of ECO/pABCA4 nanoparticle formulations for stability, size, zeta potential and morphology with lipofectamine 2000/pABCA4 formulations as controls. (A) Agarose gel electrophoresis of nanoparticle formations. (B) DLS measurements of nanoparticle size, (C) zeta potential and (D) size distribution. (E) TEM images of nanoparticle formulations. Scale bar represents 100 nm...... 118 Figure 4.3. In vitro and in vivo transfection of ECO/pCMV-GFP, ECO/pRHO- DsRed nanoparticles in ARPE-19 cells and wild type 129S1/SvImJ mice. (A) Confocal images of GFP and DsRed expression in ARPE-19 cells. (B) Quantification of mean fluorescence intensities of GFP and DsRed expression in ARPE-19 cells by flow cytometry. (C) mRNA levels of GFP and DsRed expression in ARPE-19 cells by PCR analysis. (D) Confocal images of retinal tissue slides 3 days after subretinal injection compared with non-treated controls. (E) mRNA levels of GFP and DsRed in the retinal tissue of 129S1/SvImJ mice 3 days after subretinal injection. mRNA levels were normalized to levels in the RPE. (**p < 0.005). .. 121 Figure 4.4. Characterization, in vitro and in vivo transfection efficiency of ECO/pRHO-ABCA4 and ECO/pCMV-ABCA4 nanoparticles in Abca4-/- mice. A. Agarose gel of ECO/pRHO-ABCA4, ECO/pCMV-ABCA4 nanoparticles and free plasmids; B. mRNA levels of ABCA4 expression in ARPE-19 cells transfected by ECO/pCMV-ABCA4 or control Lipofectamine/pCMV-ABCA4 nanoparticles. C. mRNA levels of ABCA4 expression in the retinal tissue of Abca4-/- mice 7 days after subretinal injections of ECO/pRHO-ABCA4 and ECO/pCMV-ABCA4 nanoparticles. D. Confocal images of ABCA4 expression (green) in Abca4-/- mice 7 days after subretinal injections of ECO/pRHO-ABCA4 and ECO/pCMV-ABCA4 nanoparticles, with the expression in wild type
10 129s mice as the control...... 124 Figure 4.5. Gene therapy in Abca4-/- mice with ECO/pCMV-ABCA4 and ECO/pRHO-ABCA4 nanoparticles. (A) A2E accumulations in Abca4-/- mice of different ages characterized by HPLC analysis. (B) Characteristic spectrum of A2E taken from the HPLC analysis of A2E accumulations in Abca4-/- mice. (C) A2E levels in Abca4-/- mice 6 month after treatment with ECO/pCMV-ABCA4 and ECO/pRHO-ABCA4 nanoparticles analyzed by HPLC. (D) Quantification of A2E levels of Abca4-/- mice 6 month after treatment. A2E levels were normalized to SHAM (PBS injected) eyes...... 126 Figure 4.6. Safety of ECO/pABCA4 treatment in wild type mice. ERG a-wave and b-wave amplitudes analyzed 7 and 30 days after injection with PBS (sham), ECO/pCMV-ABCA4, or ECO/pRHO-ABCA4 in one eye. Amplitudes are normalized to the contralateral uninjected eye. Error bars = ± std (* p < 0.05 relative to no injection)...... 126 Figure 5.1. Design of ECO isotypic derivatives by head modification and new amino acid functional linkers introduction...... 136 Figure 5.2. Plasmid maps of CRISPR/Cas9 system that targets GFP gene.137 Figure 5.3. Synthetic route of new carriers...... 137 Figure 5.4. Evaluation of GFP-targeting CRISPR/Cas9 system. (A) TEM images of ECO/psgRNA and ECO/pCas9 nanoparticles. (B) Size distribution of ECO/psgRNA and ECO/pCas9 nanoparticles by DLS measurements. (C) Size and Zeta potential of ECO/psgRNA and ECO/pCas9 by DLS measurements. (D) In vitro transfection of ECO/psgRNA and ECO/pCas9 (particle ratio 1:1) in NIH3T3-GFP cells, with the dose of each plasmid at 1 μg, 1.5 μg, 2 μg and 2.5 μg in the transfection media. Cas9 expression (Blue), gRNA expression (Red) and GFP knockdown (Green) were imaged by confocal microscopy. (E) Quantitative flow cytometry measurements of GFP knockdown efficiency represented by percentage reduction of GFP fluorescent intensities. (F) Quantitative flow cytometry measurements of sgRNA expression represented by percentage of mCherry positive cells. (G) Cas9 protein expression demonstrated by western blot (NS=nonspecific control, β-actin expression as inner control). (H) GFP knockdown
efficiency in mRNA levels measured by qRT-PCR. Error bars = ± std (* p < 0.05 relative to untreated control)...... 151 Figure 5.5. Agarose gel electrophoresis of the encapsulation and stability of new carriers-CRISPR/Cas9 nanoparticles at N/P ratios of 6, 8 and 10...... 152 Figure 5.6. DLS size measurements of the new carriers-CRISPR/Cas9 nanoparticles. The size distribution of nanoparticles formulated between (A) ECO, (B) iECO, (C) iEKCO, (D) iECKO, (E) iEHCO, (F) iECHO and plasmids of GFP targeting CRISPR/Cas9 system...... 154
11 Figure 5.7. DLS zeta potential measurements of the new carriers- CRISPR/Cas9 nanoparticles at N/P ratio of 6, 8 and 10...... 156 Figure 5.8. pH-dependent hemolytic activities of all carriers at N/P ratio of 10. Rat blood cells were diluted 1:50 in PBS and incubated with each formulation at pH = 7.4, 6.5, and 5.4 for 2 h at 37 °C. Triton X-100 (1% v/v) was implemented as a positive control...... 157 Figure 5.9. In vitro transfection of CRISPR/Cas9 system using new carriers in NIH3T3-GFP cells. For each carrier, the lipid/psgRNA and lipid/pCas9 particle ratio was 1:1, with the dose of each plasmid at 1 μg, in the transfection media. Cas9 expression (blue), gRNA expression (red) and GFP knockdown (green) were imaged by confocal microscopy 72 h after tranfection...... 159 Figure 5.10. In vitro transfection of CRISPR/Cas9 system using new carriers in NIH3T3-GFP cells. For each carrier, the lipid/psgRNA and lipid/pCas9 particle ratio was 1:1, with the dose of each plasmid at 1 μg, in the transfection media. (A) Quantitative flow cytometry measurements of GFP knockdown efficiency represented by percentage reduction of GFP fluorescent intensities. (B) MTT assay of cytotoxicity in NIH3T3-GFP cells 72 h after transfection. (C) Quantitative flow cytometry measurements of Cas9 expression represented by percentage of GFP positive cells. (D) Quantitative flow cytometry measurements of sgRNA expression represented by percentage of mCherry positive cells. Error bars = ± std (* p < 0.05 relative to untreated control)...... 160 Figure 6.1. Chemical structure of ACU4429...... 166 Figure 6.2. The Design of ACU4429-PEG-HZ-MAL Targeting Ligand. (A) Chemical synthesis route of ACU4429-PEG-HZ-MAL targeting ligand. (B) MALDI-TOF mass spectra of ACU4429-PEG-HZ-MAL and intermediate products...... 168 Figure 6.3. Confocal fluorescence image of cytosolic delivery of ACU4429- PEG-HZ-ECO/ECO/Cy3-pDNA and PEG-ECO/ECO/Cy3-pDNA nanoparticles at N/P ratio 10 in ARPE-19 Cells. Late endosomes were stained with LysoTracker Green (green), nuclei were stained with Hoechst 33342 (blue), and DNA plasmid was labeled with Cy3 (red)...... 169 Figure 6.4. In vivo evaluation of ACU4429 targeting ligand to the RPE. Nanoparticle distribution of Non-targeting ECO/Cy3-plasmid particles and ACU-PEG-HZ-ECO/ECO/Cy3-plasmid particles in the subretinal space of Abca4-/- mice after subretinal injection. SHAM (PBS injected)...... 170 Figure 6.5. Chemical structure of the ester type ECO derivative (eECO). . 172
12 Acknowledgements
Over the past five years, many individuals have helped me in both my professional and personal development. First and foremost, I would like to thank my advisor, Dr. Zheng-Rong Lu, for all his help and advice in my research and career development. I also want to thank Dr. Lu and his wife Dr. Gao as friends. I am grateful for their kindness and hospitality to me, my wife and my family.
I would like to thank my committee members, Dr. Eben Alsberg, Dr. Stathis
Karathanasis, Dr. Timothy Kern, and Dr. Akiko Maeda, for all the guidance and help in my research and life. I would like to thank my collaborators, who have provided me with the resources and supports for my research. I would like to thank
Dr. Krzysztof Palczewski and Palczewski lab members for providing the animal models and injection techniques. I would like to thank Dr. Akiko Maeda and Maeda lab members for the support in all aspects of my research. I would like to thank Dr.
Songqi Gao for performing ERG tests, and Dr. Hiroshi Maeno, Dr. Bhubanananda
Sahu and Mr. Avery Sears for performing subretinal injections.
I would like to thank all my labmates for creating a Lu lab family and sharing all the memorable moments with me. To my “foreign brother” Becky, thanks for the help in research and letting me be part of your family. I will cherish the friendship for life. To all the lab members that I have worked with, Dr. Songqi Gao, Dr. Amita
Vaidya, Dr. Xujie Liu, Dr. Zhanhu Sun, Dr. Cheng Han, Dr. Xiaohui Wu, Dr. Jenny
Parvani, Dr. Yajuan Li, Dr. Zhuxian Zhou, Dr. Erlei Jin, Dr. Xueming Wu, and Dr.
Guanping Yu, Sarah Roelle, Zhen Ye, Anthony Malamas, Anthony Puntel, Maneesh
13 Gujrati, Zheng Han, Li Sheng, Nadia Ayat, Andrew Schilb, Peter Qiao, Jingcan Qin,
Hongfa Jiang, and Runjie Yang, thanks for all the support and mentorship. To my undergraduate student Rui Xin, thanks for being inspiring and all the solid work you have done. I wish to see more success from you in the future.
Finally, I would like to thank my parents Fengyi Sun and Wenru Dong for the support and encouragement. I would not achieve what I have without both of you.
To my wife Weiqian Wang, thanks for being with me for 14 years. You are always supportive, inspiring and hardworking. I can’t wait to accomplishing more with you in the future.
14 Development of Gene Therapies for Inherited Retinal Degenerations: A Non-
Viral Approach
By
DA SUN
Inherited retinal degenerations (IRDs) are a major cause of blindness, which can be caused by mutations in a single gene occurred in rod/cone photoreceptor cells (PR) or retinal pigment epithelium (RPE). IRDs can be treated with gene replacement therapy (GRT) due to easy localized delivery into the retina and its immune privilege. Development of safe and effective gene GRT is critical in this field.
In this work, we evaluated three non-viral GRTs for treatments of IRDs. First, a hybrid system of a multifunctional lipid ECO and a G4 nanoglobule was designed for efficient gene delivery into RPE cells at low charge ratios. This system formed stable nanoparticles at low N/P ratios, exhibited low cytotoxicity, and induced high
GFP expression in ARPE-19 cells at N/P = 6. The hybrid nanoparticles mediated significant reporter gene GFP expression ex-vivo in the retina from wild type
C57BL/6J mice and in vivo in BALB/c mice.
Second, we developed a targeted non-viral GRT using a multifunctional lipid
ECO for treating Leber’s congenital amaurosis type 2 (LCA2). ECO formed stable nanoparticles with plasmid DNA (pDNA) at a low N/P ratio and mediated high gene transfection efficiency in ARPE-19 cells. All-trans-retinylamine was incorporated
15 into the nanoparticles for targeted delivery of pDNA into the RPE. The targeted
ECO/pDNA nanoparticles provided high GFP expression in the RPE of Rpe65-/- mice after subretinal injection. GRT using targeted ECO system increased electroretinographic activity for at least 120 days. A safety study in wild-type
BALB/c mice indicated no irreversible retinal damage following subretinal injection of these targeted nanoparticles.
Third, we developed an ECO based GRT for Stargardt’s Disease (STGD).
ECO could stably encapsulate large ABCA4 gene and induce efficient gene transfection. A rhodopsin promoter was incorporated into the plasmid to generate specific gene expression to the photoreceptors. Evaluations with reporter genes demonstrated enhanced and tissue specific expressions in vitro and in vivo.
Subretinal treatment of ECO/pABCA4 GRT demonstrated high and photoreceptor specific expression of therapeutic ABCA4 gene in Abca4-/- mice, and prevented A2E accumulations at least for 6 months. Safety studies demonstrated excellent in vitro and in vivo safety profiles of ECO/pABCA4 based GRT.
16 Chapter I. Gene Therapy for Inherited Retinal Degenerations: A Non-Viral
Approach
Inherited retinal degenerations (IRDs) are a major cause of blindness, with the prevalence of 1 out of 2000 globally1. Among all the IRDs, retinitis pigmentosa
(RP)2, Leber’s congenital amaurosis (LCA)3, and Stargardt’s disease (STGD)4 are the most frequent and severe. Normally, these diseases are caused by mutations in single gene occurred in rod/cone photoreceptor cells (PR) or retinal pigment epithelium (RPE)5. Gene replacement therapy holds great promise for the treatment of many IRDs6, as gene replacement therapy with adeno-associated viral vectors
(AAVs) has been approved for clinical use for LCA27. However, gene replacement therapies are still under development for broader applications in other IRDs. Non- viral gene delivery systems have received great attention because they have no limitations in cargo capacity, better safety, are low cost and easy to prepare, which have shown successful retinal gene transductions with different non-viral gene delivery systems, and are a promising gene replacement strategy for a broader range of retinal dystrophies8.
In this chapter, we will review:
1. Anatomy and function of the vertebrate retina,
17 2. Inherited retinal diseases (IRDs) and their gene therapy development,
3.Non-viral gene therapy development for IRDs, and
4. The challenges of non-viral gene therapy for IRDs.
1. The Eye and Retina
The eye serves as our window to the surrounding world, which captures, integrates and processes all visual information from light. This process is the fundamental driver of our daily functions and behaviors. Vision is considered the most important sense out of the five senses, which is complex process that requires coordinated activity of different cells in the retina and the brain. The vertebrate retina is one of the most complexed organs that consists of a retinal pigmented epithelium (RPE) monolayer, six major types of neurons (rod and cone photoreceptors, bipolar cells, amacrine cells, horizontal cells, and ganglion cells), and Müller glial cells9 shown in Figure 1. The RPE cells are the outside layer of the retina, which are a single layer of hexagonal cells tightly packed together. The
RPE helps to protect the retina, provide structural components and energy source to the retina, and regenerate visual chromophore10. The photoreceptor layer contains light sensitive rods and cones, which connect to the RPE. These photoreceptors are responsible to transform light to electrical signal in coordination with the RPE. The inner layer of retina contains ganglion neurons, which are responsible of transmitting the signal to the brain11.
18
Figure 1.1. Structure of the retina: cellular organization within the retina.
1.1 Photo transduction and the Retinoid Cycle
When a photon reaches the retina and is absorbed by a pigment molecule in the photoreceptor cells, a step called visual/photo transduction is initiated, which involves in a sequence of photochemical, biochemical and electrophysiological events that convert light to electrical cellular response in the photoreceptor with the help of the RPE12. Photo transduction is required regeneration of photo-sensitive opsin GPCR, and this regeneration process is called the visual/retinoid cycle,
Figure 2. Specifically, visual chromophore 11-cis-retinal in opsin GPCR undergoes photoisomerization to all-trans-retinal upon hit by photons, which changes the conformation of the GPCR located on the outer segment of photoreceptors. The conformational change then initiates a signal transduction cascades that causes the
19 closure of the cyclic GMP-gated sodium channel, and the hyperpolarization of the photoreceptor cell. All-trans-retinal is further reduced to all-trans retinol and is transported to the RPE, where it is transformed back to 11-cis-retinol with the help of two key enzymes, lecithin retinol acyltransferase (LRAT) and the isomerohydrolase RPE65. Finally, 11-cis-retinol is oxidized back to 11-cis-retinal and conjugated to opsin to form functional visual pigment. The generated electrochemical signals are then transmitted through bipolar cells to ganglion cells, where they are converted into action potentials that are sent to the brain13.
20 Figure 1.2. Photo transduction and retinoid cycle14.
2. Inherited Retinal Diseases (IRDs) and Gene Therapy
2.1 Gene therapy development for IRDs
For human, vision represents a basic but decisive factor of quality of life, and its impairment creates a highly incapacitating condition. As is shown previously, vision is a combination of complex chemical and biological processes that involves in numerous proteins with different functions. As the major causes of vision loss, inherited retinal diseases (IRDs), are caused by various mutations in genes associated with photo transduction, retinoid cycle and other essential processes for vision15. Great efforts have been paid to understand the molecular and genetic mechanisms associated with the IRDs, which has revealed novel targets for their therapeutic development16. Gene therapy is a promising solution, which involves transfer of nucleic acids into target cells to correct or supplant genes with normal function that will alter the disease outcomes17. Gene therapy has its advantages.
First, the therapeutic effect can maintain for long time without repeat administrations. Second, precise delivery to desired tissue can be reached by the combination of targeted gene delivery vectors and cell type specific promoters.
Retina is an ideal candidate for gene therapy. The retina is easily accessible for surgical injection through different administration routes such as intravitreal injection and subretinal injection, which target either inner or outer retinal tissue.
The blood-retinal barrier allows immune-privilege to the retinal tissue18. Several
21 gene therapy strategies have been developed and tested in different animal models of retinal degenerations over the past few decades. As most autosomal recessive and X-linked retinal degenerations are caused by the lack of a functional protein19.
Gene replacement therapy became a common strategy to treat these type of IRDs, which involves in the delivery of a normal copy of the defective gene to the targeted-cells20. Retinal gene replacement therapy has received great attention and experiencing fast development with several clinical trials (Table 2). Recently,
LUXTURNA, the gene replacement therapy using adeno-associated virus vectors
(AAVs) to treat RPE65 associated Leber’s Congenital Amaurosis (LCA), has been approved by Food and Drug Administration (FDA), which became the first gene therapy available for clinical use7. Compared to recessive diseases, dominant diseases are more challenging for gene therapy treatments, because the aberrant protein is impaired in function and also alters the expression or the function of the wild-type protein21. Gene therapy treatment requires the suppression of the dominant allele to diminish the toxicity while preserving the expression of the wild- type allele to ensure proper gene function after treatment22. Therefore, RNAi and
CRISPR/Cas9 have been applied for gene suppression, which, in some cases, is in combination with gene replacement therapy to achieve the optimal therapeutic effects22, 23. Due to the high degree of allelic heterogeneity, this approach can hardly extend to all patients with little probability of carrying the same mutation.
CRISPR/Cas9 has shown potential of targeting only mutated allele without
22 disturbing the wild type allele, which can be a promising strategy for the treatment of autosomal dominant IRDs23.
Table 1. List of gene mutations associated with IRDs with available animal models.
Inheritanc Location Gene e Animal model Disease
Photoreceptors CRX Recessive Crx-/- mouse LCA
Photoreceptors, retinal neurons NDP X-linked Ndp-/- mouse ND
Photoreceptors AIPL1 Recessive Aipl1-/- mouse LCA
Retinal pigmented Rpe65-/- rd12 mouse, RPE65 epithelium RPE65 Recessive dog LCA
Photoreceptors ABCA4 Recessive Abca4-/- mouse Stargardt's
ACHM, Photoreceptors CNGB3 Recessive Cngb3-/- mouse CRD
GUCY2 Photoreceptors D Recessive Gucy2d-/- mouse LCA
Retinal pigmented epithelium LRAT Recessive Lrat-/- mouse LCA
Retinal pigmented epithelium MERTK Recessive Mertk-/- rat (RCS rat) LCA
Photoreceptors PDE6B Recessive rd10 mouse RP
Photoreceptors RHO Recessive Rho-/- rat, RHO T4R dog RP
RPGRIP Photoreceptors 1 Recessive Rpgrip1-/- mouse LCA, CRD
Photoreceptors TULP1 Recessive Tulp1-/- mouse ARRP
23 The availability of animal models for the IRDs, which have the same mutation in the gene of interest, is a prerequisite for gene therapy development in order to evaluate the efficacy of the treatment. The development in small and large animal models for human IRDs enables rapid implementation of gene therapy strategies for different ocular conditions. Common genetic mutations are listed in Table 1 as potential targets for gene therapy treatments with available animal models.
2.2 Clinical Investigations of Gene Therapies for IRDs
Retinal gene therapy clinical trials are underway for various gene including
RPE65, MERTK, CHM, ABCA4, RS1, MYO7A, GNGB, CNGA3 and ND4, which are summarized in Table 2.
Table 2. Clinical trials of gene therapies for the treatment of IRDs.
Formulations Trial ID Phase Disease
rAAV2tYF-PR1.7-hCNGB324 NCT02599922 Ⅰ,Ⅱ Achromatopsia rAAV.hCNGA325 NCT02610582 Ⅰ,Ⅱ
rAAV.sFlt-126 NCT01494805 Ⅰ,Ⅱ Age-Related Macular Degeneration AAV2-sFLT0127 NCT01024998 Ⅰ
AAV2.REP1 NCT02553135 Ⅱ
AAV2.REP128 NCT01461213 Ⅰ,Ⅱ Choroideremia rAAV2.REP129 NCT02671539 Ⅱ
AAV2-hCHM30 NCT02341807 Ⅰ,Ⅱ
AAV2-hRPE65v231 NCT00999609 Ⅲ Leber's Congenital Amaurosis
AAV2-hRPE65v2 NCT01208389 Ⅰ,Ⅱ
24 AAV2-hRPE65v2 NCT00516477 Ⅰ rAAV2/e.hRPE65p.hRPE65(tgAAG76)31 NCT00643747 Ⅰ,Ⅱ
rAAV2-CB-hRPE6532 NCT00749957 Ⅰ,Ⅱ
rAAV2/4.hRPE6533 NCT01496040 Ⅰ,Ⅱ
AAV2/5OPTIRPE65 NCT02781480 Ⅰ
rAAV2-CBSB-hRPE6532 NCT00481546 Ⅰ
scAAV2-P1ND4v234 NCT02161380 Ⅰ Leber Hereditary Optic Neuropathy
rAAV2-VMD2-hMERTK35 NCT01482195 Ⅰ Retinitis Pigmentosa
AAV8-scRS/IRBPhRS36 NCT02317887 Ⅰ,Ⅱ X-linked Retinoschisis rAAV2tYF-CB-hRS137 NCT02416622 Ⅰ,Ⅱ
NCT01505062 Ⅰ,Ⅱ EIAV-CMV-MYO7A38 Usher Syndrome NCT02065011 Ⅰ,Ⅱ
Phase Ⅰ trials for LCA2 showed safe treatments and excellent efficacy of stable improvement in visual function of all patients participated that received unilateral subretinal injections of AAV2-hRPE65v2. A following up study of the same patents, in which patients received subretinal injection of the contralateral eye, also demonstrated safe and stable improvement in visual and retinal function of the contralateral eye, which remained at least 3 years. A Phase III clinical trial was also completed, in which intervention patients demonstrated functional improvements in vision. Due to the success of this randomized controlled Phase III trial, the viral
RPE65 based gene therapy was approved by FDA for clinical use. Other RPE65 based viral gene therapy trials using different gene constructs and viral vector formulations also demonstrated improvements in retinal function with different efficacy durability.
25 Phase I trial for MERTK associated retinitis pigmentosa was completed, in which MERTK gene was subretinally administered using AAV2 vectors. Patients demonstrated gains of visual functions up to 2 years. However, the disease progression of cannot be excluded after treatments. This gene therapy also demonstrated good safety profile.
Phase I/II trials for choroideremia of subretinal delivery of CHM gene were completed and demonstrated excellent safety and improvements in visual acuity for some participated patients with low baseline visual function. Several more phase I and II clinical studies were also initiated and currently on going.
Phase I/II clinical trial for age-related macular degeneration (AMD) was completed to evaluated safety and efficacy o f r A AV. s F LT 0 1 formulations after subretinal injection in 19 patients. After 52-week trial period, two patients in cohort experienced pyrexia and intraocular inflammation that resolved with a topical steroid. Six patient demonstrated substantial fluid reduction and improvement in visual function.
Phase I/II clinical trial for NADH dehydrogenase subunit 4 Complex I (ND4) associated Leber hereditary optic neuropathy was completed, in which scAAV2-
P1ND4v2 formulations were intravitreally injected. After 9 months of following up,
6 out of 9 patients demonstrated improvements in visual acuity.
More phase I/II clinical studies for achromatopsia and MYO7A-associated
Usher syndrome have been initiated due to excellent preclinical results demonstrated in animal models.
26 2.3 Vectors for Gene Delivery to the Retina
2.3.1 Viral Vectors
Gene delivery to different retinal cell types in vitro and in vivo has been attempted mostly using viral vectors. For example, recombinant adeno-associated virus (rAAV) has been used to transfer RPE65 cDNA to the RPE65-deficient animal models and human patients, which have shown improvement in visual function and lead to the first approved gene therapy for clinical use39. Lentiviruses have evolved the ability to infect non-dividing cells40, an attribute that significantly broadens the utility of lentiviral vectors to numerous target tissue and cell types. Lentivirus- mediated delivery has shown efficient reporter gene expression41,42. Gene therapy using lentiviral vectors in a rat model of autosomal recessive retinitis pigmentosa
(RP) has shown a long-term preservation of retinal function with a slowed-down photoreceptor cell loss43. Retrovirus has also been tried to treat proliferative vitreoretinopathy (PVR) and gene therapy with a retrovirus was able to introduce sufficient platelet-derived growth factor α receptor (αPDGFR) expression, which attenuated PVR in a rabbit model of the disease44.
2.3.2 Non-viral Vectors
The development of non-viral vectors has been underway for alternative gene delivery platforms to complement viral-based gene delivery systems for the treatment of genetic eye disorders. Different non-viral strategies have been tried to
27 deliver therapeutic genetic materials to the back of the eye using synthetic polymers, cationic lipids, polysaccharides, and even naked genetic materials. Successful gene transduction has been achieved from retinal tissue transfected by these non-viral gene delivery systems.
2.3.3 Viral vs Non-viral
Viral vectors have been modified to be nonpathogenic or replicative without affecting the inserted therapeutic transgenic products inside them. Through the cell transduction process, viral vectors are able to produce stable and long-term therapeutic gene expression45. However, there are still concerns and limitations for viral-based gene therapy. For example, one safety concern with AAV2 is their potential to spread to the brain through retinal ganglion cells, which has been seen in canine models46. The potential safety concerns can result in complicated processes to evaluate patients, especially on their immune systems when receiving the therapy. Another limitation is the complicated manufacturing of clinical grade
AAVs, which requires complex methods to generate, purify and characterize the vectors with the implementation of current Good Manufacturing Practice to ensure safety, purity and avoid immunological barriers to achieve long-term expression33.
Moreover, AAVs are limited by a loading capacity of 4.7 kB, including all of the functional sequences and the therapeutic gene sequence47. The limited packaging capacity will not affect applications in diseases caused by mutations of small genes such as LCA2, but applications in diseases caused by mutations in large genes are
28 greatly limited. For example, AAV vectors cannot package the full gene sequence
ABCA4 (Stargardt’s disease) and USH2A (Usher syndrome), which are also common forms of genetic eye disorders.
Non-viral retinal gene delivery systems are less immunogenic and easy to produce and thus less costly with applicable cGMPs. Moreover, non-viral approaches can be classified as drugs instead of biologic products by the regulatory authorities, which will further bring down translational costs and administrative efforts. Non-viral gene delivery systems are not limited by packaging capacity and can be applied to carry different types of genetic materials, enabling different therapeutic functions to treat many different types of diseases. However, non-viral gene delivery systems are limited by their low transfection efficiency. Most non- viral carriers are protonatable materials which are usually rich in protonatable amine groups for establishing electrostatic interactions with genetic materials.
However, the positively charged nature of non-viral carriers can reduce the extracellular stability and circulation time due to the precipitation caused by aggregations and increase the toxicity of the system48. Also, the efficiency of non- viral systems is limited by intracellular barriers that must be overcome by endosomal escape47. In most cases, non-viral formulations enter the cells through charge-mediated interactions or receptor-mediated endocytosis, which results in uptake into vesicles that ultimately become lysosomes49. The acidic environment in late endosomes and lysosomes destroys the particle formulations if they cannot escape from the entrapment. Therefore, systems that can mediate effective
29 endosomal escape and possess high transfection efficiency are in great need in the field of non-viral gene delivery.
2.4 Administration Routes of Gene Therapies for IRDs
Figure 1.3. Common administration routes for retinal drug delivery.
Intravitreal injection is a widely adopted technique to delivery therapeutics to the vitreous. It has been commonly used to delivery antibodies, growth factors and some drugs50. The delivery of genetic therapeutic formulations through intravitreal can receive extraocular biodistribution especially for AAVs, which can show up in blood and lymphatic tissue51. Intravitreal injection also has limited transduction efficiency, which is mostly confined to inner retinal cells52. However, intravitreal injection is relatively less invasive and has less complications compared to other intraocular administrations. Nucleic acids also have low intravitreal half-lives,
30 which requires repeated administrations to achieve a continuous effect in the retina for gene therapies, which might lead to more potential complications53.
Subretinal injection is a preferable administration route for gene therapy, since subretinal space is an excellent target for gene therapy. Subretinal injection allows the interactions of injected materials with the plasma membrane of the photoreceptor and the RPE. Compared with intravitreal injection, subretinal approach allows higher transduction efficiency in the outer retinal space, which makes it a better option for gene therapy targeting the photoreceptor cells and the
RPE. However, subretinal injection is more invasive, which potentially introduces complications including trauma to cornea, iris, retina and lens. Transscleral and transchoroidal approaches have also been tried to delivery therapeutics to the subretinal, which have shown effective delivery but also with risk of hemorrhages.
Administration routes other than local injections have also been tried for retinal gene therapy, such as intravenous administration and topical administration.
Delivery of nucleic acid-based formulations through intravenous administration is associated with poor pharmacokinetics53. Therefore, high doses of the drug are needed to be administered due to only a small fraction of the amount administered can reach the targeted retinal tissue. Topical administration suffers from limited permeation of the nanoformulations from the ocular surface to the retina, which involves penetration of the cornea and diffusion through the vitreous to reach the retina against the normal flow of the vitreous.
31 The selection of administration route for gene therapy relies on the target retinal tissue for the therapy. For gene therapies targeting the inner retina, intravitreal administration is a better approach. While for therapies targeting the outer retina and the RPE, subretinal administration is preferable, which allows better interactions of genetic therapeutics and the targeted retinal tissue.
3. Development of Non-Viral Gene Therapies for IRDs
Non-viral gene delivery systems have been designed addressing the complexity of the delivery of therapeutic genetic materials using a comprehensive multifunctional approach, while minimizing complexity of the system. At the same time, the non-viral materials have to be biocompatible and should cause minimum adverse effects. Different non-viral gene delivery systems have been designed and developed following various strategies, which involved in wide scope of materials and designs addressing different components associated with gene delivery processes. The most common non-viral gene delivery strategies are the formation of lipid/DNA complexes (lipoplexes) and cationic polymer/DNA complexes
(polyplexes)54.
3.1 Polymer Based Retinal Gene Therapies
Both natural and synthetic polymers were developed as gene delivery systems for the treatments of IRDs, due to the high availability and ease of preparation of
32 polymer based materials. The structures of some commonly used polymer based gene delivery systems were listed in Figure 1.4.
Figure 1.4. Chemical structure of commonly used polymer based non-viral gene delivery systems.
33 3.1.1 Chitosan
Chitosan is an unaceylated derivative of a natural cationic polysaccharide consisting of repeated glucosamine units, which has the advantages of renewability, biodegradability and excellent biological activity. The amino groups on chitosan with a pKa value of ~6.5 make it protonated in acidic to neutral solution depending on the pH value and the degree of deaceylation, which allows chitosan for gene therapy applications55. Chitosan-based cationic materials have been established as a safe DNA condensing agent, which can deliver genetic therapeutics both in vitro and in vivo. Moreover, chitosan can be easily chemically modified by targeting ligand or other functional groups to allow multiple functions56. The stability and transfection efficiency of chitosan based vectors are affected by the molecular weight (Mw) and the degree of deacetylation. The Mw of chitosan influences the size and binding stability of the formulations between the polymer and nucleic acid.
The key to develop chitosan based gene therapy or any non-viral gene therapy is to find the balance between the DNA protection and intracellular DNA unpacking.
Formulations of higher Mw chitosan and gene materials will have better protection for genetic materials because of stronger binding, but lower release efficiency in the cytoplasm57.
Low Mw chitosan (Mw < 150 kDa) has been shown to efficiently complex genetic materials, protect them from DNase degradation and transfect cells. Low
Mw chitosan is also more soluble at physiological pH, and have antimicrobial, inmunostimulant, and antioxidant effects58. Low Mw oligochitosan was used to
34 deliver nucleic acids in the rat retina. The formulation of oligochitosan and DNA plasmid was able to retain, protect, and release the plasmid, which induced GFP reporter gene expression in the retinal tissue through both intravitreal and subretinal injections59. A combination of chitosan oligomers and hyaluronic acid formulated with GFP reporter showed the potential for targeting and further transferring genes to the retinal tissue60.
High Mw chitosan is limited by its insolubility in neutral aqueous solution, which restricts the application of chitosan as effective gene delivery vehicle. As a result, different chemical modifications have been applied on the side chains of chitosan to increase solubility, which can improve its performance in gene delivery61. For example, high Mw Chitosan molecule has been conjugated with ethylene glycol branches to make glycol chitosan (GCS), which confers water solubility at a neutral pH. GCS nanoparticles formulated with GFP reporter gene were able to transfect the RPE and generate expression 14 days after subretinal injection without significant toxicity effect62. Thiol groups were also introduced to chitosan molecule, which formed disulfide bonds to improve gene delivery efficiency, because they could be cleaved by the action of intracellular glutathione and promote a faster release of the genetic material63.
