Gene Therapy

Part I: Technologies & Markets

by

Prof. K. K. Jain

MD, FRACS, FFPM

Jain PharmaBiotech Basel, Switzerland

May 2020

A Jain PharmaBiotech Report

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A U T H O R ' S B I O G R A P H Y

Professor K. K. Jain is a neurologist/neurosurgeon with specialist qualifications including Fellowships of the Royal Colleges of Surgeons in Australia and Canada. He has trained, practiced and held academic positions in several countries including Switzerland, Germany Canada and USA. After his retirement from neurosurgery, he is working in the biotechnology/biopharmaceuticals industry and is a Fellow of the Faculty of Pharmaceutical Medicine of the Royal College of Physicians of UK. Currently, he is a consultant at Jain PharmaBiotech.

Prof. Jain has edited "Drug Delivery Systems" (1st edn 2008, 2nd edn 2014, 3rd edn 2019) and "Drug Delivery to the Central Nervous System" (2010), both published by Humana/Springer. His other recent books include "Handbook of Nanomedicine" (Springer 2008; Chinese edition, Peking University Press 2011; 3rd ed Springer 2017), “Textbook of Personalized Medicine” (Springer 2009; Japanese ed 2012; 2nd ed Springer, 2015, 3rd ed 2020, in preparation), "Handbook of Biomarkers" (Springer 2010; Chinese edition. Chemical Industry Press 2016, 2nd ed Springer 2017) and "Handbook of Neuroprotection" (Springer 2011, 2nd edn 2019). Books that contain chapters on gene therapy include: Applications of Biotechnology in Cardiovascular Therapeutics (Springer 2011), Applications of Biotechnology in Neurology (Springer 2013), and Applications of Biotechnology in Oncology (Springer 2014). Prof. Jain has also edited “Applied Neurogenomics” (Springer 2015).

A B O U T T H I S R E P O R T

Prof. Jain wrote the first commercial report on Gene Therapy in 1995, which was published by PJB Publication, London. This was followed by reports on Gene Therapy Vectors and Cancer Gene Therapy (1996). A report on Gene Therapy of Neurologal Disorders was published by Decision Resources in 1997. In 1998, Prof. Jain wrote a Textbook of Gene Therapy, which was the first book on this subject to be translated into the Chinese language in 2000. A book on gene therapy companies was published in 2000 by John Wiley & Sons and continued to be updated online at Wiley's web site until 2003. In 2004, when the copyright reverted to Prof. Jain, it became part II of the Gene Therapy report. This report supercedes and updates all the previous books as well as reports.

May 2020 Copyright © 2020 by

Jain PharmaBiotech Bläsiring 7 CH-4057 Basel Switzerland

Tel & Fax: +4161-6924461 Email: info@pharmabiotech.ch Web site: http://pharmabiotech.ch/

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, or otherwise without the prior written permission of the Publisher. This report may not be lent, resold or otherwise traded in any manner without the consent of the Publisher. While all reasonable steps have been taken to ensure the accuracy of the information presented, the Publisher cannot accept responsibility for inadvertent errors or omissions.

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T A B L E O F C O N T E N T S

0. Executive Summary .............................................................................. 23

1. Introduction ......................................................................................... 25 Definitions ......................................................................................................................... 25 Historical evolution of gene therapy .................................................................................. 25 Relation of gene therapy to other biotechnologies............................................................. 27 Molecular biological basics for gene therapy ..................................................................... 28

Genome .......................................................................................................................... 28 DNA ............................................................................................................................... 29 RNA ............................................................................................................................... 29

RNA splicing ............................................................................................................... 30 Genes ............................................................................................................................. 31

Silent gene mutations .................................................................................................. 31 Gene regulation ............................................................................................................... 32 Gene expression .............................................................................................................. 32 ENCODE ......................................................................................................................... 33 Chromosomes ................................................................................................................. 34 Telomeres ....................................................................................................................... 35 Mitochondria ................................................................................................................... 35 Proteins .......................................................................................................................... 36

2. Gene Therapy Technologies .................................................................. 37 Classification of gene therapy techniques .......................................................................... 37 Ex vivo and in vivo gene therapy ....................................................................................... 38

Ex vivo gene therapy ........................................................................................................ 39 In vivo gene therapy ........................................................................................................ 39

Physical methods of gene transfer ..................................................................................... 39 Electroporation ................................................................................................................ 40 Applications of electroporation ........................................................................................... 40

Clinical applications of electroporation ........................................................................... 41 Advantages of electroporation ...................................................................................... 42 Limitations of electroporation ....................................................................................... 42

Hydrodynamic ................................................................................................................. 42 Microinjection .................................................................................................................. 43 Particle bombardment ...................................................................................................... 43 Ultrasound-mediated transfection ...................................................................................... 45 Molecular vibration ........................................................................................................... 46 Application of pulsed magnetic field and superparamagnetic nanoparticles .............................. 46 Gene transfection using laser irradiation ............................................................................. 46 Photochemical transfection ................................................................................................ 47

Chemical methods of gene transfer ................................................................................... 47 Vectors for gene therapy ................................................................................................... 47

Basic considerations ......................................................................................................... 47 Use of genes as pharmaceuticals ....................................................................................... 48 The ideal vector for gene therapy ....................................................................................... 48 Viral vectors .................................................................................................................... 49

Adenovirus vectors ...................................................................................................... 49 Adeno-associated virus vectors ..................................................................................... 51 Alphavirus vectors....................................................................................................... 54 Baculovirus vectors ..................................................................................................... 55 Foamy virus vectors .................................................................................................... 55 Herpes simplex virus vectors ........................................................................................ 56 Lentiviral vectors ........................................................................................................ 58 Multicistronic retroviral vectors ..................................................................................... 59 Retroviral vectors ........................................................................................................ 59 Oncogenic potential of retroviral vectors ........................................................................ 61 Quantification of viral vectors ....................................................................................... 61 Future of viral vectors ................................................................................................. 62 Companies using viral vectors ...................................................................................... 62

Nonviral vectors for gene therapy ...................................................................................... 63 Anionic lipid-DNA complexes ........................................................................................ 64 Cationic lipid-DNA complexes ....................................................................................... 65 Effects of shape of DNA molecules on delivery with nonviral vectors .................................. 65 Electrostatic modifications of surface to improve gene delivery ......................................... 65 Liposomes for gene therapy ......................................................................................... 65 Liposome-nucleic acid complexes .................................................................................. 67 Liposome-HVJ complex ................................................................................................ 67

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Transposons DNA vectors............................................................................................. 68 Polycation-DNA complexes (polyplexes) ......................................................................... 69 Plasmid DNA vs minicircle DNA ..................................................................................... 70 Polymer vectors for gene therapy.................................................................................. 70 Synthetic biology and DNA vectors ................................................................................ 71 Synthetic peptide complexes ........................................................................................ 71 Future of nonviral vs viral vectors ................................................................................. 72

Nanobiotechnology for gene therapy .................................................................................. 72 Antisense nanoparticles ............................................................................................... 72 Dendrimers ................................................................................................................ 73 Cochleates ................................................................................................................. 73 Calcium phosphate nanoparticles as nonviral vectors ....................................................... 73 DNA nanoparticles as nonviral vectors ........................................................................... 73 Gelatin nanoparticles for gene delivery .......................................................................... 74 Lipid nanoparticles for nucleic acid delivery .................................................................... 74 Nanoparticles as nonviral vectors for gene therapy .......................................................... 74 Nanoparticles with virus-like function as gene therapy vectors .......................................... 75 Nanobiolistics for nucleic acid delivery ........................................................................... 75 Nonionic polymeric micelles for oral gene delivery ........................................................... 76 Silica nanoparticles as a nonviral vector for gene delivery ................................................ 76 Virus-like particles as nonviral vector ............................................................................ 76

Receptor-mediated endocytosis ......................................................................................... 76 Artificial viral vectors ........................................................................................................ 78 Directed evolution of AAV to create efficient gene delivery vectors ......................................... 78 Bacterial ghosts as DNA delivery systems ........................................................................... 78 Bacteria plus nanoparticles for gene delivery into cells .......................................................... 79 Chromosome-based vectors for gene therapy ...................................................................... 80

Mammalian artificial chromosomes (MACs) ..................................................................... 80 Human artificial chromosomes (HACs) ........................................................................... 80 ΦC31 integrase system ................................................................................................ 81

Companies using nonviral vectors ...................................................................................... 81 Concluding remarks about vectors ..................................................................................... 82

Gene repair and replacement ............................................................................................. 83 Gene repair by single-stranded oligonucleotides................................................................... 83 History and current status of chimeraplasty ........................................................................ 84

Genome editing.................................................................................................................. 84 Gene editing by zinc finger nucleases ................................................................................. 84 Gene editing by using short synthetic oligonucleotides .......................................................... 85 Genome engineering by using TALENs ................................................................................ 86 Genome editing by homologous recombination .................................................................... 86

GeneRide technology for genome editing ....................................................................... 86 Gene editing by homing endonucleases .............................................................................. 87

ARCUS genome editing technology ................................................................................ 87 Genome editing by using CRISPR system ............................................................................ 88

