基因治疗：技术・市场・企业

Abstract

Summary

Gene therapy can be broadly defined as the transfer of defined genetic material to specific target cells of a patient for the ultimate purpose of preventing or altering a particular disease state. Genes and DNA are now being introduced without the use of vectors and various techniques are being used to modify the function of genes in vivo without gene transfer. If one adds to this the cell therapy particularly with use of genetically modified cells, the scope of gene therapy becomes much broader. Gene therapy can now combined with antisense techniques such as RNA interference (RNAi), further increasing the therapeutic applications. This report takes broad overview of gene therapy and is the most up-to-date presentation from the author on this topic built-up from a series of gene therapy report written by him during the past decade including a textbook of gene therapy and a book on gene therapy companies. This report describes the setbacks of gene therapy and renewed interest in the topic

Gene therapy technologies are described in detail including viral vectors, nonviral vectors and cell therapy with genetically modified vectors. Gene therapy is an excellent method of drug delivery and various routes of administration as well as targeted gene therapy are described. There is an introduction to technologies for gene suppression as well as molecular diagnostics to detect and monitor gene expression.

Clinical applications of gene therapy are extensive and cover most systems and their disorders. Full chapters are devoted to genetic syndromes, cancer, cardiovascular diseases, neurological disorders and viral infections with emphasis on AIDS. Applications of gene therapy in veterinary medicine, particularly for treating cats and dogs, are included.

Research and development is in progress in both the academic and the industrial sectors. The National Institutes of Health (NIH) of the US is playing an important part. As of 30 July 2007, over 1340 gene therapy clinical trials have been completed, are ongoing or have been approved in 28 countries. The total number of human gene transfer trials in the US that are registered with NIH Office of Biotechnology Activities is 885 as of December 2007. A breakdown of these trials is shown according to the areas of application. The largest number of clinical trial protocols is for cancer. The report also identifies the areas for future research.

Since the death of Jesse Gelsinger in the US following a gene therapy treatment, the FDA has further tightened the regulatory control on gene therapy. A further setback was the reports of leukemia following use of retroviral vectors in successful gene therapy for adenosine deaminase deficiency. Several clinical trials were put on hold and many have resumed now. The report also discusses the adverse effects of various vectors, safety regulations and ethical aspects of gene therapy including germline gene therapy.

The markets for gene therapy are difficult to estimate as there is only one approved gene therapy product and it is marketed in China since January 2004. At least two products are expected to be approved by 2008 and gene therapy markets are estimated for the years 2007-2017. The estimates are based on epidemiology of diseases to be treated with gene therapy, the portion of those who will be eligible for these treatments, competing technologies and the technical developments anticipated in the next decades. In spite of some setbacks, the future for gene therapy is bright.The markets for DNA vaccines are calculated separately as only genetically modified vaccines and those using viral vectors are included in the gene therapy markets

The voluminous literature on gene therapy was reviewed and selected 670 references are appended in the bibliography.The references are constantly updated. The text is supplemented with 71 tables and 13 figures.

Profiles of 192 companies involved in developing gene therapy are presented along with 213 collaborations. There were only 44 companies involved in this area in 1995. In spite of some failures and mergers, the number of companies has increased more than 4-fold within a decade. These companies have been followed up since they were the topic of a book on gene therapy companies by the author of this report. John Wiley & Sons published the book in 2000 and from 2001 to 2003, updated versions of these companies (approximately 160 at mid-2003) were available on Wiley' s web site. Since that free service was discontinued and the rights reverted to the author, this report remains the only authorized continuously updated version on gene therapy companies.

Table of Contents

0. Executive Summary 19

1. Introduction 21

• Definitions 21
• Historical evolution of gene therapy 21
• Relation of gene therapy to other biotechnologies 23
• Molecular biological basics for gene therapy 23
• Genome 23
• DNA 24
• RNA 24
• Alternative RNA splicing 25
• Genes 26
• Gene regulation 26
• Gene expression 28
• Chromosomes 28
• Telomeres 29
• Mitochondrial DNA 29
• Proteins 30