Chitosan based materials for gene delivery to the retina are limited by their hydrogelation property, strong binding and low releasing efficiency of the gene therapeutics, which results in low transfection efficiency. To overcome this problem, a colloidal system that combines liposomes and chitosan nanoparticles were
35 developed. Through the combination, the gene therapy increased the interaction with cell membranes and delivered therapeutics to selected retinal tissues64. Coating chitosan by anionic polymers such as alginate can also reduce the strength of the interaction between chitosan and DNA, which results in less stable particle, improves the transfection efficiency and its dissociation within the cell65. However, these solutions haven’t been tested in vivo for retinal gene therapy efficiency.
Chitosan based gene therapies for IRDs have shown promising reporter gene expression in the retinal tissue, but no functional treatments have been evaluated yet.
3.1.2 Synthetic Polymers
Poly(lactic-co-glycolic acid) (PLGA) is one of the most successfully developed biodegradable polymers due to its hydrolysis leads to lactic acid and glycolic acid, which are endogenous and easily metabolized by the body66. The degradation time can be manipulated by changing the molecular weight and copolymer ratio, which can vary from several months to several years67. PLGA is approved by the FDA and European Medicine Agency (EMA) in several drug delivery systems in humans and are commercially available68. PLGA/plasmid nanoparticles were extensively applied to treat angiogenesis and fibrosis in various animal models of age-related macular degeneration69, 70. For example, nanoparticle formulations of PLGA with anti-VEGF intraceptor plasmid modified with RGD peptide were intravenously injected into primate and murine AMD models, a
36 significantly improvement of nearly 40% restoration of visual loss induced by choroidal neovascularization71. However, PLGA is not water soluble, the preparation of PLGA/genetic therapeutics formulation involves in complicated process, which result in potentially low loading efficiency and damage of genetic therapeutics.
Polyethylenimine has been considered one of the most effective polymer- based transfection agents either in its branched or linear structures72. PEI/plasmid complexes coated with anionic polymers were used to deliver genes to the retinal tissue via intraviteal injection. Successful gene transfection was observed 24 h after injection73. Polyethylenimine has suffered from high cytotoxicity, when the molecular weight is greater than 25,000 Da74. Various strategies have been tried to overcome to reduce the cytotoxicity, such as introducing disulfide bond and anionic molecules coating.
Poly(beta-amino) esters (PBAEs) (Figure 1.5) have great potential as gene delivery reagents, which are easily synthesized and can induce efficiently transfection into wide range of cell types75. PBAE can spontaneously form positively-charged nanoparticles with plasmid DNA under buffer of pH=5. After facilitating an endosomal escape, PBAE are degraded by hydrolysis of the ester bonds, allowing reduced cytotoxicity. Subretinal injections of lyophilized
PBAE/pGFP nanoparticles resulted significant increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina76.
37
Figure 1.5. Schematic showing polymerization of poly(beta-amino ester)s (PBAEs) 76.
Synthetic polymers have great potentials for retinal gene therapy, because of high availabilities of polymers with different structures and flexibility of chemical modification possibilities. However, many polymers suffer from low water solubility, high cytotoxicity and low transfection efficiency. The development of synthetic polymers based retinal gene therapy still needs to overcome these problems.
38 3.1.3 Polypeptides
The advantage of peptide-based DNA delivery systems is its flexibility. The composition of the final peptide sequence can be easily modified in response to experimental results in vitro and in vivo to take advantage of specific peptide sequences, overcoming extra- and intracellular barriers to gene delivery77. Peptide- based DNA delivery systems can induce receptor mediated gene delivery, which provides an opportunity to achieve tissue specific delivery of DNA therapeutics78.
Poly-L-arginine (PA) (Figure 1.4) has been shown to be significantly effective at entering cells, which, consequently, makes poly-L-arginine a good candidate for transporting a variety of therapeutics into cells and tissues79. PA/plasmid nanoparticles were prepared to treat autosomal dominant retinitis pigmentosa (adRP) with the help of therapeutic PRPF31 gene. PA/plasmid nanoparticles were subretinally injected to prpf31A216P/+ mice and significant improvement in visual acuity and retinal thickness were observed80. Peptides with protein transduction domains (PTDs) have also been applied for ocular gene delivery. A PTD peptide for ocular delivery (POD) was prepared with a sequence of GGG(ARKKAAKA)4.
POD was able to penetrate and deliver fluorophores, siRNA, DNA and quantum dots to the retinal tissue. After modification with polyethylene glycol (PEG), PEG-
POD/pDNA nanoparticles were able to transfect retinal tissue with human FLT1, an isoform of vascular endothelial growth factor receptor 1, which achieved a 50% reduction in choroidal neovascularization in a murine model of AMD81, 82. Lysine peptide conjugated with PEG (CK30PEG) has also shown successful gene therapies
39 in animal models of IRDs, including adRP, LCA2 and Stargardt’s disease83, 84, 85.
Biochemical experiments demonstrated that CK30PEG nanoparticles can bind to cell surface nucleolin specifically with high affinity, which serves as cell surface receptor for CK30PEG nanoparticles. Nucleolin is also known to shuttle between cellular compartments, including the plasma membrane and the nucleus86.
Therefore, CK30PEG nanoparticles has enhanced passage through several steps that limiting traditional transfection vectors including escape from the endosomal pathway, cytoplasmic diffusion of delivered DNA, extensive exposure to cellular
DNases, and access to the nucleus, by nucleolin-mediated intracellular trafficking.
O O O
O H2 H S C HN CH C OH O N C C n NH2
O
H2N 30
CK30PEG
Figure 1.6. Chemical structure of lysine peptide gene delivery system (CK30PEG).
3.1.4 Dendrimers
As a unique kind of polymers, dendrimers have gained increasing interest in drug delivery. Dendrimers contain three components in their structures: an inner core, branched repeating units (layer of generations) and peripheral multivalent functional groups87, which allows them to have monodispersity, well-defined
40 shapes, multivalency and flexibility for modifications. For gene delivery, dendrimers can form polycations with nucleic acids, which can interact with cell membrane and facilitate cellular uptake of the polycation formulations. Currently, gene therapy using dendrimer systems (Figure 1.7) are mainly tested in vitro, the in vivo therapies for IRDs are associated with only delivery for therapeutic drugs, such as anti-VEGF oligodeoxynucleotide88. Dendrimers suffer from low transfection efficiencies, mainly due to the entrapment in the endosomal compartments in cells89. Therefore, in order to develop an effective gene therapy for IRDs using a dendrimer based system, biochemical and structural modifications are greatly needed, in order to enhance the cellular uptake and endosomal escape, making it more efficient in gene transfection of retinal tissue.
41 Figure 1.7. Chemical structure of fourth-generation poly(amidoamine) dendrimer (PAMAM- G4).90
3.2 Cationic lipids
After the first report of utilization of N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), a cationic lipid, as synthetic carrier for gene delivery to cells, many cationic lipid formulations have been tested to deliver nucleic acids both in vitro and in vivo91. Cationic lipids are simple and quick to formulate, are not biological hazardous and can be easily adapted for specific applications92. Cationic lipids for gene delivery applications are composed of three basic domains: a positively charged head group, hydrophobic tail group(s), and a linker that joins the polar head and non-polar tail. The head group interacts with nucleic acids, which leads to the condensation. The linker group determines the chemical stability, availability of modification, and biodegradability of the lipid.
The chemical structures of commonly used cationic lipid based gene delivery systems are listed in Figure 1.8.
42
Figure 1.8. Chemical structures of common cationic lipid based gene delivery systems.
Solid lipid nanoparticles (SLNs) formulated by cationic lipid N-(1-(2,3-
Dioleoyloxy) propyl)-N,N,N trimethyl ammonium methyl sulfate (DOTAP) and therapeutic plasmid were tested for transfection in ARPE-19 cells. Successful expression of retinoschisin was observed in cells93. Gene therapy for X-linked juvenile retinoschisis (XLRS) using these SLNs were further tested in Rs1h- deficient mice (XLRS model). The structural analysis of the treated eyes demonstrated partial recovery of the retina related to the production of retinoschisin94.
43 Gene replacement therapy using liposomes-protamine-DNA (LPD) formulated by DOTAP (1, 2-dioleoyl-3-trimethylammonium-propane), DOPE (1,
2-dioleoyl-sn-glycero-3-phosphoethanolamine) and cholesterol with therapeutic
RPE65 cDNA, were evaluated in Rpe65-/- mice. With the help of cell penetrating and nuclear localization signaling peptides, gene replacement therapy using LPD promoted cell-specific, long term RPE65 gene expression, leading to in vivo correction of blindness in mice95.
Compared with other cationic systems, cationic lipid based gene therapies have shown more promising results to conduct successful treatments for IRDs.
However, further modifications of cationic lipid based systems addressing potential toxicity, physicochemical stability, formulation stability, transfection efficiency and treatment specificity are needed.
4. Challenges of Non-Viral Gene Therapy for IRDs
Although non-viral gene therapy for IRDs holds a lot of promise, the major impediment is still the low efficiency resulting from the barriers of retinal gene delivery and the design of non-viral gene delivery systems.
4.1 Barriers of Retinal Gene Delivery
The vitreous presents a barrier for non-viral gene delivery, because it is a gel- like material made of fibrils consist of collagen, hyaluronic acid, glycosaminoglycans (GAGs), structural proteins and serum proteins96.
44 Nanoparticles can be immobilized by the proteoglycan filament network. The interactions and bindings between nanoparticles and GAGs cause the decrease of zeta potential, which further induce aggregations, destabilization of the formulations and reduction in transfection efficiency97.
The blood retinal barrier (BRB) is another impediment for retinal non-viral gene delivery. BRB is the barrier between the endothelia of retinal capillaries and the retinal pigment epithelium (RPE). Tight junctions in these cell layers greatly limit the penetration of molecules from systemic blood circulation to the retina and from the vitreous to blood stream98. The vitreous clearance transportations across
BRB are either active transport or passive diffusion. Hydrophilic molecules have low permeability through BRB, while lipophilic substances can permeate more easily. Therefore, gene transfers through BRB require designs addressing the bypass of BRB.
Transfection of retinal tissue using non-viral gene therapy can also be limited by administration routes. Treatments by intravitreal injections are hurdled by the vitreous environment, making the therapeutics less likely to arrive at the target tissue. As a preferred administration route for gene therapy of IRDs, subretinal injection has the advantages of local deliver of therapeutic genes to the subretinal space and enhancement of interactions of therapeutic genes with the targeted tissue.
However, due to the nature of subretinal injection, gene therapeutics cannot diffuse laterally through the entire subretinal space, resulting in only local expression of the therapeutic genes and limited efficacy of gene therapy.
45 4.2 Ideal Design of Non-viral Gene Therapy for IRDs
Ideal non-viral gene therapy should induce extensive, efficient, and long-term expression of therapeutic genes in targeted tissue, and promote phenotypic improvement without causing significant immune response, inflammation, toxicity, or other adverse physiological effects. To achieve this goal, the design of non-viral gene delivery systems should address the complexity of the delivery of therapeutic genetic materials to the subretinal tissue using a comprehensive multifunctional approach.
First, non-viral gene delivery systems should induce enough cellar uptake of therapeutic genes. To achieve this, formulations of non-viral gene carriers and therapeutic genes should be stable under physiological conditions, while having positive surface charge, which allows enough interactions with negatively charged cell membrane to cause either plasma membrane fusion or endocytosis. For polymer based non-viral gene therapy, stable electrostatic bindings are easy to form between carriers and therapeutic genes, due to high availability of protonatable amino groups (Figure 1.9). For cationic lipid based gene therapy, it has been shown that lipids with multivalent head groups are more effective for gene transfection, because of better interactions between the formulations and cell membrane91.
Another way to enhance cellular uptake is to induce specific receptor-mediated endocytosis pathways or subcellular trafficking pathways, in which the non-viral gene delivery systems can be modified with short peptides that target receptors on the cell surface or transporting proteins in the cells.
46
Figure 1.9. Three main strategies to package genetic materials by non-viral polycations.99
Second, non-viral gene delivery systems should induce efficient escape of formulations from the endosomal entrapment. In many cases, non-viral formulations are internalized into the cells but therapeutic genes are not well expressed100. The non-viral gene delivery systems may have difficulties in endosomal escape processes, since most non-viral gene delivery systems are strong polycations, which don’t have pH-sensitivity inside endosomal compartment and cannot induce osmotic pressure change and the release of non-viral formulations from the endosomal compartment (Figure 1.10). Therefore, pH-sensitivity is crucial for non-viral gene delivery system design, which allows better endosomal escape ability of non-viral formulations. However, pH-sensitivity is hard to be tuned for polymer based non-viral gene delivery systems, which involves complicated
47 physical and chemical modifications. For cationic lipid based non-viral gene delivery systems, pH-sensitivity can be manipulated by selecting head groups and linkers that have amino groups with wide range of pKa values, including primary, secondary, tertiary, quaternary amino groups, imidazolium and pyridinium groups.
Third, non-viral gene delivery systems should protect gene therapeutics from enzymatic degradations in the cytosol. Most non-viral gene delivery systems, either polymer based or lipid based, have good protection over therapeutic genes, because of the strong electrostatic interactions. However, the problem is the interactions are too strong to allow therapeutic genes to release when they are needed to function.
To overcome this, modifications by introduction of disulfide bonds into non-viral gene delivery system was used to enhance the stability of non-viral formulations, at the same time, induce reductive cytosolic release of the gene therapeutics (Figure
1.10).
48
Figure 1.10. pH-sensitive amphiphilic endosomal escape and Reductive cytosolic release of cationic lipid/siRNA nanoparticles. 101
Last, non-viral gene therapy for IRDs should have high specificity for targeted retinal tissues. As is discussed previously, vertebrate retina is one of the most complexed organs, where different cell types with different functions are closely connecting to each other. As a result, off-target expression from gene therapy may result in malfunction of cells. Therefore, specificity of non-viral gene therapies for
IRDs should be addressed when they are designed. Two strategies are commonly used to target retina tissue: modification of non-viral carriers with targeting ligands and genetic engineering of therapeutic plasmid with tissue specific promoters.
Folate group and hyaluronan were used to modify nanoparticle systems to mediate receptor based uptakes into the RPE, which demonstrated improved specificity and
49 efficacy102, 103. Other studies tried to modify therapeutic plasmids with tissue specific promoters either targeting photoreceptor cells or the RPE and enhanced tissue specific expressions were observed for all the treatments84, 85.
In this work, we present the design and development of non-viral gene therapies for the treatment of both autosomal recessive and dominant IRDs. In
Chapter II, we describe a multifunctional lipid with core-shell dendrimer DNA nanoparticle system to deliver plasmid DNA to the retinal tissue. In Chapter III, we present a RPE targeted non-viral gene therapy based on multifunctional lipid ECO plasmid DNA nanoparticles for the treatment of Leber’s congenital amaurosis. In
Chapter Ⅳ we present non-viral gene therapy formulation for ABCA4 delivery using pH-sensitive cationic lipid ECO to encapsulate large therapeutic gene into stable nanoparticles. We have also constructed a therapeutic ABCA4 plasmid with a photoreceptor-specific promoter region, which has demonstrated cell-specific expression of ABCA4 and slowing down of the disease progression. In Chapter Ⅴ, we present the development of a library of pH-sensitive multifunctional lipids for the gene therapy of autosomal dominant IRDs using CRISPR/Cas9 technology. We have shown knockdown effect of GFP reporter gene using these cationic lipid/CRISPR/Cas9 formulations. To conclude, we explore the relative merits and challenges of non-viral gene therapies for the treatment of IRDs, which induce tissue specific therapeutic gene expression and the alteration of phenotypes from the targeted diseases.
50 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
1. Background
Gene therapy requires safe and efficient delivery of therapeutic nucleic acids into target cells. Non-viral gene delivery systems hold great promise for safe treatment of human genetic diseases. Nucleic acid-based therapeutics are negatively charged, making it difficult for them to enter cells through negatively charged cell membranes. Thus, an efficient non-viral delivery system should possess at least three key features: (i) a robust transfection efficiency of the genetic cargo into target cells; (ii) an ability to penetrate or bypass biological barriers during the in vivo delivery process; and (iii) minimal adverse side effects in healthy cells and tissues104.
Cationic polymers and lipids have been extensively used to condense or encapsulate nucleic acids to form nanoparticles through electrostatic interactions. A large excess of cationic materials is often used to enhance the delivery of non-viral systems 105,
106. Despite promising results demonstrated by cationic lipid and cationic polymer- based systems, excessive positive charges of these cationic non-viral systems could
51 be cytotoxic and unsafe for in vivo applications due to the high positive to negative charge ratio involved in nanoparticle formulation107. Therefore, a non-viral delivery system that has low charge ratio but good transfection efficiency is desirable.
2. Delivery System Design
We intended to design an efficient non-viral gene delivery system with a low positive to negative charge ratio (N/P ratio) suitable for safe gene therapy of retinal diseases. Previously, we designed core-shell polylysine dendrimers with a cubic octa(3-aminopropyl) silsesquioxane (POSS) core or nanoglobules with a relatively rigid spheric structure to mimic histones, a class of natural proteins involved in
DNA packing108, 109. These core-shell dendrimers have well-defined nanostructures and are highly efficient to condense plasmid DNA at low N/P ratios. Recently, we also have developed multifunctional pH-sensitive lipids that form stable nanoparticles with nucleic acids and possess the capability to facilitate cellular uptake, pH-responsive endosomal escape, and cytosolic delivery of nucleic acids110,
111. Therefore, we hypothesized that the combination of a core-shell nanoglobule and a multifunctional lipid could result in a safe and highly efficient hybrid delivery system with a low N/P ratio by utilizing the distinct and advantageous features of both the nanoglobules and lipids for in vivo gene therapy of retinal diseases.
52
Figure 2.1. Formation of G4/ECO/pDNA nanoparticles. Hybrid G4/ECO/pDNA nanoparticles are formed following two stepwise electrostatic complexations: plasmid DNA first is condensed by G4 nanoglobules and lipid ECO then is incorporated into the delivery system through electrostatic interactions between the cationic head group of ECO and the negatively charged surface of the G4/pDNA complexes.
In this chapter, we designed a hybrid delivery system featuring the combination of the generation 4 (G4) nanoglobule with a multifunctional lipid ECO for gene delivery into RPE cells (Figure 2.1). The G4 nanoglobule has a relatively rigid globular structure and a molecular weight of 16 283 Da, similar to that of histones112, 113. ECO is a highly efficient multifunctional lipid carrier for cytosolic delivery of nucleic acids. We investigated the formulation of hybrid G4/ECO/DNA nanoparticles over a range of N/P ratios and their physicochemical properties, including morphology and stability. The effect of the composition of G4/ECO/DNA nanoparticles on transfection efficiency in ARPE-19 cells was determined in vitro.
53 The in vivo gene expression of a lead nanoparticle formulation in retina and RPE cells was assessed in mice via subretinal injection.
3. Materials and Methods
3.1 Cell Culture
ARPE-19 cells were cultured in Dulbecco's modified Eagle's medium and supplemented with 10% fetal bovine serum, 100 μg · mL-1 streptomycin, and
100 U · mL-1 penicillin (all reagents were from Invitrogen, Waltham, MA). Cells were maintained in a humidified incubator at 37 °C and 5% CO2.
3.2 Animal
BALB/c and C57BL/6J wild type mice were obtained from the Jackson
Laboratory (Bar Harbor, ME). All mice were housed and cared for in the animal facility at the School of Medicine, Case Western Reserve University. All animal procedures and experiments were approved by CWRU Institutional Animal Care and Use Committee.
3.3 Preparation of G4/ECO/DNA Nanoparticles
G4 nanoglobule and cationic lipid ECO were synthesized as previously reported114,115. G4/ECO/pDNA nanoparticles were prepared by a stepwise self- assembly of the G4 nanoglobule and ECO with plasmid DNA at N/N/P ratios of
2/1/1, 2/2/1, 2/3/1, 3/1/1, 3/2/1, and 3/3/1 for G4/ECO/DNA. The G4 nanoglobule
54 stock solution (1 mg · mL-1) and plasmid DNA stock solution (0.5 mg · mL-1) at predetermined amounts based on desired N/N/P ratios were diluted into equal volumes with nuclease-free water, mixed and shaken for 30 min at room temperature. Then ECO stock solution (2.5 mM in ethanol) was added to the
G4/DNA mixture, and the resulting mixture was shaken for another 30 min prior to each experiment. The amount of G4 and ECO was determined by N/P ratios in the formulations. G4/pDNA and ECO/pDNA nanoparticles used as controls were prepared by mixing plasmid DNA with G4 or ECO solutions for 1 h on a shaker.
Lipofectamine 2000 (Invitrogen, Waltham, MA)/DNA nanoparticles were prepared according to the manufacturer's recommendation.
3.4 Nanoparticle Characterization
The sizes and zeta potentials of G4/pDNA and G4/ECO/pDNA nanoparticles at different N/P or N/N/P ratios were determined by dynamic light scattering with a Brookhaven ZetaPALS Particle Size and Zeta Potential Analyzer (Brookhaven
Instruments, Holtsville, NY).
3.5 Atomic Force Microscope
Atomic force microscopy (AFM) images were obtained on a Veeco DI Atomic
Force Microscope (Veeco, Plainview, NY) at room temperature under ambient conditions, by employing a tapping mode oscillating imaging technique.
G4/ECO/pDNA nanoparticles at N/N/P ratio of 3/3/1 were evaluated in this
55 experiment. The nanoparticle solution (10 μL) was deposited onto a glass slide and left to dry for 24 h in a lyophilizer. AFM images then were obtained by scanning the slide surface.
3.6 Transmission Electron Microscope
The morphology of G4/ECO/pDNA N/N/P ratios 3/3/1 nanoparticles were also checked with transmission electron microscope (Zeiss Libra 200EF). The sample for TEM observation was prepared by depositing 20 μL of the particle solution onto a 300-mesh copper grid for electron microscopy covered by thin amorphous carbon film (20 nm). Immediately after deposition, the excess of liquid was removed by touching the grid to filter paper. The sample was dried and images were taken.
3.7 Gel Electrophoresis for Particle Stability
Solutions (15 μL) of G4/ECO/pDNA nanoparticles with the N/N/P ratios of either 2/1/1, 2/2/1, 2/3/1, 3/1/1, 3/2/1, or 3/3/1 were mixed with 3 μL of loading dye
(Promega) and loaded onto a 1% agarose gel containing ethidium bromide. The gel was submerged in 0.5× Tris/Borate/EDTA (TBE) buffer at room temperature and run at 100 V for 25 min. Free pDNA was used as a control. DNA plasmid bands were visualized with an AlphaImager ultraviolet imaging system (Biosciences,
USA).
56 3.8 Cell Viability
ARPE-19 cells were incubated with G4/ECO/pDNA nanoparticles at a pDNA concentration of 1 μg · mL-1 in a 12-well plate with a seeding density of 4 × 104 cells/well. After 48 h, MTT reagent (Invitrogen) was added to the cells for 4 h followed by the addition of SDS-HCl and incubation at 37 °C for another 4 h. The absorbance was measured at 570 nm with a SpectraMax spectrophotometer
(Molecular Devices, Sunnyvale, CA). Cellular viability was calculated as the average of triplicates for each N/N/P ratio normalized to the untreated control.
3.9 Cellular Uptake
Cellular uptake of G4/ECO/pDNA nanoparticles was quantitatively evaluated by flow cytometry. G4/ECO/pDNA nanoparticles of Cy3-labeled plasmid DNA
(Mirus Bio, Madison, WI) were similarly prepared at different N/N/P ratios as described above. ARPE-19 cells (4 × 104 well-1) were seeded onto 12-well plates and grown until they reached 90% confluence. These cells were transfected with the G4/ECO/Cy3-pDNA nanoparticles at a pDNA concentration of 0.125 μg · mL-1 in serum-free or 10% serum media for 4 h. The transfection media then was removed and each well was washed twice with PBS (1.06 mM KH2PO4, 155 mM
NaCl, and 2.96 mM Na2HPO4 · 7H2O). Cells were harvested after treatment with
0.25% trypsin containing 0.26 mM EDTA (Invitrogen, Waltham, MA) for 5 min at
37 °C by centrifugation at 1000 rpm for 5 min, and fixed in 750 μL PBS containing
4% paraformaldehyde, and finally passed through a 35 μm cell strainer (BD
57 Biosciences, San Jose, CA). Cellular internalization of G4/ECO/Cy3-pDNA nanoparticles was quantified by the fluorescence intensity measurement for 10 000 cells per each sample by using a BD FACSCalibur flow cytometer (BD
FACSCalibur flow cytometer). Nanoparticles at each N/P ratio were assayed in triplicates.
3.10 Endosomal Escape
-1 ARPE-19 cells (4 × 104 well ) were seeded onto glass-bottom micro-well dishes and allowed to grow to 90% confluence. The cells were stained with
4 μg · mL-1 Hoechst 33342 (Invitrogen) and 100 mM LysoTracker Green (Life
Technologies, Carlsbad, CA). Cells were then treated with G4/ECO/Cy3-pDNA
(N/P ratio 3/3/1) nanoparticles in serum-free medium. After culturing for 8 h in serum-free medium before the transfection medium was removed and replaced with fresh serum-containing medium (10% FBS). Cells were cultured for 24 h when the medium was removed and they were washed with PBS for three times before fixation with PBS containing 4% paraformaldehyde. Fluorescence images were taken with an Olympus FV1000 confocal microscope.
3.11 In Vitro Transfection
ARPE-19 cells were seeded in 12-well plates at a density of 4 × 104 cells per well and allowed to grow for 24 h at 37 °C. Transfections were carried out in serum- free or 10% serum media with the nanoparticles of GFP plasmid DNA (Altogen
58 Biosystems, Las Vegas, NV) at a DNA concentration of 1 μg · mL-1. G4/ECO/pGFP nanoparticles were incubated with ARPE-19 cells for 8 h at 37 °C. The media then was replaced with fresh serum-containing media (10% serum) and the cells were then cultured for an additional 48 h. GFP expression was monitored with an
Olympus FV1000 confocal microscope (Olympus, Center Valley, PA). After the culture media was removed, each well was washed twice with PBS. Cells were harvested after treatment with 0.25% trypsin containing 0.26 mM EDTA,
(Invitrogen) by centrifugation at 1000 rpm for 5 min, fixed in 750 μL PBS containing 4% paraformaldehyde, and finally passed through a 35 μm cell strainer
(BD Biosciences, San Jose, CA). A BD FACSCalibur flow cytometer (BD
Biosciences) was used to determine GFP expression based on quantified by the fluorescence intensity in a total of 10, 000 cells for each sample.
3.12 Ex Vivo Retinal Transfection
Mouse eyes were enucleated, washed with penicillin–streptomycin solution
(Sigma, St. Louis, MO), and rinsed with Hanks' balanced solution (Hyclone,
Waltham, MA). Eye cups were prepared and removed retinas were flattened by making retinal flaps. Flattened retinas were transferred onto filter paper, where the retinas were gently peeled away from the RPE layer. All these procedures were performed under a surgical microscope. Each retina and RPE layer on a filter paper was placed in a separate well of a 12-well plate filled with 0.5 mL DMEM containing 10% serum and incubated for 16 h at 37 °C. After the retinas and RPE
59 layers were washed twice with 0.5 mL of fresh DMEM with 10% serum, they were incubated with G4/ECO/pDNA nanoparticles (N/N/P ratio 3/3/1) of 4.5 μg pDNA expressing GFP for 8 h at 37 °C. The culture media then was replaced by fresh culture media and the retinas and RPE layers were further cultured for either 4 or 6 days when GFP expression was determined with an Olympus FV1000 confocal microscope.
3.13 In Vivo Subretinal Transfection With G4/ECO/pDNA Nanoparticles
All surgical manipulations were carried out under a surgical microscope (Leica
M651 MSD). Mice were anesthetized by intraperitoneal injection of 20 μL · g-1 of body weight of 6 mg · mL-1 ketamine and 0.44 mg · mL-1 xylazine in 10 mM sodium phosphate and 100 mM NaCl buffer solution (pH = 7.2). Pupils were dilated with 1.0% tropicamide ophthalmic solution (Bausch & Lomb, Rochester, NY). A
33-gauge beveled needle (World Precision Instruments, Sarasota, FL) was used as a lance to make a full thickness cut through sclera at 1.0 mm posterior to the limbus.
This needle was replaced with a 36-gauge beveled needle attached to an injection system (UMP-II microsyringe pump and Micro4 controller with a footswitch,
World Precision Instruments). This needle was aimed toward the inferior nasal area of the retina, and a G4/ECO/pDNA (3/3/1) nanoparticles solution was injected at a pDNA (GFP plasmid) dose of 18 ng into the subretinal space. Successful administration was confirmed by observing bleb formation. The tip of the needle remained in the bleb for 10 sec after bleb formation, when the needle was gently
60 withdrawn. A solution with pDNA alone (18 ng) was also injected into the subretinal space of the contra eye served as a control (mock group). After 3 or 5 days, eyes were collected, washed with penicillin–streptomycin solution (Sigma), and rinsed with Hanks' balanced solution (Hyclone, Waltham, MA). Eye cups were prepared just as previously described. The retina and RPE layers were placed in glass bottom confocal plate and fixed with 1 mL of PBS with 4% paraformaldehyde.
An Olympus FV1000 confocal microscope was used to assess GFP expression.
3.14 Histology
The eye cups for histology were fixed in 2% glutaraldehyde, 4% paraformaldehyde, and processed for visualization by OCT (optimum cutting temperature formulation). Sections were cut at 1 μm. Slides samples were permeabilized and fixed sequentially with 4% PFA and 0.25% Triton X-100 followed by blocking with 0.5% BSA blocking solution for 1 h at room temperature.
Antibodies were applied at with proper concentrations for 1 h at room temperature and washed three times with a 0.1% TBST, 5 min each wash. Slides were counter- stained with DAPI and mounted with coverslip using the Prolong Gold regent
(Invitrogen) before imaging. Stained tissue was imaged with an Olympus FV1000 confocal microscope.
61 3.15 Statistical Analysis
Experiments were performed in triplicate and presented as the means and standard deviations. Statistical analysis was conducted with ANOVA using a 95% confidence interval. Statistical significance was accepted when P ≤ 0.05.
4. Results
A two-step self-assembly process was used to form the hybrid G4/ECO/DNA nanoparticles. Plasmid DNA (pDNA) was first condensed by the G4 nanoglobule.
Lipid ECO was then added to form a lipid shell surrounding the G4/DNA plasmid complexes (Figure 2.1). Six hybrid formulations were prepared with different N/P
(positive/negative) charge ratios. Stabilities of all formulations were evaluated by agarose gel electrophoresis (Figure 2.2). When only ECO and pDNA were in the formulation, they could not form stable nanoparticle at N/P ratios of 3 and lower.
For formulations that had only G4 and pDNA, DNA was efficiently condensed at the N/P ratio of 3. For the G4/ECO/pDNA formulations, when N/N/P ratios for
G4/ECO/pDNA were 2/1/1, 2/2/1, and 2/3/1, the hybrid formulations were unable to effectively condense pDNA and form stable nanoparticles. When N/N/P ratios were 3/1/1, 3/2/1, and 3/3/1, the formulations were able to effectively condense pDNA and form stable hybrid nanoparticles. These results indicate that the hybrid systems were able to form stable nanoparticles at low overall N/P ratios with the
N/P ratio of G4/pDNA as 3.
62
Figure 2.2. Agarose gel electrophoresis showing retardation of G4/ECO/pDNA nanoparticles over a range of N/N/P ratios as compared with free pDNA, G4/pDNA nanoparticles, and ECO/pDNA nanoparticles.
The sizes and surface charges of G4/pDNA and G4/ECO/pDNA nanoparticles were determined by dynamic light scattering measurements (Figure 2.3). The size of G4/pDNA nanoparticles was around 300 nm at N/P ratio 2 and 3. The complexation with ECO resulted in a significant reduction of the size of the nanoparticles. The sizes for G4/ECO/pDNA were distributed from 50 to 200 nm dependent of the N/N/P ratios. All of our stable G4/ECO/pDNA nanoparticles (3/1/1,
3/2/1, and 3/3/1) had sizes smaller than 200 nm, which were suitable sizes for efficient cellular uptake104. All formulations displayed positive surface charges except for that with N/N/P ratio 2/1/1 and also G4/pDNA particles (Figure 2.3).
The negative surface charge of those particles might be caused by incomplete condensation, which resulted in more coiled DNA plasmid molecules than positively charged dendrimer or lipid. The net positive charge on the particle surface is an important factor affecting cellular uptake. Because cell membranes carry net negative charges, positive surface charges of the nanoparticles mediate their
63 interactions with cell membrane, facilitating efficient endocytosis of the nanoparticles. Our stable nanoparticles possessed a moderate amount of positive charges with zeta potentials in the range of 25–30 mV. Taken together, the hybrid nanoparticles with N/N/P of 3/1/1, 3/2/1, and 3/3/1 had the size and zeta potential needed for efficient cellular gene transfection.
Figure 2.3. Size and zeta potential of G4/pDNA and G4/ECO/pDNA nanoparticles over a range of N/P or N/N/P ratios.
The morphology of the nanoparticles with the N/N/P ratio of 3/3/1 was imaged with AFM and TEM (Figure 2.4). The size of the particles was around 200 nm as shown in the AFM image, comparable to what was measured by dynamic light scattering. It appears that some of the nanoparticles formed loose aggregates in the dry state. It is known that aggregation of plasmid DNA/lipid complexes is a function of the plasmid DNA/cationic lipid ratio and the DNA concentrations. Also, when these ratios and concentrations are low, there is non-uniformity in particle
64 morphology116, 117. The amount of cationic lipid and nanoglobule was greatly reduced in our particle formulation. Thus, we expected a non-uniform morphology for some of G4/ECO/pDNA nanoparticles. The morphological characteristic was also confirmed by TEM (Figure 2.4c). Single particle and particle aggregations were both observed in the TEM image. The sizes turned out to be smaller because of the shrinkage caused by air-drying process.