A method for generating genome-wide gRNA libraries for CRISPR ..................................... 89 CRISPR-genome organization system ............................................................................ 89 CRISPR vs other gene editing tools ............................................................................... 90 CRISPR-FRT ............................................................................................................... 90 CRISPR for studying human embryogenesis ................................................................... 91 CRISPER system for creating animal models of human diseases ........................................ 91 CRISPR/Cas9 technology for drug discovery ................................................................... 91 CRISPR/Cas9-mediated epigenetic editing system ........................................................... 92 Expanding the range of Cas9s ...................................................................................... 92 Inducible CRISPR/Cas9 for multiple gene targeting ......................................................... 93 Lipid nanoparticles for delivery of CRISPR-Cas9 for genome-editing .................................. 93 Nanocapsules for delivery of CRISPR-Cas9 for genome editing .......................................... 93 Nano-CRISPR ............................................................................................................. 93 Photoactivatable CRISPR-Cas9 ...................................................................................... 94 Protein delivery and genome editing .............................................................................. 94

Potential clinical applications of CRISPR systems .................................................................. 94 Autosomal dominant retinitis pigmentosa ....................................................................... 95 Clinical trial of CRISPR in human cancer......................................................................... 95 Correction of a gene mutation in the human embryo ....................................................... 96 Glaucoma .................................................................................................................. 96 PERV blockage by CRISPR to facilitate xenotransplantation .............................................. 96 RNA editing with CRISPR.............................................................................................. 97 Use of CRISPR for β-hemoglobinopathies ....................................................................... 98 Use of CRISPR to restore hereditary hearing loss ............................................................ 98 Complications and limitations of CRISPR/Cas9 technology ................................................ 99 Companies developing CRISPR technology ................................................................... 100

Clinical studies of gene editing ......................................................................................... 101

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Targeted genome editing by artificial nucleases ................................................................. 102 Ex vivo gene editing ....................................................................................................... 102 Future gene editing tools ................................................................................................ 102 DNA medicines based on gene editing .............................................................................. 102 Comparison of gene editing with techniques for suppression of gene mutations ..................... 103

Base editing of the genome ............................................................................................. 105 Base editing by protein engineering.................................................................................. 105 Adenine base editors for genomic DNA ............................................................................. 105

Messenger RNA gene therapy .......................................................................................... 106 Spliceosome mediated RNA trans-splicing ......................................................................... 106

Cell-mediated gene therapy ............................................................................................. 106 Fibroblasts .................................................................................................................... 107 Skeletal muscle cells ...................................................................................................... 107 Vascular smooth muscle cells .......................................................................................... 108 Keratinocytes ................................................................................................................ 108 Hepatocytes .................................................................................................................. 108 Lymphocytes ................................................................................................................. 109

Regulating protein delivery by genetically encoded lymphocytes ..................................... 109 Implantation of microencapulated genetically modified cells ................................................ 109 Stem cell gene therapy ................................................................................................... 110

Combination of gene therapy with therapeutic cloning ................................................... 110 Expansion of transduced HSCs in vivo ......................................................................... 111 Gene delivery to stem cells by artificial chromosome expression ..................................... 111 Improving delivery of genes to stem cells .................................................................... 111 In utero gene therapy using stem cells ........................................................................ 111 Lentiviral vectors for gene transfer to marrow stem cells ............................................... 112 Linker based sperm-mediated gene transfer technology ................................................. 112 Mesenchymal stem cells for gene therapy .................................................................... 112 Microporation for transfection of MSCs ......................................................................... 112 Preventing immune rejection of transplanted stem cells ................................................. 113 Therapeutic applications for hematopoietic stem cell gene transfer .................................. 113 Transdermal gene therapy for drug addiction ............................................................... 113 The future of hematopoietic stem cell gene therapy ...................................................... 113

Chimeric antigen receptor T cells in relation to gene therapy .......................................... 114 Basics of CAR-T cell ........................................................................................................ 114 Basis of anticancer effect of CAR-T cells ............................................................................ 114

Smart T-cells ............................................................................................................ 114 CAR-T cell manufacture .................................................................................................. 115 Companies developing CAR-T cell therapy ......................................................................... 115 CAR NK cells derived from human iPSCs ........................................................................... 117 Genome editing of CAR-T cells ......................................................................................... 118 Safety and efficacy of CAR-T cell therapy .......................................................................... 118

Cytokine release syndrome ........................................................................................ 119 Precautions for CAR-T cell therapy .............................................................................. 120

Role of genetically modified bacteria in gene therapy ..................................................... 120 Routes of administration for gene therapy ...................................................................... 121

Direct injection of naked DNA .......................................................................................... 121 Intramuscular injection ................................................................................................... 122 Intravenous DNA injection .............................................................................................. 122 Intraarterial delivery ...................................................................................................... 122 Companies with gene delivery devices/technologies ........................................................... 122

Targeted gene therapy ..................................................................................................... 123 Targeted integration ....................................................................................................... 124 Bacteriophage integrase system for site-specific gene delivery ............................................ 124 Controlled-release delivery of DNA ................................................................................... 125

Controlled gene therapy .................................................................................................. 125 Controlled delivery of genetic material .............................................................................. 125 Controlled induction of gene expression ............................................................................ 126 Drug-inducible systems for control of gene expression ........................................................ 126 Timed activation of gene therapy by a circuit based on signaling network ............................. 127 Small molecules for post-transcriptional regulation of gene expression ................................. 127 Light Activated Gene Therapy .......................................................................................... 127 Spatial control of gene expression via local hyperthermia .................................................... 128 Companies with regulated /targeted gene therapy ............................................................. 128

Gene marking .................................................................................................................. 129 Germline gene therapy .................................................................................................... 129

Potential applications of human germline genome modification ............................................ 129 Pros and cons of human germline genome modification ...................................................... 130

Role of gene transfer in antibody therapy ........................................................................ 131 Genetically engineered vaccines ...................................................................................... 132

DNA vaccines ................................................................................................................ 132

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DNA inoculation technology ........................................................................................ 132 Methods for enhancing the potency of DNA vaccines ..................................................... 133 Advantages of DNA vaccines ...................................................................................... 133

Vaccine vectors ............................................................................................................. 134 Challenges and limitations of genetically engineered vaccines .............................................. 134 RNA vaccines................................................................................................................. 135

Nonviral delivery of self-amplifying RNA vaccine ........................................................... 135 Vaccines based on reverse genetics .................................................................................. 135

Technologies for gene suppression .................................................................................. 135 Antisense oligonucleotides .............................................................................................. 136 Transcription factor decoys ............................................................................................. 136 Aptamers ...................................................................................................................... 137 Ribozymes .................................................................................................................... 137 Peptide nucleic acid ........................................................................................................ 137

Intracellular delivery of PNAs ...................................................................................... 138 Locked nucleic acid ........................................................................................................ 138

Zorro-LNA ................................................................................................................ 138 Gene silencing ............................................................................................................... 139 Post-transcriptional gene silencing ................................................................................... 139 Definitions and terminology of RNAi ................................................................................. 139 RNAi mechanisms .......................................................................................................... 139 Inhibition of gene expression by antigene RNA .................................................................. 141 RNAi gene therapy ......................................................................................................... 141 microRNA gene therapy .................................................................................................. 141 Viral vectors for RNAi and miRNA gene therapy ................................................................. 141

Application of molecular diagnostic methods in gene therapy ......................................... 143 Use of PCR to study biodistribution of gene therapy vector .................................................. 143 PCR for verification of the transcription of DNA .................................................................. 143 In situ PCR for direct quantification of gene transfer into cells ............................................. 143 Detection of retroviruses by reverse transcriptase (RT)-PCR ................................................ 144 Confirmation of viral vector integration ............................................................................. 144 Monitoring of gene expression ......................................................................................... 144

Monitoring of gene expression by green fluorescent protein ............................................ 144 Monitoring in vivo gene expression by molecular imaging ............................................... 145 Advantages of gene therapy compared with protein therapy ........................................... 145

Formulation, transport and storage of materials for gene therapy .................................. 145

3. Clinical Applications of Gene Therapy ................................................. 147 Introduction .................................................................................................................... 147 Aging-related disorders ................................................................................................... 147

Telomerase gene therapy in aging ................................................................................... 147 Combination gene therapy for treatment of multiple age-related diseases ............................. 147

Bone and joint disorders .................................................................................................. 148 Bone fractures ............................................................................................................... 148 Gene therapy for intervertebral disc degeneration .............................................................. 149 Spinal fusion ................................................................................................................. 149 Osteogenesis imperfecta ................................................................................................. 150 Rheumatoid arthritis ....................................................................................................... 150

Local or systemic treatment ....................................................................................... 151 In vivo or ex vivo gene therapy of RA .......................................................................... 151 Clinical trials of gene therapy for rheumatoid arthritis .................................................... 152

Gene therapy for osteoarthritis ........................................................................................ 153 Gene therapy strategies for osteoarthritis .................................................................... 153 Clinical trials of gene therapy for osteoarthritis ............................................................. 154

Sports injuries ............................................................................................................... 154 Repair of articular cartilage defects ............................................................................. 155

Regeneration and replacement of bone by gene therapy ..................................................... 155 Bacterial infections .......................................................................................................... 156

Antisense approach to bacterial infections ......................................................................... 156 Dentistry .......................................................................................................................... 157