2. Gene Therapy Technologies 31

• Classification of gene therapy techniques 31
• Ex vivo and in vivo gene therapy 32
• Ex vivo gene therapy 32
• In vivo gene therapy 33
• Physical methods of gene transfer 33
• Electroporation 33
• Applications of electroporation 34
• Clinical applications of electroporation 35
• Advantages of electroporation 35
• Limitations of electroporation 36
• Hydrodynamic 36
• Microinjection 36
• Particle bombardment 37
• Ultrasound-mediated transfection 39
• Molecular vibration 40
• Application of pulsed magnetic field and superparamagnetic nanoparticles 40
• Gene transfection using laser irradiation 40
• Photochemical transfection 41
• Chemical methods of gene transfer 41
• Gene repair and replacement 41
• Gene repair by single-stranded oligonucleotides 42
• History and current status of chimeraplasty 42
• Spliceosome-mediated RNA trans-splicing 42
• mRNA gene therapy 43
• Vectors for gene therapy 43
• Basic considerations 43
• Use of genes as pharmaceuticals 44
• The ideal vector for gene therapy 44
• Viral vectors 45
• Retroviral vectors 45
• Oncognic potential of retroviral vectors 47
• Adenovirus vectors 48
• Adeno-associated virus vectors 50
• Herpes simplex virus vectors 52
• Lentiviral vectors 54
• Baculovirus vectors 55
• Alphavirus vectors 55
• Multicistronic retroviral vectors 56
• Future prospects of viral vectors 56
• Companies using viral vectors 57
• Nonviral vectors for gene therapy 58
• Liposomes for gene therapy 59
• Liposome-nucleic acid complexes 60
• Cationic lipid-DNA complexes 61
• Anionic lipid-DNA complexes 61
• HVJ-liposome method 61
• Polycation-DNA complexes (polyplexes) 62
• Synthetic peptide complexes 63
• Polymer molecules 63
• Effects of shape of DNA molecules on delivery with nonviral vectors 63
• Plasmid DNA vector for treatment of chronic inflammatory disease 63
• Nanobiotechnology for gene transfer 64
• Nanoparticles as non-viral vectors for gene therapy 64
• Dendrimers 64
• Cochleates 65
• Calcium phosphate nanoparticles as non-viral vectors 65
• Lipid nanoparticles for nucleic acid delivery 66
• Silica nanoparticles as a nonviral vector for gene delivery 66
• Gelatin nanoparticles for gene delivery 67
• Nonionic polymeric micelles for oral gene delivery 67
• Biological nanoparticle technology 67
• Receptor-mediated endocytosis 67
• Artificial viral vectors 68
• Directed evolution of AAV to create efficient gene delivery vectors 69
• Bacterial ghosts as DNA delivery systems 69
• Bacteria plus nanoparticles for gene delivery into cells 70
• Chromosome-based vectors for gene therapy 71
• Companies using non-viral vectors 72
• Concluding remarks about vectors 73
• Cell-mediated gene therapy 74
• Fibroblasts 75
• Skeletal muscle cells 75
• Vascular smooth muscle cells 76
• Keratinocytes 76
• Hepatocytes 77
• Lymphocytes 77
• Regulating protein delivery by genetically encoded lymphocytes 77
• Implantation of microencapulated genetically modified cells 77
• Stem cell gene therapy 78
• Therapeutic applications for hematopoietic stem cell gene transfer 78
• Improving delivery of genes to stem cells 79
• Lentiviral vectors for gene transfer to marrow stem cells 79
• Use of mesenchymal stem cells for gene therapy 79
• In utero gene therapy using stem cells 79
• Gene delivery to stem cells by artificial chromosome expression 80
• Linker based sperm-mediated gene transfer technology 80
• Combination of gene therapy with therapeutic cloning 80
• Expansion of transduced HSCs in vivo 80
• The future of hematopoietic stem cell gene therapy 81
• Routes of administration for gene therapy 81
• Direct injection of naked DNA 82
• Intramuscular injection 82
• Intravenous DNA injection 82
• Intraarterial delivery 82
• Companies with gene delivery devices/ technologies 83
• Targeted gene therapy 84
• Targeted integration 84
• Bacteriophage integrase system for site-specific gene delivery 84
• Controlled-release delivery of DNA 85
• Controlled gene therapy 85
• Controlled delivery of genetic material 85
• Controlled induction of gene expression 86
• Drug-inducible systems for control of gene expression 86
• Timed activation of gene therapy by a circuit based on signaling network 87
• Small molecules for post-transcriptional regulation of gene expression 87
• Light Activated Gene Therapy 87
• Engineered zinc finger DNA binding proteins for gene correction 88
• Companies with regulated /targeted gene therapy 88
• Gene marking 89
• Germline gene therapy 90
• Potential applications of human germline genome modification 90
• Pros and cons of human germline genome modification 90
• Role of gene transfer in antibody therapy 92
• Genetically engineered vaccines 92
• DNA vaccines 92
• DNA inoculation technology 93
• Methods for enhancing the potency of DNA vaccines 93
• Advantages of DNA vaccines 94
• Vaccine vectors 94
• Challenges and limitations of genetically engineered vaccines 95
• Vaccines based on reverse genetics 95
• Technologies for gene suppression 96
• Antisense oligonucleotides 96
• Transcription factor decoys 97
• Aptamers 97
• Ribozymes 97
• Peptide nucleic acid 98
• Intracellular delivery of PNAs 98
• Locked nucleic acid 98
• Zorro-LNA 99
• Gene silencing 99
• Post-transcriptional gene silencing 99
• Definitions and terminology of RNAi 100
• RNAi mechanisms 100
• Inhibition of gene expression by antigene RNA 101
• RNAi gene therapy 101
• Application of molecular diagnostic methods in gene therapy 102
• Use of PCR to study biodistribution of gene therapy vector 102
• PCR for verification of the transcription of DNA 102
• In situ PCR for direct quantification of gene transfer into cells 102
• Detection of retroviruses by reverse transcriptase (RT)-PCR 103
• Confirmation of viral vector integration 103
• Monitoring of gene expression 103
• Monitoring of gene expression by green fluorescent protein 103
• Monitoring in vivo gene expression by molecular imaging 104
• Advantages of gene therapy compared with protein therapy 104