Figure 2.4. Morphological characterization of a G4/ECO/pDNA N/N/P ratio 3/3/1 formulation. (a) 2D image from a tapping mode AFM scan. (b) Corresponding 3D image from the same tapping mode AFM scan. (c) TEM image of dry form G4/ECO/pDNA N/N/P ratio 3/3/1 particles.
The toxicity of the G4/ECO/pDNA nanoparticles, along with the controls of lipofectamine 2000/pDNA, ECO/pDNA particles, G4/pDNA nanoparticles, and
PBS were evaluated in ARPE-19 cell line (human retinal pigmented epithelium)
(Figure 2.5). Our hybrid nanoparticles exhibited significantly less cytotoxic effects on ARPE-19 cells in 10% serum media than lipofectamine 2000. With serum free media, ECO/pDNA (N/P = 20/1) and lipofectamine 2000/pDNA nanoparticles exhibited low cell viability (ca. 40%), while the G4/ECO/pDNA nanoparticles had at least 60% viability. Cell viability was greatly improved for the G4/ECO/pDNA nanoparticles in serum media.
65
Figure 2.5. Viability of ARPE-19 cells incubated without or with 10% serum transfection media. G4/ECO/pDNA nanoparticles were tested over a range of N/N/P ratios with lipofectamine 2000, ECO/pDNA nanoparticle and G4/pDNA nanoparticle serving as controls. Error bars = ± std (* p < 0.05 relative to cell viability of lipofectamine 2000 under 10% serum; # p < 0.05 relative to cell viability of lipofectamine 2000 under no serum. Statistical analysis followed one-way AN OVA).
Cellular uptake of G4/ECO/pDNA nanoparticles was evaluated in the ARPE-
19 cells. Cellular uptake studies were carried out with a Cy3-labeled plasmid DNA.
The percentage of cells transfected with G4/ECO/pDNA nanoparticles after incubation for 4 h was quantified by flow cytometry (Figure 2.6a). Generally, an increase in pDNA uptake associated with the increase in lipid ECO amount. pDNA uptake was higher in 10% serum transfection media than in serum-free media as
66 reflected by the greater cell viability in the former media. The nanoparticles with
N/N/P of 3/3/1 exhibited transfection efficiency over 95% in 10% serum media.
Figure 2.6. (a) Cellular uptake of G4/ECO/Cy3 pDNA nanoparticles cultured with ARPE-19 cells for 4 h. A range of N/P ratios were tested under serum-free and 10% serum culture media. (b) Cellular uptake of G4/ECO/pDNA N/P ratio 3/3/1 particles cultured with ARPE-19 cells for 4 h in serum-free media under different inhibitory conditions (cytochalasin D (5 μg · mL-1), nocodazole (20 μM), and 4 °C). (c) Confocal fluorescence image of cytosolic delivery of G4/ECO/pDNA N/P ratio 3/3/1 particles. Late endosomes were stained with LysoTracker Green (green), nuclei were stained with Hoechst 33342 (blue), and DNA plasmid was labeled with Cy3 (red). At 24 h, nanoparticles mostly escape endosomal entrapment, shown by the red signals dispersed throughout the cytoplasm. Green signals represent late endosomes/lysosomes. Co-localization with late endosomes (yellow) appears minor. Error bars = ± std (* p < 0.05 relative to uptake under 4 °C. Statistical analysis followed one way ANO VA).
Cellular uptake was further investigated using G4/ECO/pDNA nanoparticles with N/N/P ratio of 3/3/1 in the presence of different inhibitors to understand the
67 pathway through which the formulations are internalized. The nanoparticles were cultured with either cytochalasin D, a phagocytosis inhibitor, or nocodazole, an inhibitor of actin and microtubules polymerization118, 119 . Another group of cells was cultured under 4 °C, which inhibits all energy-dependent trafficking pathways.
The results showed that the groups cultured with inhibitors had a slightly lower uptake than the control group at 37 °C (Figure 2.6b). However, a much larger difference was observed between the group cultured under 4 °C and the other groups, suggesting that G4/ECO/pDNA nanoparticles were mainly internalized through an energy-dependent pathway. The positively charged nanoparticles might electrostatically interact with the negatively charged cellular membrane, followed by their non-specific absorptive endocytosis120.
The cytosolic delivery of G4/ECO/pDNA nanoparticles was studied using the nanoparticle with an N/N/P ratio of 3/3/1, which was captured by 3D confocal microscopy at 24 h (Figure 2.6c). An excellent endosomal escape property was observed, which was shown by a predominant red fluorescence signals over yellow signals, in the cytoplasm. The yellow color indicated the co-localization of the nanoparticles of pDNA labeled with red fluorescence with late endosomes/lysosomes labeled with green fluorescence, while red signals suggested the endosomal escape of the nanoparticles in the cytoplasm.
Intracellular transfection efficiency of the G4/ECO/pDNA nanoparticles, along with some controls, such as lipofectamine 2000, ECO/pDNA particles, and
G4/pDNA particles, was tested with GFP plasmid DNA. GFP expression was tested
68 in transfection media with no serum and with 10% serum using confocal microscopy and flow cytometry (Figure 2.7). GFP expression was increased in 10% serum transfection media samples relative to those with serum-free media for the hybrid nanoparticles. For hybrid nanoparticles, an increase of the ECO ratio in the formulation resulted in higher GFP expression in 10% serum media. The nanoparticles with N/N/P ratios of 2/3/1 and 3/3/1 showed the higher GFP expression in 10% serum transfection media than in the other formulations.
ECO/pDNA (N/P = 12/1) nanoparticles exhibited high GFP expression in all transfection media. Whereas, the G4/pDNA (N/P = 20/1) control group failed to evoke GFP expression in the ARPE-19 cells, although G4/pDNA nanoparticles are reportedly effective in transfecting the MDA-MB-231 cells114. The commercial control lipofectamine 2000 group evidenced high GFP expression in the absence of serum but this system was not as effective in 10% serum transfection media.
Moreover, cell viability was also low with lipofectamine 2000 in 10% serum media as compared with G4/ECO/pDNA formulations (Figure 2.5).
69
Figure 2.7. (a) Confocal microscopic image of GFP expression 48 h post transfection in ARPE-19 cell line under serum-free and 10% serum transfection media. Transfection of G4/ECO/pDNA N/N/P ratios 2/1/1, 2/2/1, 2/3/1, 3/1/1, 3/2/1, and 3/3/1 nanoparticles were evaluated. (b) Flow cytometry of GFP expression 48 h post-transfection of ARPE-19 cell line under serum-free and 10% serum transfection media. G4/ECO/pDNA nanoparticles were tested over a range of N/N/P ratios with lipofectamine 2000, ECO/pDNA particles and G4/pDNA particles used as controls. Error bars = ± std (* p < 0.05 relative to GFP expression of lipofectamine 2000 under 10% serum media. Statistical analysis followed by one-way ANO VA).
Some of the ocular genetic disorders involve monogenetic mutations in the photoreceptor cells or retinal pigmented epithelium (RPE) cells. Introduction of a therapeutic gene to the photoreceptor cells or RPE cells has the potential to correct
70 the monogenetic disorders. The hybrid G4/ECO/pDNA nanoparticles of
N/N/P = 3/3/1 showed a good combination of stability, safety, and gene transfection, which were selected for gene delivery with ex vivo tissue culture and an animal model. An organotypic culture experiment was carried out with retina and RPE tissue from C57BL/6J wild type mice. The RPE layer and retinal layer were cultured with the nanoparticles expressing GFP for 8 h and the culture media was replaced with fresh media. GFP expression then was assessed by confocal microscopy after
4 and 6 days later, Figure 2.8a. GFP expression was noted from both RPE and retina layers 4 and 6 days post-transfection indicating good transfection properties for live tissue.
71 Figure 2.8. (a) Ex-vivo transfection of RPE and retinal tissues accomplished with an organotypic culture method. G4/ECO/pDNA N/N/P ratio 3/3/1 nanoparticles were cultured with RPE and retinal tissue from C57/BL6J wild type mice for 8 h. Confocal microscopic images of GFP expression (green) after either 4 or 6 days are shown. (b) In vivo transfection. G4/ECO/pDNA N/N/P ratio 3/3/1 particles were subretinally injected into the eyes of wild type BALB/c mice. Confocal microscopic images of GFP expression (green) in flat mounted retina and the RPE layer are shown 3 and 5 days after the injection. Control groups consisted of mice treated with only DNA plasmid at the same dose. (c) Histology of eye cups from BALB/c mice 7 days post- transfection. GFP antibody and a secondary fluorescent labeling antibody (red) were applied to identify GFP expression.
The nanoparticles were also subretinally injected to the eyes of BALB/c mice.
After 3, 5, and 7 days, eyes were collected and eyecups were prepared. GFP expression of the flat mounted RPE layer and retina layer was visualized with a confocal microscope after 3 and 5 days. GFP expression was found in both the RPE and retina layers, Figure 2.8b. GFP expression was further validated by immunohistochemistry with an anti-GFP antibody, a secondary red fluorescence- labeled antibody at 7 days after injection. Figure 2.8c shows immunofluorescence staining of GFP in histological slides from eye cups. Green fluorescent protein was shown in the RPE layer. The results suggest that the hybrid G4/ECO/pDNA nanoparticles are able to mediate in vivo gene expression in retina and RPE cells after subretinal injection.
5. Discussion
The design of hybrid G4/ECO/DNA delivery system is to take the advantages of both dendrimer and cationic lipid. The fourth generation nanoglobule dendrimer
(G4) has 128 surface primary amine groups, allowing effective electrostatic
72 complexation with the DNA cargo. Due to the multifunctionality of the POSS core, the G4 nanoglobule has eight branches, and a size and globular shape reminiscent of histones. Histones are a class of natural proteins with molecular weights in the range of 10, 000-22, 000 Da and condensate DNA molecules via electrostatic interactions of their positively charged Lys residue rich regions and negatively charged genetic materials113. This process helps to pack DNA in high efficiency, regulates DNA transcription and replication, and protects genetic materials from degradation. G4 nanoglobule can condense DNA cargo in the similar manner.
Results also indicated that the hybrid systems were able to form stable nanoparticles at low overall N/P ratios with the N/P ratio of G4/pDNA as 3.
The other component in our delivery system is a multifunctional cationic lipid
ECO. Our previous studies have shown that ECO can complex with nucleic acids forming nanoparticles through charge complexation and hydrophobic condensation of the lipid tails. The thiol groups in ECO can be oxidized for disulfide bond cross- links to further stabilize the nanoparticle formulations, which are cleaved through a glutathione-dependent reduction inside cytosol to release nucleic acids after escaping from endosomal-lysosomal pathway121, 122. These features of ECO facilitate cellular uptake and endosomal escape for efficient cytosolic delivery of nucleic acids in response to the pH change during subcellular trafficking123, 101.
Therefore, the incorporation of ECO into the G4/DNA condensates would result in a hybrid plasmid DNA delivery system to overcome the barriers for efficient intracellular gene at low N/P ratios.
73 The presence of ECO in the hybrid nanoparticles enhanced transfection of the hybrid nanoparticles, but ECO/pDNA nanoparticles resulted in relatively low cell viability at a high N/P ratio (Figure 2.5). The combination of the G4 nanoglobule and ECO resulted in the hybrid nanoparticle formulation with high transfection efficiency in low serum concentration at low N/P ratios, which was critical for high cell viability. Notably, GFP expression for G4/ECO/pDNA N/N/P ratio 2/3/1 and
3/3/1 formulations was much higher than lipofectamine 2000 in 10% serum media and less toxic. Overall, the G4/ECO/pDNA nanoparticles showed high gene expression with less cytotoxicity as compared with the commercialized delivery system.
In vivo subretinal injections of the hybrid G4/ECO/pGFP nanoparticles induced promising GFP expression. However, it appears that GFP expressions happened both in the RPE and retina, which was mainly at the site of injection and.
The efficiency of the hybrid G4/ECO/pGFP nanoparticles was still limited and the transfection was not tissue specific. Therefore, for future disease treatment applications, the system should be optimized with better efficiency and specificity for retina tissue. The hybrid nanoparticles can be modified with targeting moieties specific to retina or RPE cells to further improve gene transfection and expression.
Plasmid DNA also can be constructed with a specific promoter to control gene expression specifically in retina or RPE cells.
74 In summary, this chapter demonstrates the development of a hybrid
G4/ECO/pDNA nanoparticle gene delivery system for retinal gene transduction.
The hybrid G4/ECO/pDNA with N/N/P of 3/3/1 showed high stability and in vitro and in vivo gene transfection efficiency. The overall N/P ratio of the nanoparticles was only 6/1, which was much lower than some reported non-viral gene delivery systems. Consequently, the nanoparticles demonstrated lower cytotoxicity to
ARPE-19 cells in serum media than a commercial transfection agent. These findings provide a paradigm to enhance transfection efficiency through the design of an improved delivery system. Subretinal injection is a commonly used approach of gene therapy with both viral and non-viral gene delivery systems. It avoids the complications associated with systemic gene therapy approaches. We have shown in this study that the hybrid nanoglobule/ECO/pDNA nanoparticles are promising for subretinal delivery of genes to retina or RPE cells.
75 Chapter Ⅲ. Targeted Multifunctional Lipid ECO Plasmid DNA
Nanoparticles as Efficient Non-viral Gene Therapy for Leber’s Congenital
Amaurosis
1. Background
Leber’s congenital amaurosis (LCA) is a genetic disease causing retinal degeneration with severe vision loss at an early age that affects 1 in 80,000 subjects124, 125. One molecular form of this disease, LCA type 2 (LCA2), is caused by mutations in the RPE65 gene that encodes the RPE65 protein (retinal pigment epithelium-specific protein 65-kDa) predominantly expressed in the retinal pigmented epithelium (RPE). RPE65, a key enzyme in retinoid metabolism, catalyzes the hydrolysis and isomerization of all-trans-retinyl esters to 11-cis- retinal. RPE65 deficiency results in the accumulation of all-trans-retinyl esters and causes rod and cone photoreceptor dysfunction126, 127, 128.
As a monogenic disease, LCA2 is a good candidate for gene therapy because the photoreceptor cells and the RPE do not show extensive pathological abnormalities in the early stages of this disease129. Recently, gene replacement therapy with adeno-associated viral vectors (AAVs) has demonstrated considerable
76 therapeutic efficacy in improving vision in RPE65-deficient animal models and human patients130, 131, 132, 133, 32. Although clinical trials have validated the overall benefit of gene replacement therapy, their success is limited by several drawbacks associated with viral delivery systems. The possibility of an immune response induced by viral vectors greatly compromises the efficiency of gene transfection and can cause complications in patients134. Studies have shown that vector DNA is detectable in the optic nerve and brain following subretinal injections, which raises additional safety concerns135.
Non-viral gene delivery systems that employ cationic lipids, dendrimers, polycations, and polysaccharides have also been developed for gene delivery72, 136,
137, 138, 139. Non-viral systems generally exhibit advantages such as ease of production, good safety profiles, and unlimited cargo capacity. However, their clinical translation is still hindered by their low transfection efficiency and transient gene expression140. Novel designs of highly effective non-viral delivery systems are needed to overcome the limitations of existing non-viral delivery systems for gene therapy of inherited monogenic visual disorders to become effective and practical.
2. Delivery System Design
77 Figure 3.1. Molecular structure of pH-sensitive multifunctional lipid (1-aminoethyl)iminobis[N- (oleicylcysteinyl-1-amino-ethyl)propionamide] (ECO).
In this work, we designed a multifunctional lipid, (1-aminoethyl)iminobis[N-
(oleicylcysteinyl-1-amino-ethyl)propionamide] (ECO), as a simple and smart gene delivery carrier based on its mechanism of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC) of nucleic acids. ECO contains a protonable ethylenediamine (E) head group, two cysteine (C) functional linkers, and two oleoyl (O) lipophilic tails. The thiol groups of the cysteine residues can form disulfide bonds to stabilize particle formulations and can also be chemically modified with targeting ligands. Following cellular uptake, cytosolic release of the gene cargo is facilitated by pH-sensitive amphiphilic endosomal membrane destabilization through protonation of the head group of ECO in the acidic endosomal-lysosomal compartment (pH = 5-6) and dissociation of the nanoparticles by reduction of the disulfide bonds in the cytoplasm.
Figure 3.2. Chemical structure of all-trans-retinylamine targeting ligand.
We also designed and prepared all-trans-retinylamine modified ECO plasmid
DNA (pDNA) nanoparticles with a polyethylene glycol (PEG) spacer to target interphotoreceptor retinoid-binding protein (IRBP) for enhanced gene delivery into the retina. All-trans-retinoids have a high binding affinity for retinoid binding
78 proteins, which play important roles in visual transduction141. IRBP is a major protein in the interphotoreceptor matrix (IPM) that selectively transports 11-cis- retinal to photoreceptor outer segments and all-trans-retinol to the RPE142, 143, 144,
145, 146, 147. Such selective transport mechanism can increase the transfection efficiency directly into the RPE with the ECO/pDNA nanoparticles conjugated with all-trans-retinylamine.
In this chapter, we first evaluated the in vitro transfection efficiency of
ECO/pDNA nanoparticles in ARPE-19 cells, a human RPE cell line. The in vivo transfection efficiency of targeted ECO/pDNA nanoparticles to the RPE was then evaluated in wild-type BALB/c mice using GFP plasmids. Finally, the efficacy of gene therapy with the targeted nanoparticles was determined by electroretinography
(ERG) in the Rpe65-/- mouse model of human LCA2.
3. Materials and methods
3.1 Cell Cultures
ARPE-19 cells were cultured in DMEM and supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 units/mL penicillin (all reagents were from Invitrogen). Cells were maintained in a humidified incubator at 37°C and 5% CO2.
79 3.2 Animals
BALB/c wild-type mice were purchased from Jackson Laboratory. Rpe65-/- deficient C57BL/6J mice were obtained from Michael Redmond (National Eye
Institute, NIH) and genotyped as described previously148. All mice were housed and cared for in the animal facility at the School of Medicine, Case Western Reserve
University, and all animal procedures were approved by the Case Western Reserve
University (CWRU) Institutional Animal Care and Use Committee.
3.3 Synthesis of Ret-PEG-MAL
All-trans-retinylamine (15 mg) and MAL-PEG-succinimidyl carboxymethyl
(SCM) (molecular weight [MW] = 3,400 Da, NANOCS) (55 mg) were added to 15 mL dimethylformamide. The solution was stirred at room temperature overnight.
The product Ret-PEG-MAL was precipitated in 50 mL diethyl ether and washed three times. The product was dried under vacuum to give Ret-PEG-MAL (yield,
89%).
3.4 Preparation of ECO/pDNA and Ret-PEG-ECO/pDNA Nanoparticles
Multifunctional pH-sensitive lipid ECO was synthesized as reported previously115. The ECO/pDNA nanoparticles were prepared by self-assembly of
ECO with plasmid DNA at an amine/phosphate (N/P) ratio of 6. The ECO stock solution (2.5 mM in ethanol) and plasmid DNA stock solution (0.5 mg/mL) at predetermined amounts based on the N/P ratio were diluted into equal volumes with
80 nuclease-free water, mixed, and shaken for 30 min at room temperature. The Ret-
PEG-MAL solution (0.4 mM in 50% DMSO and water) was then added to the mixture at 2.5 mol % and shaken for another 30 min to facilitate the reaction between the maleimide functional group and free thiols on ECO. A different ECO stock solution (25 mM) was used for in vivo formulations. Lipofectamine 2000
(Invitrogen)/DNA nanoparticles were prepared according to the manufacturer’s recommendations.
3.5 TEM
The morphology of ECO/pDNA (N/P = 6) and Ret-PEG-ECO/pDNA (N/P =
6) nanoparticles was imaged with a transmission electron microscope (JEOL
JEM2200FS). Samples for TEM were prepared by depositing 20 μL of the particle solution onto a 300-mesh copper grid covered by a thin amorphous carbon film (20 nm). Immediately after deposition, the excess liquid was removed by touching the grid with filter paper. Samples were stained twice by adding 3 μL 2% uranyl acetate aqueous solution; the excess of staining solution was removed after each addition.
Images of the nanoparticles were acquired by TEM after the samples were dried.
3.6 DLS
The sizes and zeta potentials of ECO/pDNA (N/P=6) and Ret-PEG-
ECO/pDNA (N/P=6) nanoparticles were determined by DLS with an Anton Paar
81 Litesizer 500 (Anton Paar USA). Three measurements were performed and averaged for each sample at 20°C.
3.7 In Vitro Transfection
ARPE-19 cells were seeded onto 12-well plates at a density of 4 × 104 cells/well and allowed to grow for 24 h at 37°C. Transfections were conducted in
10% fetal bovine serum medium with the ECO nanoparticles of GFP plasmid DNA
(Altogen Biosystems) at a DNA concentration of 1 μg/mL. ECO/pGFP nanoparticles were incubated with ARPE-19 cells for 8 h at 37°C. The medium was then replaced with fresh serum-containing medium (10% serum), and cells were then cultured for an additional 48 h. GFP expression was evaluated with an
Olympus FV1000 confocal microscope (Olympus). After removal of the culture medium, each well was washed twice with PBS (10 mM sodium phosphate [pH 7.2] and 100 mM NaCl). Cells were harvested after treatment with 0.25% trypsin containing 0.26 mM EDTA (Invitrogen), followed by centrifugation at 1,000 rpm for 5 min and fixation in 750 μL PBS containing 4% paraformaldehyde, and finally passed through a 35-μm cell strainer (BD Biosciences). A BD FACSCalibur flow cytometer (BD Biosciences) was used to determine GFP expression based on the fluorescence intensity from a total of 10,000 cells for each sample.
82 3.8 Intracellular Uptake
ARPE-19 cells (4 ×104/well) were seeded onto glass-bottom micro-well dishes and allowed to grow for 24 h at 37°C before they were stained with 4 μg/mL
Hoechst 33342 (Invitrogen) and 100 mM LysoTracker Green (Life Technologies).
Cells then were treated with ECO/Cy3-pDNA (Mirus Bio, catalog number
MIR7904, N/P=6) nanoparticles in 10% fetal bovine serum medium. Cells were cultured with nanoparticles for 1, 4, and 24 h (media were replaced by fresh media after 4 h), and then the media were removed, and cells were washed with PBS three times before fixation with PBS containing 4% paraformaldehyde. Fluorescence images were acquired with an Olympus FV1000 confocal microscope.
3.9 In Vivo Subretinal Transfection with ECO/pDNA and Ret-PEG-
ECO/pDNA Nanoparticles
All surgical manipulations were carried out under a surgical microscope (Leica
M651 MSD). Mice were anesthetized by intraperitoneal injection of a cocktail (15
μL/g body weight) comprised of ketamine (6 mg/mL) and xylazine (0.44 mg/mL) in PBS buffer (10 mM sodium phosphate and 100 mM NaCl [pH 7.2]). Pupils were dilated with 1.0% tropicamide ophthalmic solution (Bausch & Lomb). A 33G beveled needle (World Precision Instruments) was used as a lance to make a full- thickness cut through the sclera 1.0 mm posterior to the limbus. This needle then was replaced with a 36G beveled needle attached to an injection system (UMP-II microsyringe pump and a Micro4 controller with a footswitch, World Precision
83 Instruments). The needle was aimed toward the inferior nasal area of the retina, and an ECO/pDNA or Ret-PEG-ECO/pDNA nanoparticles solution (2.4 μL) was injected to deliver either a pRPE65 (OriGene) or pGFP at the dose of 240 ng into the subretinal space. Successful administration was confirmed by bleb formation.
The tip of the needle remained in the bleb for 10 sec after bleb formation, when it was gently withdrawn. A solution (2.4 μL) of Ret-PEG-ECO carrier alone with the same concentration as Ret-PEG-ECO/pDNA nanoparticles was also injected into the subretinal space of the contra eye to serve as a control. Each of the group included at least three eyes with successful subretinal injection. To assess GFP expression, the mice were sacrificed, and eyes were collected 3 days after injection, washed with penicillin-streptomycin solution (Sigma), and rinsed with Hank’s balanced salt solution (HyClone). Eye cups were prepared as described previously149. The retina and RPE layers were placed in glass-bottom confocal plates and fixed with 1 mL of PBS containing 4% paraformaldehyde. GFP expression in the RPE layer was evaluated with an Olympus FV1000 confocal microscope.
3.10 qRT-PCR
Rpe65-/- mice were sacrificed 15 days after subretinal injection with Ret-PEG-
ECO/pRPE65 nanoparticles, and RNA was isolated from their eyes. cDNA was synthesized with the QuantiTect reverse transcription kit (QIAGEN) following the manufacturer’s instructions. qRT-PCR amplification was performed with SYBR
84 Green I Master mix (Roche Diagnostics). Fold changes were calculated after normalizing the data to glyceraldehyde 3-phosphate dehydrogenase. Rpe65-/- mice without treatment were used as controls.
3.11 Electroretinograms
Electroretinograms were acquired according to a reported method150. Animals were anesthetized by intraperitoneal injection of a cocktail (15 μL/g body weight) comprised of ketamine (6 mg/mL) and xylazine (0.44 mg/mL) in PBS buffer (10 mM sodium phosphate and 100 mM NaCl [pH 7.2]). Pupils were dilated with 1% tropicamide for imaging. Experiments were performed in a dark room. Three electrodes were placed on each mouse: a contact lens electrode on the eye, a reference electrode underneath the skin between the ears, and a ground electrode underneath the skin of the tail. Electroretinograms were recorded with the universal electrophysiologic system UTAS E-3000 (LKC Technologies).
3.12 Histology
Eye cups were fixed in 2% glutaraldehyde and 4% paraformaldehyde and processed with optimum cutting temperature (OCT) formulation. Sections were cut at 1 μm. The sample slides were permeabilized and fixed sequentially with 4% paraformaldehyde (PFA) and 0.25% Triton X-100, followed by treatment with 0.5%
BSA blocking solution for 1 h at room temperature. The fluorescence labeled peanut agglutinin (PNA) was applied at a concentration of 12.5 μg/mL for 1 h at room
85 temperature and washed three times with a 0.1% Tris-buffered saline with Tween
20 (TBST) solution for 5 min each time. Slides were counter-stained with DAPI and mounted with a coverslip using the Prolong Gold reagent (Invitrogen) before imaging. Stained tissue was imaged with an Olympus FV1000 confocal microscope.
3.13 Statistical Analysis
Experiments were performed in triplicate and presented as the means and standard deviations. Statistical analysis was conducted with ANOVA and two-tailed
Student's t-tests using a 95% confidence interval. Statistical significance was accepted when P ≤ 0.05.
4. Results
4.1 In Vitro Transfection with ECO/pDNA Nanoparticles
To determine the transfection efficiency of ECO in vitro, human RPE cells
(ARPE-19) were transfected with ECO/plasmid GFP (pGFP; amine to phosphate
[N/P] ratio=6/1) nanoparticles, and confocal microscopy was used to determine
GFP expression 48 h after transfection (Figure 3.3A). ECO/pGFP nanoparticles produced significant GFP expression, with 69.7% of cells expressing GFP, whereas the Lipofectamine control transfected only 14.4% of the cells, as determined by flow cytometry (Figure 3.3B). The high gene expression efficiency of ECO/pDNA nanoparticles correlated positively with their efficient intracellular uptake. Figure
3.3C shows the intracellular uptake of ECO/pDNA nanoparticles as imaged by 3D
86 confocal microscopy 1, 4, and 24 h post-transfection with Cy3-pDNA as the tracker.
After 1 h incubation, ECO/Cy3-pDNA nanoparticles (red) were aligned at the surface of the cell membrane because of electrostatic interactions of these positively charged nanoparticles with the negatively charged cell membrane. After 4 h, the nanoparticles entered the cell and co-localized with late endosomes, indicated by their yellow color. After 24 h, the nanoparticles escaped endosomal entrapment, as shown by the red fluorescence in the cytoplasm and the diminished overlap with endosomes. Efficient cytosolic pDNA delivery of ECO/pDNA nanoparticles resulted in high gene expression efficiency in RPE cells in vitro.
87 Figure 3.3. In Vitro Transfection of ARPE-19 Cells with ECO/pDNA Nanoparticles (A and B) Confocal microscopy images (A) and flow cytometry analysis (B) of ARPE-19 cells transfected with ECO/pGFP (N/P = 6) nanoparticles and Lipofectamine 2000/pGFP nanoparticles for 48 h (**p < 0.005). Each bar represents the mean ± expression level of GFP (n = 3). (C) Confocal fluorescence microscopy images demonstrating intracellular trafficking of ECO/Cy3-pDNA nanoparticles in ARPE-19 cells. Cells were treated with LysoTracker Green (1:2,500 dilution) and Hoechst 33342 (1:10,000 dilution) and then transfected with ECO/Cy3-labeled nanoparticles at N/P = 6. After 1, 4, and 24 h of transfection, cells were fixed and imaged. Green, endosomes; blue, nuclei; red, Cy3-labeled pDNA. Arrows denote the ECO/Cy3-pDNA nanoparticles and Cy3- pDNA. Scale bars, 20 μm. Error bars = ± std (** p < 0.05 relative to the GFP expression level of lipofectamine 2000. Significance was analyzed by two-tailed student t-test.)
4.2 Preparation of Retinylamine-Targeted ECO/pDNA Nanoparticles
To target IRBP, an all-trans-retinoid structure was introduced onto the surface of ECO/pDNA nanoparticles via a PEG (3.4-kDa) spacer. All-trans-retinylamine
(all-trans-retinylamine [Ret]-NH2) was first reacted with the N- hydroxysuccinimide (NHS)-activated ester of NHS-PEG-malaimido (MAL) to yield Ret-PEG-MAL, which was characterized by MALDI-TOF mass spectroscopy
(Figures 3.4 A and B). To form targeted ECO/pDNA nanoparticles, Ret-PEG-MAL was first reacted with the 2.5 mol % ECO via Michael addition between the thiol and maleimide. The targeted nanoparticles were then formed by self-assembly with pDNA. Figure 3.4C shows the transmission electron microscopy (TEM) images of the untargeted and targeted ECO/pDNA nanoparticles. The average size of
ECO/pDNA nanoparticles was approximately 100 nm based on TEM. The average size of Ret-PEG-ECO/pDNA nanoparticles was around 120 nm, a slight increase after surface modification with Ret-PEG. The result was consistent with that measured by dynamic light scattering (DLS). Slight aggregation of ECO/pDNA
88 nanoparticles might result in wider size distribution than Ret-PEG-ECO/pDNA nanoparticles in the size distribution curve, as shown in Figure 3.4D. After conjugation with the targeting ligand, little aggregation was observed for the Ret-
PEG-ECO/pDNA nanoparticles with a narrow distribution of the particle size. The sizes and zeta potentials of ECO/pDNA and Ret-PEG-ECO/pDNA nanoparticles are depicted in Figure 3.4E. The average size of the ECO/pDNA nanoparticles (117 nm) was slightly smaller than that of the Ret-PEG-ECO/pDNA nanoparticles (131 nm) based on DLS measurements. After targeting ligand conjugation, the average zeta potential of Ret-PEG-ECO/pDNA nanoparticles dropped from 26 mV to 18 mV, which not only reduced the cytotoxicity but also stabilized the delivery system.
89
90 Figure 3.4. Preparation of the Targeting Ligand and Characterization of Ret-PEG-ECO/pDNA Nanoparticles. (A) Synthesis route. (B) MALDI-TOF mass spectrum of Ret-PEG-MAL. (C) TEM images of Ret-PEG-ECO/pDNA nanoparticles. (D) Size distribution of Ret-PEG-ECO/pDNA nanoparticles measured by DLS. (E) Sizes and zeta potentials of Ret-PEG-ECO/pDNA nanoparticles. Each bar represents the mean ± size of particles and each dot represents the mean ± zeta potential of particles (n = 3).
4.3 In Vivo Transfection with Targeted Ret-PEG-ECO/pGFP Nanoparticles in
Wild-Type BALB/c Mice
The Ret-PEG-ECO/pGFP nanoparticles were subretinally injected in BALB/c mice to determine in vivo gene delivery and expression efficiency with GFP as a reporter gene. Significant GFP expression was observed in RPE flat mounts with both unmodified ECO/pGFP nanoparticles and targeted Ret-PEG-ECO/pGFP nanoparticles 3 days post-injection. However, Ret-PEG-ECO/pGFP nanoparticles produced greater GFP expression than ECO/pGFP nanoparticles (Figure 3.5A).
ZO-1 staining of tight junction proteins in RPE flat mounts further confirmed that the enhanced GFP expression emanated from RPE cells (Figure 3.5B).
91
Figure 3.5. In Vivo Gene Transfection with Targeted Ret-PEG-ECO/pGFP Nanoparticles in Wild- Type BALB/c Mice. Mice (1 month old) were subretinally injected with ECO/pGFP or Ret-PEG- ECO/pGFP nanoparticles. RPE flat mounts were obtained 3 days post-transfection. (A) Fluorescence microscopic images showing enhanced GFP expression with Ret-PEG-ECO/pGFP nanoparticles in the RPE 3 days post-injection. (B) Confocal fluorescence microscopic images revealing GFP expression specifically in the RPE with anti-ZO-1 antibody staining (white). The tight junction protein ZO-1 represents the borders of the RPE cells.