Tissue engineering in dental implant defects ..................................................................... 157 Endocrine and metabolic disorders .................................................................................. 157

Introduction .................................................................................................................. 157 Gene therapy of obesity .................................................................................................. 158

AAV vector-mediated delivery of GDNF for obesity ........................................................ 158 Oligopeptide for targeted nonviral gene delivery to adipocytes ........................................ 158 Viral vector-mediated leptin gene therapy .................................................................... 159

Diabetes mellitus ........................................................................................................... 159 Methods of gene therapy of diabetes mellitus .................................................................... 160

Viral vector-mediated gene transfer in diabetes ............................................................ 160 Endogenous reprogramming of α cells into cells ......................................................... 161

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Gene delivery with ultrasonic microbubble destruction technology ................................... 161 Genetically engineered cells for diabetes mellitus .......................................................... 162 Genetically altered liver cells ...................................................................................... 162 Genetically modified stem cells ................................................................................... 162 Genetically engineered dendritic cells .......................................................................... 163 Glucokinase and insulin co-expression ......................................................................... 163 Leptin gene therapy .................................................................................................. 163 Concluding remarks about cell and gene therapy of diabetes .......................................... 163

Gene therapy of growth-hormone deficiency ..................................................................... 164 Gastrointestinal disorders................................................................................................ 165

Introduction .................................................................................................................. 165 Methods of gene transfer to the gastrointestinal tract ......................................................... 165

Direct delivery of genes ............................................................................................. 165 Naked plasmid DNA into the submucosa ...................................................................... 166 Viral vectors ............................................................................................................. 166 Receptor-mediated endocytosis .................................................................................. 166

Indications for gastrointestinal gene therapy ..................................................................... 166 Gene therapy for inflammatory disorders of the bowel ................................................... 167

Gene transfer to the salivary glands ................................................................................. 167 Potential clinical applications of salivary gene therapy ................................................... 168

Hematology ..................................................................................................................... 168 Hemophilias .................................................................................................................. 169

Gene therapy of hemophilia ....................................................................................... 169 Hemophilia A ............................................................................................................ 170 Hemophilia B ............................................................................................................ 171 Concluding remarks about gene therapy of hemophilias ................................................. 173

Hemoglobinopathies ....................................................................................................... 174 Gene therapy for β-thalassemia .................................................................................. 174 Gene therapy for sickle cell disease ............................................................................. 176 HSC-targeted gene therapy for SCD ............................................................................ 176 Gene therapy and RNAi for SCD based on stem cells ..................................................... 176 Gene editing in sickle cell disease ............................................................................... 177 Gene editing using ZFN as treatment for both SCD and β-thalassemia ............................. 177 Gene editing using CRISPR-Cas9 for both SCD and β-thalassemia ................................... 178 Gene therapy of Fanconi's anemia ............................................................................... 178

Acquired hematopoietic disorders ..................................................................................... 179 Chronic acquired anemias .......................................................................................... 179 Neutropenia ............................................................................................................. 179 Thrombocytopenia .................................................................................................... 180

Concluding remarks about gene therapy of hemoglobinopathies .......................................... 181 Companies involved in gene thery of hematological disorders .............................................. 181

In utero/fetal gene therapy ............................................................................................. 181 Fetal gene transfer techniques ......................................................................................... 182 Animal models of fetal gene therapy ................................................................................ 183 Potential applications of fetal gene therapy ....................................................................... 183

Fetal gene therapy for cystic fibrosis ........................................................................... 183 Fetal intestinal gene therapy ...................................................................................... 184 Gene therapy for fetal growth restriction ...................................................................... 184

Hearing disorders ............................................................................................................ 184 Potential of gene therapy ................................................................................................ 185 Vectors for gene therapy of hearing disorders .................................................................... 185 Auditory hair cell replacement and hearing improvement by gene therapy ............................ 186

Kidney diseases ............................................................................................................... 186 End-stage renal disease .................................................................................................. 186 Methods of gene delivery to the kidney ............................................................................. 187

Bone marrow stem cells for renal disease .................................................................... 187 Gene transfer into kidney by viral vectors .................................................................... 188 Gene transfer into the glomerulus by HVJ-liposome ....................................................... 188 Gene transfer to tubules with cationic polymer polyethylenimine ..................................... 188 Liposome-mediated gene transfer into the tubules ........................................................ 188 Mesangial cell therapy ............................................................................................... 189 Non-viral gene transfer to the kidneys ......................................................................... 189

Gene therapy in animal experimental models of renal disease ............................................. 189 Genetic manipulations of the embryonic kidney ................................................................. 190 Antisense intervention in glomerulonephritis ..................................................................... 190 Gene therapy for renal fibrosis ........................................................................................ 190 Use of genetically engineered cells for uremia due to renal failure ........................................ 191 Concluding remarks ....................................................................................................... 191

Liver disorders ................................................................................................................. 191 Techniques of gene delivery to liver ................................................................................. 192

Direct injection of DNA into liver ................................................................................. 193

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Local gene delivery by isolated organ perfusion ............................................................ 193 Liposome-mediated direct gene transfer ...................................................................... 193 Retroviral vector for gene transfer to liver .................................................................... 193 Adenoviral vectors for gene transfer to liver ................................................................. 193 Receptor-mediated approach ...................................................................................... 194

Cell therapy for liver disorders ......................................................................................... 194 Transplantation of genetically modified hepatocytes ...................................................... 194 Genetically modified hematopoietic stem cells .............................................................. 195 Gene therapy by ex vivo transduced liver progenitor cells .............................................. 195

Gene therapy of genetic diseases affecting the liver ........................................................... 195 Crigler-Najjar syndrome ............................................................................................ 195 Hereditary tyrosinemia type I (HT1) ............................................................................ 196 Hereditary tyrosinemia type 3 .................................................................................... 196 Wilson’s disease ........................................................................................................ 197

Gene therapy of acquired diseases affecting the liver ......................................................... 197 Cirrhosis of liver ....................................................................................................... 197

Ophthalmic disorders ....................................................................................................... 197 Introduction to gene therapy of ophthalmic disorders ......................................................... 197 Methods of gene therapy for ophthalmic disorders ............................................................. 198

Delivery of gene therapy by intravitreal injection .......................................................... 199 DNA nanoparticles for nonviral gene transfer to the eye ................................................. 200 Optogenetic gene therapy for blindness due to retinal degeneration ................................ 200

Age-related macular degeneration ................................................................................... 201 Inherited disorders of optic nerve affecting vision .............................................................. 202

Leber hereditary optic neuropathy ............................................................................... 202 Gene therapy for LHON .............................................................................................. 203

Inherited retinal degenerations ........................................................................................ 203 Choroideremia .......................................................................................................... 205 Leber congenital amaurosis ........................................................................................ 205 Monogenic macular degeneration due to mutations in the BEST1 gene ............................ 207 Retinitis pigmentosa .................................................................................................. 207 Stargardt disease ...................................................................................................... 209

Other inherited disorders affecting vision .......................................................................... 209 Color blindness ......................................................................................................... 209 Usher syndrome ....................................................................................................... 210 X-linked retinoschisis ................................................................................................. 211

Diabetic retinopathy ....................................................................................................... 211 Future of gene therapy of inherited retinal degenerations ................................................... 212

Combining cell and gene therapies for retinal disorders.................................................. 212 Prevention of complications associated with eye surgery ..................................................... 212

Prevention of proliferative retinopathy by gene therapy ................................................. 212 Posterior capsule opacification after cataract surgery ..................................................... 213

Autoimmune uveitis ....................................................................................................... 213 Retinal ischemic injury .................................................................................................... 213 Corneal disorders ........................................................................................................... 214 Glaucoma ..................................................................................................................... 214 Corneal scarring ............................................................................................................ 214 Companies developing gene therapy for eye disorders ........................................................ 215

Organ transplantation ...................................................................................................... 215 Introduction .................................................................................................................. 215 DNA vaccines for transplantation ..................................................................................... 216 Gene therapy for prolonging allograft survival ................................................................... 216

Genetically modified Tregs expressing CAR for preventing GVHD .................................... 216 Gene therapy in lung transplantation ................................................................................ 216 Role of gene therapy in liver transplantation ..................................................................... 217 Gene therapy in kidney transplantation............................................................................. 217 Veto cells and transplant tolerance ................................................................................... 217

Pulmonary disorders ........................................................................................................ 218 Techniques of gene delivery to the lungs .......................................................................... 218

Adenoviral vectors .................................................................................................... 219 Nonviral vectors ........................................................................................................ 220 Aerosolization as an aid to gene transfer to lungs. ........................................................ 220

Cystic fibrosis ................................................................................................................ 220 Genetics and clinical features ..................................................................................... 220 Gene therapy for CF .................................................................................................. 221 CFTR gene transfer in CF ........................................................................................... 221 Concluding remarks about gene therapy of CF .............................................................. 222

Miscellaneous pulmonary disorders .................................................................................. 223 Gene therapy for pulmonary arterial hypertension ......................................................... 223 Gene therapy for bleomycin-induced pulmonary fibrosis ................................................ 224 Gene therapy of emphysema due to α1-antitrypsin deficiency ........................................ 224