3. Clinical Applications of Gene Therapy 105

• Introduction 105
• Bone and joint disorders 105
• Bone fractures 105
• Gene therapy for intervertebral disc degeneration 106
• Spinal fusion 106
• Osteogenesis imperfecta 107
• Rheumatoid arthritis 107
• Local or systemic treatment 108
• In vivo or ex vivo gene therapy of RA 108
• Clinical trials 109
• Gene therapy for osteoarthritis 110
• Sports injuries 111
• Repair of articular cartilage defects 111
• Regeneration and replacement of bone by gene therapy 112
• Dentistry 113
• Tissue engineering in dental implant defects 113
• Endocrine disorders 113
• Introduction 113
• Diabetes mellitus 113
• Methods of gene therapy of diabetes mellitus 114
• Viral vector-mediated gene transfer in diabetes 115
• Gene delivery with ultrasonic microbubble destruction technology 115
• Genetically engineered cells for diabetes mellitus 115
• Genetically altered liver cells 116
• Genetically modified stem cells 116
• Genetically engineered dendritic cells 116
• Insertion of gene encoding for IL-4 117
• Concluding remarks about cell and gene therapy of diabetes 117
• Gene therapy of growth-hormone deficiency 118
• Gene therapy of obesity 118
• Ad viral vector-mediated transfer of leptin gene 118
• AAV vector-mediated delivery of GDNF for obesity 119
• Gastrointestinal disorders 120
• Introduction 120
• Methods of gene transfer to the gastrointestinal tract 120
• Direct delivery of genes 120
• Naked plasmid DNA into the submucosa 120
• Viral vectors 120
• Receptor-mediated endocytosis 121
• Indications for gastrointestinal gene therapy 121
• Gene therapy for inflammatory disorders of the bowel 122
• Gene transfer to the salivary glands 122
• Potential clinical applications of salivary gene therapy 123
• Hematology 123
• Hemophilias 123
• Gene therapy of hemophilia 124
• Hemophilia A 124
• Hemophilia B 125
• Concluding remarks about gene therapy of hemophilias 126
• Hemoglobinopathies 126
• Gene therapy for β-thalassemia 127
• Gene therapy of Fanconi' s anemia 129
• Acquired hematopoietic disorders 129
• Chronic acquired anemias 129
• Neutropenia 130
• Thrombocytopenia 131
• Concluding remarks about gene therapy of hemoglobinopathies 131
• Companies involved in gene thery of hematological disorders 132
• In utero gene therapy 132
• Fetal gene transfer techniques 132
• Animal models of fetal gene therapy 133
• Potential applications of fetal gene therapy 133
• Fetal gene therapy for cystic fibrosis 134
• Fetal intestinal gene therapy 134
• Hearing disorders 134
• Potential of gene therapy 135
• Vectors for gene therapy of hearing disorders 135
• Auditory hair cell replacement and hearing improvement by gene therapy 136
• Kidney diseases 136
• End-stage renal disease 136
• Methods of gene delivery to the kidney 137
• Gene transfer into kidney by adenoviral vectors 137
• Non-viral gene transfer to the kidneys 137
• Gene transfer into the glomerulus by HVJ-liposome 138
• Bone marrow stem cells for renal disease 138
• Mesangial cell therapy 138
• Liposome-mediated gene transfer into the tubules 139
• Gene transfer to tubules with cationic polymer polyethylenimine 139
• Gene therapy in animal experimental models of renal disease 139
• Genetic manipulations of the embryonic kidney 140
• Antisense intervention in glomerulonephritis 140
• Gene therapy for renal fibrosis 140
• Use of genetically engineered cells for uremia due to renal failure 141
• Concluding remarks 141
• Liver disorders 141
• Techniques of gene delivery to liver 142
• Direct injection of DNA into liver 143
• Local gene delivery by isolated organ perfusion 143
• Liposome-mediated direct gene transfer 143
• Retroviral vector for gene transfer to liver 143
• Adenoviral vectors for gene transfer to liver 143