4.4 Gene Replacement Therapy with Ret-PEG-ECO/pRPE65 Nanoparticles in
Rpe65-/- Mice
Gene therapy with all-trans-retinylamine modified ECO nanoparticles was carried out in Rpe65-/- mice in which Rpe65 was completely knocked down. Rpe65-
92 /- mice exhibit phenotypic features similar to human LCA2 patients151. Ret-PEG-
ECO/pRPE65 nanoparticles were injected into the subretinal space of 1-month-old
Rpe65-/- mice. 15 days post-injection, treatment with Ret-PEG-ECO/pRPE65 nanoparticles produced higher mRNA levels of RPE65 in the treated group than in the untreated control group (Figure 3.6A). This finding demonstrates successful introduction of the therapeutic gene. ERG was performed at an intensity of 1.6 log cd × s/m2 to determine the efficacy of the nanoparticle treatment based on the electrical responses to light from the retina152. Figure 3.6B shows significant scotopic and photopic ERG response waveforms in the nanoparticle treatment group 7 days post-treatment, whereas there was almost no response in control group mice injected with Ret-PEG-ECO. The amplitudes of the major waves from all
ERG tests were calculated 3, 7, 30, and 120 days post-treatment (Figures 3.6C–
3.6F). Significant increases in the amplitudes of scotopic a-waves and b-waves were observed for nanoparticle-treated groups but not for control groups (vehicle- injected). Introduction of the exogenous RPE65 gene increased about 50% of the scotopic ERG amplitude throughout all time points up to 120 days (Figures 3.6C and 3.6E), which demonstrated improved function of rod photoreceptors. Cone function also improved, represented by a 3- to 5-fold increase in photopic b-wave amplitude in the first 7 days after treatment. Although the amplitude decreased at later time points, the photopic b-wave amplitude of the treatment group was 2-fold that of the control, even at 120 days. Photopic a-waves were higher in the treatment groups than in the controls, but the difference was not statistically significant.
93
Figure 3.6. Gene Replacement Therapy with Ret-PEG-ECO/pRPE65 Nanoparticles in Rpe65-/- mice. Mice were subretinally injected with Ret-PEG-ECO/pRPE65 nanoparticles or Ret-PEG- ECO. (A) Relative RPE65 mRNA levels in treated (pRPE65-injected) versus control groups 15 days after treatment. Each bar represents the mean ± mRNA expression level (n = 3). (**p < 0.005). (B) Representative scotopic and photopic electroretinograms acquired from Rpe65-/- mice under the light intensity of 1.6 log cd × s/m2 7 days after treatment. (C–F) Amplitudes of scotopic a-waves (C), photopic a-waves (D), scotopic b-waves (E), and photopic b-waves (F) of treated and control Rpe65-/- mice 3, 7, 30, and 120 days post-injection (**p < 0.005). Each bar represents the mean ± wave amplitude (n ≥ 3). Significance analysis was performed using ANOVA and student t- test.
94
4.5 Cone Preservation after Gene Replacement Therapy with Ret-PEG-
ECO/pRPE65 Nanoparticles in Rpe65-/- mice
To determine whether Ret-PEG-ECO/pRPE65 nanoparticles could rescue cone cells in Rpe65-/- mice, we prepared cryo-sections of the whole retina at 120 days post-injection and stained cone cells with peanut agglutinin (green). Compared with the control group (Figure 3.7A), the treatment group (Figure 3.7B) revealed substantial green fluorescence staining, representing a greater number of healthy cone photoreceptors. This result also explains the increase in photopic wave amplitudes in the ERG. Interestingly, fewer cone cells were observed away from the injection site (Figure 3.7C), suggesting local rescue in this gene therapy approach.
95 Figure 3.7. Cone Preservation after Gene Replacement Therapy with Ret-PEG-ECO/pRPE65 Nanoparticles in Rpe65-/- mice 120 Days after Treatment. (A–C) Peanut agglutinin (green) was used to stain cone photoreceptors. Nuclei were stained with DAPI (blue).
4.6 Therapeutic Effect of Gene Replacement Therapy with Ret-PEG-
ECO/pRPE65 Nanoparticles in 3-Month-Old Rpe65-/- mice Mice
To determine the optimal timing for gene replacement therapy of LCA2 with the targeted nanoparticles, we initiated RPE65 gene therapy with Ret-PEG-
ECO/pRPE65 nanoparticles in 3-month-old Rpe65-/- mice and performed ERG tests to evaluate its therapeutic efficacy. According to the ERG responses measured 7 and 30 days post-treatment, no differences were observed for scotopic and photopic waveforms between the treatment and control groups (Figure 3.8), indicating no observable improvement of retinal function. This result suggests that gene replacement therapy with targeted nanoparticles in these older mice was not as effective in restoring vision as in younger mice, likely because of the progression of irreversible retinal degeneration in older animals.
96
Figure 3.8. Therapeutic Effect of Gene Replacement Therapy with Ret-PEG-ECO/pRPE65 Nanoparticles in 3-Month-old Rpe65-/- Mice. Shown are ERG amplitudes of major response waveforms. (A–D) Scotopic a-waves (A), photopic a-waves (B), scotopic b-waves (C), and photopic b-waves (D) in the treatment and control groups of Rpe65-/- mice. Each bar represents the mean ± wave amplitude (n ≥ 3).
4.7 Safety Assessment of Ret-PEG-ECO/pRPE65 Nanoparticles in BALB/c
Mice
To evaluate the safety of Ret-PEG-ECO/pRPE65 nanoparticles in gene therapy, the nanoparticles were injected into the subretinal space of healthy 1-month-old
BALB/c mice, and ERG tests were carried out 7 and 30 days post-injection. ERG responses for both the nanoparticle-injected group and the un-injected group were comparable at each light intensity (Figure 3.9A). A slight decrease in response
97 amplitudes was observed for some major waveforms 7 days post-injection because of the induced inflammation. Eye function after nanoparticle injection became normal at 30 days, and no deleterious effects were noted in the ERG major wave amplitudes (Figures 3.9B–3.9E). This result indicates that Ret-PEG-ECO/pRPE65 nanoparticles are safe for subretinal injection in gene replacement therapy.
98 Figure 3.9. Safety Assessment of Ret-PEG-ECO/pRPE65 Nanoparticles in 1-Month-old BALB/c Mice. (A) Representative ERG traces of scotopic waveforms in the PEG-ECO/pRPE65-treated group and untreated mice 30 days post-injection. (B–E) ERG amplitudes of scotopic a-waves (B), photopic a-waves (C), scotopic b-waves (D), and photopic b-waves (E) in treated and control animals. Each bar represents the mean ± wave amplitude (n ≥ 3).
5. Discussion
Gene replacement therapy holds great promise for treating monogenic vision disorders. Thus, establishing a gene delivery system with high transfection efficiency, good therapeutic efficacy, and a high safety profile is critical for broad clinical applications of this treatment. Gene therapy with AAV1 has been extensively investigated for treatment of LCA2, a monogenic genetic disease.
Although gene delivery by AAV has been reported as successful in LCA2, its application is limited for treating other monogenic ocular diseases because some therapeutic genes are too large to be loaded into this viral vector. Design of a safe and efficient non-viral gene delivery system has the potential to circumvent this limitation in gene therapy of monogenic visual disorders.
ECO is a multifunctional lipid that has demonstrated excellent efficiency for cytosolic delivery of a variety of genetic materials because of its intrinsic PERC effect. This study has shown that ECO is also effective for delivering therapeutic pDNA for non-viral gene replacement therapy in Rpe65-/- mice. The superior transfection properties of ECO/pDNA nanoparticles result from the multifunctional properties of the lipid carrier, including self-assembly formation of stable nanoparticles with pDNA without helper lipids, pH-sensitive amphiphilic cell
99 membrane destabilization and endosomal escape, as well as reductive dissociation of the nanoparticles to release nucleic acids in the cytoplasm. Here we tested a targeting mechanism that involves the use of IRBP to enhance pDNA delivery with
ECO into RPE cells (Figure 3.10). In the retina, the interphotoreceptor matrix fills the space between rod outer segment and RPE cells, where IRBP is the major carrier that selectively transports all-trans-retinol from photoreceptor cells to RPE cells.
Modification of ECO/pDNA nanoparticles with this all-trans-retinoid can then facilitate their binding to IRBPs for enhanced delivery to the RPE. When injected into the subretinal space, IRBPs will quickly bind the targeting ligand and help to transport and release the particles near the apical side of the RPE before being internalized by the RPE cells147. In vivo transfection with pGFP demonstrated the enhanced gene transfer and expression efficiency of ECO/pDNA nanoparticles with this targeting mechanism in the RPE.
100 Figure 3.10. Targeting Mechanism of All-trans-Retinylamine-Modified ECO Nanoparticles. When injected into the subretinal space, all-trans-retinylamine-modified ECO nanoparticles will bind to IRBP in the interphotoreceptor matrix. IRBP binding helps to retain the nanoparticles in the space and transports the nanoparticles to the target cells in the RPE. Following cellular uptake by endocytosis, the nanoparticles escape from the endosomal compartment and release the RPE65 plasmid DNA via the PERC mechanism. Finally, the RPE65 gene is expressed by the RPE cell, where it slows cone cell degeneration and preserves visual function.
Gene replacement therapy using Ret-PEG-ECO/pRPE65 nanoparticles successfully introduced the expression of exogenous therapeutic RPE65 genes in the RPE layer. ERG results in treated Rpe65-/- mice demonstrated a significant increase in function of both rod and cone photoreceptors, with a therapeutic effect comparable to that of viral gene delivery systems152. In addition to protecting the
RPE, Ret-PEG-ECO/pRPE65 treatment protected cone photoreceptors adjacent to the injection sites in these mice, slowing cone degeneration for at least 4 months.
This therapeutic effect is similar to that reported for viral delivery of RPE65, which also delayed cone degeneration 4 months post-injection in Rpe65-/- mice153.
Furthermore, gene therapy with Ret-PEG-ECO/pRPE65 nanoparticles has demonstrated a safe profile that does not irreversibly damage the retina. The slight drop in ERG major wave amplitudes caused by the injection’s inflammatory effect recovered shortly after the injection.
Similar to the phenotype of human LCA2 patients, mice with RPE65 gene knockout have diminished ERG responses because the supply of the 11-cis-retinal visual chromophore cannot be regenerated. Rather than producing the chromophore, the impaired visual cycle accumulates the intermediate product all-trans-retinyl ester in the RPE, which gradually damages the retina. Massive degeneration of cone
101 cells can occur as early as 1 month of age in Rpe65-/- mice. Photoreceptor outer segment abnormalities are commonly visible at 1 or 2 months of age, and the outer nuclear layer is thinner than normal at 3 months of age. After introduction of the exogenous RPE65 gene to the RPE layer of 1-month-old mice, even in a small number of RPE cells, the visual cycle can supply sufficient 11-cis-retinal for the adjacent photoreceptor cell layer to provide improved visual function154.
Rescue of cone cells because of Ret-PEG-ECO/pRPE65 nanoparticle gene therapy could also be attributed to RPE65 expression in such cells as well as in RPE cells. This effect was also reported in a clinical trial that demonstrated a cone rescue effect after gene therapy131. Although the treatment was effective in 1-month-old mice, treatment of 3-month-old mice with Ret-PEG-ECO/pRPE65 nanoparticles had no effect because the photoreceptor cells had begun to degenerate by this age.
The observation is consistent with human patients with LCA2. During the first few years of life, children with LCA2 are less visually responsive than healthy children.
Older LCA2 patients demonstrate more severe retinal degeneration that makes the retina less responsive to RPE65 gene therapy.
Other strategies have been reported previously to target the RPE layer. For example, hyaluronan has been applied to target CD44 receptors expressed by RPE cells. Folate has been tested as a targeting ligand for folate receptors associated with the RPE102, 103. However, the expression of CD44 receptors is more restricted in inflammatory tissue, and folate receptors reside predominantly in the basal rather than apical membranes of RPE cells. Therefore, the distribution of these receptors
102 greatly restricts the use of these ligands to target the RPE. By comparison, targeting
IRBPs with the all-trans-retinyl group can avoid the restrictions of the targeting mechanisms reported above and provide efficient gene delivery to RPE cells after subretinal injection.
In summary, we have demonstrated the efficacy of a targeted gene delivery system for gene replacement in LCA2. However, improvements are needed to optimize the system prior to clinical translation. These include prolonging gene expression, identifying the appropriate disease stage for maximal effectiveness of therapy, and exploring alternate routes of injection to transfect the whole RPE layer.
In future work, we can address the transience of gene expression with these nanoparticles by modifying the DNA plasmid with sequences that prolong gene expression84. The targeted ECO/pDNA nanoparticles can also be further optimized by introducing a pH-sensitive spacer between PEG and ECO to enhance endosomal escape of the ECO/pDNA nanoparticles155.
103 Chapter Ⅳ. pH-Sensitive Multifunctional Lipid ECO Plasmid DNA
Nanoparticles as Efficient Non-viral Gene Therapy for Stargardt’s Disease
1. Background
Stargardt’s disease (STGD), as first illustrated by Stargardt in 1909, is a bilateral progressive atrophic macular dystrophy caused by mutations in ABCA4 gene which encodes a 210 kDa ATP binding cassette protein that is responsible for transportation of retinoid compounds across the outer segment disk membrane during photo transduction156,157. The absence of ABCA4 results in accumulation of all-trans-retinal, which will further react with ethanolamine to form photo toxin
A2E158. Patients of STGD exhibit delayed dark adaptation and loss of visual clarity, followed by gradual loss of central vision, atrophy of foveal RPE and photoreceptor cells159. Currently, there is no effective therapy to completely cure STGD even though gene replacement therapy holds great promise. Due to the size of ABCA4 gene (6.8 kB), it has been challenging to develop viral delivery system to treat
STGD. Therefore, gene therapy strategies that can facilitate delivery of large genes are in great need to treat diseases involving delivery of large genes.
104 Non-viral gene delivery systems have been developed to overcome the problems of viral gene delivery systems, due to their wide range of products, flexibility of application, ease of production, safe profile and low toxicity. Non- viral approaches can be classified as drugs instead of biologic products by the regulatory authorities, which will further bring down translational costs and administrative efforts. More importantly, for the treatment of genetic disorders caused by large gene, non-viral gene delivery systems are not limited by packaging capacity compared with viral based delivery systems. Successful treatments have been achieved in animal models of inherited retinal diseases (IRDs) using different non-viral gene delivery systems covering wide range of materials. However, non- viral gene delivery systems are still suffering from transient expression, low transfection efficiency and uptake limitation. Therefore, systems that can mediate effective endosomal escape and possess high transfection efficiency are greatly needed in this field.
In previous chapters, we demonstrated the design of a multifunctional lipid
ECO, as a simple and smart gene delivery carrier based on its mechanism of pH- sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC) of nucleic acids. In this chapter, we aim at developing gene replacement therapy for
Stargardt’s disease caused by mutations in the large ABCA4 gene using ECO lipids.
Specifically, a nanoparticle based gene delivery system was formulated by pH- sensitive cationic lipid ECO and engineered tissue specific ABCA4 therapeutic plasmids.
105
2. Delivery System Design
In this chapter, we designed and evaluated a gene therapy for STGD using
ECO based non-viral system to deliver therapeutic ABCA4 gene specifically in photoreceptor cells. In order to target photoreceptors, we created the ABCA4 plasmid to induce gene expression only to photoreceptor cells by promoter modification. Some promoters, such as cytomegalovirus (CMV) promoter, are considered generic or common constitutive promoters and will initiate transcription across all eukaryotic cell types in any condition160. Therefore, one of our therapeutic plasmids with CMV promoter would have ABCA4 expression in any retinal cell that transfected with the plasmid (pCMV-ABCA4). However, modifying our therapeutic plasmid with a tissue-specific promoter can limit the expression to a narrow range of cells. Rod cells express rhodopsin, the visual chromophore, while other cells such as RPE do not express this protein. Previously, the promoter region of the bovine rhodopsin gene was isolated161. Selective gene expression of the
DsRed reporter gene in the photoreceptor cell layer has been achieved with this bovine rhodopsin promoter (RHO) sequence162. Therefore, therapeutic ABCA4 plasmid was constructed with the bovine RHO promoter located upstream of human
ABCA4 gene (Figure 4.1). The plasmid was condensed by ECO and formed stable
ECO/pABCA4 nanoparticles for transfection into the Abca4-/- mouse, a mouse model to human STGD. Transfection with reporter and therapeutic genes expressed
106 with the CMV or RHO promoters demonstrated differential expression indicative of tissue specificity for the RHO plasmid both in vitro and in vivo. Gene replacement therapy treatments by the same formulations in Abca4-/- mice demonstrated a slowdown of STGD progression, indicated by reductions in A2E levels.
Figure 4.1. Map of therapeutic ABCA4 plasmid with photoreceptor specific bovine rhodopsin promoter (RHO).
3. Materials and Methods
3.1 Plasmid Preparation
Reporter gene plasmids were purchased from the Addgene repository. pCMV-
GFP and pRHO-DsRed were developed by Constance L.Cepko (Harvard
University., Boston, MA) (Addgene plasmids # 11153 & 11156, respectively)162.
107 pCMV-ABCA4 was a gift from Robert S. Molday (University of British Columbia,
BC, CA), and contained the full-length human ABCA4 cDNA sequence (NCBI
Accession # NM_000350.2) on a pCEP4 backbone. Plasmids were transformed in
MAX Efficiency DH5α competent E. coli (Life Technologies, Carlsbad, CA, USA) and grown overnight on LB agar plates containing ampicillin. Selected colonies were amplified overnight and purified using a QIAGEN Plasmid Maxi Kit according to the manufacturer’s protocol. Plasmid purity and size were verified by gel electrophoresis. The pRHO-ABCA4 plasmid was constructed by molecular cloning of the cDNA for the ABCA4 gene into the linearized pRHO-DsRed plasmid with the DsRed reporter gene removed. All enzymes were purchased from New
England Biolabs (Ipswich, MA) cDNA for the ABCA4 gene was amplified by polymerase chain reaction (PCR) with the Q5 High-Fidelity DNA Polymerase enzyme, and using the forward primer 5’-
AATACCGGTATGGGCTTCGTGAGACAGATA-3’ and the reverse primer 5’-
TATATAGCGGCCGCTAGCTCAGTCTGCTGTTT-3’ to add the AgeI and NotI restriction sites to the 5’ and 3’ ends of ABCA4, respectively. cDNA was purified using a QIAquick PCR Purification Kit (QIAGEN) according to the manufacturer’s instructions. The amplified ABCA4 cDNA and pRHO-DsRed plasmid were cut using the NotI and AgeI restriction endonucleases and purified by gel electrophoresis using the QIAGEN gel extraction kit according to the manufacturer’s instructions. Calf intestinal alkaline phosphatase (CIP) was added to the endonuclease reaction with the pRHO vector in order to dephosphorylate
108 vector termini and prevent self-annealing of the open ends. For the ligation step, the cut pRHO vector and ABCA4 cDNA were incubated overnight at 16 °C with the
T4 DNA Ligase enzyme in the supplied reaction buffer. Ligation products were verified by gel electrophoresis and transformed and amplified as described above.
Final plasmid stocks were verified by Sanger sequencing.
3.2 Cell Culture
ARPE-19 cells were cultured in Dulbecco’s modified Eagle’s medium and supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 units/mL penicillin (all reagents were from Invitrogen, Waltham, MA). Cells were maintained in a humidified incubator at 37 ºC and 5% CO2.
3.3 Animal
129S1/SvImJ mice were obtained from Jackson Laboratory (Bar Harbor, ME).
Abca4-/- mice were generated by standard procedures as previously described163, and were maintained with either pigmented 129Sv/Ev or C57BL/6 mixed backgrounds, and their siblings were used for future experiments. All mice were housed and cared for in the animal facility at the School of Medicine, Case Western
Reserve University, and animal procedures were approved by CWRU Institutional
Animal Care and Use Committee.
109 3.4 Gel Electrophoresis
ECO/pDNA nanoparticles were prepared, and 4 μL aliquots of nanoparticles,
4 μL of loading dye (Promega) and 16 uL were mixed. The mixture (20 μL) was loaded onto a 0.7 % agarose gel containing ethidium bromide. The gel was submerged in 0.5× Tris/Borate/EDTA (TBE) buffer and run at 100 V for 25 min.
Plasmid DNA bands were visualized using an AlphaImager ultraviolet imaging system (Biosciences).
3.5 Synthesis of A2E
A mixture of all-trans-retinal (100 mg, 352 μmol) and ethanolamine (9.5 mg,
155 μmol) in ethanol (EtOH) (3.0 ml) was stirred in the presence of acetic acid (9.3
μL, 155 μmol) at room temperature with a sealed cap in the dark for 2 days. After the mixture was concentrated in vacuo, the residue was purified by silica gel column chromatography. After elution with methanol (MeOH):CH2Cl2 (5:95), further elution with MeOH:CH2Cl2:trifluoroacetic acid (TFA) (8:92:0.001) gave A2E. Pure samples were obtained by HPLC purification [ZORBAX 300 SB-C18, 9.4 × 250 mm, 84-100% H2O/ACN for 30 min, 1.0 mL/min flow detected at UV 430 nm].
A2E was detected at retention time (tR= 35.2 min). Collection of the fraction provided pure A2E for further analysis. A2E was identical in all respects to material previously described.
110 3.6 HPLC Analysis of A2E Samples
Mice were euthanized by cervical dislocation and eyes were excised with curved scissors. Eyes were deep-frozen in liquid nitrogen immediately after removal and stored at −80 °C until used. A2E was extracted from the eyes of each mouse in 1 ml of acetonitrile after homogenization with a Brinkmann Politron homogenizer (Kinematica, Lucerne, Switzerland). After evaporation of solvent, extracts were dissolved in 120 μl acetonitrile with 0.1% TFA and then filtered through a Teflon syringe filter. Samples (100 μl) were loaded on a C18 column
(ultrasphere ODS, 4.6 × 250 mm) (Beckman Coulter, Indianapolis, IN) and analyzed by reverse-phase HPLC. A2E was eluted with the following gradients of acetonitrile in water (containing 0.1% trifluoroacetic acid): 85–96% (10 min), 96%
(5 min), 96–100% (2 min), and 100% (13 min) (flow rate, 1 mL/min), and they were monitored at 430 nm. For A2E quantitation by reverse phase HPLC, the area of the A2E peak was normalized to the standard, A2E, which was synthesized as previously described.
3.7 Preparation of ECO/pDNA Nanoparticles
Multifunctional pH-sensitive lipid ECO were synthesized as previously reported164. The ECO/pDNA nanoparticles were prepared by self-assembly of ECO with plasmid DNA at an amine/phosphate (N/P) ratio of 10. The ECO stock solution
(2.5 mM in ethanol) and plasmid DNA stock solution (0.5 mg/mL) at predetermined amounts based on the N/P ratio were diluted into equal volumes with nuclease-free
111 water, mixed and shaken for 30 min at room temperature. A different ECO stock solution (25 mM) was used for in vivo formulations. Lipofectamine 2000
(Invitrogen, Waltham, MA)/DNA nanoparticles were prepared according to the manufacturer’s recommendation, while the pDNA amount was kept the same as the one in ECO/pDNA nanoparticles.
3.8 Transmission Electron Microscope
The morphology of ECO/pCMV-ABCA4 (N/P=10) and Lipofectamine
2000/pCMV-ABCA4 nanoparticles were imaged with a transmission electron microscope (JEOL JEM2200FS). Samples for TEM were prepared by depositing
20 μL of the particle solution onto a 300-mesh copper grid covered by a thin amorphous carbon film (20 nm). Immediately after deposition, the excess liquid was removed by touching the grid with filter paper. Samples were stained twice by adding 3 μL 2% uranyl acetate aqueous solution. The excess of staining solution was removed. Images of the nanoparticles were acquired with TEM after the samples were dried.
3.9 Dynamic Light Scattering (DLS)
The sizes and zeta potentials of ECO/pCMV-ABCA4 (N/P=10) and
Lipofectamine 2000/pCMV-ABCA4 nanoparticles were determined by dynamic light scattering with an Anton Paar Litesizer 500 (Anton Paar USA Inc, Ashland,
VA). Three measurements were performed for each sample at 20 ºC.
112 3.10 In Vitro Transfection
ARPE-19 cells were seeded onto 12-well plates at a density of 5 × 104 cells per well and allowed to grow for 24 h at 37 ºC. Transfections were conducted in
10% serum media with the ECO/pDNA nanoparticles at a DNA concentration of 1
μg/mL. ECO/pDNA nanoparticles were incubated with ARPE-19 cells for 4 h at 37
ºC. The media then was replaced with fresh serum-containing media (10% serum) and cells were then cultured for additional 48 h. For reporter genes, GFP and DsRed expressions were evaluated with an Olympus FV1000 confocal microscope
(Olympus, Center Valley, PA). After the culture media was removed, each well was washed twice with PBS (10 mM sodium phosphate, pH 7.2, and 100 mM NaCl).
Cells were harvested after treatment with 0.25% trypsin containing 0.26 mM EDTA
(Invitrogen), followed by centrifugation at 1000 rpm for 5 min, fixation in 750 μL
PBS containing 4% paraformaldehyde, and finally passed through a 35 μm cell strainer (BD Biosciences, San Jose, CA). A BD FACSCalibur flow cytometer (BD
Biosciences) was used to determine GFP and DsRed expressions based on the fluorescence intensity in a total of 10,000 cells for each sample. For the evaluation of ABCA4 gene expression, qRT-PCR analysis was carried out.
3.11 In Vivo Subretinal Transfection with ECO/pDNA nanoparticles
All surgical manipulations were carried out under a surgical microscope
(Leica M651 MSD). Mice were anesthetized by intraperitoneal injection of a cocktail (15 μL/g body weight) comprised of ketamine (6 mg/mL) and xylazine
113 (0.44 mg/mL) in PBS buffer (10 mM sodium phosphate, 100 mM NaCl, pH 7.2,).
Pupils were dilated with 1.0 % tropicamide ophthalmic solution (Bausch & Lomb,
Rochester, NY). A 33-gauge beveled needle (World Precision Instruments, Sarasota,
FL) was used as a lance to make a full thickness cut through sclera at 1.0 mm posterior to the limbus. This needle was replaced with a 36-gauge beveled needle attached to an injection system (UMP-II microsyringe pump and a Micro4 controller with a footswitch, World Precision Instruments). This needle was aimed toward the inferior nasal area of the retina, and an ECO/pDNA nanoparticles solution (2.0 μL) was injected at a pDNA dose of 200 ng into the subretinal space.
Successful administration was confirmed by observing bleb formation. The tip of the needle remained in the bleb for 10 s after bleb formation, when the needle was gently withdrawn. A solution (2.0 μL) of PBS was also injected into the subretinal space of the contralateral eye to serve as a treatment control. Each of the treatment group included 5 mice.
3.12 Quantitative RT-PCR (qRT-PCR)
Cell lysis and homogenization in cell lines was performed using a cell scraper in lysis buffer. For animal tissues, mice were sacrificed and eyes enucleated.
Eyecups were dissected in PBS buffer and retinas and RPE were separated. Tissues were homogenized manually using a glass rod in lysis buffer. RNA extraction was performed using a QIAGEN RNeasy kit according to the manufacturer’s instructions. mRNA transcripts were converted to cDNA using the miScriptII
114 reverse transcriptase kit (QIAGEN). qRT-PCR was performed with SYBR Green
Master mix (AB Biosciences, Allston, MA) in an Eppendorf Mastercycler machine.
Fold changes were normalized to 18S, with SHAM eyes as controls.
3.13 Electroretinograms
Animals were anesthetized by intraperitoneal injection of a cocktail (15 μL/g body weight) comprised of ketamine (6 mg/mL) and xylazine (0.44 mg/mL) in PBS buffer (10 mM sodium phosphate, 100 mM NaCl, pH 7.2). Pupils were dilated with
1% tropicamide for imaging. Experiments were performed in a dark room. Three electrodes were placed on the animal: a contact lens electrode on the eye, a reference electrode underneath the skin between the ears, and a ground electrode underneath the skin of the tail. Full field electroretinograms were recorded with the universal electrophysiologic system UTAS E-3000 (LKC Technologies, Inc.,
Gaithersburg, MD, USA).
3.14 Histology
To assess gene expression and distribution in throughout the retina, eyecups were prepared for histological analysis as previously published164. Mice were sacrificed by cervical dislocation, and eyes were enucleated and washed in PBS buffer prior to fixation in 4% paraformaldehyde (PFA) in PBS buffer. After 2 h, eye cups were removed and fixed in 4% PFA overnight. The next day, samples were transferred to 20% sucrose/OCT (Tissue-Tek optimal cutting temperature
115 compound, Sakura) via a gradual sucrose gradient. Eyecups were incubated in 20% sucrose/OCT overnight, and then imbedded in cryomolds and frozen in OCT.
Frozen slides were cut in 10 μm-thick slices and adhered to glass slides.
Prior to immunohistochemistry, slides were warmed to room temperature and washed in tris buffered saline with 1% tween 20 (TBST) buffer. Heat-induced antigen retrieval was conducted in 10 mM citrate buffer (pH 6.0) in a pressure cooker for 20 min. and slides were allowed to slowly cool to room temperature.
Primary antibodies used for IHC were rabbit anti-GFP for GFP, rabbit polyclonal anti-RFP (Rockland, Limerick, PA) for DsRed, and the mouse monoclonal 3F4
(Abcam, Cambridge, MA) for ABCA4. Fluorescent secondary antibodies were donkey anti-rabbit or anti-mouse IgG Alexa488 (Jackson ImmunoResearch, West
Grove, PA), and these slides were imaged using an Olympus FV1000 confocal microscope.
3.15 Statistical Analysis
Experiments were performed in triplicate and presented as the means and standard deviations. Statistical analysis was conducted with ANOVA and two-tailed
Student's t-tests using a 95% confidence interval. Statistical significance was accepted when P ≤ 0.05.
116 4. Results
4.1 Nanoparticle Characterization
Stability of nanoparticle formulations was verified by agarose gel electrophoresis for both ECO and the commercially available transfection agent lipofectamine 2000 (Figure 4.2A). When formulated in either nuclease free water or 10% serum medium, free ABCA4 plasmids migrated down the gel, while
ECO/pABCA4 an lipofectamine/pABCA4 nanoparticles stayed inside of the wells when voltage was applied. However, slight DNA smears were observed for lipofectamine nanoparticles for both conditions. These results demonstrated stable formulations were formed between ECO and large ABCA4 plasmid (16 kb).
Compared with ECO/pABCA4 nanoparticles, lipofectamine formed less stable formulations indicated by the DNA smears.
Dynamic light scattering (DLS) measurements (Figure 4.2B) of nanoparticle size showed that both ECO and lipofectamine formed nanoparticles with large pABCA4 under either nuclease free water or 10% serum media, with the size of
118.0 ± 1.8 nm and 149.5 ± 4.3 nm respectively in water, and 192.4 ± 8.0 nm and
174.6 ± 3.9 nm respectively in media. For both conditions, ECO/pABCA4 formulations demonstrated better uniformity than lipofectamine/pABCA4 formulations, indicated by narrower particle size distributions shown in Figure
4.2D, which was also confirmed by TEM images (Figure 4.2E) with more aggregations observed for lipofectamine/pABCA4 nanoparticles. Zeta potentials
117 (Figure 4.2C) of ECO/pABCA4 and lipofectamine/pABCA4 were positive for both nanoparticles in nuclease free water (26.3 ± 2.1 mV and 26.0 ± 0.3 mV, respectively). Under 10% serum media, zeta potentials became negative for both nanoparticles because of the interactions with serum proteins (-15.1 ± 1.0 mV for
ECO/pABCA4 and -12.5 ± 1.0 mV for lipofectamine/pABCA4).
Figure 4.2. Characterization of ECO/pABCA4 nanoparticle formulations for stability, size, zeta potential and morphology with lipofectamine 2000/pABCA4 formulations as controls. (A) Agarose gel electrophoresis of nanoparticle formations. (B) DLS measurements of nanoparticle size, (C) zeta potential and (D) size distribution. (E) TEM images of nanoparticle formulations. Scale bar represents 100 nm.
4.2 Differential Expression of Reporter Genes In Vitro and In Vivo
In order to generate photoreceptor-specific expression of transfected genes, plasmid constructs of reporter genes were evaluated for expression of either GFP driven by the common CMV promotor (pCMV-GFP) or DsRed driven by the
118 bovine rhodopsin promoter (pRHO-DsRed). In vitro transfections of reporter genes in cultured human RPE cells (ARPE-19) were carried out. Confocal images in
Figure 4.3A demonstrated high expression of GFP in cells transfected with pCMV-
GFP in contrast to low expression of DsRed in cells transfected with the pRHO-
DsRed, suggesting the RHO promoter is specific for rod photoreceptors. Confocal images of GFP demonstrated no fluorescence in untreated controls, widely distributed fluorescence in ECO/pCMV-GFP transfected cells, and a narrower distribution of fluorescent cells in the lipofectamine/pCMV-GFP transfected samples, suggesting better transfection efficiency of pH-sensitive lipid ECO gene delivery system than lipofectamine control. Quantification of fluorescence intensity by flow cytometry was in agreement with the results of confocal microscopy
(Figure 4.3B). Compared to untreated controls, green fluorescent intensity was approximately 35-fold higher in ECO/pCMV-GFP transfected cells, while only a two-fold increase was measured after transfection with lipofectamine/pCMV-GFP.
Transfection with ECO/pRHO-DsRed generated an approximate two-fold increase in red fluorescence intensity, while the intensity decreased with lipofectamine/pRHO-DsRed. Measurements of mRNA levels corresponding to gene expression by qRT-PCR were in agreement with fluorescence output from the translated reporter genes (Figure 4.3C). Compared to lipofectamin/pCMV-GFP transfection, PCR results indicated a 93-fold increase in mRNA expression of GFP after transfection with ECO/pCMV-GFP. Expression of DsRed was the same between lipofectamine/pRHO-DsRed and ECO/pRHO-DsRed transfections.
119 In order to evaluate the efficiency of photoreceptor specific expression driven by RHO promoter in vivo, a co-delivery experiment was carried out in wild type
129S1/SvImJ mice. The in vivo expression of reporter genes was evaluated following subretinal injections of 200 ng pDNA formulated in a 1:1 ratio of
ECO/pCMV-GFP and ECO/pRHO-DsRed nanoparticles at N/P = 10 in 4-week old wild type 129S1/SvImJ mice. Immunohistochemistry for GFP and DsRed expression revealed expression of transfected genes in the photoreceptor segments for both genes, as well as GFP expression in the outer plexiform layer (Figure 4.3D).
The expression in mRNA levels was evaluated separately for the retina and the RPE
(Figure 4.3E). GFP expression was higher in the RPE compared to the retina, whereas DsRed expression was higher in the retina compared to the RPE, indicating that we have achieved tissue-specific expression in the retina using the bovine rhodopsin promoter (RHO) on the transfected plasmid DNA.