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Gene therapy for asthma ........................................................................................... 224 Gene therapy for adult respiratory distress syndrome .................................................... 225 Gene therapy for lung injury ...................................................................................... 225 Gene therapy for bronchopulmonary dysplasia .............................................................. 225

Concluding remarks about gene therapy of lungs ............................................................... 226 Companies involved in pulmonary gene therapy ................................................................ 226

Skin and soft tissue disorders .......................................................................................... 227 Gene transfer to the skin ................................................................................................ 227

Electroporation for transdermal delivery of plasmid DNA ................................................ 227 Electroporation for transdermal delivery of DNA vaccines ............................................... 227 Liposomes for transdermal gene delivery ..................................................................... 228 Ultrasound and topical gene therapy ........................................................................... 228

Gene therapy in skin disorders ........................................................................................ 228 Gene therapy of hair loss ........................................................................................... 228 Gene therapy for epidermolysis bullosa ........................................................................ 229 Gene therapy for xeroderma pigmentosa ..................................................................... 230 Gene therapy for lamellar ichthyosis ............................................................................ 231

Gene transfer techniques for wound healing ...................................................................... 231 Urogenital disorders ........................................................................................................ 231

Gene therapy for urinary tract dysfunction ........................................................................ 232 Gene therapy for erectile dysfunction ............................................................................... 232

NOS gene transfer for erectile dysfunction ................................................................... 232 Clinical trial of hMaxi-K Gene transfer in erectile dysfunction .......................................... 232 Gene therapy for erectile dysfunction due to nerve injury............................................... 233 Concluding remarks on gene therapy for erectile dysfunction .......................................... 233

Gene therapy of miscellaneous disorders ........................................................................ 233 Primary Sjögren's syndrome ............................................................................................ 233

Veterinary gene therapy .................................................................................................. 233 Gene therapy for mucopolysaccharidosis VII in dogs .......................................................... 234 Gene therapy to increase disease resistance...................................................................... 234 Gene therapy for infections ............................................................................................. 234 Gene therapy for chronic anemia ..................................................................................... 235 Gene therapy for endocrine disorders ............................................................................... 235 Gene therapy for arthritis................................................................................................ 235 Cancer gene therapy ...................................................................................................... 236

Brain tumors in cats and dogs .................................................................................... 236 Breast cancer in dogs ................................................................................................ 236 Canine hemangiosarcoma .......................................................................................... 237 Canine melanoma ..................................................................................................... 237 Canine soft tissue sarcoma ......................................................................................... 238

Melanoma in horses ....................................................................................................... 238

4. Gene Therapy of Genetic Disorders ..................................................... 240 Introduction .................................................................................................................... 240 Primary immunodeficiency disorders ............................................................................... 241

Severe combined immune deficiency ................................................................................ 242 Gene therapy for SCID .............................................................................................. 242 Lentiviral gene therapy combined with low-dose busulfan for SCID-X1 ............................ 245

Chronic granulomatous disease ....................................................................................... 245 Wiskott-Aldrich syndrome ............................................................................................... 245 Purine nucleoside phosphorylase deficiency ....................................................................... 246 Major histocompatibility class II deficiency ........................................................................ 246 Future prospects of gene therapy of inherited immunodeficiencies ....................................... 247

Metabolic disorders.......................................................................................................... 247 Alpha1-antitrypsin deficiency ........................................................................................... 248

AAV mediated gene therapy for α1-antitrypsin deficiency ............................................... 248 iPSCs for targeted gene correction of α1-antitrypsin deficiency ....................................... 249 Gene editing for AAT deficiency .................................................................................. 250

Adrenoleukodystrophy .................................................................................................... 251 Canavan disease ............................................................................................................ 252 Lesch-Nyhan syndrome .................................................................................................. 252 Lipoprotein lipase deficiency ............................................................................................ 253

Alipogene tiparvovec ................................................................................................. 253 Ornithine transcarbamylase deficiency .............................................................................. 254

Gene therapy for OTCD .............................................................................................. 254 AAV-mediated gene editing for OTCD using CRISPR-Cas9 .............................................. 254

Phenylketonuria ............................................................................................................. 255 Porphyrias .................................................................................................................... 255 Tetrahydrobiopterin deficiency ......................................................................................... 256

Lysosomal storage disorders. .......................................................................................... 257 Batten disease ............................................................................................................... 258

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Fabry's disease .............................................................................................................. 258 Farber’s disease ............................................................................................................. 259 Gaucher disease ............................................................................................................ 259

Animals models of Gaucher's disease........................................................................... 259 Gene therapy of Gaucher's disease .............................................................................. 260

Glycogen storage disorders ............................................................................................. 261 Glycogen storage disease type I ................................................................................. 261 Gene therapy of GSD1a ............................................................................................. 261

Hunter syndrome ........................................................................................................... 261 Krabbe's disease ............................................................................................................ 262 Metachromatic leukodystrophy ........................................................................................ 263 Mucopolysaccharidosis type 1 (Hurler syndrome) ............................................................... 263 Niemann-Pick type A disease ........................................................................................... 264 Pompe disease .............................................................................................................. 264 Sanfilippo A syndrome .................................................................................................... 265 Sly syndrome ................................................................................................................ 266 Tay-Sachs disease/Sandhoff disease ................................................................................ 266 Future of gene therapy of lysosomal storage disorders ....................................................... 267

Trinucleotide repeat disorders ......................................................................................... 267 Muscular dystrophies ....................................................................................................... 268

Duchenne muscular dystrophy (DMD) ............................................................................... 268 Animal models for gene therapy of DMD ...................................................................... 268 Antisense approaches to DMD..................................................................................... 269 CRISPR/Cas9 gene editing for DMD ............................................................................. 269 Exon-skipping for DMD .............................................................................................. 269 Galgt2 gene therapy .................................................................................................. 271 Liposome-mediated gene transfer ............................................................................... 271 Microdystrophin gene therapy ..................................................................................... 271 Myoblast-based gene transfer in DMD .......................................................................... 271 Plasmid-mediated gene therapy .................................................................................. 272 Purinoreceptor P2RX7 ablation .................................................................................... 272 Post-transcriptional modulation of gene expression in DMD ............................................ 272 Repair/Editing of dystrophin gene ............................................................................... 273 Routes of administration of gene therapy in DMD .......................................................... 275 Types of dystrophin constructs ................................................................................... 276 Viral vectors for DMD ................................................................................................ 276 Conclusions and future of gene therapy of DMD ............................................................ 277

Limb-girdle muscular dystrophy ....................................................................................... 278 Myotonic dystrophy ........................................................................................................ 278 Spinal muscular atrophy ................................................................................................. 279

Gene therapy strategies for SMA ................................................................................. 279 Zolgensma® gene therapy for SMA ............................................................................. 279 Antisense approaches for SMA .................................................................................... 280 Nusinersen ............................................................................................................... 280 Nusinersen vs Zolgensma® gene therapy for SMA ........................................................ 281

X-linked myotubular myopathy (XLMTM)........................................................................... 281 Hereditary neuropathies .................................................................................................. 281

Charcot-Marie-Tooth disease ........................................................................................... 281 Hereditary axonal neuropathies of the peripheral nerves ..................................................... 282

Gene therapy of mitochondrial disorders ......................................................................... 282 Techniques for mitochondrial replacement therapy ............................................................. 283 Status of mitochondrial replacement and transfer therapies ................................................ 284 Barth syndrome ............................................................................................................. 285

Companies involved in gene therapy of genetic disorders ............................................... 285

5. Gene Therapy of Cancer ...................................................................... 287 Strategies for cancer gene therapy .................................................................................. 287 Direct gene delivery to the tumor .................................................................................... 288

Injection into tumor ....................................................................................................... 288 Direct injection of adenoviral vectors ........................................................................... 288 Direct injection of a plasmid DNA-liposome complex ...................................................... 289 A polymer approach to local gene therapy for cancer ..................................................... 289

Electroporation for cancer gene therapy ............................................................................ 289 Control of gene expression in tumor by local heat .............................................................. 290 Radiation-guided gene therapy of cancer .......................................................................... 290 Radioprotective gene therapy of cancer ............................................................................ 290 Nanoparticles to facilitate combination of hyperthermia and gene therapy ............................. 291

Immunogene therapy of cancer ....................................................................................... 291 Cell-based cancer gene therapy ....................................................................................... 292

Adoptive cell therapy ...................................................................................................... 292 CAR-T cell therapy for cancer .......................................................................................... 293

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CAR-T cells for tumor targeting ................................................................................... 293 CAR-T cells targeting both CD19 and CD22 .................................................................. 293 Remote control of CAR-T cells for cancer immunotherapy .............................................. 293 CAR-T cell therapy for leukemia .................................................................................. 294 CAR-T cell therapy for multiple myeloma ..................................................................... 295 CAR-T cell therapy for lymphoma ................................................................................ 295 CAR-T cell therapy for solid tumors ............................................................................. 296

Cytokine gene therapy .................................................................................................... 297 Genetic modification of human hematopoietic stem cells ..................................................... 300

Cancer vaccines ............................................................................................................... 300 Genetically modified cancer cell vaccines .......................................................................... 300

GVAX cancer vaccines ............................................................................................... 301 Genetically modified dendritic cells .............................................................................. 301