• Receptor-mediated approach 144
• Cell therapy for liver disorders 144
• Transplantation of genetically modified hepatocytes 144
• Genetically modified hematopoietic stem cells 145
• Gene therapy by ex vivo transduced liver progenitor cells 145
• Gene therapy of genetic diseases affecting the liver 145
• Crigler-Najjar syndrome 145
• Hereditary tyrosinemia type I (HT1) 146
• Hereditary tyrosinemia type 3 146
• Gene therapy of acquired diseases affecting the liver 146
• Cirrhosis of liver 146
• Ophthalmic disorders 147
• Introduction to gene therapy of ophthalmic disorders 147
• Degenerative retinal disorders 148
• Inherited retinal degenerations 148
• Leber' s congenital amaurosis 148
• X-linked juvenile retinoschisis 149
• Retinitis pigmentosa 149
• Age-related macular degeneration 150
• Proliferative retinopathies 151
• Methods of gene transfer to retinal cells 151
• DNA nanoparticles for nonviral gene transfer to the eye 153
• Prevention of complications associated with eye surgery 153
• Prevention of proliferative retinopathy by gene therapy 153
• DNA nanoparticles for gene therapy of retinal degenerative disorders 153
• Posterior capsule opacification after cataract surgery 153
• Autoimmune uveitis 154
• Retinal ischemic injury 154
• Corneal disorders 155
• Glaucoma 155
• Disorders of hearing 156
• Gene therapy for hearing loss 156
• Organ transplantation 156
• Introduction 156
• Veto cells and transplant tolerance 157
• Gene therapy for prolonging allograft survival 157
• Gene therapy in lung transplantation 158
• Role of gene therapy in liver transplantation 158
• Gene therapy in kidney transplantation 158
• Pulmonary disorders 159
• Techniques of gene delivery to the lungs 159
• Adenoviral vectors 159
• Non-viral vectors 160
• Aerosolization as an aid to gene transfer to lungs 160
• Cystic fibrosis 161
• Genetics and clinical features 161
• Gene therapy for CF 161
• CFTR gene transfer in CF 162
• Concluding remarks about gene therapy of CF 163
• Miscellaneous pulmonary disorders 163
• Gene therapy for pulmonary arterial hypertension 163
• Gene therapy for bleomycin-induced pulmonary fibrosis 164
• Pulmonary complications of a1-antitrypsin deficiency 165
• Gene therapy for asthma 165
• Gene therapy for adult respiratory distress syndrome 166
• Gene therapy for lung injury 166
• Gene therapy for bronchopulmonary dysplasia 167
• Concluding remarks about gene therapy of lungs 167
• Companies involved in pulmonary gene therapy 167
• Skin and soft tissue disorders 168
• Gene transfer to the skin 168
• Electroporation for transdermal delivery of DNA vaccines 168
• Ultrasound and topical gene therapy 169
• Gene therapy in skin disorders 169
• Gene therapy of hair loss 169
• Gene therapy for xeroderma pigmentosa 170
• Gene therapy for lamellar ichthyosis 170
• Gene therapy for epidermolysis bullosa 170
• Gene transfer techniques for wound healing 171
• Urogenital disorders 171
• Gene therapy for urinary tract dysfunction 172
• Gene therapy for erectile dysfunction 172
• NOS gene transfer for erectile dysfunction 172
• Clinical trial of hMaxi-K Gene transfer in erectile dysfunction 172
• Gene therapy for erectile dysfunction due to nerve injury 173
• Concluding remarks on gene therapy for erectile dysfunction 173
• Veterinary gene therapy 173
• Gene therapy for mucopolysaccharidosis VII in dogs 173
• Gene therapy to increase disease resistance 174
• Gene therapy for infections 174
• Gene therapy for chronic anemia 174
• Gene therapy for endocrine disorders 175
• Gene therapy for arthritis 175
• Cancer gene therapy 175
• Brain tumors in cats and dogs 176
• Breast cancer in dogs 176
• Canine hemangiosarcoma 177
• Canine melanoma 177
• Canine soft tissue sarcoma 178
• Melanoma in horses 178