120
Figure 4.3. In vitro and in vivo transfection of ECO/pCMV-GFP, ECO/pRHO-DsRed nanoparticles in ARPE-19 cells and wild type 129S1/SvImJ mice. (A) Confocal images of GFP and DsRed expression in ARPE-19 cells. (B) Quantification of mean fluorescence intensities of GFP and DsRed expression in ARPE-19 cells by flow cytometry. (C) mRNA levels of GFP and DsRed expression in ARPE-19 cells by PCR analysis. (D) Confocal images of retinal tissue slides 3 days after subretinal injection compared with non-treated controls. (E) mRNA levels of GFP and DsRed in the retinal tissue of 129S1/SvImJ mice 3 days after subretinal injection. mRNA levels were normalized to levels in the RPE. (**p < 0.005). Significance analysis was performed using two-tailed student t-test.
4.3 In Vitro and In Vivo Transfection with ECO/pABCA4 Nanoparticles
In order to evaluated the ability of pH-sensitive multifunctional lipid ECO gene delivery system in delivering large gene, particle characterization and gene
121 expression evaluation in vitro and in vivo were carried out for ECO/pCMV-ABCA4 and ECO/pRHO-ABCA4 nanoparticles. Gel electrophoresis (Figure 4.4A) indicated that stable lipid-DNA complexes had formed and were retained at the top of the gel between ECO and both large therapeutic plasmids for gene therapy treatments. ABCA4 expression was demonstrated in ARPE19 cells in vitro by qRT-
PCR analysis (Figure 4.4B) performed with RNA harvested from ARPE19 cells 48 hours after transfection with 1 μg/mL pCMV-ABCA4. Results indicated high expression levels of ABCA4 in ARPE-19 cells, suggesting the success of introducing expression of a large gene in vitro by ECO gene delivery system.
Compared with the mRNA level transfected by lipofectamine, ECO generated a near 800-fold higher expression of the ABCA4 gene in ARPE-19 cells.
In order to test the viability of ECO/pABCA4 to treat Stargardt’s disease
(STGD), it was necessary to demonstrate successful transfection of the gene in vivo.
ECO/pABCA4 nanoparticles (both CMV and RHO) were delivered by subretinal injection in Abca4-/- mice, the orthologous rodent model to human STGD. The in vivo transfection generated expression of ABCA4 mRNA with both pCMV-ABCA4 and pRHO-ABCA4 (Figure 4.4C). ABCA4 expression levels driven by the RHO promoter were 10-fold higher in the retina compared to the RPE, while ABCA4 expression from the CMV promoter were similar in the retina and the RPE. To further confirm these results, immunohistochemical (IHC) staining was carried out.
IHC for the ABCA4 protein stained with anti-ABCA4 antibody (3F4, Green)
(Figure 4.4D) revealed protein expression in the outer segments. The green
122 fluorescence intensity was higher in the transfected eye than in non-transfected controls. Compared the ABCA4 expression driven by RHO promoter with the one driven by CMV promoter, more tissue specific expression was observed in the photoreceptor outer segments for RHO promoter, while the one driven by CMV promoter showed ABCA4 expression in both photoreceptors and other cell types in the retina. These results indicated that with ECO/pABCA4 nanoparticles, we have successfully formulated a synthetic gene therapy platform for STGD that forms stable complexes with the ABCA4 gene and efficiently and specifically transfects photoreceptor cells in vivo.
123 Figure 4.4. Characterization, in vitro and in vivo transfection efficiency of ECO/pRHO-ABCA4 and ECO/pCMV-ABCA4 nanoparticles in Abca4-/- mice. A. Agarose gel of ECO/pRHO-ABCA4, ECO/pCMV-ABCA4 nanoparticles and free plasmids; B. mRNA levels of ABCA4 expression in ARPE-19 cells transfected by ECO/pCMV-ABCA4 or control Lipofectamine/pCMV-ABCA4 nanoparticles. C. mRNA levels of ABCA4 expression in the retinal tissue of Abca4-/- mice 7 days after subretinal injections of ECO/pRHO-ABCA4 and ECO/pCMV-ABCA4 nanoparticles. D. Confocal images of ABCA4 expression (Green) in Abca4-/- mice 7 days after subretinal injections of ECO/pRHO-ABCA4 and ECO/pCMV-ABCA4 nanoparticles, with the expression in wild type 129S1/SvImJ mice as the control. (**p < 0.005). Significance analysis was performed using two- tailed student t-test.
4.4 Gene Replacement Therapy using ECO/pABCA4 Nanoparticles
The accumulation of lipofuscin in the RPE is a common phenotype in retinal diseases associated with mutations in the ABCA4 gene165. Lipofuscin accumulates as a byproduct of cumulative damage during aging166. Along with A2E, other dimers of vitamin A and other uncharacterized orange pigments have been identified in lipofuscin granules and/or RPE extracts167. Therefore, the amount of
A2E in the retina extracts was commonly used as an indication for disease progression of Stargardt’s disease168.
Here, we first analyzed the disease progression of Abca4-/- mice (STGD model). A2E levels were analyzed using HPLC in untreated Abca4-/- mice aging from 2 months up to 6 months. In order to achieve best resolution in HPLC chromatograms, 4 retinas were combined together and extracted with ACN. A2E levels were read as the peaks eluted at 10~11 min in the chromatograms. A2E quantification was carried out by integrating the areas of A2E peaks in the chromatograms. As is shown in Figure 4.5A, A2E levels increased with the mice age for untreated Abca4-/-, which indicated a clear phenotype in the studied Abca4-
124 /-mouse model. Furthermore, to evaluate the gene therapy efficacy with
ECO/pABCA4 nanoparticles, Abca4-/-mice were subretinally injected with PBS
(SHAM) in one of the eyes and injected with either ECO/pCMV-ABCA4 or
ECO/pRHO-ABCA4 nanoparticles in the contralateral eye at 1 month of age. A2E levels were analyzed 6 months after gene therapy. A reduction in A2E levels (Figure
4.5C) were observed in the treatment groups (CMV and RHO) compared with
SHAM, shown by HPLC chromatograms. A2E levels were also quantified and normalized to the level in SHAM (Figure 4.5D), which demonstrated a 30% reduction of A2E levels in the treatment groups (CMV and RHO). However, there was no noticeable difference between the treatment by ECO/pCMV-ABCA4 nanoparticles and ECO/pRHO-ABCA4 nanoparticles.
125 Figure 4.5. Gene therapy in Abca4-/- mice with ECO/pCMV-ABCA4 and ECO/pRHO-ABCA4 nanoparticles. (A) A2E accumulations in Abca4-/- mice of different ages characterized by HPLC analysis. (B) Characteristic spectrum of A2E taken from the HPLC analysis of A2E accumulations in Abca4-/- mice. (C) A2E levels in Abca4-/- mice 6 months after treatment with ECO/pCMV- ABCA4 and ECO/pRHO-ABCA4 nanoparticles analyzed by HPLC. (D) Quantification of A2E levels of Abca4-/-mice 6 months after treatment. A2E levels were normalized to SHAM (PBS injected) eyes.
4.5 Safety of Gene Replacement Therapy using ECO/pABCA4 Nanoparticle
Figure 4.6. Safety of ECO/pABCA4 treatment in wild type mice. ERG a-wave and b-wave amplitudes analyzed 7 and 30 days after injection with PBS (sham), ECO/pCMV-ABCA4, or ECO/pRHO-ABCA4 in one eye. Amplitudes are normalized to the contralateral uninjected eye. Error bars = ± std (* p < 0.05 relative to no injection). (**p < 0.005). Significance analysis was performed using ANOVA.
In order to evaluate the safety of gene replacement therapy using
ECO/pABCA4 nanoparticles, wild type 129S1/SvImJ mice were subretinally injected in one eye with either PBS (sham), ECO/pCMV-ABCA4, or ECO/pRHO-
ABCA4, while the other eye was left without injection. Due to the invasive nature
126 of the subretinal injection route, the contralateral control was used to establish a baseline for normal eye function, and the sham injection allowed us to distinguish the effects of the injection from the treatment. ERGs recorded 7 and 30 days after treatment indicated moderate loss of visual function in some injected eyes, represented by decreased scotopic a- and b- wave amplitudes in some groups.
Photopic wave amplitudes did not change significantly compared to the no injection controls across all injections.
5. Discussion
In this chapter, we have developed a gene replacement therapy using
ECO/pABCA4 nanoparticles for STGD. Two main challenges for ABCA4 delivery to the retinal tissue have been addressed: stable encapsulation of large ABCA4 therapeutic plasmid and photoreceptor specific expression of the ABCA4 gene. By using the pH-sensitive multifunctional cationic lipid ECO as gene carrier, stable nanoformulations were obtained with large ABCA4 therapeutic plasmids.
Photoreceptor specific expressions of both reporter and therapeutic gene were generated by bovine rhodopsin promoter (RHO). Gene replacement therapy using
ECO/pABCA4 nanoparticles demonstrated a slowdown effect of disease progression in Abca4-/- mice. A safety study demonstrated ECO/pABCA4 based gene replacement therapy could be a safe strategy for the treatment of STGD.
Recently, with the FDA approval of the viral gene therapy treating an IRD, development of gene replacement therapies targeting monogenic IRDs is receiving
127 the most attention. As one of the most common forms of IRD, Stargardt’s disease
(STGD) is a good candidate for gene replacement therapy, because it is a monogenic disease caused by mutations in single ABCA4 gene. However, none of the viral vectors is effective for gene replacement therapy treatment of STGD, because the size of ABCA4 gene is as large as 6.8 kB, which already exceed the loading capacity of most viral vectors without including other functional sequences169. Several groups tried to compacted large ABCA4 genes into oversize vectors and observed gene expression in rodent and canine models170, 171. However, the mechanism of transducing oversize AAVs is still elusive, which raises many safety concerns.
Others also exploited gene-splitting methods to transfer large genes using viral vectors, but complications are still introduced by the heterogeneous mix of DNA products172.
Non-viral retinal gene delivery systems, on the other hand, are not limited by payload capacity173. In this chapter a pH-sensitive multifunctional lipid ECO was used to encapsulate large ABCA4 therapeutic plasmids. As discussed in previous chapters, ECO contains a protonatable ethylenediamine (E) head group, two cysteine (C) functional linkers and two oleoyl (O) lipophilic tails, which not only promotes the packaging of therapeutic genetic materials, but also facilitates the pH- sensitive amphiphilic endosomal membrane destabilization through protonation of the head group of ECO in the acidic endosomal-lysosomal compartment (pH = 5-
6) and dissociation of the nanoparticles by reduction of the disulfide bonds in the cytoplasm. Compared with the commercialized gene delivery system lipofectamine
128 2000 which is designed universally for DNA and RNA transfection, ECO demonstrated better stability in both water and serum environment formulating with large ABCA4 plasmid (16.9 KB). Compared with lipofectamine, ECO and ABCA4 plasmid formed nanoparticles with narrower size distribution, which demonstrated a more uniformed and reliable nanoparticle-based gene delivery system. Moreover,
ECO/pABCA4 nanoparticles were able to induce significantly more ABCA4 expression in ARPE-19 cells than lipofectamine 2000, showing excellent ability introducing large gene expression in cells.
The retina is a light-sensitive tissue, which is functioning by complex processes requiring coordinated activity of different cells including the photoreceptor cells, different neurons and the retinal pigment epithelium (RPE)174.
Therefore, gene replacement therapy for retinal genetic disorders requires high specificity, otherwise, failure of functions and death of cells due to mis-expression of therapeutic genes could cause further damages to vision. Since STGD is associated with a photoreceptor mutation, incorporation of rhodopsin specific promoter (RHO) could induce therapeutic gene expression only in the photoreceptor outer segment. The tissue specific expression was demonstrated using both reporter and therapeutic genes, which strong selectivity of expression was generated in photoreceptor cells. However, compared with the wild type
ABCA4 expression levels, the expression levels of ABCA4 was still low.
Modifications are still needed to further increase the transfection efficiency of ECO based system.
129 The Abca4-/- mouse is currently the only available animal model for STGD.
Abca4-/- mice has some limitations, including the lack of a macula, the primary area affected by STGD, and a slower disease progression than that seen in patients175.
However, the accumulation of the major fluorophore of lipofuscin, A2E, is at a 5- to 10-fold higher rate175. Therefore, as the Abca4-/- mice do not present with other clinically relevant phenotypic feature, A2E accumulation is the direct measure of the protein function, which is also the measure of efficiency of the therapeutic intervention176. Six months after gene replacement therapy using ECO/pABCA4 nanoparticles, there was a 30% reduction of A2E accumulation in the treated eyes compared to the control eyes injected with PBS. The treatment effect is comparable to the treatments by a lentiviral gene delivery system for STGD177, suggesting ECO based gene replacement therapy can be a promising strategy for the treatment of
STGD.
For every retinal gene replacement therapy, safety is always the primary concern. Here, ECO based gene delivery system didn’t show cytotoxicity in ARPE-
19 cells, which indicated excellent in vitro safety. Furthermore, there was no significant difference between treatment groups and SHAM group for in vivo safety studies, demonstrating an excellent in vivo therapeutic safety. However, gene replacement therapy using ECO/pABCA4 nanoparticles has caused slight reduction in the ERG response, which was due to the subretinal injection route. The functional reduction was commonly observed for subretinal injections, where there was often a significant injury to the retina and function reduction178. Similar effects were also
130 observed in previous therapy for RPE disease shown in previous chapter. In order to minimize the damage, elaborated injection skills are greatly required for subretinal gene therapy.
Compared to the gene replacement therapy for LCA2 which is an RPE based genetic disorder, development of gene replacement therapy for the photoreceptor based STGD is more challenging. Rather than generating effective amount of
ABCA4 expression in the rod photoreceptors of which there are millions in the retina to improve the visual function, gene transduction of a smaller number RPE are able to supply 11-cis retinal for their attachment photoreceptors. This greatly eases the burden of transfection requirements for the RPE delivery. Gene replacement therapy using ECO/pABCA4 nanoparticles for STGD revealed gene expression in the photoreceptor outer segments. Even though, the expression levels were lower than wild type, the gene replacement therapy was able to show therapeutic effect at current levels of ABCA4 expression. Therefore, ECO based gene replacement therapy can be a promising gene replacement therapy strategy for
STGD and a broader range of IRDs.
131 Chapter Ⅴ. Efficient Delivery of CRISPR/Cas9 via pH-Sensitive
Multifunctional Lipids for the Treatment of Autosomal Dominant Retinal
Genetic Diseases
1. Background
Gene replacement therapy has shown great promise in clinical investigations for the treatments of recessive IRDs. However, the application in autosomal dominant diseases is limited because of the requirement of silencing or ablating the gain-of-function alleles22. The combination of RNAi and gene replacement therapy has been developed to treat dominant genetic disorders, but it is still challenging to achieve high specificity, long-term expression and effective regulation22.
The CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas9 (CRISPR associated protein 9) system first discovered in bacteria and archaea as adaptive immune system has become one of the most powerful gene editing methods, which presents an alternative approach to address the challenges of gene therapies for autosomal dominant diseases23. CRISPR/Cas9 system relies on uptake of foreign DNA fragments into CRISPR loci and subsequent transcription and processing of these RNA transcripts into short CRISPR RNAs (crRNAs), which
132 in turn anneal to a trans-activating crRNA (tracrRNA) and direct sequence-specific silencing of foreign nucleic acids by Cas proteins179. Recent in vitro work showed that a synthetic single guide RNA (sgRNA) consisting of a fusion of crRNA and tracrRNA can direct Cas9 endonuclease-mediated cleavage of target DNA180. The
20 nucleotide guide portion of the sgRNA recognizes complementary DNA sequences flanked by a protospacer adjacent motif (PAM), and Cas9 cleaves the recognized DNA.
CRISPR/Cas9 systems can be good strategies to treat autosomal dominant diseases, because when the disease‐causing allele includes unique PAM sequences which are not present in its wild‐type counterpart, allele‐specific genome editing by selectively targeting and permanently inactivating the mutant allele can be achieved while leaving the wild‐type allele functionally intact181. Therefore, CRISPR/Cas9 systems were established to treat autosomal dominant retinal genetic disorders.
Allele specific edition and disease phenotype alleviation was observed in animal models23, 182, 183.
As the CRISPR/Cas9 system is developing further from bench to bedside, delivery modality is becoming a major challenge184. Since both components, Cas9 and sgRNA can be incorporated in DNA plasmids sequences, conventional gene delivery modalities including both viral and non-viral delivery approaches have been developed for the delivery of CRISPR/Cas9 gene editing systems185. Viral gene delivery systems have limitations in payload capacities, especially when the sequence size of Cas9 protein easily passes 10 kb. Moreover, for some viral vectors,
133 the DNA can randomly integrate into the genome, potentially causing cancer and other diseases169. Compared with viral delivery systems, non-viral gene delivery systems are not limited by cargo capacities and have better safety profile. The preparation of non-viral gene delivery systems is less expensive and easier186.
Cationic lipids are widely used as a non-viral gene delivery strategy187.
Previously, we designed a multifunctional lipid, (1-aminoethyl)iminobis[N-
(oleicylcysteinyl-1-amino-ethyl)propionamide] (ECO), as a simple and smart gene delivery carrier for the development of gene replacement therapies to treat retinal genetic disorders115. Gene replacement therapy using ECO/therapeutic plasmid nanoparticles successfully rescued the visual functions in animal models of inherited retinal degenerations (IRDs)164. Like most cationic lipids, ECO contains a protonatable ethylenediamine (E) head group, two cysteine (C) functional linkers, and two oleoyl (O) lipophilic tails. ECO is highly efficient in gene transfection because of its mechanism of pH-sensitive amphiphilic endosomal escape and reductive cytosolic release (PERC) of nucleic acids. The pH-sensitive amphiphilicity of lipid ECO allows the carrier to change its amphiphilic structure at endosomal/lysosomal pH (5.0–6.0), resulting in disruption of endosomal– lysosomal membranes and escape of gene delivery system188. The incorporation of degradable disulfide bonds into ECO lipid delivery system by cysteine residues promotes the release of nucleic acids in the reductive environment of cytosol.
Following this mechanism, a series of ECO lipid derivatives have been designed, which have more compact and simpler head structure, cysteine residues to provide
134 disulfide bonds, protonatable amines with different pKaʼs and lipophilic tails. These derivatives were hypothesized to have similar gene delivery behaviors as ECO lipid and were evaluated in CRISPR/Cas9 delivery, in order to develop gene therapy for autosomal dominant retinal genetic disorders.
2. Delivery System Design
In this chapter, a series of new pH-sensitive multifunctional cationic lipids were designed based on previously designed ECO, addressing two main structural components from ECO: the protonatable head group and the functional amino acid linkers. First, the head group structure was simplified from a 3,3'-[(2-
Aminoethyl)imino]bis[N-(2-aminoethyl)propanamide] structure to a 2,2',2''-
Triaminotriethylamine one. Second, lysine and histidine were introduced as functional linkers to the structure. Following this idea, new isotypic ECO carriers were generated by head modifications and different sequence combinations of new functional linkers with cysteine. The design of new pH-sensitive multifunctional cationic lipids was demonstrated in Figure 5.1.
135
Figure 5.1. Design of ECO isotypic derivatives by head modification and new amino acid functional linkers introduction.
The CRISPR/Cas9 system targeting eGFP sequence was used to evaluate the delivery efficiency of the new carriers by monitoring GFP knockdown efficiency.
The CRISPR/Cas9 system contains two components: a plasmid expressing sgRNAs
(psgRNA) and a plasmid expressing Cas9 protein (pCas9). The sequence of psgRNA contains two guide RNAs and a mCherry reporter gene. The sequence of pCas9 contains Cas9 gene and GFP reporter gene. The plasmid maps are shown in
Figure 5.2.
136
Figure 5.2. Plasmid maps of CRISPR/Cas9 system that targets GFP gene.
3. Materials and Methods
3.1 Synthesis of New Carriers
O R R R R Fmoc NH NH HN Fmoc NH 2 N-Fmoc HN HN NH2 NH HN 2 amino acid 1 O N-Fmoc O O 20% piperidine Boc2O, CH2Cl2 N amino acid 2 N N N N in DMF BocHN O BocHN O H2N BocHN EDC·HCl, HOBt BocHN O HN EDC·HCl, HOBt HN NH NH DMF, DIPEA HN 2 2 NH DMF, DIPEA NH HN Fmoc NH 2 Fmoc R R ′ R O R ′ O R ′ ′ O R O R R R R NH HN NH HN NH NH2 HN O HN HN O CH Cl /TFA/EDT/TIBS O piperidine 2 2 O O oleoyl chloride N /DMF N N BocHN O O DMAP, K CO SO H2N O O BocHN O 2 3, Na2 4 HN THF HN HN NH HN NH HN NH NH2 R ′ R R O R ′ ′ O R O R
Figure 5.3. Synthetic route of new carriers.
Carrier ECO was prepared and purified according to our previous report.189
The synthetic route was described briefly, taking synthesis of iEHCO as an example. Tert-butyl 2-(bis(2-aminoethyl)amino)ethylcarbamate (BocTren) was prepared with a good yield (72%) as reported previously190. Then, BocTren reacted with Fmoc-His(Trt)-OH to provide ipEH in the presence of EDC⋅HCl, HOBt and
137 DIPEA191. To a DMF (40 mL) solution of BocTren (246 mg, 1.0 mmol) and Fmoc-
His(Trt)-OH (1859 mg, 3.0 mmol) were successively added 1-hydroxybenzotriazol hydrate (HOBt⋅H2O, 306.2 mg, 2.0 mmol), 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDC⋅HCl, 383.4 mg, 2.0 mmol) and diisopropylethylamine (DIPEA, 348 μL, 2.0 mmol). The solution was stirred for 9 h at room temperature under N2 atmosphere, and the solvent was evaporated to dryness. The residue was dissolved in methylene chloride and washed twice with
5% aqueous sodium carbonate. The organic extract was dried over Na2SO4 and filtered off. Solvent was evaporated to dryness, and the residue was purified by recrystallization from methylene chloride/diethyl ether twice. The raw product ipEH was directly used to next step reaction.
A solution of ipEH (363 mg, 0.25 mmol) in DMF (10 mL) containing 20% piperidine was stirred under N2 atmosphere at room temperature for 2 h and the reaction was monitored by MS. Once no ipEH remained, the solution was evaporated to dryness, washed with 5% aqueous sodium carbonate, and extracted with methylene chloride. The organic extract was dried over sodium sulfate, filtered off, evaporated to dryness. The residue was purified on silica gel using a gradient eluent (chloroform to chloroform/methanol, v/v, 30/1). The product was obtained
1 in a yield of 78% for two step reactions. H NMR (500 MHz, CDCl3) δ 7.93, 7.33,
7.12, 6.66, 6.00, 4.67, 3.64, 3.24, 3.12, 3.07, 2.71, 2.56, 1.38. 13C NMR (126 MHz,
CDCl3) δ 142.42, 138.48, 129.74, 128.05, 119.43, 55.82, 53.68, 36.85, 28.46. MS
(ESI): m/z (%) = 1027 (100), [M+Na]+, 1005 (81), [M+H]+.
138 Intermediate iEH reacted with Fmoc-Cys(Trt)-OH to provide ipEHC in the presence of EDC⋅HCl, HOBt and DIPEA. To a DMF (40 mL) solution of iEH (503 mg, 0.5 mmol) and Fmoc- Cys (Trt)-OH (879 mg, 1.5 mmol) were successively added 1-hydroxybenzotriazol hydrate (HOBt⋅H2O, 153 mg, 1.0 mmol), 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC⋅HCl, 192 mg, 1.0 mmol) and diisopropylethylamine (DIPEA, 192 μL, 1.0 mmol). The solution was stirred for 9 h at room temperature under N2 atmosphere, and the solvent was evaporated to dryness. The residue was dissolved in methylene chloride and washed twice with 5% aqueous sodium carbonate. The organic extract was dried over
Na2SO4 and filtered off. Solvent was evaporated to dryness, and the residue was purified by recrystallization from methylene chloride/diethyl ether twice. The raw product ipEHC was directly used to next step reaction.
A solution of ipEHC (535 mg, 0.25 mmol) in DMF (10 mL) containing 20% piperidine was stirred under N2 atmosphere at room temperature for 2 h and the reaction was monitored by MS. Once no ipEHC remained, the solution was evaporated to dryness, washed with 5% aqueous sodium carbonate, and extracted with methylene chloride. The organic extract was dried over sodium sulfate, filtered off, evaporated to dryness. The residue was purified from recrystallization of methylene chloride/hexane twice and direct used into next step reaction.
To a THF (20 mL) solution of iEHC (170 mg, 0.1 mmol) was successively added 4-(dimethylamino)pyridine (12.2 mg, 0.1 mmol), sodium sulfate (340 mg) and potassium carbonate (340 mg). After cooling to 0 °C, a THF (10 mL) solution
139 of oleoyl chloride (90.3 mg, 0.3 mmol) was added drop by drop. The reaction was monitored by MS and once no iEHC remained, the salts were filtered off and evaporated to dryness. The residue was washed with water and centrifuged to separate the supernatant and precipitate. The precipitate was lyophilized to provide ipEHCO.
To a methylene chloride (3 mL) solution of ipEHCO (100 mg, 0.045 mmol) was added a freshly prepared cocktail solution of trifluoroacetic acid/water/1,2- ethanedithiol/triisobutylsilane (1.5 mL/75 uL/75 uL/150 uL) in ice/water bath. The solution was stirred for 1 hand monitored by MS. Once no ipEHCO remained, the reaction solution was evaporated to dryness and purified via Biotage flash column
(eluents: water and methanol) to provide EHCO in a yield of 20%. 1H NMR (500
MHz, DMSO-d6) δ 8.93 (s, 2H), 8.25 (m, 4H), 8.17 (b, 2H), 8.07 (b, 2H), 7.31 (m,
2H), 5.26 (m, 4H), 4.54 (m, 4H), 3.13 (m, 8H), 2.93 (m, 4H), 2.80 (m, 8H), 2.11 (m,
4H), 1.91 (m, 10H), 1.41 (b, 6H), 1.17 (b, 38H), 0.79 (t, J = 7.0 Hz, 6H). MS
(MALDI-TOF), [M + H]+, calculated: 1155.79, measured: 1155.84
Other carriers were obtained in a very similar manner.
Intermediate iEC for iECO, iECHO and iECKO. Yield: 72%. 1H NMR (500
MHz, CDCl3) δ 7.42 (b, 2H), 7.34 (m, 12H), 7.19 (m, 12H), 7.12 (m, 12H), 5.46 (t,
J = 5.0 Hz), 3.08 (m, 8H), 2.61 (m, 2H), 2.41 (m, 8H), 1.33 (s, 9). 13C NMR (126
MHz, CDCl3) δ 175.77, 172.67, 155.94, 144.60, 129.60, 128.00, 126.82, 66.95,
54.06, 53.56, 53.14, 38.66, 37.37, 36.96, 28.53. MS (ESI): m/z (%) = 937 (100),
[M+H]+, 959 (66), [M+Na]+.
140 1 Carrier iECO. Yield: 40%. H NMR (500 MHz, DMSO-d6) δ 8.22 (m, 4H),
5.33 (m, 4H), 3.20 (m, 4H), 2.83 (m, 4H), 2.18 (b, 6H), 1.98 (b, 8H), 1.49 (b, 8H),
1.25 (b, 46H), 0.87 (t, J = 5.5 Hz, 6H). MS (MALDI-TOF), [M+H]+, Calcd: 881.67, measured, 881.82
Carrier iECHO. Yield: 32%. 1H NMR (500 MHz, DMSO) δ 8.94 (s, 2H), 8.80
(b, 2H), 8.18 (b, 4H), 7.73 (b, 2H), 7.31 (b, 2H), 5.32 (m, 4H), 4.56 (m, 4H), 3.18
(m, 10H), 2.74 (m, 10h), 2.07 (M, 12H), 1.41 (m, 8H), 1.23 (b, 38H), 0.85 (t, J =
6.0 Hz, 6H). MS Calcd: [M + H]+, 1155.79, measured, 1155.60.
1 Intermediate iEK. Yield: 81%. H NMR (500 MHz, CDCl3) δ 7.73 (b 2H),
5.54 (b, 1H), 4.75 (b, 2H), 3.39 (b, 2H), 3.31 (m, 4H), 3.12 (m, 6H), 2.61 (m, 6),
13 1.84 (m, 2H), 1.76 (b, 4H), 1.53 (m, 6H), 1.45 (s, 27H). C NMR (126 MHz, CDCl3)
δ 175.20, 156.08, 55.31, 54.01, 53.56, 40.21, 38.72, 37.16, 34.74, 29.94, 28.38,
23.02. MS (MALDI-TOF): [M+Na]+, Calcd: 725.49, Measured: 725.56; [M+H]+,
Calcd: 703.51 , Measured: 703.54.
Carrier iEKCO: Yield: 35%. 1H NMR (500 MHz, DMSO) δ 8.08 (m, 4H), 7.79
(m, 2H), 5.34 (m, 4H), 4.31 (m, 4H), 3.13 (m, 10H), 2.77 (m, 6H), 2.14 (m, 6H),
1.99 (m, 6H), 1.25 (m, 62H), 0.87 (t, J = 5.5 Hz, 6H). MS (MALDI-TOF): calculated: 1137.86, measured, 1138.13.
Carrier iECKO: Yield: 39%. 1H NMR (500 MHz, DMSO) δ 7.83 (m, 4H), 7.67
(m, 2H), 5.37 (m, 4H), 4.23 (m, 4H), 3.20 (m, 8H), 2.76 (m, 6H), 2.11(m, 6H), 1.95
(m, 6H), 1.51 (m, 18H), 1.20 (b, 46H), 0.86 (t, J = 7.0 Hz, 6H). MS (MALDI-TOF):
[M+H]+, calculated:1137.86, measured, 1137.83.
141 3.2 Cell Culture
An NIH3T3 stable cell line expressing GFP was prepared as previous described192. The NIH3T3-GFP cells were cultured in DMEM and supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 units/mL penicillin
(all reagents were from Invitrogen). Cells were maintained in a humidified incubator at 37°C and 5% CO2.
3.3 Particle Formulation and Characterization
CRISPR/Cas9 plasmids were purchased from the Addgene repository. psgRNA and pCas9 were gifts from Roderic Guigo and Rory Johnson (Addgene plasmids #78535 & 78547, respectively)193. The cationic lipid carriers are dissolved in ethanol at a stock concentration of 2.5 mM, while the plasmids of CRISPR/Cas9 system are reconstituted in nuclease-free water at a 0.5 μg/mL concentration.
Nanoparticles are formulated by mixing the carriers with plasmid DNA for a period of 30 min in nuclease-free water at pre-specified N/P ratios (N = number of protonable amines in lipid, P = number of phosphate groups in plasmid DNA). The diameters (as determined by dynamic light scattering) and zeta potentials of all nanoparticles were analyzed using an Anton Paar Litesizer 500 instrument (Anton
Paar USA Inc, Ashhland, VA) in nuclease free water.
The ability of all lipid carriers to complex and condense CRISPR/cas9 plasmids was assessed by gel electrophoresis. Lipid/plasmid DNA nanoparticles were prepared, and 4 μL aliquots of nanoparticles, 4 μL of loading dye (Promega)
142 and 16 uL were mixed. The mixture (20 μL) was loaded onto a 0.7 % agarose gel containing ethidium bromide. The gel was submerged in 0.5× Tris/Borate/EDTA
(TBE) buffer and run at 100 V for 25 min. Plasmid DNA bands were visualized using an AlphaImager ultraviolet imaging system (Biosciences). Lipid/plasmid
DNA complexes were prepared at N/P ratios 6, 8 and 10 and run on the gel as described above.
3.4 Evaluation of pH-Sensitive Membrane Disruption
Hemolytic activity of each carrier was determined to verify the pH-sensitive membrane disruption capabilities of each carrier in the delivery system. Red blood cells (RBCs) extracted from rats were purchased from Innovative Research Inc.
(Novi, MI) and diluted 1:50 in PBS solutions of pHs = 5.4, 6.5, or 7.4. A total of
100 μL of nanoparticles were created at N/P ratios of 10 and then incubated with
100 μL of diluted RBCs at 37 °C for 2 h. Nanoparticles were formulated so that the final amine concentration for all the samples, after mixing with the RBCs, was 150
μM. Multiple pHs were tested to predict if the nanoparticles could disrupt membranes of the endosomal-lysosomal compartments (pH = 5-6), without affecting the integrity of the outer cell membrane prior to endocytosis (pH = 7.4).
The absorbance of each test sample was measured on a SpectraMax spectrophotometer at a wavelength of 540 nm in order to determine the amount of hemoglobin released from the RBCs, due to membrane destabilization, relative to
143 that achieved by a positive control treatment of 1% (v/v) Triton-X100 surfactant during the 2-hour incubation period.
3.5 In vitro Transfection
NIH3T3-GFP cells were seeded either onto 12-well plates at a density of 5 ×
104 cells per well or confocal dish at a density of 1× 105 cells and allowed to grow for 24 h at 37 ºC. Transfections were conducted in 10% serum media with the
ECO/pDNA nanoparticles at different DNA concentrations of 1, 1.5, 2, and 2.5
μg/mL. ECO/pDNA nanoparticles were incubated with NIH3T3 cells for 8 h at 37
ºC. The media then was replaced with fresh serum-containing media (10% serum) and cells were then cultured for additional 72 h. The expression of Cas9, gRNA and
GFP knockdown were evaluated with an Olympus FV1000 confocal microscope
(Olympus, Center Valley, PA). The quantitative analysis was performed with flow cytometry. The cells on 12-well plate were harvested by treatment with 0.25% trypsin containing 0.26 mM EDTA (Invitrogen), collected by centrifugation at 1500 rpm for 5 min, resuspended in 500 μL of PBS containing 4% paraformaldehyde, and finally passed through a 35 μm cell strainer (BD Biosciences). Cas9 expression, gRNA expression and GFP knockdown were quantified by the fluorescence intensity measurement for a total of 10,000 cells per sample using a BD
FACSCalibur flow cytometer. Each sample was conducted in triplicate.