Nucleic acid-based cancer vaccines .................................................................................. 302 DNA cancer vaccines ................................................................................................. 302 RNA vaccines ............................................................................................................ 302

Viral vector-based cancer vaccines ................................................................................... 302 Intradermal delivery of cancer vaccines by Ad vectors ................................................... 303

Vaccines based on genetically engineered nonviral vectors .................................................. 303 Future of cancer vaccines ................................................................................................ 303 Companies involved in nucleic acid-based cancer vaccines .................................................. 304

Monoclonal antibody gene transfer for cancer ................................................................. 305 Transfer and expression of intracellular adhesion-1 molecules ....................................... 305 Other gene therapy techniques for immunotherapy of cancer ......................................... 305

Chemokines .................................................................................................................. 305 CRISPR for immunogene therapy ..................................................................................... 306 Engineered viruses as anticancer immunotherapy vectors ................................................... 307 Fas (Apo-1) ................................................................................................................... 307 Major Histocompatibility Complex (MHC) Class I ................................................................ 307 IGF (Insulin-Like Growth Factor) ...................................................................................... 307

Cancer immunotherapies targeting multiple mechanisms................................................ 308 Inhibition of immunosuppressive function in cancer ....................................................... 308 Delivery of toxic genes to tumor cells for eradication ...................................................... 308

Gene-directed enzyme prodrug therapy ............................................................................ 308 Combination of gene therapy with radiotherapy .............................................................. 309 Correction of genetic defects in cancer cells .................................................................... 309 Targeted gene therapy for cancer .................................................................................... 310

Antiangiogenic therapy for cancer .................................................................................... 310 Bacteria as novel anticancer gene vectors ......................................................................... 311 Cancer-specific gene expression ...................................................................................... 311 Cancer-specific transcription............................................................................................ 312 Delivery of retroviral particles hitchhiking on T cells ........................................................... 312 Electrogene and electrochemotherapy .............................................................................. 312 Epidermal growth factor-mediated DNA delivery ................................................................ 312 Gene expression in hypoxic tumor cells ............................................................................ 313 Genetically modified T cells for targeting tumors ................................................................ 313 Genetically engineered stem cells for targeting tumors ....................................................... 314 Hematopoietic stem cells for targeted cancer gene therapy ................................................. 314 Immunolipoplex for delivery of p53 gene .......................................................................... 315 Nanomagnets for targeted cell-based cancer gene therapy .................................................. 315 Nanoparticles for targeted site-specific delivery of anticancer genes ..................................... 316 Targeted cancer therapy using a dendrimer-based synthetic vector ...................................... 316 Tumor-targeted gene therapy by receptor-mediated endocytosis ......................................... 316

Virus-mediated oncolysis ................................................................................................. 317 Cytokine-induced killer cells for delivery of an oncolytic virus .............................................. 317 Monitoring of viral-mediated oncolysis by PET ................................................................... 317 Oncolytic adenoviruses ................................................................................................... 318 Oncolytic HSV ................................................................................................................ 319 Oncolytic measles viruses ............................................................................................... 319 Oncolytic vaccinia virus................................................................................................... 320 Oncolytic vesicular stomatitis virus ................................................................................... 320 Targeted cancer treatments based on oncolytic viruses....................................................... 320 Concluding remarks on oncolytic gene therapy .................................................................. 321

Companies developing oncolytic viruses ....................................................................... 321 Apoptotic approach to improve cancer gene therapy ....................................................... 322 Tumor suppressor gene therapy ...................................................................................... 323

P53 gene therapy........................................................................................................... 323 BRIT1 gene therapy ....................................................................................................... 323

Nitric oxide-based cancer gene therapy ........................................................................... 323 Anticancer effect of nitric oxide synthase .......................................................................... 323 Gene therapy for radiosensitization of cancer .................................................................... 324

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Gene therapy of cancer of selected organs ...................................................................... 324 Gene therapy for bladder cancer ...................................................................................... 324 Gene therapy for glioblastoma ......................................................................................... 325

Adenoviral vectors for treatment of brain tumors .......................................................... 326 Antiangiogenic gene therapy ...................................................................................... 326 Autophagy induced by conditionally replicating adenoviruses .......................................... 327 Baculovirus vector for diphtheria toxin gene therapy ..................................................... 327 Cerepro® (sitimagene ceradenovec) ........................................................................... 327 Gene therapy targeting hepatocyte growth factor .......................................................... 328 Genetically engineered MSCs for gene delivery to intracranial gliomas ............................. 328 Intravenous gene delivery with nanoparticles into brain tumors ...................................... 328 Ligand-directed delivery of dsRNA molecules targeted to EGFR ....................................... 329 Olig2 targeting to hinder growth of treatment-resistant glioblastomas ............................. 329 RNAi gene therapy of brain cancer .............................................................................. 329 Targeting normal brain cells with an AAV vector encoding interferon-β ............................ 330 Viral oncolysis of glioblastoma multiforme .................................................................... 330

Gene therapy for breast cancer ........................................................................................ 332 Gene vaccine for breast cancer ................................................................................... 332 Recombinant adenoviral ErbB-2/neu vaccine ................................................................ 333

Gene Therapy for ovarian cancer ..................................................................................... 333 Gene therapy for malignant melanoma ............................................................................. 334

Immunogene therapy ................................................................................................ 334 Nonviral immunogene therapy for malignant melanoma ................................................. 335 Oncogene targeted therapy ........................................................................................ 336 Targeted gene therapy for malignant melanoma ........................................................... 336

Gene therapy of lung cancer............................................................................................ 336 Intravenous nanoparticle formulation for delivery of FUS1 gene ...................................... 336 Aerosol gene delivery for lung cancer .......................................................................... 337

Gene therapy for cancer of prostate ................................................................................. 337 Experimental studies ................................................................................................. 337 Nanoparticle-based gene therapy for prostate cancer .................................................... 338 Tumor suppressor gene therapy in prostate cancer ....................................................... 338 Vaccines for prostate cancer ....................................................................................... 338 Viral oncolysis for prostate cancer ............................................................................... 338 Clinical trials of gene therapy for prostate cancer .......................................................... 339

Gene therapy of head and neck cancer ............................................................................. 339 Adenoviral vector based P53 gene therapy ................................................................... 339 Gene therapy as adjunct to 5-FU for nasopharangeal carcinoma ..................................... 340

Gene therapy of pancreatic tumors .................................................................................. 340 Pancreatic neuroendocrine tumors .............................................................................. 340 Pancreatic ductal adenocarcinoma ............................................................................... 340 Editing of altered genes ............................................................................................. 341 Targeted gene therapy .............................................................................................. 341 Targeting in pancreatic adenocarcinoma with cell surface antigens .................................. 342 Targeted Expression of BikDD gene ............................................................................. 342 Viral oncolysis in pancreatic cancer ............................................................................. 342 Concluding remarks on gene therapy of pancreatic cancer ............................................. 342

Gene therapy of renal cancer........................................................................................... 343 Viral onclolytic therapy for renal cancer ....................................................................... 343

Gene therapy of hematological malignancies ..................................................................... 343 Genetically engineered T lymphocytes ......................................................................... 343

Cancer gene therapy companies ...................................................................................... 344

6. Gene Therapy of Neurological Disorders ............................................. 347 Indications ...................................................................................................................... 347 Gene transfer techniques for the nervous system ............................................................ 348

Methods of gene transfer to the nervous system ................................................................ 348 Ideal vector for gene therapy of neurological disorders ....................................................... 348 Promoters of gene transfer .............................................................................................. 348 Lentivirus-mediated gene transfer to the CNS ................................................................... 349 AAV vector mediated gene therapy for neurogenetic disorders ............................................. 349 Gene transfer to the CNS using recombinant SV40-derived vectors ...................................... 350 Routes of delivery of genes to the nervous system ............................................................. 350

Direct injection into CNS ............................................................................................ 350 Introduction of the genes into cerebral circulation ......................................................... 351 Introduction of genes into cerebrospinal fluid ............................................................... 351 Intravenous administration of vectors .......................................................................... 352

Delivery of gene therapy to the peripheral nervous system ................................................. 352 Cell-mediated gene therapy of neurological disorders ......................................................... 352

Neuronal cells ........................................................................................................... 352 Neural stem cells and progenitor cells .......................................................................... 352

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Astrocytes ................................................................................................................ 353 Cerebral endothelial cells ........................................................................................... 353 Implantation of genetically modified encapsulated cells into the brain .............................. 353

Gene transfer for neuromodulation ................................................................................... 354 Monitoring of CNS gene therapy ...................................................................................... 354 Gene therapy of neurodegenerative disorders ................................................................. 354

Gene therapy for Parkinson disease .................................................................................. 354 Rationale ...................................................................................................................... 355

Techniques of gene therapy for PD .............................................................................. 356 Augmenting CNS glucocerebrosidase activity ................................................................ 359 Delivery of neurotrophic factors by gene therapy .......................................................... 359 Delivery of parkin gene .............................................................................................. 360 Gene editing in PD .................................................................................................... 360 Introduction of functional genes into the brain of patients with PD .................................. 360 Nanoparticle-based gene therapy for PD ...................................................................... 361 Mitochondrial gene therapy for PD ............................................................................... 361 RNAi approach to PD ................................................................................................. 361 Viral vector-based ubiquitination to prevent spread of α-synuclein .................................. 362 Prospects of gene therapy for PD ................................................................................ 362 Concluding remarks about gene therapy of PD .............................................................. 363 Companies developing gene therapy for PD .................................................................. 363