4. Gene Therapy of Genetic Disorders 179

• Introduction 179
• Primary immunodeficiency disorders 180
• Severe combined immune deficiency 181
• Chronic granulomatous disease 183
• Wiskott-Aldrich syndrome 183
• Purine nucleoside phosphorylase deficiency 184
• Major histocompatibility class II deficiency 184
• Future prospects of gene therapy of inherited immunodeficiencies 184
• Metabolic disorders 185
• Adrenoleukodystrophy 185
• Canavan disease 186
• Lesch-Nyhan syndrome 186
• Ornithine transcarbamylase deficiency 186
• Phenylketonuria 187
• Porphyrias 187
• Tetrahydrobiopterin deficiency 188
• Lysosomal storage disorders 189
• Batten disease 190
• Fabry' s disease 190
• Gaucher disease 190
• Animals models of Gaucher' s disease 191
• Gene therapy of Gaucher' s disease 191
• Hunter syndrome 192
• Combination of cell and gene therapy for Krabbe' s disease 192
• Metachromatic leukodystrophy 193
• Mucopolysaccharidosis type 1 (Hurler syndrome) 194
• Niemann-Pick type A disease 194
• Pompe disease 194
• Sanfilippo A syndrome 195
• Sly syndrome 195
• Tay-Sachs disease 195
• Future prospects of gene therapy of lysosomal storage disorders 196
• Trinucleotide repeat disorders 196
• Muscular dystrophies 197
• Duchenne muscular dystrophy (DMD) 197
• Animal models for gene therapy of DMD 197
• Types of dystrophin constructs 197
• Antisense approach to DMD 198
• Post-transcriptional modulation of gene expression in DMD 199
• Myoblast-based gene transfer in DMD 199
• Plasmid-mediated gene therapy 199
• Liposome-mediated gene transfer 200
• Viral vectors for DMD 200
• Routes of administration of gene therapy in DMD 201
• Conclusions and future prospects of gene therapy of DMD 201
• Limb-girdle muscular dystrophy 202
• Spinal muscular atrophy 203
• Hereditary neuropathies 203
• Charcot-Marie-Tooth disease 203
• Hereditary axonal neuropathies of the peripheral nerves 204
• Companies involved in gene therapy of genetic disorders 204