144 3.6 Cytotoxicity
Cytotoxicity of the new carriers was investigated using an MTT colorimetric assay measuring cellular metabolic activity (Invitrogen). The transfection procedure was identical to that presented for the CRISPR/Cas9 transfection studies previously. After transfection, the cells were allowed to grow for an additional 72 h. At that point, they were incubated with MTT for 4 h, followed by an additional
4-hour incubation with an SDS-HCl solution to dissolve any insoluble formazan crystals formed by the reduction of MTT by NAD(P)H-dependent enzymes in the cells. The absorbance of each sample was measured at 570 nm using a SpectraMax microplate reader. Cellular viability was calculated by averaging the signal intensities over three replicates and then normalizing the results relative to the negative control data
3.7 TEM
The morphology of ECO/pCas9 and ECO/psgRNA nanoparticles were imaged with a transmission electron microscope (JEOL JEM2200FS). Samples for TEM were prepared by depositing 20 μL of the particle solution onto a 300-mesh copper grid covered by a thin amorphous carbon film (20 nm). Immediately after deposition, the excess liquid was removed by touching the grid with filter paper.
Samples were stained twice by adding 3 μL 2% uranyl acetate aqueous solution.
The excess of staining solution was removed. Images of the nanoparticles were acquired with TEM after the samples were dried.
145 3.8 qRT-PCR
Total RNA was extracted from cells and tissues using the RNeasy Plus Mini
Kit (Qiagen, Germantown, MD), according to manufacturer’s instructions. Reverse transcription was performed using the miScript II RT Kit (Qiagen) and qPCR was performed using the SyBr Green PCR Master Mix (Applied Biosystems, CA). Gene expression was analyzed by the 2-ΔΔCt method with 18S expression as the control.
The following primer sequences were used- mCherry: Fwd 5ʹ-
GAACGGCCACGAGTTCGAGA-3ʹ and Rev 5ʹ-
CTTGGAGCCGTACATGAACTGAGG-3ʹ; eGFP: Fwd 5ʹ-
ACGTAAACGGCCACAAGTTC-3ʹ and Rev 5ʹAAGTCGTGCTGCTTCATGTG-
3ʹ; and 18S: Fwd 5ʹ-TCAAGAACGAAAGTCGGAGG-3ʹ and Rev 5ʹ-
GGACATCTAAGGGCATCACA-3ʹ.
3.9 Western Blot
Total cellular protein was extracted as previously described194. Protein extracts
(40 µg) were separated by SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with primary antibodies overnight. Both Anti-Cas9 and anti-β- actin (used as loading control) were purchased from Cell Signaling Technology,
Danvers, MA.
146 4. Results
4.1 Evaluation of GFP-targeting CRISPR/Cas9 System.
The CRISPR/Cas9 system selected in this study includes two components, which are a plasmid expressing two sgRNAs targeting GFP gene sequence and a plasmid expressing Cas9 nuclease. In order to evaluate whether this CRISPR/Cas9 system was suitable for evaluations of the new carriers. The efficiency of the
CRISPR/Cas9 system was first evaluated with our previously developed pH- sensitive multifunctional cationic lipid ECO.
First, the nanoparticle formulations were characterized for its morphology, size and zeta potential. ECO formed nanoparticles with both plasmids (psgRNA and pCas9) at N/P ratio of 10, with plasmid amount of 0.5 μg in the formulation. As shown in Figure 5.4A, ECO could form uniformed nanoparticles with both plasmids, which are round shape with the sizes around 100 nm in the TEM images.
The uniformity of the nanoparticles was confirmed by the size distribution in
Figure 5.4B, which showed single and narrow distributions for both particles. The measured nanoparticle sizes and surface charges were demonstrated in Figure 5.4C.
The sizes for ECO/psgRNA and ECO/pCas9 were 108.43±2.16 nm and
108.87±3.89 nm, respectively. The zeta potential for ECO/psgRNA was 25.6±5.81 mV and 29.6±4.91 mV for ECO/pCas9. These results demonstrated the potential of
CRISPR/cas9 system to form positively charged and uniformed nanoparticles with cationic lipids.
147 The knockdown efficiency of GFP-targeting CRISPR/Cas9 system was evaluated using ECO lipid as the transfection agent. Both ECO/psgRNA and
ECO/pCas9 nanoparticles were formulated at N/P ratio of 10. The CRISPR/Cas9 system was transfected with the particle ratio of ECO/psgRNA and ECO/pCas9 nanoparticles at 1:1. Different doses were used to transfect NIH3T3-GFP cells, starting from 1 μg for each plasmid in the transfection media up to 2.5 μg. With the dose increase of CRISPR/Cas9 system, higher expression levels of Cas9 (Blue) and sgRNA (Red) were observed under confocal microscope 72 h after transfection
(Figure 5.4D). A reduction of green fluorescent intensity was also observed, indicating GFP knockdown effects were generated after transfection of
CRISPR/Cas9 system. This GFP knockdown was quantified using flow cytometry,
Figure 5.4E. A continuous reduction of GFP fluorescent intensity with the increase of the dose of CRISPR/Cas9 system was observed, with the knockdown efficiency saturated at around 50% at the dose of 2.5μg. The sgRNA expression increased with the dose (Figure 5.4F), with 40~52% of cells were positive in sgRNA expression.
Western blot and qRT-PCR tests were used to further confirm the expression of
Cas9 nuclease and the GFP knockdown at the dose of 2 μg (Figure 5.4G and H).
Clear protein expression band for Cas9 nuclease was observed in the western blot, with no band showed up for untreated and nonspecific controls (Figure 5.4G).
Significant reduction of GFP mRNA levels were demonstrated by qRT-PCR, 24 and
48 h after transfection (Figure 5.4H). These results demonstrated good efficiency of selected CRISPR/Cas9 system. In order to minimize the toxicity by
148 CRISRP/Cas9 system and demonstrate differentiable knockdown efficiency, the dose for the characterization and evaluation of new carriers was selected as 1 μg for each plasmid in the transfection media for the following tests.
149
150 Figure 5.4. Evaluation of GFP-targeting CRISPR/Cas9 system. (A) TEM images of ECO/psgRNA and ECO/pCas9 nanoparticles. (B) Size distribution of ECO/psgRNA and ECO/pCas9 nanoparticles by DLS measurements. (C) Size and Zeta potential of ECO/psgRNA and ECO/pCas9 by DLS measurements. (D) In vitro transfection of ECO/psgRNA and ECO/pCas9 (particle ratio 1:1) in NIH3T3-GFP cells, with the dose of each plasmid at 1 μg, 1.5 μg, 2 μg and 2.5 μg in the transfection media. Cas9 expression (blue), gRNA expression (red) and GFP knockdown (green) were imaged by confocal microscopy. (E) Quantitative flow cytometry measurements of GFP knockdown efficiency represented by percentage reduction of GFP fluorescent intensities. (F) Quantitative flow cytometry measurements of sgRNA expression represented by percentage of mCherry positive cells. (G) Cas9 protein expression demonstrated by western blot (NS=nonspecific control, β-actin expression as inner control). (H) GFP knockdown efficiency in mRNA levels measured by qRT-PCR. Error bars = ± std (* p < 0.05 relative to untreated control). (**p < 0.005). Significance analysis was performed using ANOVA.
4.2 Gel Electrophoresis of Encapsulation and Stability of New Carriers-
CRISPR/Cas9 Nanoparticle Formulations.
Agarose gel electrophoresis was used to evaluate the encapsulation and stability of nanoparticles formulated between new carriers and both plasmids from
CRISPR/Cas9 system. Nanoparticles of each new carrier were formulated at N/P ratio of 6, 8 and 10, with nanoparticles formulated by ECO and CRISPR/Cas9 system as controls (Figure 5.5). At N/P ratio of 6, good encapsulation and stability were observed for iECO, iEHCO and ECO control. For iEKCO, iECKO and iECHO, low encapsulation and stability were shown by faint and unclear bands. At
N/P ratio of 8, almost all the new carriers and ECO were able to encapsulate plasmids from CRISPR/Cas9 system and the nanoparticles showed good stability, indicated by bands maintaining at the top. At N/P ratio of 10, most new carriers and
ECO showed efficient encapsulation and formed stable particles. Interestingly, for iECKO and iEKCO, faint bands were also observed.
151
Figure 5.5. Agarose gel electrophoresis of the encapsulation and stability of new carriers- CRISPR/Cas9 nanoparticles at N/P ratios of 6, 8 and 10.
4.3 Size Evaluation of New Carriers-CRISPR/Cas9 Nanoparticle
Formulations.
To evaluate quality of the new carriers-CRISPR/Cas9 nanoparticle formulations, dynamic light scattering was used to characterize system size distributions. Normally, the size distribution curve is an indication of particle quality. A narrower nanoparticle distribution often represents better uniformity of nanoparticles, which is an important factor for nanoparticle based therapeutics. The
152 DLS measurements were summarized in Figure 5.6, shown by size distribution curves for nanoparticles formulated by each carrier either with psgRNA or pCas9.
For ECO control and new carrier iECO, good quality nanoparticles with sizes between 100 nm and 150 nm were obtained for both plasmids in the CRISPR/Cas9 system across all tested N/P ratios, demonstrated by narrow nanoparticle size distributions. (Figure 5.6A, B). For new carriers iEKCO and iECKO, large aggregations were observed for nanoparticles formulated at N/P ratio of 6 with both psgRNA and pCas9, demonstrated by either size distribution peaks around 1000 nm or a wider size distribution (Figure 5.6C and D). For new carriers iEHCO and iECHO, nanoparticles with larger sizes and wider distribution were observed for psgRNA and pCas9 at N/P ratio of 6, compared with nanoparticles formulated at
N/P ratio of 8 and 10 (Figure 5.6E and F). All new carriers were able to form nanoparticles with single and narrow size distributions at N/P ratio of 8 for both psgRNA and pCas9. However, when the N/P ratio increased to 10, increases in size were observed for iEKCO, iECKO, iEHCO and iECHO, which indicated that more aggregations were formed.
153
Figure 5.6. DLS size measurements of the new carriers-CRISPR/Cas9 nanoparticles. The size distribution of nanoparticles formulated between (A) ECO, (B) iECO, (C) iEKCO, (D) iECKO, (E) iEHCO, (F) iECHO and plasmids of GFP targeting CRISPR/Cas9 system.
154 4.4 Zeta Potential Evaluation of New Carriers-CRISPR/Cas9 Nanoparticle
Formulations.
The zeta potential of new carriers-CRISPR/Cas9 nanoparticle formulations were measured using dynamic light scattering, and results were shown in Figure
5.7. In general, an increase in zeta potential with the increase of N/P ratios was observed for all carriers. At N/P ratio of 6, ECO and the new carriers formed nanoparticles with both pCas9 an psgRNA either have negative zeta potentials or slight positive zeta potentials, which were in the range between. Only iECO was able to form nanoparticles with psgRNA that had a zeta potential larger than +30 mV (45.9±1.5 mV). At N/P ratio of 8, ECO/psgRNA, ECO/pCas9, iECO/psgRNA, iECO/pCas9 and iECHO/psgRNA nanoparticles were formed with zeta potentials higher than +30 mV. Other nanoparticles formed by iEKCO, iECKO, iEHCO and iECHO had zeta potentials slightly above 0 mV or showed great fluctuation around
0 mV. At N/P ratio of 10, ECO, iECO, iECKO, and iEHCO were able to form nanoparticles with both plasmids with zeta potentials over +30 mV. However, iEKCO still formed nanoparticles with slight positive zeta potentials (<+10 mV) for both plasmids. For new carrier iECHO, the iECHO/psgRNA had a zeta potential of 39.7±1.7 mV, while iECHO/pCas9 had a zeta potential of 14.7±0.8 mV.
155
Figure 5.7. DLS zeta potential measurements of the new carriers-CRISPR/Cas9 nanoparticles at N/P ratio of 6, 8 and 10.
156 4.5 pH-Dependent Hemolytic Activities of New Carriers
A hemolysis assay was used in to evaluate membrane disruptive capabilities of each new carrier. Fig. 5.8 demonstrated that all new carriers exhibited pH- sensitive hemolytic activity at N/P ratio of 10. At pH=7.4, ECO and all new carriers have low hemolytic activities that were less than 30%. At pH=6.5, slight increase of hemolytic activities was observed for ECO, iECO and iEHCO. For other carriers, the hemolytic activities remained low and no significant difference was observed against activities under pH 7.4. At pH=5.5, significant increase of hemolytic activities was observed for ECO, iECO, iEKCO and iECKO. However, the hemolytic activity increase was not obvious for iEHCO and iECHO.
Figure 5.8. pH-dependent hemolytic activities of all carriers at N/P ratio of 10. Rat blood cells were diluted 1:50 in PBS and incubated with each formulation at pH = 7.4, 6.5, and 5.4 for 2 h at 37 °C. Triton X-100 (1% v/v) was implemented as a positive control.
157 4.6 In Vitro Transfection of CRISPR/Cas9 System Using New Carriers in
NIH3T3-GFP Cells.
To evaluate the transfection efficiency of new carriers in delivery of
CRISPR/Cas9 system, in vitro transfection of GFP targeting CRISPR/Cas9 system by the new carriers were tested in NIH3T3-GFP cells, results were shown in Figure
5.9 and Figure 5.10. At N/P ratio of 6, reductions of GFP signal intensity were observed for both ECO and iECO, with expressions of both Cas9 (blue) and sgRNA
(red) shown in the confocal images (Figure 5.9). The quantified knockdown efficiencies for ECO and iECO were 16% and 21% respectively at N/P ratio of 6
(Figure 5.10A). The knockdown efficiencies for other carriers were not obvious, which were between 4% and 13% (Figure 5.10A). At N/P ratio of 8, increased knockdown efficiency, Cas9 nuclease and sgRNA expressions were observed across almost all the carriers, indicated by the fluorescent images and quantitative flow cytometry data (Figure 5.9 and 5.10). ECO and iECO had the most knockdown efficiencies, which are 25% and 24% respectively. The efficiencies for iEKCO, iECKO, and iEHCO were ranging from 11% to 17%. Interestingly, at N/P of 10, only iEKCO and iECHO demonstrated increases in GFP knockdown efficiency, 17% to 23% for iEKCO and 0% to 16% for iEHCO. While the knockdown efficiencies for other carriers remained similar levels as N/P ratio of 8 or slightly decreased. The expression levels for Cas9 and sgRNA showed similar trend as GFP knockdown efficiency, which reductions were observed for ECO, iECO and iEHCO from N/P ratio of 8 to 10.
158
Figure 5.9. In vitro transfection of CRISPR/Cas9 system using new carriers in NIH3T3-GFP cells. For each carrier, the lipid/psgRNA and lipid/pCas9 particle ratio was 1:1, with the dose of each plasmid at 1 μg, in the transfection media. Cas9 expression (blue), gRNA expression (red) and GFP knockdown (green) were imaged by confocal microscopy 72 h after transfection.
The cell viability of in vitro transfection of CRISPR/Cas9 system using new carriers were also evaluated using an MTT assay. Overall, all the tested carriers exhibited excellent cell viabilities, which were more than 75% across all N/P ratios.
Reductions in cell viabilities with the increase of the N/P ratios were observed for iEKCO, iECKO, iEHCO and iECHO (Figure 5.10B). No significant cytotoxicity was observed for all the carriers at N/P ratio of 8.
159
Figure 5.10. In vitro transfection of CRISPR/Cas9 system using new carriers in NIH3T3-GFP cells. For each carrier, the lipid/psgRNA and lipid/pCas9 particle ratio was 1:1, with the dose of each plasmid at 1 μg, in the transfection media. (A) Quantitative flow cytometry measurements of GFP knockdown efficiency represented by percentage reduction of GFP fluorescent intensities. (B) MTT assay of cytotoxicity in NIH3T3-GFP cells 72 h after transfection. (C) Quantitative flow cytometry measurements of Cas9 expression represented by percentage of BFP positive cells. (D) Quantitative flow cytometry measurements of sgRNA expression represented by percentage of mCherry positive cells. Error bars = ± std (* p < 0.05 relative to untreated control. Significance analysis was performed using ANOVA).
5. Discussion
In this chapter, we designed and optimized a new library of pH-sensitive multifunctional cationic lipid carriers for CRISRPR/Cas9 delivery for potential applications in gene therapy for autosomal dominant IRDs. New carriers were modified based on the structure of previously designed ECO. In the new design, the
160 head group structure was simplified from 3,3'-[(2-Aminoethyl)imino]bis[N-(2- aminoethyl)propanamide] to 2,2',2''-Triaminotriethylamine structure, and new functional linker amino acids (lysine and histidine) were introduced into the structure.
To demonstrate the transfection efficiencies of CRISPR/Cas9 system using the new carriers, a two-component GFP targeting CRISPR/Cas9 system was selected, which included a plasmid expressing sgRNA (9.4 kb) and a plasmid expressing
Cas9 nuclease (13.6 kb). The sgRNA plasmid (psgRNA) contains two sgRNAs in its sequence, in order to induce efficient gene suppression195, because small indel mutations caused by single sgRNAs are less likely to ablate function. A pair of sgRNAs that recruit Cas9 to sites flanking the target region can induce simultaneous strand break, following by non-homologous end joining (NHEJ) activity to repair the lesion, which becomes an efficient deletion strategy196, 197. When delivered by our previously designed ECO carriers, the GFP-targeting CRISPR/Cas9 could induce efficient GFP sequence knockdown, which had a dose dependent manner with the highest knockdown efficiency of 50%. In previous research, it was shown that increasing sgRNA and Cas9 concentrations decreased DNA-cleaving specificity and off-target cleavage was attributed to excess dosing198, 199. Therefore, the dose of 1 μg for both plasmids and a number ratio of 1:1 for both nanoparticles in the transfection media were selected for the characterization and evaluation of the new carriers.
161 Compared with the head group of ECO, which is 3,3'-[(2-
Aminoethyl)imino]bis[N-(2-aminoethyl)propanamide], the head group for new carriers were simplified to 2,2',2''-Triaminotriethylamine structure, which also provided protonatable amino groups from ethylenediamine based structure.
Compared the characterizations and evaluations of ECO and iECO, the electrostatic interactions between the carriers and plasmids didn’t demonstrate significantly change. Nanoparticles formulated by ECO and iECO with CRISPR/Cas9 plasmids showed similar size and zeta potential profiles at high N/P ratios. However, iECO induced more Cas9 and sgRNA expression for in vitro transfections, demonstrating that a compact head group could facilitate transfection efficiency increase, which was also observed in previous work200.
ECO is highly efficient in gene delivery applications due to the ability of facilitating pH-sensitive amphiphilic endosomal escape and reductive cytosolic release of therapeutic genetic materials. Following cellular uptake of ECO/pDNA nanoparticles, cytosolic release of the gene cargo is facilitated by pH-sensitive amphiphilic endosomal membrane destabilization through protonation of the head group of ECO in the acidic endosomal-lysosomal compartment (pH=5-6) and dissociation of the nanoparticles by reduction of the disulfide bonds in the cytoplasm188. The pH-sensitive amphiphilicity can be fine-tuned by providing protonatable amino groups with different pKa values. Therefore, new amino acids with protonatable amino groups were introduced in the new design while maintaining the cysteine linker. Histidine and histidine-containing dipeptides are
162 known to act as endogenous buffers, which are commonly used as a modifying group to improve the endosomal escaping of several non-viral gene carriers201, 202,
203 . With a pKa around 6, the imidazole ring of histidine is a weak base that can acquire a cationic charge when the pH of the environment drops below 6 in the endosomal compartments after cellular uptake. In the unprotonated state, the imidazole has one donor and one acceptor site for hydrogen bonding, thus stabilizing the nanoparticle structure following particle formation, while after protonation, the heterocycle possesses two donor sites for hydrogen bonding, which induces fusogenic characteristic of the formulation204. After introduction of histidine in the lipid structure, new carriers iEHCO and iECHO were able to form stable nanoparticles at N/P of 10, when the lipid amount was reduced by half because of the doubled charge density resulted from the introduction of protonatable imidazole group. At N/P ratios of 6 and 8, both iEHCO and iECHO struggled forming stable formulations, possibly due to the sizes of CRISPR/Cas9 plasmids. Even previously, excellent transfection efficiency and hemolytic activities were observed for EHCO (same head group as ECO) in siRNA delivery115, we didn’t observe as efficient hemolytic activities or GFP knockdown efficiency.
This could also be a result from the size of plasmids, which created less positive surface charge to facilitate hemolytic activities. To note, iEHCO did induce high sgRNA expression at N/P ratio 8.
Efficient non-viral gene delivery systems also require enough electrostatic interactions. In order to increase charge density in the pH-sensitive multifunctional
163 lipids, lysine was introduced to the design, which could potentially increase the electrostatic interactions and reduce the amount of cationic lipid needed for stabilizing nanoparticle formulations. Lysine based materials have been extensively used in gene delivery, due to its primary amino residue, which has a pKa value of
10.778, 205, 206 and remain protonated under physiological and endosomal- lysosomal pH conditions. Therefore, lysine can facilitate strong interactions with genetic materials207. After introduction of lysine to lipid structure, the interactions between cationic lipid and plasmid greatly changed. More aggregations tended to form at N/P ratios of 6, 8 and 10 for iECKO and iEKCO, because of the increase in charge density and reduction in molecular amount of lipid. The aggregations were not tightly packed and demonstrated negative zeta potentials at low N/P ratios. Both iEKCO and iECKO nanoparticles demonstrated good hemolytic activities at N/P ratio of 10, because of the high charge densities. However, these hemolytic activities didn’t transform to excellent transfection efficiency of CRISPR/Cas9 system. The reason could be the reduced pH-sensitivities of lysine introduction, which prevent the nanoparticles from endosomal escaping, which is also a common problem for lysine based gene delivery systems.
The effect of combinations of the newly added amino acids with cysteine were evaluated here as well. However, the effect of which amino acid located closer to the head group was not clear in nanoparticle formulations and transfection efficiency of the CRISPR/Cas9 system.
164 Chapter Ⅵ. Optimization of pH-sensitive Multifunctional Cationic Lipid
Gene Delivery Systems
As the first gene therapy for the treatment of LCA is approved by the FDA, the development of gene therapies for treatments of IRDs has received the highest attention. Various gene therapies have been designed and evaluated following either viral or non-viral approach. Compared with viral based gene therapies, non-viral strategies are less immunogenic, easy to produce, and not limited by packaging capacity, thus have broader applications in treating IRDs involving gene delivery of different sizes. Promising results have been seen from many works, which have not only shown therapeutic gene expression but also the alteration of disease phenotypes. However, a non-viral gene therapy to treat IRDs has yet to be successfully translated from bench to bedside, mainly due to low transfection efficiencies and transient therapeutic gene expression compared with viral approaches. Therefore, future directions will focus on elevating the gene transfection efficiency and promoting prolonged gene expression of therapeutic genes.
165 1. Targeting Ligand Development
Targeted drug delivery systems have the promise to expand therapeutic windows of therapeutics by increasing delivery to the target tissue as well as the target/non-target tissue ratio, which will in turn lead to a reduction in the minimum effective dose of the therapeutic and the accompanying toxicity, and an improvement in therapeutic efficacy208, 209, 210. In this work, we tried to use all-trans- retinylamine as targeting ligand for the RPE to enhance gene transfection efficiency and specificity. Although, enhanced GFP expression was observed in the RPE flatmount, all-trans-retinylamine is not an optimized targeting ligand, because of its low stability and high light sensitivity211.
ACU4429 (Emixustat) has similar chemical structure as all-trans-retinylamine and also inhibits retinal pigment epithelium 65 (RPE65). ACU4429 serves as small- molecule visual cycle modulator prevents the accumulation A2E212, 213, 214.
Compared with all-trans-retinylamine, ACU4429 has more stable structure, which makes it a potential ligand to target the RPE (Figure 6.1).
Figure 6.1. Chemical structure of ACU4429.
To design a targeting ligand, a spacer is always needed between the ligand and the non-viral carrier. Polyethylene glycol (PEG) spacers are commonly involved in
166 targeting ligand designs, due to the advantages of increasing nanoparticle circulations and reducing toxicity effect215, 216. However, PEGylation compromises the efficiency of nanoparticle delivery in cellular uptake and endosomal escape properties. Therefore, PEGylation strategies that also address the delivery and uptake pathways are needed.
As previously showed, a pH-sensitive spacer between the PEG chain of the targeting ligand and ECO could enhance endosomal escape of the ECO/siRNA nanoparticles in cancer RNAi therapy217. The incorporation of a pH-sensitive hydrazone spacer will shed the PEG layer via pH-sensitive hydrolysis of hydrazone in acidic endosomes, which will expose the core ECO/pDNA nanoparticles to enhance endosomal escape. This strategy can solve the issues of low cytosolic gene delivery efficiency and low expression levels of the therapeutic gene caused by
PEGylations that hinder the endosomal escape of non-viral nanoparticle formulations.
Following these concepts, ACU4429 targeting ligand connected by a PEG- hydrazone linker (PEG-HZ) was designed to target the RPE (Figure 6.2).
ACU4429-PEG-HZ-MAL targeting ligand was made following three-step reactions, the molecular weight of the final targeting ligand and intermediate products were shown in Figure 6.2B. The targeting ligand could bind to the surface free thiol groups on ECO after nanoparticle formulation by MAL (maleimido) group.
167
Figure 6.2. The Design of ACU4429-PEG-HZ-MAL Targeting Ligand. (A) Chemical synthesis route of ACU4429-PEG-HZ-MAL targeting ligand. (B) MALDI-TOF mass spectra of ACU4429- PEG-HZ-MAL and intermediate products.
168 After transfection with ACU4429-PEG-HZ-ECO/ECO/Cy3-pDNA and PEG-
ECO/ECO/Cy3-pDNA nanoparticles at N/P ratio 10 in ARPE-19 Cells, clear differences were observed on their endosomal trafficking behaviors. At 1 h, both nanoparticles were sticking to the cell surfaces and starting to get internalized. At 2 h, both nanoparticles entered the cytosol. At 4 h and 24 h, more nanoparticles escaped from endosomal entrapment for ACU4429-PEG-HZ targeting nanoparticles, demonstrated by bright red signals. Therefore, the PEG-HZ linker demonstrated excellent ability facilitating endosomal-lysosomal escape of targeted
ECO/pDNA nanoparticles.
Figure 6.3. Confocal fluorescence image of cytosolic delivery of ACU4429-PEG-HZ- ECO/ECO/Cy3-pDNA and PEG-ECO/ECO/Cy3-pDNA nanoparticles at N/P ratio 10 in ARPE-19 Cells. Late endosomes were stained with LysoTracker Green (green), nuclei were stained with Hoechst 33342 (blue), and DNA plasmid was labeled with Cy3 (red).
ACU4429 targeting ligand also showed excellent targeting efficiency in vivo after subretinal injections of ACU-PEG-HZ-ECO/ECO/Cy3-plasmid particles. As is shown in Figure 6.4, for non-targeting ECO/Cy3-plasmid nanoparticles, wider
169 distributions of nanoparticles were observed in both the outer and inner retinal layers. However, ACU-PEG-HZ-ECO/ECO/Cy3-plasmid nanoparticles remained in the interphotoreceptor matrix (IPM), which could enhance the uptake of therapeutic plasmids by the RPE.
Figure 6.4. In vivo evaluation of ACU4429 targeting ligand to the RPE. Nanoparticle distribution of Non-targeting ECO/Cy3-plasmid particles and ACU-PEG-HZ-ECO/ECO/Cy3-plasmid particles in the subretinal space of Abca4-/- mice after subretinal injection. SHAM (PBS injected).
Therefore, the targeting ligand design involving a more stable ligand and a hydrazone modified PEGylation could potentially increase the RPE targeting specificity and enhance transfection efficiency in vivo by improving circulation time and endosomal-lysosomal escape efficiency.
2. Enhancers for Prolonged and Efficient Therapeutic Gene Expression
Transient and inefficient therapeutic gene expression have raised problems such as repeated invasive injections, and potential safety issues218. For non-viral gene therapies, these problems have greatly limited their applications. Therefore, strategies to provide prolonged and efficient gene expression of the therapeutic gene are greatly needed. To address this, therapeutic DNA plasmid can be modified with
170 enhancers or sequences for enhanced and prolonged gene expression. The sequence poly(dT-dG)poly(dC-dA) (TG-element) which is a ubiquitous component of eukaryotic genomes has demonstrated the ability modulating gene expression by initiating 2 to 10 times more chloramphenicol acetyltransferase activity219. P elements which are 2.9 kb in length with inverted terminal repeat structures also showed enhance the efficiency of mutagenesis, transformation and mobilization220,
221. For non-viral retinal gene therapy applications, scaffold/matrix attachment regions (S/MARs) has been incorporated into the therapeutic plasmid sequence.
The inclusion of S/MARs could augment transcription using AT-rich sequences to modulate superhelical stress that arises during transcription, and play a role in targeting gene domains to matrix-associated transcription centers prior to gene expression, which has shown to support efficient and long-term gene expression222.
Gene replacement therapy using a non-viral based system carrying therapeutic plasmid containing S/MAR sequence was able to generate prolonged (2.5 years) gene expression in the RPE and mediate phenotypic improvement in Rpe65-/- mice.
Therefore, modifying therapeutic plasmid with enhancer sequences can potentially induce long term gene expression and persistent therapeutic effect for non-viral gene therapies for the treatment of IRDs.
3. Modification of Current ECO Based Carrier
In this work, modifications of current ECO based pH-sensitive multifunctional cationic lipids were tested in varying the head structure and functional linkers.
171 Further modification of ECO based cationic lipids can focus on improving the biodegradability. Ester bonds have been extensively applied to improve biocompatibility and biodegradability of biomaterials and nanoparticles223, 224. The biodegradation of ester bonds is considered to be a hydrolytic process. The cleavage of an ester bond yields a carboxyl end group and a hydroxyl group225. Ester bonds can also be introduced in the ECO based structure through the connections between functional linkers and lipophilic tails (Figure 6.5). Through previous evidence, slight changes in the structure of ECO didn’t cause significant change in ECO function. The ester type ECO (eECO) is hoping to have similar efficiency and better biocompatibility compared with ECO.
O
HS O HN O
N H2N
O HN O
O SH
Figure 6.5. Chemical structure of the ester type ECO derivative (eECO).
172 Chapter Ⅶ. Outlook for Non-Viral Gene Therapy for IRDs
The eye is a promising organ for gene therapy, due to its small, enclosed structure, immune privilege, and easy accessibility. The high availability of various animal models and non-invasive evaluation technologies allow us to receive outstanding success in retinal gene therapy development for IRDs. More importantly, the success in LCA2 clinical investigations have encouraged even broader applications in other gene mutations in various retinal tissue layers.
However, the challenges for retinal gene therapy for IRDs still remain and require future efforts to overcome them.
1. Administration Routes
Retinal gene therapy treatments have been delivered to the retinal tissue layers mainly through intraocular injections, including subretinal and intravitreal injections. Subretinal injection has demonstrated high efficiency delivery to photoreceptor cells and the RPE. However, subretinal injection is technically challenging and has extremely raised concerns from patients. Especially for IRD
173 patients with severe retinal degenerations, the injection itself may cost irreversible damage to the retina, which has been shown in several LCA2 trails where foveal thinning, macular holes, choroidal effusions and hypertensions were observed131,
226. Compared with subretinal injection, intravitreal injection is less invasive and can be a more popular choice from patients receiving retinal gene therapy. However, intravitreal injection is more likely inducing gene transfection in the inner retinal tissue, while most IRDs are caused by mutations in the outer retinal tissue. The failure of delivery of therapeutic genes to the outer retinal tissue by intravitreal injection greatly limited its application. Recently, efforts have been made to develop delivery systems that favor the diffusion to the outer retinal tissue from vitreous.
Results demonstrated the feasibility of enhanced delivery to the outer retinal tissue trough intravitreal injection227.
2. Gene Therapy Development for Autosomal Dominant IRDs
Currently, most gene therapy treatments have been developed for autosomal recessive IRDs. Giving about one-third of IRD patients are affected by dominant form of IRDs, the development of effective gene therapy for autosomal dominant
IRDs is in great need. Currently, gene therapy developments have mainly focused on retinitis pigmentosa (RP) caused by mutations in RHO gene. Results have demonstrated suppressing the toxic products provides excellent efficacy. However, there are more than 100 different forms of mutations associated with autosomal dominant RP. Therefore, in order to benefit more patient groups, more efficient
174 strategies are needed to suppress the mutated allele without affecting the wild type allele. CRISPR/Cas9 systems are promising to accomplish this. However, there is still a long way to provide efficient CRISPR/Cas9 systems with minimal side effects, and to develop efficient delivery systems for CRISPR/Cas9.
3. Disease and Patient Selection
Regardless of how optimal the gene therapy is, the success of gene therapy treatments cannot be guaranteed, because successful gene transfer also relies on viability of targeted cells and early identification of patients when they have better condition to maximize the benefits by gene therapy treatments. For example, for
LCA2, patients represent preserved RPE and retina structure for decades after diagnosis. If patients receive treatments at early stage, gene therapy will maximize the benefit. To facilitate this, advancement in genotyping techniques and accurate non-invasive monitoring of retinal and visual function are needed. With the help of these technologies, disease diagnosis can achieve identification of conditions with high genetic heterogeneity at molecular level. Functional evaluation can provide early and accurate retinal functions. The information collected with these technologies can further ensure a successful gene therapy treatment.
4. Unexpected Results
Clinical investigations of gene therapy for LCA2 have revealed several unexpected results. First, after receiving gene therapy treatments, the reconstituted
175 retinoid cycle was not completely normal, which demonstrated slower rod kinetics than fully functional retinoid cycle. Second, a pseudo-fovea was observed from a patient 1 year after treatment, which helped the patient further improve visual function. Third, patients kept showing continued disease progression and retinal degeneration independent of gene therapy. Unexpected results need better understanding, which helps modifying of current gene therapy. As the first gene therapy for LCA2 is approved by the FDA, more and more patients can receive gene therapy treatments. More clinical practice can improve the understandings of unexpected results of current gene therapy treatments, which can further improve the quality of gene therapy.