Gene therapy for Alzheimer disease ................................................................................. 364 Rationale ................................................................................................................. 364 NGF gene therapy for AD ........................................................................................... 364 FGF2 gene transfer in AD ........................................................................................... 365

Gene therapy for restoring brain cholesterol metabolism ..................................................... 365 Neprilysin gene therapy ............................................................................................. 366 Viral gene transfer of APPsα for rescuing synaptic failure in AD ....................................... 366 Gene vaccination ...................................................................................................... 367 Combination of gene therapy with other treatments for AD ............................................ 367

Gene therapy of Huntington disease ................................................................................. 367 Encapsulated genetically engineered cellular implants.................................................... 367 Viral vector mediated administration of neurotrophic factors .......................................... 368 RNAi gene therapy .................................................................................................... 368

Gene therapy of amyotrophic lateral sclerosis .................................................................... 368 Rationale ................................................................................................................. 368 Technique of gene therapy of ALS ............................................................................... 368 Other approaches to gene therapy of ALS .................................................................... 370

Gene therapy of cerebrovascular diseases ....................................................................... 370 Preclinical research in gene therapy for cerebrovascular disease .......................................... 370 Animal models of stroke relevant to gene therapy .............................................................. 371

Transgenic mice as models for stroke .......................................................................... 371 Animal models for gene therapy of arteriovenous malformations ..................................... 371

Gene transfer to cerebral blood vessels ............................................................................ 371 Gene therapy for vasospasm following subarachnoid hemorrhage ........................................ 372

NOS gene therapy for cerebral vasospasm ................................................................... 373 Gene therapy for stroke .................................................................................................. 374

Gene therapy for stroke using neurotrophic factors ....................................................... 375 Gene therapy of strokes with a genetic component ....................................................... 375 Gene therapy for intracranial aneurysms ...................................................................... 375 RNAi-based gene silencing for neuroprotection in cerebral ischemia ................................ 376

Concluding remarks about gene therapy for stroke............................................................. 376 Gene therapy of injuries to the nervous system ............................................................... 376

Traumatic brain injury .................................................................................................... 376 Spinal cord injury ........................................................................................................... 377 Peripheral nerve injuries ................................................................................................. 378

Gene therapy of epilepsy ................................................................................................. 378 Gene therapy for control of seizures ................................................................................. 378 Gene therapy for neuroprotection in epilepsy .................................................................... 379 Gene therapy for genetic forms of epilepsy ....................................................................... 380

Gene therapy for multiple sclerosis ................................................................................. 380 Gene therapy for impairment of special senses ............................................................... 380

Gene therapy for hearing loss .......................................................................................... 381 Gene therapy for olfactory impairment ............................................................................. 381

Gene therapy for relief of pain ......................................................................................... 382 Rationale of gene therapy for pain ................................................................................... 382 Vectors for gene therapy of pain ...................................................................................... 382 Methods of gene delivery for pain .................................................................................... 383

Endogenous analgesic production for cranial neuralgias ................................................. 383 Gene delivery by intrathecal route .............................................................................. 384 Gene transfer for delivery of analgesics to the spinal nerve roots .................................... 384

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Gene therapy of peripheral neuropathic pain ................................................................ 385 Gene transfer by injections into the brain substance ...................................................... 386

Targets for gene therapy of pain ...................................................................................... 386 Zinc finger DNA-binding protein therapeutic for chronic pain .......................................... 386 Gene therapy for producing enkephalin to block pain signals .......................................... 387 Targeting nuclear factor-B ........................................................................................ 387 Gene therapy targeted to neuroimmune component of chronic pain ................................ 387

Potential applications of gene therapy for management of pain ............................................ 388 Concluding remarks on gene therapy for pain .................................................................... 388

Gene therapy for psychiatric disorders ............................................................................ 389 Gene therapy for depression ........................................................................................... 389 Gene therapy for enhancing cognition after stress .............................................................. 390 Gene therapy against fear disorders ................................................................................. 390

Companies involved in gene therapy of neurological disorders ........................................ 391

7. Gene Therapy of Cardiovascular Disorders ......................................... 393 Introduction .................................................................................................................... 393 Techniques of gene transfer to the cardiovascular system .............................................. 393

Direct plasmid injection into the myocardium .................................................................... 394 Catheter-based systems for vector delivery ....................................................................... 394 Ultrasound microbubbles for cardiovascular gene delivery ................................................... 395 Vectors for cardiovascular gene therapy ........................................................................... 395

AAV vectors for therapeutic delivery to the heart .......................................................... 395 Adenoviral vectors for cardiovascular diseases .............................................................. 396 Molecular cardiac surgery with recirculating delivery of AAV vectors ................................ 396 Plasmid DNA-based delivery in cardiovascular disorders ................................................. 396

Gene therapy for counteracting hypoxia in myocardial ischemia ........................................... 396 Therapeutic angiogenesis vs vascular growth factor therapy ................................................ 397 Gene painting for delivery of targeted gene therapy to the heart ......................................... 397 Gene delivery to vascular endothelium ............................................................................. 398 Targeted plasmid DNA delivery to the cardiovascular system with nanoparticles .................... 398 Vascular stents for gene delivery ..................................................................................... 399

Gene therapy for genetic cardiovascular disorders .......................................................... 399 Catecholaminergic polymorphic ventricular tachycardia....................................................... 399 Genetic disorders predisposing to atherosclerosis ............................................................... 400 Familial hypercholesterolemia .......................................................................................... 401 Apolipoprotein E deficiency .............................................................................................. 402 Hypertension ................................................................................................................. 402 Genetic factors for myocardial infarction ........................................................................... 403

Acquired cardiovascular diseases .................................................................................... 404 Coronary artery disease with angina pectoris ..................................................................... 404

Ad5FGF-4................................................................................................................. 404 Gene therapy for improving long-term CABG patency rates ................................................. 405 Ischemic heart disease with myocardial infarction .............................................................. 405

Gene therapy and angiogenesis in ischemic heart disease .............................................. 405 Induction of angiogenesis in myocardium by HEXIM1 re-expression ................................ 406 Myocardial repair with IGF-1 therapy ........................................................................... 407 Metalloproteinase-2 inhibitor gene therapy ................................................................... 407 miRNA gene therapy for ischemic heart disease ............................................................ 408

Congestive heart failure .................................................................................................. 408 Rationale of gene therapy in CHF ................................................................................ 408 AAV-mediated gene transfer for CHF ........................................................................... 408 AngioCell gene therapy for CHF .................................................................................. 409 β-ARKct gene therapy................................................................................................ 410 Elevating cardiac dATP by gene therapy to improve cardiac function ................................ 410 Elevating cardiac adenyl cylase type 6 to improve cardiac function .................................. 411 Intracoronary adenovirus-mediated gene therapy for CHF .............................................. 411 nNOS gene transfer in CHF......................................................................................... 412

Cardiomyopathies .......................................................................................................... 412 Cardiac arrhythmias ....................................................................................................... 412

Gene transfer approaches for biological pacemakers ...................................................... 413 Genetically engineered biological pacemakers ............................................................... 414 Gene therapy for ventricular arrhythmia ...................................................................... 414

Gene therapy and heart transplantation ............................................................................ 414 Hyperlipidemia and hypercholesterolemia ......................................................................... 415

Antisense approach to hypertriglyceridemia.................................................................. 415 Cholesterol reduction by genetic engineering of PCSK9 gene .......................................... 415 Inactivating variants in ANGPTL4 for lowering circulating triglycerides ............................. 416

Peripheral arterial disease ............................................................................................... 416 Incidence and clinical features ......................................................................................... 416 Current management ..................................................................................................... 416

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Gene therapy for peripheral arterial disease ...................................................................... 417 Angiogenesis by gene therapy .................................................................................... 417 HIF-1 gene therapy for peripheral arterial disease ....................................................... 417 HGF gene therapy for peripheral arterial disease ........................................................... 418

Prevention of restenosis after angioplasty ......................................................................... 418 Antisense approaches ................................................................................................ 418 Gene therapy to prevent restenosis after angioplasty .................................................... 419 hTIMP-1 gene therapy to prevent intimal hyperplasia .................................................... 420 miRNA-based gene therapy for restenosis .................................................................... 420 NOS gene therapy for restenosis ................................................................................. 421 Techniques of gene therapy for restenosis.................................................................... 421

Maintaining vascular patency after surgery ....................................................................... 422 Companies involved in gene therapy of cardiovascular diseases ..................................... 422

Future of gene therapy of cardiovascular disorders ............................................................. 423

8. Gene therapy of viral infections .......................................................... 425 Introduction .................................................................................................................... 425 Acquired Immunodeficiency Syndrome (AIDS) ................................................................ 425

Current management of AIDS .......................................................................................... 425 Gene therapy strategies in HIV/AIDS ................................................................................ 426 Cell/gene therapies for HIV/AIDS ..................................................................................... 427