5. Gene Therapy of Cancer 205

• Strategies for cancer gene therapy 205
• Direct gene delivery to the tumor 206
• Injection into tumor 206
• Direct injection of adenoviral vectors 206
• Direct injection of a plasmid DNA-liposome complex 207
• A polymer approach to local gene therapy for cancer 207
• Electroporation for cancer gene therapy 207
• Control of gene expression in tumor by local heat 208
• Radiation-guided gene therapy of cancer 208
• Nanoparticles to facilitate combination of hyperthermia and gene therapy 209
• Cell-based cancer gene therapy 209
• Adoptive cell therapy 209
• Cytokine gene therapy 210
• Genetic modification of human hematopoietic stem cells 213
• Immunogene therapy 213
• Cancer vaccines 214
• Genetically modified cancer cell vaccines 214
• GVAX cancer vaccines 214
• Genetically modified dendritic cells 215
• Nucleic acid-based cancer vaccines 215
• DNA cancer vaccines 216
• RNA vaccines 216
• Viral vector-based cancer vaccines 216
• Intradermal delivery of cancer vaccines by Ad vectors 217
• Future prospects of cancer vaccines 217
• Companies involved in nucleic acid-based cancer vaccines 218
• Monoclonal antibody gene transfer for cancer 219
• Transfer and expression of intracellular adhesion-1 molecules 219
• Other gene-based techniques of immunotherapy of cancer 219
• Fas (Apo-1) 219
• Chemokines 220
• Major Histocompatibility Complex (MHC) Class I 220
• IGF (Insulin-Like Growth Factor) 220
• Inhibition of immunosuppressive function in cancer 221
• Delivery of toxic genes to tumor cells for eradication 221
• Gene-directed enzyme prodrug therapy 221
• Combination of gene therapy with radiotherapy 222
• Correction of genetic defects in cancer cells 222
• Targeted gene therapy for cancer 223
• Bacteria as novel anticancer gene vectors 223
• Cancer-specific gene expression 223
• Cancer-specific transcription 223
• Delivery of retroviral particles hitchhiking on T cells 224
• Electrogene and electrochemotherapy 224
• Epidermal growth factor-mediated DNA delivery 224
• Gene-based targeted drug delivery to tumors 225
• Gene expression in hypoxic tumor cells 225
• Genetically modified T cells for targeting tumors 226
• Genetically engineered stem cells for targeting tumors 226
• Hematopoietic stem cells for targeted cancer gene therapy 227
• Immunolipoplex for delivery of p53 gene 227
• Nanomagnets for targeted cell-based cancer gene therapy 228
• Nanoparticles for targeted site-specific delivery of anticancer genes 228
• Targeted cancer therapy using a dendrimer-based synthetic vector 229
• Tumor-targeted gene therapy by receptor-mediated endocytosis 229
• Virus-mediated oncolysis 229
• Targeted cancer treatments based on oncolytic viruses 229
• Oncolytic HSV 230
• Oncolytic adenoviruses 230
• Oncolytic vesicular stomatitis virus 232
• Oncolytic paramyxovirus 232
• Oncolytic vaccinia virus 232
• Cancer terminator virus 232
• Cytokine-induced killer cells for delivery of an oncolytic virus 233
• Monitoring of viral-mediated oncolysis by PET 233
• Oncolytic gene therapy 234
• Companies developing oncolytic viruses 234
• Apoptotic approach to improve cancer gene therapy 235
• Tumor suppressor gene therapy 235
• P53 gene therapy 236
• BRIT1 gene therapy 236
• Nitric oxide-based cancer gene therapy 236
• Nitric oxide synthase II DNA injection 236
• Gene therapy for radiosensitization of cancer 236
• Gene therapy of cancer of selected organs 237
• Gene therapy for bladder cancer 237
• Gene therapy for glioblastoma multiforme 238
• Targeted adenoviral vectors 239
• Targeting normal brain cells with an AAV vector encoding interferon-β 239
• Viral oncolysis of brain tumors 240
• Autophagy induced by conditionally replicating adenoviruses 240
• Oncolytic virus targeted to brain tumor stem cells 240
• Antiangiogenic gene therapy 241
• Baculovirus vector for diphtheria toxin gene therapy 241
• Intravenous gene delivery with nanoparticles into brain tumors 242
• Gene therapy targeting hepatocyte growth factor 242
• RNAi gene therapy of brain cancer 242
• Ligand-directed delivery of dsRNA molecules targeted to EGFR 243
• Gene therapy for breast cancer 243
• Intratumoral injection of Ad5CMV-p53 (Advexin) 243
• Gene vaccine for breast cancer 244
• Recombinant adenoviral ErbB-2/neu vaccine 244
• Gene Therapy for ovarian cancer 245
• Gene therapy for malignant melanoma 246
• Gene therapy of lung cancer 247
• Intravenous nanoparticle formulation for delivery of FUS1 gene 247
• Aerosol gene delivery for lung cancer 248
• Gene therapy for cancer of prostate 248
• Experimental studies 248
• Nanoparticle-based gene therapy for prostate cancer 249
• Tumor suppressor gene therapy in prostate cancer 249
• Vaccine for prostate cancer 249
• Clinical trials 249
• Gene therapy of head and neck cancer 250
• Adenoviral vector based P53 gene therapy 250
• Gene therapy of pancreatic cancer 250
• Adenovirus-mediated transfer of vasostatin gene 251
• Rexin-G™ for targeted gene delivery in cancer 251
• Targeted Expression of BikDD gene 251
• Cancer gene therapy companies 252