5. Manufacture
Scale-up and manufacturing of non-viral gene therapy nanoformulations present great challenges in pharmaceutical development. Traditional pharmaceutical manufacturing does not associate with systems in nanometer scale.
Even though several nanomedicines have been in the market for several years, and promising therapies have been tested in clinical trials, there is still a lack of information on scaling up production of nanoformulations. Currently, most clinical and pre-clinical investigations have been using small scale production of their nanoformulations, which is compatible with the lab settings. To scale up from lab to mass production, all the parameters need to re-tested and all the reaction facilities need to be re-designed to ensure same quality of product. Moreover, information on
176 scaling up production of nanoformulations is greatly limited. Very tiny change to the production process can result in completely different nanoformualtion system with different therapeutic effects. Even if the production of nanoformulaitons is reproducible at lab scale, the manufacturing of nanoformulations in much bigger scale is uncertain. Therefore, future efforts are needed to achieve the goal to overcome the drawbacks and obstacles in industrial manufacture.
In conclusion, we have presented three gene therapy strategies for the treatment of IRDs caused by mutations in different sizes of genes. First we demonstrated that a hybrid nanoglobule/ECO/pDNA nanoparticle system could effectively condense DNA at low N/P ratios and mediate efficient cellular uptake and gene expression. The G4/ECO/pDNA nanoparticles with an N/N/P ratio of
3/3/1 showed high stability, low cytotoxicity, and efficient intracellular gene transfection and expression in ARPE-19 cells in 10% serum media. The nanoparticles also mediated significant gene transfection in both mouse retina and
RPE layers ex vivo. Subretinal injection of the nanoparticles also resulted in significant gene expression in both retina and RPE cells in mice for at least 7 days.
These findings indicated that the hybrid G4/ECO/pDNA nanoparticles provided a promising platform for safe and efficient delivery of gene therapeutics to treat genetic eye diseases.
177 Second, gene replacement therapy using all-trans-retinylamine targeted multifunctional lipid ECO-based gene delivery system significantly enhanced transfection efficiency in the RPE in vivo. Treatments with Ret-PEG-ECO/pRPE65 nanoparticles significantly improved the ERG activity and vision of Rpe65-/- mice, and this therapeutic effect continued for at least 120 days. Ret-PEG-ECO/pRPE65 nanoparticles were also safe for subretinal injection, as shown in wild-type BALB/c mice. These findings suggested that all-trans-retinylamine-modified pH-sensitive
ECO/pDNA nanoparticles comprise a promising non-viral platform for safe, efficient, and targeted delivery of gene therapeutics to treat RPE tissue-specific monogenic eye diseases, including LCA2.
Third, we designed ECO based gene replacement therapy to deliver large
ABCA4 therapeutic gene to treat STGD. To achieve for targeted expression of
ABCA4 in the photoreceptors, tissue-specific promoter was incorporated in the therapeutic plasmid sequence, and we demonstrated stable nanoparticle formation and tissue specific gene expression for both reporter and therapeutic genes in the retina. Successful gene therapy treatment of STGD using nanoparticles formulated by ECO and therapeutic ABCA4 plasmids was achieved as demonstrated by tissue specific expression of ABCA4, slowing down of A2E accumulation and STGD progression up to six months. The non-viral ECO/therapeutic gene formulations are a promising gene replacement therapy strategy for a broad range of visual dystrophies caused by mutations in different sizes of genes.
178 References
1. Colella, P.; Trapani, I.; Cesi, G.; Sommella, A.; Manfredi, A.; Puppo, A.; Iodice, C.; Rossi, S.; Simonelli, F.; Giunti, M.; Bacci, M. L.; Auricchio, A., Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors. Gene Therapy 2014, 21, 450. 2. Rivolta, C.; Sharon, D.; DeAngelis, M. M.; Dryja, T. P., Retinitis pigmentosa and allied diseases: numerous diseases, genes, and inheritance patterns. Human Molecular Genetics 2002, 11 (10), 1219-1227. 3. Perrault, I.; Rozet, J.-M.; Gerber, S.; Ghazi, I.; Leowski, C.; Ducroq, D.; Souied, E.; Dufier, J.-L.; Munnich, A.; Kaplan, J., Leber Congenital Amaurosis. Molecular Genetics and Metabolism 68 (2), 200-208. 4. Noble, K. G.; Carr, R. E., Stargardt's disease and fundus flavimaculatus. Archives of Ophthalmology 1979, 97 (7), 1281-1285. 5. Sullivan, L. S.; Daiger, S. P., Inherited retinal degeneration: exceptional genetic and clinical heterogeneity. Molecular Medicine Today 1996, 2 (9), 380-386. 6. Trapani, I.; Banfi, S.; Simonelli, F.; Surace, E. M.; Auricchio, A., Gene Therapy of Inherited Retinal Degenerations: Prospects and Challenges. Human Gene Therapy 2015, 26 (4), 193-200. 7. Smalley, E., First AAV gene therapy poised for landmark approval. Nature Biotechnology 2017, 35, 998. 8. Issa, P. C.; MacLaren, R. E., Non-viral retinal gene therapy: a review. Clinical & Experimental Ophthalmology 2012, 40 (1), 39-47. 9. Weale, R. A., The Vertebrate Retina. Principles of Structure and Function. The British Journal of Ophthalmology 1974, 58 (11), 948-949. 10. Strauss, O., The Retinal Pigment Epithelium in Visual Function. Physiological Reviews 2005, 85 (3), 845-881. 11. Lamb, T. D.; Collin, S. P.; Pugh Jr, E. N., Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup. Nature Reviews Neuroscience 2007, 8, 960.
179 12. Chabre, M.; Deterre, P., Molecular mechanism of visual transduction. European Journal of Biochemistry 1989, 179 (2), 255-266. 13. Kiser, P. D.; Golczak, M.; Palczewski, K., Chemistry of the Retinoid (Visual) Cycle. Chemical Reviews 2014, 114 (1), 194-232. 14. McBee, J. K.; Palczewski, K.; Baehr, W.; Pepperberg, D. R., Confronting Complexity: the Interlink of Phototransduction and Retinoid Metabolism in the Vertebrate Retina. Progress in Retinal and Eye Research 2001, 20 (4), 469-529. 15. Ratnapriya, R.; Swaroop, A., Genetic architecture of retinal and macular degenerative diseases: the promise and challenges of next-generation sequencing. Genome Medicine 2013, 5 (10), 84-84. 16. Zhang, K.; Zhang, L.; Weinreb, R. N., Ophthalmic drug discovery: novel targets and mechanisms for retinal diseases and glaucoma. Nature Reviews Drug Discovery 2012, 11, 541. 17. Friedmann, T., Progress toward human gene therapy. Science 1989, 244 (4910), 1275-1281. 18. Caspi, R. R., Ocular autoimmunity: the price of privilege? Immunological Reviews 2006, 213 (1), 23-35. 19. Boye, S. E.; Boye, S. L.; Lewin, A. S.; Hauswirth, W. W., A Comprehensive Review of Retinal Gene Therapy. Molecular Therapy 2013, 21 (3), 509-519. 20. Smith, A. J.; Bainbridge, J. W.; Ali, R. R., Prospects for retinal gene replacement therapy. Trends in Genetics 2009, 25 (4), 156-165. 21. Sung, C. H.; Schneider, B. G.; Agarwal, N.; Papermaster, D. S.; Nathans, J., Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Proceedings of the National Academy of Sciences 1991, 88 (19), 8840-8844. 22. Millington-Ward, S.; Chadderton, N.; O'Reilly, M.; Palfi, A.; Goldmann, T.; Kilty, C.; Humphries, M.; Wolfrum, U.; Bennett, J.; Humphries, P.; Kenna, P. F.; Farrar, G. J., Suppression and Replacement Gene Therapy for Autosomal Dominant Disease in a Murine Model of Dominant Retinitis Pigmentosa. Molecular Therapy 2011, 19 (4), 642-649.
180 23. Bakondi, B.; Lv, W.; Lu, B.; Jones, M. K.; Tsai, Y.; Kim, K. J.; Levy, R.; Akhtar, A. A.; Breunig, J. J.; Svendsen, C. N.; Wang, S., In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa. Molecular Therapy 2016, 24 (3), 556-563. 24. Ye, G.-j.; Budzynski, E.; Sonnentag, P.; Nork, T. M.; Miller, P. E.; McPherson, L.; Ver Hoeve, J. N.; Smith, L. M.; Arndt, T.; Mandapati, S.; Robinson, P. M.; Calcedo, R.; Knop, D. R.; Hauswirth, W. W.; Chulay, J. D., Safety and Biodistribution Evaluation in CNGB3-Deficient Mice of rAAV2tYF-PR1.7- hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia. Human Gene Therapy. Clinical Development 2016, 27 (1), 27-36. 25. Gootwine, E.; Ofri, R.; Banin, E.; Obolensky, A.; Averbukh, E.; Ezra-Elia, R.; Ross, M.; Honig, H.; Rosov, A.; Yamin, E.; Ye, G.-j.; Knop, D. R.; Robinson, P. M.; Chulay, J. D.; Shearman, M. S., Safety and Efficacy Evaluation of rAAV2tYF- PR1.7-hCNGA3 Vector Delivered by Subretinal Injection in CNGA3 Mutant Achromatopsia Sheep. Human Gene Therapy Clinical Development 2017, 28 (2), 96-107. 26. Lai, C.-M.; Estcourt, M. J.; Wikstrom, M.; Himbeck, R. P.; Barnett, N. L.; Brankov, M.; Tee, L. B. G.; Dunlop, S. A.; Degli-Esposti, M. A.; Rakoczy, E. P., rA AV. s Fl t -1 Gene Therapy Achieves Lasting Reversal of Retinal Neovascularization in the Absence of a Strong Immune Response to the Viral Vector. Investigative Ophthalmology & Visual Science 2009, 50 (9), 4279-4287. 27. Heier, J. S.; Kherani, S.; Desai, S.; Dugel, P.; Kaushal, S.; Cheng, S. H.; Delacono, C.; Purvis, A.; Richards, S.; Le-Halpere, A.; Connelly, J.; Wadsworth, S. C.; Varona, R.; Buggage, R.; Scaria, A.; Campochiaro, P. A., Intravitreous injection o f A AV 2 -sFLT01 in patients with advanced neovascular age-related macular degeneration: a phase 1, open-label trial. The Lancet 390 (10089), 50-61. 28. Tolmachova, T.; Tolmachov, O. E.; Barnard, A. R.; de Silva, S. R.; Lipinski, D. M.; Walker, N. J.; MacLaren, R. E.; Seabra, M. C., Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo. Journal of Molecular Medicine 2013,
181 91 (7), 825-837. 29. Barnard, A. R.; Tolmachova, T.; Seabra, M. C.; MacLaren, R. E., Assessment of Visual Function by Electroretinography following rAAV2-CHM/REP1 Gene Therapy in a Mouse Model of Choroideremia. Investigative Ophthalmology & Visual Science 2012, 53 (14), 1933-1933. 30. Vasireddy, V.; Mills, J. A.; Gaddameedi, R.; Basner-Tschakarjan, E.; Kohnke, M.; Black, A. D.; Alexandrov, K.; Zhou, S.; Maguire, A. M.; Chung, D. C.; Mac, H.; Sullivan, L.; Gadue, P.; Bennicelli, J. L.; French, D. L.; Bennett, J. , A AV- Mediated Gene Therapy for Choroideremia: Preclinical Studies in Personalized Models. PLoS ONE 2013, 8 (5), e61396. 31. Russell, S.; Bennett, J.; Wellman, J. A.; Chung, D. C.; Yu, Z.-F.; Tillman, A.; Wittes, J.; Pappas, J.; Elci, O.; McCague, S.; Cross, D.; Marshall, K. A.; Walshire, J.; Kehoe, T. L.; Reichert, H.; Davis, M.; Raffini, L.; George, L. A.; Hudson, F. P.; Dingfield, L.; Zhu, X.; Haller, J. A.; Sohn, E. H.; Mahajan, V. B.; Pfeifer, W.; Weckmann, M.; Johnson, C.; Gewaily, D.; Drack, A.; Stone, E.; Wachtel, K.; Simonelli, F.; Leroy, B. P.; Wright, J. F.; High, K. A.; Maguire, A. M., Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. The Lancet 390 (10097), 849-860. 32. Hauswirth, W. W.; Aleman, T. S.; Kaushal, S.; Cideciyan, A. V.; Schwartz, S. B.; Wang, L.; Conlon, T. J.; Boye, S. L.; Flotte, T. R.; Byrne, B. J.; Jacobson, S. G., Treatment of Leber Congenital Amaurosis Due to RPE65 Mutations by Ocular Subretinal Injection of Adeno-Associated Virus Gene Vector: Short-Term Results of a Phase I Trial. Human Gene Therapy 2008, 19 (10), 979-990. 33. Wright, J. F.; Jennifer, W.; Katherine, A. H., Manufacturing and Regulatory Strategies for Clinical AAV2-hRPE65. Current Gene Therapy 2010, 10 (5), 341- 349. 34. Feuer, W. J.; Schiffman, J. C.; Davis, J. L.; Porciatti, V.; Gonzalez, P.; Koilkonda, R. D.; Yuan, H.; Lalwani, A.; Lam, B. L.; Guy, J., Gene Therapy for Leber Hereditary Optic Neuropathy. Ophthalmology 123 (3), 558-570.
182 35. Conlon, T. J.; Deng, W.-T.; Erger, K.; Cossette, T.; Pang, J.-j.; Ryals, R.; Clément, N.; Cleaver, B.; McDoom, I.; Boye, S. E.; Peden, M. C.; Sherwood, M. B.; Abernathy, C. R.; Alkuraya, F. S.; Boye, S. L.; Hauswirth, W. W., Preclinical Potency and Safety Studies of an AAV2-Mediated Gene Therapy Vector for the Treatment of MERTK Associated Retinitis Pigmentosa. Human Gene Therapy. Clinical Development 2013, 24 (1), 23-28. 36. Marangoni, D.; Bush, R. A.; Zeng, Y.; Wei, L. L.; Ziccardi, L.; Vijayasarathy, C.; Bartoe, J. T.; Palyada, K.; Santos, M.; Hiriyanna, S.; Wu, Z.; Colosi, P.; Sieving, P. A., Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice. Molecular Therapy. Methods & Clinical Development 2016, 5, 16011. 37. Ye, G.-J.; Budzynski, E.; Sonnentag, P.; Miller, P. E.; Sharma, A. K.; Ver Hoeve, J. N.; Howard, K.; Knop, D. R.; Neuringer, M.; McGill, T.; Stoddard, J.; Chulay, J. D., Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF- CB-hRS1, a Recombinant Adeno-Associated Virus Vector Expressing Retinoschisin. Human Gene Therapy. Clinical Development 2015, 26 (3), 165-176. 38. Zallocchi, M.; Binley, K.; Lad, Y.; Ellis, S.; Widdowson, P.; Iqball, S.; Scripps, V.; Kelleher, M.; Loader, J.; Miskin, J.; Peng, Y.-W.; Wang, W.-M.; Cheung, L.; Delimont, D.; Mitrophanous, K. A.; Cosgrove, D., EIAV-Based Retinal Gene Therapy in the shaker1 Mouse Model for Usher Syndrome Type 1B: Development of UshStat. PLOS ONE 2014, 9 (4), e94272. 39. Bennett, J.; Ashtari, M.; Wellman, J.; Marshall, K. A.; Cyckowski, L. L.; Chung, D. C.; McCague, S.; Pierce, E. A.; Chen, Y.; Bennicelli, J. L.; Zhu, X.; Ying, G.-s.; Sun, J.; Wright, J. F.; Auricchio, A.; Simonelli, F.; Shindler, K. S.; Mingozzi, F.; High, K. A.; Maguire, A. M., AAV2 Gene Therapy Readministration in Three Adults with Congenital Blindness. Science Translational Medicine 2012, 4 (120), 120ra15-120ra15. 40. Naldini, L.; Blömer, U.; Gage, F. H.; Trono, D.; Verma, I. M., Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proceedings of the National Academy of Sciences
183 1996, 93 (21), 11382-11388. 41. Miyoshi, H.; Takahashi, M.; Gage, F. H.; Verma, I. M., Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proceedings of the National Academy of Sciences 1997, 94 (19), 10319-10323. 42. Loewen, N.; Leske, D. A.; Chen, Y.; Teo, W.-L.; Saenz, D. T.; Peretz, M.; Holmes, J. M.; Poeschla, E. M., Comparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epithelium. The Journal of Gene Medicine 2003, 5 (12), 1009-1017. 43. Tschernutter, M.; Schlichtenbrede, F. C.; Howe, S.; Balaggan, K. S.; Munro, P. M.; Bainbridge, J. W. B.; Thrasher, A. J.; Smith, A. J.; Ali, R. R., Long-term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy. Gene Therapy 2005, 12, 694. 44. Ikuno, Y.; Kazlauskas, A., An In Vivo Gene Therapy Approach for Experimental Proliferative Vitreoretinopathy Using the Truncated Platelet-Derived Growth Factor α Receptor. Investigative Ophthalmology & Visual Science 2002, 43 (7), 2406-2411. 45. During, M. J., Adeno-associated virus as a gene delivery system. Advanced Drug Delivery Reviews 1997, 27 (1), 83-94. 46. Narfström, K.; Katz, M. L.; Bragadottir, R.; Seeliger, M.; Boulanger, A.; Redmond, T. M.; Caro, L.; Lai, C.-M.; Rakoczy, P. E., Functional and Structural Recovery of the Retina after Gene Therapy in the RPE65 Null Mutation Dog. Investigative Ophthalmology & Visual Science 2003, 44 (4), 1663-1672. 47. Colella, P.; Cotugno, G.; Auricchio, A., Ocular gene therapy: current progress and future prospects. Trends in Molecular Medicine 2009, 15 (1), 23-31. 48. Li, S. D.; Huang, L., Gene therapy progress and prospects: non-viral gene therapy by systemic delivery. Gene Therapy 2006, 13, 1313. 49. Thomas, M.; Klibanov, A. M., Non-viral gene therapy: polycation-mediated DNA delivery. Applied Microbiology and Biotechnology 2003, 62 (1), 27-34. 50. Urtti, A., Challenges and obstacles of ocular pharmacokinetics and drug
184 delivery. Advanced Drug Delivery Reviews 2006, 58 (11), 1131-1135. 51. Ochakovski, G. A.; Bartz-Schmidt, K. U.; Fischer, M. D., Retinal Gene Therapy: Surgical Vector Delivery in the Translation to Clinical Trials. Frontiers in Neuroscience 2017, 11, 174. 52. Maurice, D., Review: Practical Issues in Intravitreal Drug Delivery. Journal of Ocular Pharmacology and Therapeutics 2001, 17 (4), 393-401. 53. Khar, R. K.; Jain, G. K.; Warsi, M. H.; Mallick, N.; Akhter, S.; Pathan, S. A.; Ahmad, F. J., Nano-vectors for the Ocular Delivery of Nucleic Acid-based Therapeutics. Indian Journal of Pharmaceutical Sciences 2010, 72 (6), 675-688. 54. Ramamoorth, M.; Narvekar, A., Non Viral Vectors in Gene Therapy- An Overview. Journal of Clinical and Diagnostic Research : JCDR 2015, 9 (1), GE01- GE06. 55. Borchard, G., Chitosans for gene delivery. Advanced Drug Delivery Reviews 2001, 52 (2), 145-150. 56. Raftery, R.; #039; Brien, F.; Cryan, S.-A., Chitosan for Gene Delivery and Orthopedic Tissue Engineering Applications. Molecules 2013, 18 (5), 5611. 57. Huang, M.; Fong, C.-W.; Khor, E.; Lim, L.-Y., Transfection efficiency of chitosan vectors: Effect of polymer molecular weight and degree of deacetylation. Journal of Controlled Release 2005, 106 (3), 391-406. 58. Richardson, S. W.; Kolbe, H. J.; Duncan, R., Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA. International Journal of Pharmaceutics 1999, 178 (2), 231-243. 59. Puras, G.; Zarate, J.; Aceves, M.; Murua, A.; Díaz, A. R.; Avilés-Triguero, M.; Fernández, E.; Pedraz, J. L., Low molecular weight oligochitosans for non-viral retinal gene therapy. European Journal of Pharmaceutics and Biopharmaceutics 2013, 83 (2), 131-140. 60. de la Fuente, M.; Seijo, B. a.; Alonso, M. J., Novel Hyaluronic Acid-Chitosan Nanoparticles for Ocular Gene Therapy. Investigative Ophthalmology & Visual Science 2008, 49 (5), 2016-2024.
185 61. Sang Yoo, H.; Eun Lee, J.; Chung, H.; Chan Kwon, I.; Young Jeong, S., Self- assembled nanoparticles containing hydrophobically modified glycol chitosan for gene delivery. Journal of Controlled Release 2005, 103 (1), 235-243. 62. Mitra, R. N.; Han, Z.; Merwin, M.; Al Taai, M.; Conley, S. M.; Naash, M. I., Synthesis and Characterization of Glycol Chitosan DNA Nanoparticles for Retinal Gene Delivery. ChemMedChem 2014, 9 (1), 189-196. 63. Oliveira, A. V.; Silva, A. P.; Bitoque, D. B.; Silva, G. A.; Rosa da Costa, A. M., Transfection efficiency of chitosan and thiolated chitosan in retinal pigment epithelium cells: A comparative study. Journal of Pharmacy & Bioallied Sciences 2013, 5 (2), 111-118. 64. Diebold, Y.; Jarrín, M.; Sáez, V.; Carvalho, E. L. S.; Orea, M.; Calonge, M.; Seijo, B.; Alonso, M. J., Ocular drug delivery by liposome–chitosan nanoparticle complexes (LCS-NP). Biomaterials 2007, 28 (8), 1553-1564. 65. Rafiee, A.; Alimohammadian, M. H.; Gazori, T.; Riazi-rad, F.; Fatemi, S. M. R.; Parizadeh, A.; Haririan, I.; Havaskary, M., Comparison of chitosan, alginate and chitosan/alginate nanoparticles with respect to their size, stability, toxicity and transfection. Asian Pacific Journal of Tropical Disease 2014, 4 (5), 372-377. 66. Makadia, H. K.; Siegel, S. J., Poly Lactic-co-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers 2011, 3 (3), 1377-1397. 67. Mittal, G.; Sahana, D. K.; Bhardwaj, V.; Ravi Kumar, M. N. V., Estradiol loaded PLGA nanoparticles for oral administration: Effect of polymer molecular weight and copolymer composition on release behavior in vitro and in vivo. Journal of Controlled Release 2007, 119 (1), 77-85. 68. Bobo, D.; Robinson, K. J.; Islam, J.; Thurecht, K. J.; Corrie, S. R., Nanoparticle-Based Medicines: A Review of FDA-Approved Materials and Clinical Trials to Date. Pharmaceutical Research 2016, 33 (10), 2373-2387. 69. Park, K.; Chen, Y.; Hu, Y.; Mayo, A. S.; Kompella, U. B.; Longeras, R.; Ma, J.-x., Nanoparticle-Mediated Expression of an Angiogenic Inhibitor Ameliorates Ischemia-Induced Retinal Neovascularization and Diabetes-Induced Retinal Vascular Leakage. Diabetes 2009, 58 (8), 1902-1913.
186 70. Zhang, C.; Wang, Y. S.; Wu, H.; Zhang, Z. X.; Cai, Y.; Hou, H. Y.; Zhao, W.; Yang, X. M.; Ma, J. X., Inhibitory efficacy of hypoxia-inducible factor 1α short hairpin RNA plasmid DNA-loaded poly (D, L-lactide-co-glycolide) nanoparticles on choroidal neovascularization in a laser-induced rat model. Gene Therapy 2009, 17, 338. 71. Luo, L.; Zhang, X.; Hirano, Y.; Tyagi, P.; Barabás, P.; Uehara, H.; Miya, T. R.; Singh, N.; Archer, B.; Qazi, Y.; Jackman, K.; Das, S. K.; Olsen, T.; Chennamaneni, S. R.; Stagg, B. C.; Ahmed, F.; Emerson, L.; Zygmunt, K.; Whitaker, R.; Mamalis, C.; Huang, W.; Gao, G.; Srinivas, S. P.; Krizaj, D.; Baffi, J.; Ambati, J.; Kompella, U. B.; Ambati, B. K., Targeted Intraceptor Nanoparticle Therapy Reduces Angiogenesis and Fibrosis in Primate and Murine Macular Degeneration. ACS Nano 2013, 7 (4), 3264-3275. 72. Godbey, W. T.; Wu, K. K.; Mikos, A. G., Poly(ethylenimine) and its role in gene delivery. Journal of Controlled Release 1999, 60 (2), 149-160. 73. Kurosaki, T.; Uematsu, M.; Shimoda, K.; Suzuma, K.; Nakai, M.; Nakamura, T.; Kitahara, T.; Kitaoka, T.; Sasaki, H., Ocular Gene Delivery Systems Using Ternary Complexes of Plasmid DNA, Polyethylenimine, and Anionic Polymers. Biological and Pharmaceutical Bulletin 2013, 36 (1), 96-101. 74. Al-Dosari, M. S.; Gao, X., Nonviral Gene Delivery: Principle, Limitations, and Recent Progress. The AAPS Journal 2009, 11 (4), 671. 75. Anderson, D. G.; Akinc, A.; Hossain, N.; Langer, R., Structure/property studies of polymeric gene delivery using a library of poly(β-amino esters). Molecular Therapy 2005, 11 (3), 426-434. 76. Sunshine, J. C.; Sunshine, S. B.; Bhutto, I.; Handa, J. T.; Green, J. J., Poly(β- Amino Ester)-Nanoparticle Mediated Transfection of Retinal Pigment Epithelial Cells In Vitro and In Vivo. PLOS ONE 2012, 7 (5), e37543. 77. Smith, L. C.; Duguid, J.; Wadhwa, M. S.; Logan, M. J.; Tung, C.-H.; Edwards, V.; Sparrow, J. T., Synthetic peptide-based DNA complexes for nonviral gene delivery. Advanced Drug Delivery Reviews 1998, 30 (1), 115-131. 78. Martin, M. E.; Rice, K. G., Peptide-guided gene delivery. The AAPS Journal
187 2007, 9 (1), E18-E29. 79. Mitchell, D. J.; Steinman, L.; Kim, D. T.; Fathman, C. G.; Rothbard, J. B., Polyarginine enters cells more efficiently than other polycationic homopolymers. The Journal of Peptide Research 2000, 56 (5), 318-325. 80. Pensado, A.; Diaz-Corrales, F. J.; De la Cerda, B.; Valdés-Sánchez, L.; del Boz, A. A.; Rodriguez-Martinez, D.; García-Delgado, A. B.; Seijo, B.; Bhattacharya, S. S.; Sanchez, A., Span poly-L-arginine nanoparticles are efficient non-viral vectors for PRPF31 gene delivery: An approach of gene therapy to treat retinitis pigmentosa. Nanomedicine: Nanotechnology, Biology and Medicine 2016, 12 (8), 2251-2260. 81. Johnson, L. N.; Cashman, S. M.; Kumar-Singh, R., Cell-penetrating Peptide for Enhanced Delivery of Nucleic Acids and Drugs to Ocular Tissues Including Retina and Cornea. Molecular Therapy 2008, 16 (1), 107-114. 82. Dasari, B. C.; Cashman, S. M.; Kumar-Singh, R., Reducible PEG-POD/DNA Nanoparticles for Gene Transfer In Vitro and In Vivo: Application in a Mouse Model of Age-Related Macular Degeneration. Molecular Therapy - Nucleic Acids 2017, 8, 77-89. 83. Cai, X.; Conley, S. M.; Nash, Z.; Fliesler, S. J.; Cooper, M. J.; Naash, M. I., Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa. The FASEB Journal 2009, 24 (4), 1178-1191. 84. Koirala, A.; Makkia, R. S.; Conley, S. M.; Cooper, M. J.; Naash, M. I., S/MAR- containing DNA nanoparticles promote persistent RPE gene expression and improvement in RPE65-associated LCA. Human Molecular Genetics 2013, 22 (8), 1632-1642. 85. Han, Z.; Conley, S. M.; Makkia, R. S.; Cooper, M. J.; Naash, M. I., DNA nanoparticle-mediated ABCA4 delivery rescues Stargardt dystrophy in mice. The Journal of Clinical Investigation 2012, 122 (9), 3221-3226. 86. Ginisty, H.; Sicard, H.; Roger, B.; Bouvet, P., Structure and functions of nucleolin. Journal of Cell Science 1999, 112 (6), 761-772. 87. Chaplot, S. P.; Rupenthal, I. D., Dendrimers for gene delivery – a potential
188 approach for ocular therapy? Journal of Pharmacy and Pharmacology 2014, 66 (4), 542-556. 88. Lee, I.-H.; Yu, M. K.; Kim, I. H.; Lee, J.-H.; Park, T. G.; Jon, S., A duplex oligodeoxynucleotide–dendrimer bioconjugate as a novel delivery vehicle for doxorubicin in in vivo cancer therapy. Journal of Controlled Release 2011, 155 (1), 88-95. 89. Yang, S.; Coles, D. J.; Esposito, A.; Mitchell, D. J.; Toth, I.; Minchin, R. F., Cellular uptake of self-assembled cationic peptide–DNA complexes: Multifunctional role of the enhancer chloroquine. Journal of Controlled Release 2009, 135 (2), 159-165. 90. Kesharwani, P.; Banerjee, S.; Gupta, U.; Mohd Amin, M. C. I.; Padhye, S.; Sarkar, F. H.; Iyer, A. K., PAMAM dendrimers as promising nanocarriers for RNAi therapeutics. Materials Today 2015, 18 (10), 565-572. 91. Felgner, J. H.; Kumar, R.; Sridhar, C. N.; Wheeler, C. J.; Tsai, Y. J.; Border, R.; Ramsey, P.; Martin, M.; Felgner, P. L., Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. Journal of Biological Chemistry 1994, 269 (4), 2550-2561. 92. Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J., Toxicity of cationic lipids and cationic polymers in gene delivery. Journal of Controlled Release 2006, 114 (1), 100-109. 93. Apaolaza, P. S.; Delgado, D.; Pozo-Rodríguez, A. d.; Gascón, A. R.; Solinís, M. Á., A novel gene therapy vector based on hyaluronic acid and solid lipid nanoparticles for ocular diseases. International Journal of Pharmaceutics 2014, 465 (1), 413-426. 94. Apaolaza, P. S.; del Pozo-Rodríguez, A.; Torrecilla, J.; Rodríguez-Gascón, A.; Rodríguez, J. M.; Friedrich, U.; Weber, B. H. F.; Solinís, M. A., Solid lipid nanoparticle-based vectors intended for the treatment of X-linked juvenile retinoschisis by gene therapy: In vivo approaches in Rs1h-deficient mouse model. Journal of Controlled Release 2015, 217, 273-283. 95. Rajala, A.; Wang, Y.; Zhu, Y.; Ranjo-Bishop, M.; Ma, J.-X.; Mao, C.; Rajala,
189 R. V. S., Nanoparticle-Assisted Targeted Delivery of Eye-Specific Genes to Eyes Significantly Improves the Vision of Blind Mice In Vivo. Nano Letters 2014, 14 (9), 5257-5263. 96. Le Goff, M. M.; Bishop, P. N., Adult vitreous structure and postnatal changes. Eye 2008, 22, 1214. 97. Naik, R.; Mukhopadhyay, A.; Ganguli, M., Gene delivery to the retina: focus on non-viral approaches. Drug Discovery Today 2009, 14 (5), 306-315. 98. José, C.-V.; Rui, B.; Conceição, L., Blood-Retinal Barrier. European Journal of Ophthalmology 2011, 21 (6_suppl), 3-9. 99. Wong, S. Y.; Pelet, J. M.; Putnam, D., Polymer systems for gene delivery— Past, present, and future. Progress in Polymer Science 2007, 32 (8), 799-837. 100. Conley, S. M.; Naash, M. I., Nanoparticles for Retinal Gene Therapy. Progress in retinal and eye research 2010, 29 (5), 376-397. 101. Gujrati, M.; Malamas, A.; Shin, T.; Jin, E.; Sun, Y.; Lu, Z.-R., Multifunctional Cationic Lipid-Based Nanoparticles Facilitate Endosomal Escape and Reduction-Triggered Cytosolic siRNA Release. Molecular Pharmaceutics 2014, 11 (8), 2734-2744. 102. Gan, L.; Wang, J.; Zhao, Y.; Chen, D.; Zhu, C.; Liu, J.; Gan, Y., Hyaluronan-modified core–shell liponanoparticles targeting CD44-positive retinal pigment epithelium cells via intravitreal injection. Biomaterials 2013, 34 (24), 5978-5987. 103. Suen, W.-L. L.; Chau, Y., Specific uptake of folate-decorated triamcinolone-encapsulating nanoparticles by retinal pigment epithelium cells enhances and prolongs antiangiogenic activity. Journal of Controlled Release 2013, 167 (1), 21-28. 104. Neu, M.; Fischer, D.; Kissel, T., Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives. The Journal of Gene Medicine 2005, 7 (8), 992-1009. 105. Kolli, S.; Wong, S.-P.; Harbottle, R.; Johnston, B.; Thanou, M.; Miller, A. D., pH-Triggered Nanoparticle Mediated Delivery of siRNA to Liver Cells in Vitro
190 and in Vivo. Bioconjugate Chemistry 2013, 24 (3), 314-332. 106. Remy, J.-S.; Abdallah, B.; Zanta, M. A.; Boussif, O.; Behr, J.-P.; Demeneix, B., Gene transfer with lipospermines and polyethylenimines. Advanced Drug Delivery Reviews 1998, 30 (1), 85-95. 107. Kim, S. T.; Saha, K.; Kim, C.; Rotello, V. M., The Role of Surface Functionality in Determining Nanoparticle Cytotoxicity. Accounts of Chemical Research 2013, 46 (3), 681-691. 108. Zhou, Z.; Lu, Z.-R., Dendritic nanoglobules with polyhedral oligomeric silsesquioxane core and their biomedical applications. Nanomedicine 2014, 9 (15), 2387-2401. 109. Wu, X.; Yu, G.; Luo, C.; Maeda, A.; Zhang, N.; Sun, D.; Zhou, Z.; Puntel, A.; Palczewski, K.; Lu, Z.-R., Synthesis and Evaluation of a Nanoglobular Dendrimer 5-Aminosalicylic Acid Conjugate with a Hydrolyzable Schiff Base Spacer for Treating Retinal Degeneration. ACS Nano 2014, 8 (1), 153-161. 110. Xu, R.; Lu, Z.-R., Design, synthesis and evaluation of spermine-based pH- sensitive amphiphilic gene delivery systems: Multifunctional non-viral gene carriers. Science China Chemistry 2011, 54 (2), 359-368. 111. Xu, R.; Wang, X.-L.; Lu, Z.-R., New Amphiphilic Carriers Forming pH- Sensitive Nanoparticles for Nucleic Acid Delivery. Langmuir 2010, 26 (17), 13874- 13882. 112. Canine, B. F.; Wang, Y.; Hatefi, A., Evaluation of the effect of vector architecture on DNA condensation and gene transfer efficiency. Journal of Controlled Release 2008, 129 (2), 117-123. 113. DeLange, R. J.; Smith, E. L., Histones: Structure and Function. Annual Review of Biochemistry 1971, 40 (1), 279-314. 114. Kaneshiro, T. L.; Wang, X.; Lu, Z.-R., Synthesis, Characterization, and Gene Delivery of Poly-l-lysine Octa(3-aminopropyl)silsesquioxane Dendrimers: Nanoglobular Drug Carriers with Precisely Defined Molecular Architectures. Molecular Pharmaceutics 2007, 4 (5), 759-768. 115. Malamas, A. S.; Gujrati, M.; Kummitha, C. M.; Xu, R.; Lu, Z.-R., Design and
191 evaluation of new pH-sensitive amphiphilic cationic lipids for siRNA delivery. Journal of Controlled Release 2013, 171 (3), 296-307. 116. Fischer, D.; von Harpe, A.; Kunath, K.; Petersen, H.; Li, Y.; Kissel, T., Copolymers of Ethylene Imine and N-(2-Hydroxyethyl)-ethylene Imine as Tools To Study Effects of Polymer Structure on Physicochemical and Biological Properties of DNA Complexes. Bioconjugate Chemistry 2002, 13 (5), 1124-1133. 117. Bennett, M. J.; Aberle, A. M.; Balasubramaniam, R. P.; Malone, J. G.; Nantz, M. H.; Malone, R. W., Considerations for the Design of Improved Cationic Amphiphile-Based Transfection Reagents. Journal of Liposome Research 1996, 6 (3), 545-565. 118. Ribes, S.; Ebert, S.; Czesnik, D.; Regen, T.; Zeug, A.; Bukowski, S.; Mildner, A.; Eiffert, H.; Hanisch, U.-K.; Hammerschmidt, S.; Nau, R., Toll-Like Receptor Prestimulation Increases Phagocytosis of Escherichia coli DH5α and Escherichia coli K1 Strains by Murine Microglial Cells. Infection and Immunity 2009, 77 (1), 557-564. 119. Kastl, L.; Sasse, D.; Wulf, V.; Hartmann, R.; Mircheski, J.; Ranke, C.; Carregal- Romero, S.; Martínez-López, J. A.; Fernández-Chacón, R.; Parak, W. J.; Elsasser, H.-P.; Rivera_Gil, P., Multiple Internalization Pathways of Polyelectrolyte Multilayer Capsules into Mammalian Cells. ACS Nano 2013, 7 (8), 6605-6618. 120. Erbacher, P.; Remy, J. S.; Behr, J. P., Gene transfer with synthetic virus- like particles via the integrin-mediated endocytosis pathway. Gene Therapy 1999, 6, 138. 121. Manickam, D. S.; Li, J.; Putt, D. A.; Zhou, Q.-H.; Wu, C.; Lash, L. H.; Oupický, D., Effect of innate glutathione levels on activity of redox-responsive gene delivery vectors. Journal of Controlled Release 2010, 141 (1), 77-84. 122. Won, Y.-W.; Yoon, S.-M.; Lee, K.-M.; Kim, Y.-H., Poly(oligo-D-arginine) With Internal Disulfide Linkages as a Cytoplasm-sensitive Carrier for siRNA Delivery. Molecular Therapy 2011, 19 (2), 372-380. 123. Parvani, J. G.; Gujrati, M. D.; Mack, M. A.; Schiemann, W. P.; Lu, Z.-R., Silencing β3 Integrin by Targeted ECO/siRNA Nanoparticles Inhibits EMT and
192 Metastasis of Triple-Negative Breast Cancer. Cancer Research 2015, 75 (11), 2316. 124. Hufnagel, R. B.; Ahmed, Z. M.; Corrêa, Z. M.; Sisk, R. A., Gene therapy for Leber congenital amaurosis: advances and future directions. Graefe's Archive for Clinical and Experimental Ophthalmology 2012, 250 (8), 1117-1128. 125. Koenekoop, R. K., An overview of leber congenital amaurosis: a model to understand human retinal development. Survey of Ophthalmology 2004, 49 (4), 379-398. 126. Kiser, P. D.; Palczewski, K., Membrane-binding and enzymatic properties of RPE65. Progress in Retinal and Eye Research 2010, 29 (5), 428-442. 127. Kiser, P. D.; Zhang, J.; Badiee, M.; Li, Q.; Shi, W.; Sui, X.; Golczak, M.; Tochtrop, G. P.; Palczewski, K., Catalytic mechanism of a retinoid isomerase essential for vertebrate vision. Nature Chemical Biology 2015, 11, 409. 128. Golczak, M.; Kiser, P. D.; Lodowski, D. T.; Maeda, A.; Palczewski, K., Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity. Journal of Biological Chemistry 2010, 285 (13), 9667-9682. 129. Aguirre, G. D.; Baldwin, V.; Pearce-Kelling, S.; Narfström, K.; Ray, K.; Acland, G. M., Congenital stationary night blindness in the dog: common mutation in the RPE65 gene indicates founder effect. Molecular vision 1998, 4, 23. 130. Simonelli, F.; Maguire, A. M.; Testa, F.; Pierce, E. A.; Mingozzi, F.; Bennicelli, J. L.; Rossi, S.; Marshall, K.; Banfi, S.; Surace, E. M.; Sun, J.; Redmond, T. M.; Zhu, X.; Shindler, K. S.; Ying, G.-S.; Ziviello, C.; Acerra, C.; Wright, J. F.; McDonnell, J. W.; High, K. A.; Bennett, J.; Auricchio, A., Gene Therapy for Leber's Congenital Amaurosis is Safe and Effective Through 1.5 Years After Vector Administration. Molecular Therapy 2010, 18 (3), 643-650. 131. Jacobson, S. G.; Cideciyan, A. V.; Ratnakaram, R.; et al., Gene therapy for leber congenital amaurosis caused by rpe65 mutations: Safety and efficacy in 15 children and adults followed up to 3 years. Archives of Ophthalmology 2012, 130 (1), 9-24. 132. Ginn, S. L.; Alexander, I. E.; Edelstein, M. L.; Abedi, M. R.; Wixon, J., Gene therapy clinical trials worldwide to 2012 – an update. The Journal of Gene
193 Medicine 2013, 15 (2), 65-77. 133. Cideciyan, A. V.; Hauswirth, W. W.; Aleman, T. S.; Kaushal, S.; Schwartz, S. B.; Boye, S. L.; Windsor, E. A. M.; Conlon, T. J.; Sumaroka, A.; Pang, J.-j.; Roman, A. J.; Byrne, B. J.; Jacobson, S. G., Human RPE65 Gene Therapy for Leber Congenital Amaurosis: Persistence of Early Visual Improvements and Safety at 1 Year. Human Gene Therapy 2009, 20 (9), 999-1004. 134. Jooss, K.; Chirmule, N., Immunity to adenovirus and adeno-associated viral vectors: implications for gene therapy. Gene Therapy 2003, 10, 955. 135. Provost, N.; Le Meur, G.; Weber, M.; Mendes-Madeira, A.; Podevin, G.; Cherel, Y.; Colle, M.-A.; Deschamps, J.-Y.; Moullier, P.; Rolling, F., Biodistribution of rAAV Vectors Following Intraocular Administration: Evidence for the Presence and Persistence of Vector DNA in the Optic Nerve and in the Brain. Molecular Therapy 2005, 11 (2), 275-283. 136. Wasungu, L.; Hoekstra, D., Cationic lipids, lipoplexes and intracellular delivery of genes. Journal of Controlled Release 2006, 116 (2), 255-264. 137. Martin, B.; Sainlos, M.; Aissaoui, A.; Oudrhiri, N.; Hauchecorne, M.; Vigneron, J. P.; Lehn, J. M.; Lehn, P., The Design of Cationic Lipids for Gene Delivery. Current Pharmaceutical Design 2005, 11 (3), 375-394. 138. Shcharbin, D. G.; Klajnert, B.; Bryszewska, M., Dendrimers in gene transfection. Biochemistry (Moscow) 2009, 74 (10), 1070-1079. 139. de la Fuente, M.; Raviña, M.; Paolicelli, P.; Sanchez, A.; Seijo, B.; Alonso, M. J., Chitosan-based nanostructures: A delivery platform for ocular therapeutics. Advanced Drug Delivery Reviews 2010, 62 (1), 100-117. 140. Glover, D. J.; Lipps, H. J.; Jans, D. A., Towards safe, non-viral therapeutic gene expression in humans. Nature Reviews Genetics 2005, 6, 299. 141. D’Ambrosio, D. N.; Clugston, R. D.; Blaner, W. S., Vitamin A Metabolism: An Update. Nutrients 2011, 3 (1), 63-103. 142. Gonzalez-Fernandez, F.; Ghosh, D., Focus on Molecules: Interphotoreceptor retinoid-binding protein (IRBP). Experimental Eye Research 2008, 86 (2), 169-170.