Anti-HIV ribozyme delivered in hematopoietic progenitor cells ........................................ 427 Autocrine interferon (INF)- production by somatic cell gene therapy .............................. 427 Gene editing for HIV-1 ............................................................................................... 427 Transplantation of genetically modified T cells .............................................................. 428 Transplantation of genetically modified hematopoietic cells ............................................ 429

Inhibition of HIV-1 replication by lentiviral vectors ............................................................. 430 VRX496-T ................................................................................................................ 430

Insertion of protective genes into target cells .................................................................... 431 HIV/AIDS vaccines ......................................................................................................... 431 Intracellular immunization............................................................................................... 432 Engineered cellular proteins such as soluble CD4s .............................................................. 432 Intracellular antibodies ................................................................................................... 433 Anti-rev single chain antibody fragment ............................................................................ 433 Use of genes to chemosensitize HIV-1 infected cells ........................................................... 433 Antisense approaches to AIDS ......................................................................................... 433

RNA decoys .............................................................................................................. 433 Antisense oligodeoxynucleotides ................................................................................. 434 RNA decoys .............................................................................................................. 434 Ribozymes ............................................................................................................... 434

RNAi applications in HIV/AIDS ......................................................................................... 435 siRNA-directed inhibition of HIV-1 infection .................................................................. 435 Role of the nef gene during HIV-1 infection and RNAi .................................................... 436 Bispecific siRNA constructs ......................................................................................... 436 Targeting CXCR4 with siRNAs ..................................................................................... 436 Targeting CCR5 with siRNAs ....................................................................................... 436

Companies involved in developing gene therapy for HIV/AIDS ............................................. 437 Conclusions regarding gene therapy of HIV/AIDS ............................................................... 438

Genetic vaccines for other viral infections ....................................................................... 438 Cytomegalic virus infections ............................................................................................ 438 Viral hepatitis ................................................................................................................ 439

Vaccine for hepatitis B ............................................................................................... 439 Vaccine for hepatitis C ............................................................................................... 439 Gene therapy for hepatitis C ....................................................................................... 440

Vaccine for herpes simplex virus ...................................................................................... 440 DNA vaccine against rabies ............................................................................................. 440 DNA vaccine for Ebola .................................................................................................... 441 Vaccines for avian influenza ............................................................................................ 441

Future prospects of DNA vaccines for avian influenza .................................................... 442 Human trial of a DNA vaccine for avian influenza .......................................................... 443

Companies developing genetic vaccines for infections other than AIDS ................................. 443

9. Research, Development and Future of Gene Therapy .......................... 445 Basic research in gene therapy ........................................................................................ 445 R & D in gene therapy ...................................................................................................... 445

Animal models of human diseases for gene therapy research .............................................. 446 Lentiviral transgenesis ............................................................................................... 446

Financing research and development ............................................................................... 446 Role of the NIH in gene therapy research .......................................................................... 446 National Gene Vector Laboratories ................................................................................... 446 Funding of gene therapy research in Europe ...................................................................... 447

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Gene therapy funding in Horizon 2020 of European Commission ..................................... 447 Financing by the industry ................................................................................................ 448

Clinical trials in gene therapy .......................................................................................... 448 Clinical trials worldwide .................................................................................................. 448

Clinical trials in cancer gene therapy ........................................................................... 449 Trials of gene therapy for neurological disorders ........................................................... 449 Clinical trials for genetic disorders ............................................................................... 449 Clinical trials in cardiovascular gene therapy ................................................................ 449 Clinical trials for infectious diseases ............................................................................. 449 Gene therapy for other disorders ................................................................................ 449

Clinical trials in the US .................................................................................................... 449 Vectors used in gene therapy clinical trials ........................................................................ 450

Vector analytics for clinical trials using rAAV vectors ...................................................... 451 Gene therapy in China ...................................................................................................... 451 Future of gene therapy .................................................................................................... 452

How to improve gene therapy .......................................................................................... 452 International Gene Therapy Consortium ............................................................................ 453 Future opportunities and challenges for gene therapy ......................................................... 454 Promising areas of application of gene therapy .................................................................. 455

Neurological disorders ............................................................................................... 455 Gene therapy of cardiovascular disorders ..................................................................... 456 Cancer gene therapy ................................................................................................. 457

Personalized gene therapy .............................................................................................. 458

10. Regulatory, Safety, Ethical Patent Issues of Gene Therapy ............... 459 Regulation of gene therapy in the United States .............................................................. 459

US Federal guidelines for research involving recombinant DNA molecules ............................. 459 Regulation of gene therapy in US ..................................................................................... 459 Implantation of genetically manipulated cells..................................................................... 459

Modification of oocytes for use in IVF........................................................................... 460 Clinical trials in gene therapy .......................................................................................... 460 Gene therapy INDs placed on hold by the FDA ................................................................... 461 FDA policy for advancing development of gene therapy ...................................................... 461

Future for approval of gene therapy in the US .............................................................. 463 Do-it-yourself gene therapy ............................................................................................ 465

Regulation of gene therapy in Europe .............................................................................. 465 European Union ............................................................................................................. 465 Regulation of gene therapy in Germany ............................................................................ 466

Preclinical research ................................................................................................... 466 Clinical Trials ............................................................................................................ 467

Marketing authorization .................................................................................................. 468 Regulation of gene therapy in the United Kingdom ............................................................. 468 Regulation of gene therapy in France................................................................................ 469 Regulation of gene therapy in Italy .................................................................................. 469 Regulation of gene therapy in the Netherlands................................................................... 469 Gene therapy regulation in Switzerland............................................................................. 471

Regulation of gene therapy in Australia ........................................................................... 471 Regulation of gene therapy in Japan ................................................................................ 472 Regulation of gene therapy in China ................................................................................ 472 Safety issues of gene transfer ......................................................................................... 472

Adverse effects of retroviral vectors ................................................................................. 473 Insertional mutagenesis ............................................................................................. 473

Adverse effects of HSV vectors ........................................................................................ 473 Neurotoxicity of HSV vectors ...................................................................................... 473 Hepatotoxicity of HSV-tk/ganciclovir approach .............................................................. 474

Adverse effects of adenoviral vectors................................................................................ 474 Inflammatory effects of adenoviruses in lungs .............................................................. 474 Inflammatory effects involving the liver ....................................................................... 474 Induction of immune response by adenoviral vectors ..................................................... 475 Impairment of adrenocortical steroidogenesis ............................................................... 475 Adverse effects of AAV vectors ................................................................................... 475

Toxicity associated with cationic lipid-mediated gene transfer .............................................. 476 Toxicity of lipopolysaccharides ......................................................................................... 476 Potential side effects of RNAi gene therapy........................................................................ 476 Role of molecular diagnostics in safety of gene therapy ...................................................... 477 Quality control of vectors ................................................................................................ 477

Testing for retroviruses .............................................................................................. 477 Adenoviral vectors .................................................................................................... 478 Replication competent viruses .................................................................................... 478

Genetic characteristics of viral vectors .............................................................................. 478 Concluding remarks about safety of viral vectors ............................................................... 479

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Ethical aspects of gene therapy ....................................................................................... 479 The lay consumer's view of somatic gene therapy ethics ..................................................... 480 Ethical aspects of clinical trials ......................................................................................... 480 Regulatory and ethical issues for in utero gene therapy ...................................................... 480 Ethical aspects of germline gene therapy .......................................................................... 480 Ethical aspects of mitochondrial replacement therapy ......................................................... 481 Ethical aspects of gene editing ......................................................................................... 482

Ethical aspects of clinical trials of germline editing ........................................................ 482 Gene editing in the UK ............................................................................................... 483 Gene editing in the US ............................................................................................... 484 Guidelines for gene editing by Alliance for Regenerative Medicine ................................... 485 UNESCO’s view on gene editing in humans ................................................................... 486 Concluding remarks on the ethical aspects of genome editing ......................................... 486

Germline gene therapy for genetic enhancement ............................................................... 486 Athletic enhancement by genetic engineering .................................................................... 487

Gene doping in sports ................................................................................................ 487 Gene transfer methods used for enhancing physical performance .................................... 487 Adverse effect of genetic engineering .......................................................................... 489 Problems in detecting genetic manipulations in athletes ................................................. 489 Ethical dilemma ........................................................................................................ 489

Gene therapy patents ...................................................................................................... 490 CRISP/Cas9 patents ....................................................................................................... 491

11. Markets for Gene Therapy ................................................................. 493 Introduction .................................................................................................................... 493 Gene therapy markets in various regions of the world..................................................... 493 Gene therapy markets according to therapeutic areas ..................................................... 494

Cancer gene therapy market ........................................................................................... 494 Markets for gene therapy of genetic disorders ................................................................... 495

Markets for DNA vaccines ................................................................................................ 495 DNA vaccines markets according to technologies ............................................................... 495 DNA vaccines markets according to therapeutic indications ................................................. 496 DNA vaccines markets according to geographical areas ...................................................... 496

Competing treatments ..................................................................................................... 497 Antisense ...................................................................................................................... 497 RNAi ............................................................................................................................. 497 Cell therapy .................................................................................................................. 498