6. Gene Therapy of Neurological Disorders 255

• Indications 255
• Gene transfer techniques for the nervous system 256
• Methods of gene transfer to the nervous system 256
• Ideal vector for gene therapy of neurological disorders 256
• Promoters of gene transfer 256
• Lentivirus-mediated gene transfer to the CNS 257
• AAV vector mediated gene therapy for neurogenetic disorders 257
• Gene transfer to the CNS using recombinant SV40-derived vectors 258
• Routes of delivery of genes to the CNS 258
• Cell-mediated gene therapy of neurological disorders 259
• Neuronal cells 259
• Implantation of genetically modified encapsulated cells into the brain 260
• Gene therapy of neurodegenerative disorders 261
• Gene therapy for Parkinson disease 261
• Rationale 262
• Introduction of functional genes into the brain of patients with PD 262
• Delivery of neurotrophic factors by gene therapy 262
• Delivery of parkin gene 263
• Techniques of gene therapy for PD 263
• RNAi approach to PD 266
• Nanoparticle-based gene therapy for PD 267
• Prospects of gene therapy for PD 267
• Companies developing gene therapy for PD 268
• Gene therapy for Alzheimer disease 268
• Rationale 268
• NGF gene therapy for AD 269
• Neprilysin gene therapy 270
• Targeting plasminogen activator inhibitor type-1 gene 270
• Gene vaccination 270
• Combination of gene therapy with other treatments for AD 271
• Gene therapy of Huntington disease 271
• Encapsulated genetically engineered cellular implants 271
• Viral vector mediated administration of neurotrophic factors 271
• RNAi gene therapy 272
• Gene therapy of amyotrophic lateral sclerosis 272
• Rationale 272
• Technique of gene therapy of ALS 272
• Gene therapy of cerebrovascular diseases 273
• Preclinical research in gene therapy for cerebrovascular disease 273
• Animal models of stroke relevant to gene therapy 274
• Transgenic mice as models for stroke 274
• Animal models for gene therapy of arteriovenous malformations 274
• Gene transfer to cerebral blood vessels 274
• Gene therapy for vasospasm following subarachnoid hemorrhage 276
• NOS gene therapy for cerebral vasospasm 276
• Gene therapy for stroke 277
• Gene therapy for stroke using neurotrophic factors 278
• Gene therapy of strokes with a genetic component 278
• Gene therapy for intracranial aneurysms 278
• Concluding remarks about gene therapy for stroke 279
• Gene therapy of injuries to the nervous system 279
• Traumatic brain injury 279
• Spinal cord injury 280
• Gene therapy of epilepsy 280
• Gene therapy for control of seizures 281
• Gene therapy for neuroprotection in epilepsy 281
• Gene therapy for genetic forms of epilepsy 282
• Gene therapy for multiple sclerosis 282
• Gene therapy for relief of pain 283
• Rationale of gene therapy for pain 283
• Vectors for gene therapy of pain 283
• Methods of gene delivery for pain 284
• Endogenous analgesic production for cranial neuralgias 284
• Gene delivery by intrathecal route 285
• Gene transfer for delivery of analgesics to the spinal nerve roots 285
• Gene therapy of peripheral neuropathic pain 286
• Gene transfer by injections into the brain substance 287
• Targets for gene therapy of pain 287
• Zinc finger DNA-binding protein therapeutic for chronic pain 287
• Gene therapy for producing enkephalin to block pain signals 287
• Targeting nuclear factor-yβ 287
• Gene therapy targeted to neuroimmune component of chronic pain 288
• Potential applications of gene therapy for management of pain 288
• Concluding remarks on gene therapy for pain 288
• Gene therapy for psychiatric disorders 289
• Gene therapy for depression 290
• Gene therapy for enhancing cognition after stress 290
• Companies involved in gene therapy of neurological disorders 290

7. Gene Therapy of Cardiovascular Disorders 293

• Introduction 293
• Techniques of gene transfer to the cardiovascular system 293
• Direct plasmid injection into the myocardium 294
• Catheter-based systems for vector delivery 294
• Ultrasound microbubbles for cardiovascular gene delivery 295
• Vectors for cardiovascular gene therapy 295
• Adenoviral vectors for cardiovascular diseases 295
• Plasmid DNA-based delivery in cardiovascular disorders 295
• Intravenous rAAV vectors for targeted delivery to the heart 296
• Hypoxia-regulated gene therapy for myocardial ischemia 296
• Angiogenesis and gene therapy of ischemic disorders 296
• Therapeutic angiogenesis vs vascular growth factor therapy 297
• Gene painting for delivery of targeted gene therapy to the heart 297
• Gene delivery to vascular endothelium 298
• Targeted plasmid DNA delivery to the cardiovascular system with nanoparticles 298
• Vascular stents for gene delivery 298
• Gene therapy for genetic cardiovascular disorders 299
• Genetic disorders predisposing to atherosclerosis 299
• Familial hypercholesterolemia (FH) 299
• Apolipoprotein E (apoE) deficiency 301
• Hypertension 301
• Genetic factors for myocardial infarction 302
• Acquired cardiovascular diseases 302
• Coronary artery disease with angina pectoris 302
• Ad5FGF-4 302
• Ischemic heart disease with myocardial infarction 303
• Myocardial repair with IGF-1 therapy 304
• Metalloproteinase-2 inhibitor gene therapy 304
• Congestive heart failure 305
• Rationale of gene therapy in CHF 305
• Genetic manipulation of βadrenergic receptor system in CHF 305
• Intracoronary adenovirus-mediated gene therapy for CHF 306
• AAV-mediated gene transfer for CHF 306
• AngioCell gene therapy for CHF 306
• nNOS gene transfer in CHF 307
• Cardiomyopathies 307
• Cardiac conduction disturbances 307
• Gene therapy and heart transplantation 308
• Peripheral arterial disease 308
• Incidence and clinical features 308
• Current management 309
• Gene therapy for peripheral arterial disease 309
• Angiogenesis by gene therapy 309
• HIF-1αgene therapy for peripheral arterial disease 309
• HGF gene therapy for peripheral arterial disease 310
• Ischemic neuropathy secondary to peripheral arterial disease 310
• Prevention of restenosis after angioplasty 311
• Antisense approaches 311
• Gene therapy to prevent restenosis after angioplasty 311
• Techniques of gene therapy for restenosis 313
• NOS gene therapy for restenosis 313
• hTIMP-1 gene therapy to prevent intimal hyperplasia 314
• Maintaining vascular patency after surgery 314
• Companies involved in gene therapy of cardiovascular diseases 315
• Future prospects of gene therapy of cardiovascular disorders 316