194 143. Vachali, P. P.; Besch, B. M.; Gonzalez-Fernandez, F.; Bernstein, P. S., Carotenoids as possible interphotoreceptor retinoid-binding protein (IRBP) ligands: A surface plasmon resonance (SPR) based study. Archives of Biochemistry and Biophysics 2013, 539 (2), 181-186. 144. Gonzalez-Fernandez, F., Interphotoreceptor retinoid-binding protein––an old gene for new eyes. Vision Research 2003, 43 (28), 3021-3036. 145. Jin, M.; Li, S.; Nusinowitz, S.; Lloyd, M.; Hu, J.; Radu, R. A.; Bok, D.; Travis, G. H., The Role of Interphotoreceptor Retinoid-Binding Protein on the Translocation of Visual Retinoids and Function of Cone Photoreceptors. The Journal of Neuroscience 2009, 29 (5), 1486-1495. 146. Gonzalez-Fernandez, F., Evolution of the visual cycle: the role of retinoid- binding proteins. Journal of Endocrinology 2002, 175 (1), 75-88. 147. Chen, Y.; Houghton, L. A.; Brenna, J. T.; Noy, N., Docosahexaenoic Acid Modulates the Interactions of the Interphotoreceptor Retinoid-binding Protein with 11-cis-Retinal. Journal of Biological Chemistry 1996, 271 (34), 20507-20515. 148. Redmond, T. M.; Yu, S.; Lee, E.; Bok, D.; Hamasaki, D.; Chen, N.; Goletz, P.; Ma, J.-X.; Crouch, R. K.; Pfeifer, K., Rpe65 is necessary for production of 11- cis-vitamin A in the retinal visual cycle. Nature Genetics 1998, 20, 344. 149. Sun, D.; Maeno, H.; Gujrati, M.; Schur, R.; Maeda, A.; Maeda, T.; Palczewski, K.; Lu, Z.-R., Self-Assembly of a Multifunctional Lipid With Core– Shell Dendrimer DNA Nanoparticles Enhanced Efficient Gene Delivery at Low Charge Ratios into RPE Cells. Macromolecular Bioscience 2015, 15 (12), 1663- 1672. 150. Schur, R. M.; Sheng, L.; Sahu, B.; Yu, G.; Gao, S.; Yu, X.; Maeda, A.; Palczewski, K.; Lu, Z.-R., Manganese-Enhanced MRI for Preclinical Evaluation of Retinal Degeneration Treatments. Investigative Ophthalmology & Visual Science 2015, 56 (8), 4936-4942. 151. Cideciyan, A. V., Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy. Progress in Retinal and Eye Research 2010, 29 (5), 398-427.
195 152. Lai, C.-M.; Yu, M. J. T.; Brankov, M.; Barnett, N. L.; Zhou, X.; Redmond, T. M.; Narfstrom, K.; Rakoczy, P. E., Recombinant adeno-associated virus type 2- mediated gene delivery into the Rpe65-/- knockout mouse eye results in limited rescue. Genetic Vaccines and Therapy 2004, 2 (1), 3. 153. Bemelmans, A.-P.; Kostic, C.; Crippa, S. V.; Hauswirth, W. W.; Lem, J.; Munier, F. L.; Seeliger, M. W.; Wenzel, A.; Arsenijevic, Y., Lentiviral Gene Transfer of Rpe65 Rescues Survival and Function of Cones in a Mouse Model of Leber Congenital Amaurosis. PLOS Medicine 2006, 3 (10), e347. 154. Smith, J.; Ward, D.; Michaelides, M.; Moore, A. T.; Simpson, S., New and emerging technologies for the treatment of inherited retinal diseases: a horizon scanning review. Eye 2015, 29, 1131. 155. Gujrati, M.; Vaidya, A. M.; Mack, M.; Snyder, D.; Malamas, A.; Lu, Z.-R., Targeted Dual pH-Sensitive Lipid ECO/siRNA Self-Assembly Nanoparticles Facilitate In Vivo Cytosolic sieIF4E Delivery and Overcome Paclitaxel Resistance in Breast Cancer Therapy. Advanced Healthcare Materials 2016, 5 (22), 2882-2895. 156. Allikmets, R.; Shroyer, N. F.; Singh, N.; Seddon, J. M.; Lewis, R. A.; Bernstein, P. S.; Peiffer, A.; Zabriskie, N. A.; Li, Y.; Hutchinson, A.; Dean, M.; Lupski, J. R.; Leppert, M., Mutation of the Stargardt Disease Gene (ABCR) in Age-Related Macular Degeneration. Science 1997, 277 (5333), 1805-1807. 157. Gabriele, T., Prospectives for Gene Therapy of Retinal Degenerations. Current Genomics 2012, 13 (5), 350-362. 158. Koenekoop, R. K., The gene for Stargardt disease, ABCA4, is a major retinal gene: a mini-review. Ophthalmic Genetics 2003, 24 (2), 75-80. 159. Boye, S. E.; Boye, S. L.; Lewin, A. S.; Hauswirth, W. W., A Comprehensive Review of Retinal Gene Therapy. Molecular Therapy 21 (3), 509- 519. 160. Hitoshi, N.; Ken-ichi, Y.; Jun-ichi, M., Efficient selection for high- expression transfectants with a novel eukaryotic vector. Gene 1991, 108 (2), 193- 199.
196 161. Zack, D. J.; Bennett, J.; Wang, Y.; Davenport, C.; Klaunberg, B.; Gearhart, J.; Nathans, J., Unusual topography of bovine rhodopsin promoter-IacZ fusion gene expression in transgenic mouse retinas. Neuron 1991, 6 (2), 187-199. 162. Matsuda, T.; Cepko, C. L., Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proceedings of the National Academy of Sciences 2004, 101 (1), 16-22. 163. Maeda, A.; Maeda, T.; Golczak, M.; Palczewski, K., Retinopathy in Mice Induced by Disrupted All-trans-retinal Clearance. Journal of Biological Chemistry 2008, 283 (39), 26684-26693. 164. Sun, D.; Sahu, B.; Gao, S.; Schur, R. M.; Vaidya, A. M.; Maeda, A.; Palczewski, K.; Lu, Z.-R., Targeted Multifunctional Lipid ECO Plasmid DNA Nanoparticles as Efficient Non-viral Gene Therapy for Leber’s Congenital Amaurosis. Molecular Therapy - Nucleic Acids 2017, 7, 42-52. 165. Radu, R. A.; Mata, N. L.; Bagla, A.; Travis, G. H., Light exposure stimulates formation of A2E oxiranes in a mouse model of Stargardt's macular degeneration. Proceedings of the National Academy of Sciences of the United States of America 2004, 101 (16), 5928-5933. 166. Sparrow, J. R.; Fishkin, N.; Zhou, J.; Cai, B.; Jang, Y. P.; Krane, S.; Itagaki, Y.; Nakanishi, K., A2E, a byproduct of the visual cycle. Vision Research 2003, 43 (28), 2983-2990. 167. Kennedy, C. J.; Rakoczy, P. E.; Constable, I. J., Lipofuscin of the retinal pigment epithelium: A review. Eye 1995, 9, 763. 168. Radu, R. A.; Mata, N. L.; Nusinowitz, S.; Liu, X.; Sieving, P. A.; Travis, G. H., Treatment with isotretinoin inhibits lipofuscin accumulation in a mouse model of recessive Stargardt's macular degeneration. Proceedings of the National Academy of Sciences 2003, 100 (8), 4742-4747. 169. Thomas, C. E.; Ehrhardt, A.; Kay, M. A., Progress and problems with the use of viral vectors for gene therapy. Nature Reviews Genetics 2003, 4, 346. 170. Auricchio, A.; Trapani, I.; Allikmets, R., Gene Therapy of ABCA4- Associated Diseases. Cold Spring Harbor Perspectives in Medicine 2015, 5 (5),
197 a017301. 171. Dong, B.; Nakai, H.; Xiao, W., Characterization of Genome Integrity for Oversized Recombinant AAV Vector. Molecular Therapy 2010, 18 (1), 87-92. 172. Duan, D.; Yue, Y.; Engelhardt, J. F., Expanding AAV Packaging Capacity with Trans-splicing or Overlapping Vectors: A Quantitative Comparison. Molecular Therapy 2001, 4 (4), 383-391. 173. Adijanto, J.; Naash, M. I., Nanoparticle-based technologies for retinal gene therapy. European Journal of Pharmaceutics and Biopharmaceutics 2015, 95, 353- 367. 174. Masland, R. H., The fundamental plan of the retina. Nature Neuroscience 2001, 4, 877. 175. Tanna, P.; Strauss, R. W.; Fujinami, K.; Michaelides, M., Stargardt disease: clinical features, molecular genetics, animal models and therapeutic options. The British Journal of Ophthalmology 2017, 101 (1), 25-30. 176. Molday, R. S.; Zhong, M.; Quazi, F., The role of the photoreceptor ABC transporter ABCA4 in lipid transport and Stargardt macular degeneration. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 2009, 1791 (7), 573-583. 177. Kong, J.; Kim, S. R.; Binley, K.; Pata, I.; Doi, K.; Mannik, J.; Zernant- Rajang, J.; Kan, O.; Iqball, S.; Naylor, S.; Sparrow, J. R.; Gouras, P.; Allikmets, R., Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy. Gene Therapy 2008, 15, 1311. 178. Qi, Y.; Dai, X.; Zhang, H.; He, Y.; Zhang, Y.; Han, J.; Zhu, P.; Zhang, Y.; Zheng, Q.; Li, X.; Zhao, C.; Pang, J., Trans-Corneal Subretinal Injection in Mice and Its Effect on the Function and Morphology of the Retina. PLOS ONE 2015, 10 (8), e0136523. 179. Horvath, P.; Barrangou, R., CRISPR/Cas, the Immune System of Bacteria and Archaea. Science 2010, 327 (5962), 167-170. 180. Cong, L.; Ran, F. A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P. D.; Wu, X.; Jiang, W.; Marraffini, L.; Zhang, F., Multiplex Genome Engineering Using
198 CRISPR/Cas Systems. Science 2013. 181. Christie, K. A.; Courtney, D. G.; DeDionisio, L. A.; Shern, C. C.; De Majumdar, S.; Mairs, L. C.; Nesbit, M. A.; Moore, C. B. T., Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders. Scientific Reports 2017, 7 (1), 16174. 182. Hung, S. S. C.; Chrysostomou, V.; Li, F.; Lim, J. K. H.; Wang, J.-H.; Powell, J. E.; Tu, L.; Daniszewski, M.; Lo, C.; Wong, R. C.; Crowston, J. G.; Pébay, A.; King, A. E.; Bui, B. V.; Liu, G.-S.; Hewitt, A. W., AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo. Investigative Ophthalmology & Visual Science 2016, 57 (7), 3470-3476. 183. Gori, J. L.; Hsu, P. D.; Maeder, M. L.; Shen, S.; Welstead, G. G.; Bumcrot, D., Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy. Human Gene Therapy 2015, 26 (7), 443-451. 184. Liu, C.; Zhang, L.; Liu, H.; Cheng, K., Delivery strategies of the CRISPR- Cas9 gene-editing system for therapeutic applications. Journal of Controlled Release 2017, 266, 17-26. 185. Platt, Randall J.; Chen, S.; Zhou, Y.; Yim, Michael J.; Swiech, L.; Kempton, Hannah R.; Dahlman, James E.; Parnas, O.; Eisenhaure, Thomas M.; Jovanovic, M.; Graham, Daniel B.; Jhunjhunwala, S.; Heidenreich, M.; Xavier, Ramnik J.; Langer, R.; Anderson, Daniel G.; Hacohen, N.; Regev, A.; Feng, G.; Sharp, Phillip A.; Zhang, F., CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Cell 2014, 159 (2), 440-455. 186. Sun, D.; Schur, R. M.; Lu, Z.-R., A novel nonviral gene delivery system for treating Leber's congenital amaurosis. Therapeutic Delivery 2017, 8 (10), 823- 826. 187. Mahato, R. I.; Rolland, A.; Tomlinson, E., Cationic Lipid-Based Gene Delivery Systems: Pharmaceutical Perspectives. Pharmaceutical Research 1997, 14 (7), 853-859. 188. Gujrati, M.; Vaidya, A.; Lu, Z.-R., Multifunctional pH-Sensitive Amino Lipids for siRNA Delivery. Bioconjugate Chemistry 2016, 27 (1), 19-35.
199 189. Malamas, A. S.; Gujrati, M.; Kummitha, C. M.; Xu, R.; Lu, Z. R., Design and evaluation of new pH-sensitive amphiphilic cationic lipids for siRNA delivery. J. Control. Release 2013, 171 (3), 296-307. 190. Boon, J. M.; Lambert, T. N.; Smith, B. D.; Beatty, A. M.; Ugrinova, V.; Brown, S. N., Structure/Activity Study of Tris(2-aminoethyl)amine-Derived Translocases for Phosphatidylcholine. J. Org. Chem. 2002, 67 (7), 2168-2174. 191. (a) Okuro, K.; Kinbara, K.; Tsumoto, K.; Ishii, N.; Aida, T., Molecular glues carrying multiple guanidinium ion pendants via an oligoether spacer: stabilization of microtubules against depolymerization. J. Am. Chem. Soc. 2009, 131 (5), 1626-7; (b) Sun, Z.; Gilles, A.; Kocsis, I.; Legrand, Y. M.; Petit, E.; Barboiu, M., Squalyl Crown Ether Self-Assembled Conjugates: An Example of Highly Selective Artificial K Channels. Chem. Eur. J. 2016, 22 (6), 2158-2164; (c) Sun, Z. H.; Albrecht, M.; Raabe, G.; Pan, F. F.; Rauber, C., Solvent-dependent enthalpic versus entropic anion binding by biaryl substituted quinoline based anion receptors. The journal of physical chemistry. B 2015, 119 (1), 301-6. 192. Golczak, M.; Maeda, A.; Bereta, G.; Maeda, T.; Kiser, P. D.; Hunzelmann, S.; von Lintig, J.; Blaner, W. S.; Palczewski, K., Metabolic Basis of Visual Cycle Inhibition by Retinoid and Nonretinoid Compounds in the Vertebrate Retina. The Journal of Biological Chemistry 2008, 283 (15), 9543-9554. 193. Pulido-Quetglas, C.; Aparicio-Prat, E.; Arnan, C.; Polidori, T.; Hermoso, T.; Palumbo, E.; Ponomarenko, J.; Guigo, R.; Johnson, R., Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion. PLOS Computational Biology 2017, 13 (3), e1005341. 194. Vaidya, A.; Mao, Z.; Tian, X.; Spencer, B.; Seluanov, A.; Gorbunova, V., Knock-In Reporter Mice Demonstrate that DNA Repair by Non-homologous End Joining Declines with Age. PLOS Genetics 2014, 10 (7), e1004511. 195. Chen, X.; Xu, F.; Zhu, C.; Ji, J.; Zhou, X.; Feng, X.; Guang, S., Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans. Scientific Reports 2014, 4, 7581. 196. Vidigal, J. A.; Ventura, A., Rapid and efficient one-step generation of
200 paired gRNA CRISPR-Cas9 libraries. Nature Communications 2015, 6, 8083. 197. Ho, T.-T.; Zhou, N.; Huang, J.; Koirala, P.; Xu, M.; Fung, R.; Wu, F.; Mo, Y. -Y., Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines. Nucleic Acids Research 2015, 43 (3), e17-e17. 198. Pattanayak, V.; Lin, S.; Guilinger, J. P.; Ma, E.; Doudna, J. A.; Liu, D. R., High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology 2013, 31, 839. 199. Fu, Y.; Foden, J. A.; Khayter, C.; Maeder, M. L.; Reyon, D.; Joung, J. K.; Sander, J. D., High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology 2013, 31, 822. 200. S̆misterová, J.; Wagenaar, A.; Stuart, M. C. A.; Polushkin, E.; ten Brinke, G.; Hulst, R.; Engberts, J. B. F. N.; Hoekstra, D., Molecular Shape of the Cationic Lipid Controls the Structure of Cationic Lipid/Dioleylphosphatidylethanolamine- DNA Complexes and the Efficiency of Gene Delivery. Journal of Biological Chemistry 2001, 276 (50), 47615-47622. 201. Midoux, P.; Monsigny, M., Efficient Gene Transfer by Histidylated Polylysine/pDNA Complexes. Bioconjugate Chemistry 1999, 10 (3), 406-411. 202. Shigeta, K.; Kawakami, S.; Higuchi, Y.; Okuda, T.; Yagi, H.; Yamashita, F.; Hashida, M., Novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte-selective gene transfer in human hepatoma HepG2 cells. Journal of Controlled Release 2007, 118 (2), 262-270. 203. Yang, S. R.; Lee, H. J.; Kim, J.-D., Histidine-conjugated poly(amino acid) derivatives for the novel endosomolytic delivery carrier of doxorubicin. Journal of Controlled Release 2006, 114 (1), 60-68. 204. Midoux, P.; Pichon, C.; Yaouanc, J.-J.; Jaffrès, P.-A., Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers. British Journal of Pharmacology 2009, 157 (2), 166-178. 205. Toncheva, V.; Wolfert, M. A.; Dash, P. R.; Oupicky, D.; Ulbrich, K.; Seymour, L. W.; Schacht, E. H., Novel vectors for gene delivery formed by self-
201 assembly of DNA with poly(l-lysine) grafted with hydrophilic polymers. Biochimica et Biophysica Acta (BBA) - General Subjects 1998, 1380 (3), 354-368. 206. Choi, J. S.; Lee, E. J.; Choi, Y. H.; Jeong, Y. J.; Park, J. S., Poly(ethylene glycol)-block-poly(l-lysine) Dendrimer: Novel Linear Polymer/Dendrimer Block Copolymer Forming a Spherical Water-Soluble Polyionic Complex with DNA. Bioconjugate Chemistry 1999, 10 (1), 62-65. 207. Singh, R.; Pantarotto, D.; McCarthy, D.; Chaloin, O.; Hoebeke, J.; Partidos, C. D.; Briand, J.-P.; Prato, M.; Bianco, A.; Kostarelos, K., Binding and Condensation of Plasmid DNA onto Functionalized Carbon Nanotubes: Toward the Construction of Nanotube-Based Gene Delivery Vectors. Journal of the American Chemical Society 2005, 127 (12), 4388-4396. 208. Sudimack, J.; Lee, R. J., Targeted drug delivery via the folate receptor. Advanced Drug Delivery Reviews 2000, 41 (2), 147-162. 209. Torchilin, V. P., Targeted polymeric micelles for delivery of poorly soluble drugs. Cellular and Molecular Life Sciences CMLS 2004, 61 (19), 2549-2559. 210. Qian, Z. M.; Li, H.; Sun, H.; Ho, K., Targeted Drug Delivery via the Transferrin Receptor-Mediated Endocytosis Pathway. Pharmacological Reviews 2002, 54 (4), 561-587. 211. Golczak, M.; Kuksa, V.; Maeda, T.; Moise, A. R.; Palczewski, K., Positively charged retinoids are potent and selective inhibitors of the trans-cis isomerization in the retinoid (visual) cycle. Proceedings of the National Academy of Sciences of the United States of America 2005, 102 (23), 8162-8167. 212. Petrukhin, K., Pharmacological inhibition of lipofuscin accumulation in the retina as a therapeutic strategy for dry AMD treatment. Drug discovery today. Therapeutic strategies 2013, 10 (1), e11-e20. 213. Leung, E.; Landa, G., Update on current and future novel therapies for dry age-related macular degeneration. Expert Review of Clinical Pharmacology 2013, 6 (5), 565-579. 214. Kubota, R.; Boman, N. L.; David, R.; Mallikaarjun, S.; Patil, S.; Birch, D., SAFETY AND EFFECT ON ROD FUNCTION OF ACU-4429, A NOVEL
202 SMALL-MOLECULE VISUAL CYCLE MODULATOR. RETINA 2012, 32 (1), 183-188. 215. Lee, M.; Kim, S. W., Polyethylene Glycol-Conjugated Copolymers for Plasmid DNA Delivery. Pharmaceutical Research 2005, 22 (1), 1-10. 216. Kawano, K.; Maitani, Y., Effects of Polyethylene Glycol Spacer Length and Ligand Density on Folate Receptor Targeting of Liposomal Doxorubicin In Vitro. Journal of Drug Delivery 2011, 2011. 217. Gujrati, M.; Mack, M.; Snyder, D.; Vaidya, A. M.; Malamas, A.; Lu, Z.-R., Targeted dual pH-sensitive lipid siRNA self-assembly nanoparticles facilitate in vivo cytosolic siRNA delivery in tumor and overcome drug resistance by silencing eIF4E in breast cancer therapy. Advanced healthcare materials 2016, 5 (22), 2882- 2895. 218. Han, Z.; Koirala, A.; Makkia, R.; Cooper, M. J.; Naash, M. I., Direct gene transfer with compacted DNA nanoparticles in retinal pigment epithelial cells: expression, repeat delivery and lack of toxicity. Nanomedicine 2012, 7 (4), 521-539. 219. Hamada, H.; Seidman, M.; Howard, B. H.; Gorman, C. M., Enhanced gene expression by the poly(dT-dG).poly(dC-dA) sequence. Molecular and Cellular Biology 1984, 4 (12), 2622-2630. 220. Robertson, H. M.; Preston, C. R.; Phillis, R. W.; Johnson-Schlitz, D. M.; Benz, W. K.; Engels, W. R., A stable genomic source of P element transposase in Drosophila melanogaster. Genetics 1988, 118 (3), 461-470. 221. Sonane, M.; Goyal, R.; Chowdhuri, D. K.; Ram, K. R.; Gupta, K. C., Enhanced efficiency of P-element mediated transgenesis in Drosophila: Microinjection of DNA complexed with nanomaterial. Scientific Reports 2013, 3, 3408. 222. Jackson, D. A.; Juranek, S.; Lipps, H. J., Designing Nonviral Vectors for Efficient Gene Transfer and Long-Term Gene Expression. Molecular Therapy 2006, 14 (5), 613-626. 223. Göpferich, A., Mechanisms of polymer degradation and erosion A2 - Williams, D.F. In The Biomaterials: Silver Jubilee Compendium, Elsevier Science:
203 Oxford, 1996; pp 117-128. 224. Tehrani-Bagha, A. R.; Oskarsson, H.; van Ginkel, C. G.; Holmberg, K., Cationic ester-containing gemini surfactants: Chemical hydrolysis and biodegradation. Journal of Colloid and Interface Science 2007, 312 (2), 444-452. 225. Mueller, R.-J., Biological degradation of synthetic polyesters—Enzymes as potential catalysts for polyester recycling. Process Biochemistry 2006, 41 (10), 2124-2128. 226. Maguire , A. M.; Simonelli , F.; Pierce , E. A.; Pugh , E. N. J.; Mingozzi , F.; Bennicelli , J.; Banfi , S.; Marshall , K. A.; Testa , F.; Surace , E. M.; Rossi , S.; Lyubarsky , A.; Arruda , V. R.; Konkle , B.; Stone , E.; Sun , J.; Jacobs , J.; Dell'Osso , L.; Hertle , R.; Ma , J.-x.; Redmond , T. M.; Zhu , X.; Hauck , B.; Zelenaia , O.; Shindler , K. S.; Maguire , M. G.; Wright , J. F.; Volpe , N. J.; McDonnell , J. W.; Auricchio , A.; High , K. A.; Bennett , J., Safety and Efficacy of Gene Transfer for Leber's Congenital Amaurosis. New England Journal of Medicine 2008, 358 (21), 2240-2248. 227. Kay, C. N.; Ryals, R. C.; Aslanidi, G. V.; Min, S. H.; Ruan, Q.; Sun, J.; Dyka, F. M.; Kasuga, D.; Ayala, A. E.; Van Vliet, K.; Agbandje-McKenna, M.; Hauswirth, W. W.; Boye, S. L.; Boye, S. E., Targeting Photoreceptors via Intravitreal Delivery Using Novel, Capsid-Mutated AAV Vectors. PLOS ONE 2013, 8 (4), e62097.
204