Strategies for developing gene therapy markets ............................................................. 498 Collaboration with pharmaceutical companies ............................................................... 498 Collaboration with companies developing cell-based therapies ........................................ 498 Collaboration with academic gene therapy centers ........................................................ 498 Developing safer and cost-effective gene medicines ...................................................... 498 Intellectual property and commercialization of gene therapy .......................................... 499 Overcoming obstructions to the development of gene therapy ........................................ 499

Unmet needs in gene therapy .......................................................................................... 500 ................................................................................................................................... 500

Promises and challenges for the development of gene therapy ....................................... 500 Development of gene therapy market in China .................................................................. 501 Challenges of developing gene therapy in the USA ............................................................. 501 Challenges of developing gene therapy in the European Union ............................................. 501

12. References ........................................................................................ 503

Tables

Table 1-1: Landmarks in development of gene therapy................................................................... 25 Table 2-1: Classification of methods of gene therapy...................................................................... 37 Table 2-2: A comparison of various physical methods of gene transfer ............................................. 39 Table 2-3: Experimental applications of gene transfer by electroporation .......................................... 41 Table 2-4: An overview of characteristics of commonly used viral vectors ......................................... 49 Table 2-5: Companies using viral vectors ...................................................................................... 62 Table 2-6: Companies using nonviral vectors ................................................................................ 81 Table 2-7: Target organs for nonviral gene therapy methods. ......................................................... 83 Table 2-8: CRISPR vs other gene editing tools ............................................................................... 90 Table 2-9: Companies developing CRISPR technology .................................................................. 100 Table 2-10: Companies developing CAR-T cell therapy ................................................................. 115 Table 2-11: Potential routes for administration of DNA ................................................................. 121 Table 2-12: Companies with gene delivery devices/ technologies ................................................... 122 Table 2-13: Strategies for targeted gene therapy ........................................................................ 123

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Table 2-14: Animal experimental studies of in vivo gene delivery with polymer systems ................... 125 Table 2-15: Approaches to controlling gene expression in gene therapy ......................................... 126 Table 2-16: Companies with regulated/targeted gene therapy and special techniques ...................... 128 Table 2-17: Potential applications of human germline genome modification .................................... 129 Table 2-18: Applications of molecular diagnostics in gene therapy ................................................. 143 Table 2-19: Advantages of gene therapy compared with protein therapy ........................................ 145 Table 3-1: Experimental approaches to gene therapy of rheumatoid arthritis .................................. 151 Table 3-2: Gene therapy strategies for osteoarthritis.................................................................... 153 Table 3-3: Cell and gene therapy approaches for type 1 diabetes mellitus....................................... 160 Table 3-4: Indications for gastrointestinal gene therapy ............................................................... 166 Table 3-5: Hematological disorders that can be potentially treated by gene therapy. ........................ 168 Table 3-6: Clinical trials of gene therapy for hemophilia A and B .................................................... 170 Table 3-7: Companies involved in gene therapy of hematological disorders ..................................... 181 Table 3-8: Techniques of gene transfer to the kidneys .................................................................. 187 Table 3-9: Gene therapy in animal experimental models of renal disease. ....................................... 189 Table 3-10: Applications of gene therapy in ophthalmological disorders .......................................... 198 Table 3-11: Companies developing gene therapy for eye disorders ................................................ 215 Table 3-12: Strategies for gene delivery to the lungs ................................................................... 218 Table 3-13: Companies developing gene therapy for pulmonary disorders ...................................... 226 Table 4-1: Genetic disorders that are being investigated for gene therapy ...................................... 240 Table 4-2: X-linked immunodeficiency disorders .......................................................................... 242 Table 4-3: Examples of inherited metabolic disorders amenable to gene therapy ............................. 247 Table 4-4: Gene therapy approaches to Duchenne muscular dystrophy .......................................... 268 Table 4-5: Companies involved in gene therapy of genetic/metabolic disorders ............................... 285 Table 5-1: Strategies for cancer gene therapy ............................................................................. 287 Table 5-2: Cell-based gene therapy for cancer ............................................................................ 292 Table 5-3: Companies with nucleic acids/genetically modified cell cancer vaccines ........................... 304 Table 5-4: Enzyme/prodrug combinations employed in suicide gene therapy ................................... 309 Table 5-5: Mutation compensation strategies used clinically .......................................................... 309 Table 5-6: Companies developing oncolytic viruses ...................................................................... 321 Table 5-7: Strategies for gene therapy of malignant brain tumors .................................................. 325 Table 5-8: Clinical trials of oncolytic virotherapy against glioblastoma multiforme ............................ 331 Table 5-9: Clinical trials of gene therapy in ovarian cancer ............................................................ 333 Table 5-10: Gene therapy for malignant melanoma...................................................................... 334 Table 5-11: Clinical trials of gene therapy for prostate cancer ....................................................... 339 Table 5-12: Companies involved in cancer gene therapy ............................................................... 344 Table 6-1: Example of potential indications for gene therapy of neurologic disorder ......................... 347 Table 6-2: Methods of gene transfer as applied to neurologic disorders .......................................... 348 Table 6-3: Gene therapy techniques applicable to Parkinson disease .............................................. 355 Table 6-4: Companies developing gene therapy for Parkinson's disease .......................................... 363 Table 6-5: Gene transfer in animal models of carotid artery restenosis ........................................... 372 Table 6-6: Gene therapy strategies for vasospasm ....................................................................... 373 Table 6-7: Neuroprotective gene therapy in animal stroke models ................................................. 374 Table 6-8: Experimental gene therapy approaches for relief of pain ............................................... 383 Table 6-9: Companies involved in gene therapy of neurological disorders ....................................... 391 Table 7-1: Cardiovascular disorders that are potential indications for gene therapy .......................... 393 Table 7-2: Catheter-based systems for vector delivery to the cardiovascular system ........................ 394 Table 7-3: Companies involved in gene therapy of cardiovascular diseases ..................................... 422 Table 8-1: Strategies for gene therapy of AIDS ............................................................................. 426 Table 8-2: Companies involved in developing gene therapy for HIV/AIDS ....................................... 437 Table 8-3: Companies developing genetic vaccines for infections other than AIDS ........................... 443 Table 9-1: Geographical distribution of gene therapy clinical trials ................................................. 448 Table 9-2: Opportunities and challenges for gene therapy and resources needed ............................. 455 Table 9-3: Potential applications of gene therapy in disorders of the nervous system ....................... 456 Table 10-1: Genes that may be used for performance enhancement .............................................. 487 Table 11-1: Gene therapy market according to regions/countries − 2019 to 2029 ............................ 493 Table 11-2: Gene therapy markets according to therapeutic areas − 2019 to 2029 ........................... 494 Table 11-3: Cancer gene therapy market according to type of cancer - 2019 to 2029 ....................... 494 Table 11-4: Gene therapy market for selected genetic disorders - 2019 to 2029 .............................. 495 Table 11-5: DNA vaccines markets according to technologies - 2019 to 2028 .................................. 496 Table 11-6: DNA vaccines markets according to therapeutic indications - 2019 to 2029 .................... 496 Table 11-7: DNA vaccines markets according to geographical areas - 2019 to 2029 ......................... 496

Figures

Figure 1-1: Relation of gene therapy to other biotechnologies ......................................................... 28 Figure 1-2: Relationship of DNA, RNA and protein in the cell ........................................................... 32 Figure 2-1: Ex vivo and in vivo techniques of gene therapy ............................................................. 38 Figure 2-2: Structure of the Helios gene gun ................................................................................. 44 Figure 2-3: Categories of rAAV-based gene therapy strategies ........................................................ 52

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Figure 2-4: Bacteria plus nanoparticles for drug delivery into cells ................................................... 79 Figure 2-5: Gene editing by zinc finger nucleases .......................................................................... 85 Figure 2-6: Genome engineering by transcription-activator–like-effector-nucleases............................ 86 Figure 2-7: Mechanism of action of GeneRide technology ................................................................ 87 Figure 2-8: A scheme of CRISPR/Cas9 gene editing ....................................................................... 88 Figure 2-9: Use of CRISPR-Cas9 for hereditary hearing loss ............................................................ 99 Figure 2-10: Gene editing vs suppression of gene expression ........................................................ 104 Figure 2-11: Chimeric antigen receptor (CAR)-T cells attacking tumor cells ..................................... 114 Figure 2-12: Schematic of suppression of gene expression by RNAi ................................................ 140 Figure 3-1: Retina and routes of administration of gene therapy for retinal disorders ....................... 204 Figure 4-1: Targeted gene correction of α1-antitrypsin deficiency by iPSCs ..................................... 250 Figure 4-2: Application of CRISPR-Cas9 in a mouse model of DMD ................................................. 274 Figure 4-3: Techniques for mitochondrial nuclear transfer ............................................................. 283 Figure 5-1: Gene therapy approaches for pancreatic cancer .......................................................... 341 Figure 6-1: Effect of tyrosine hydroxylase gene delivery on dopamine levels ................................... 356 Figure 6-2: Role of cell and gene therapy in stroke according to pathology and stage ....................... 376 Figure 9-1: Product development cycle in gene therapy ................................................................ 445 Figure 9-2: Proportions of therapeutic areas in clinical trials of gene therapy in the US ..................... 450 Figure 9-3: Proportions of various vectors used in gene therapy trials ............................................ 450 Figure 11-1: Unmet needs in gene therapy ................................................................................. 500