8. Gene therapy of viral infections 317

• Introduction 317
• Acquired Immunodeficiency Syndrome (AIDS) 317
• Current management of AIDS 317
• Gene therapy strategies in HIV/AIDS 318
• HIV/AIDS vaccines 318
• Insertion of protective genes into target cells 319
• Cell/gene therapies for HIV/AIDS 320
• Transplantation of genetically modified T-cells 320
• Transplantation of genetically modified hematopoietic cells 320
• Inhibition of HIV-1 replication by lentiviral vectors 321
• VRX496 321
• Intracellular immunization 321
• Engineered cellular proteins such as soluble CD4s 322
• Intracellular antibodies 322
• Anti-rev single chain antibody fragment 322
• Use of genes to chemosensitize HIV-1 infected cells 322
• Autocrine interferon (INF)-βproduction by somatic cell gene therapy 323
• Antisense approaches to AIDS 323
• RNA decoys 323
• Antisense oligodeoxynucleotides 323
• RNA decoys 323
• Ribozymes 324
• RNAi applications in HIV/AIDS 324
• siRNA-directed inhibition of HIV-1 infection 325
• Role of the nef gene during HIV-1 infection and RNAi 325
• Bispecific siRNA constructs 325
• Targeting CXCR4 with siRNAs 326
• Targeting CCR5 with siRNAs 326
• Companies involved in developing gene therapy for HIV/AIDS 327
• Conclusions regarding gene therapy of HIV/AIDS 328
• Genetic vaccines for other viral infections 328
• Cytomegalic virus infections 328
• Viral hepatitis 329
• Vaccine for hepatitis B virus 329
• Vaccine for hepatitis C virus 329
• Vaccine for herpes simplex virus 330
• DNA vaccine against rabies 330
• DNA vaccine for Ebola 331
• Vaccines for avian influenza 331
• Future prospects of DNA vaccines for avian influenza 332
• Human trial of a DNA vaccine for avian influenza 332
• Companies developing genetic vaccines for infections other than AIDS 333

9. Research, Development and Future of Gene Therapy 335

• Basic research in gene therapy 335
• R & D in gene therapy 335
• Animal models of human diseases for gene therapy research 336
• Lentiviral transgenesis 336
• Financing research and development 336
• Role of the NIH in gene therapy research 336
• National Gene Vector Laboratories 336
• Financing by the industry 337
• Clinical trials in gene therapy 337
• Clinical trials worldwide 337
• Clinical trials in cancer gene therapy 338
• Clinical trials in cardiovascular gene therapy 338
• Clinical trials in inherited monogenic diseases 338
• Clinical trials for other indications 339
• Clinical trials in the US 339
• Vectors used in gene therapy clinical trials 340
• Future prospects for the gene therapy 341
• How to improve gene therapy 341
• Promising areas of application of gene therapy 342
• Neurological disorders 342
• Gene therapy of cardiovascular disorders 343
• Cancer gene therapy 344
• Personalized gene therapy 344

10. Regulatory, Safety and Ethical Issues of Gene Therapy 347

• Regulation of gene therapy in the United States 347
• US Federal guidelines for research involving recombinant DNA molecules 347
• Regulation of gene therapy in US 347
• Office of Biotechnology Activities 347
• Implantation of genetically manipulated cells 348
• Clinical trials in gene therapy 348
• Regulation of gene therapy in Germany 348
• Preclinical research 349
• Clinical Trials 349
• Marketing authorization 350
• Regulation of gene therapy in the United Kingdom 350
• Regulation of gene therapy in France 350
• Regulation of gene therapy in the Netherlands 351
• Regulation of gene therapy in Australia 351
• Regulation of gene therapy in Japan 352
• Regulation of gene therapy in China 352
• Safety issues of gene transfer 352
• Adverse effects of retroviral vectors 353
• Insertional mutagenesis 353
• Adverse effects of HSV vectors 353
• Neurotoxicity of HSV vectors 353
• Hepatotoxicity of HSV-tk/ganciclovir approach 354
• Adverse effects of adenoviral vectors 354
• Inflammatory effects of adenoviruses in lungs 354
• Inflammatory effects involving the liver 354
• Induction of immune response by adenoviral vectors 355
• Impairment of adrenocortical steroidogenesis 355
• Adverse effects of AAV vectors 355
• Toxicity associated with cationic lipid-mediated gene transfer 356
• Toxicity of lipopolysaccharides 356
• Potential side effects of RNAi gene therapy 356
• Role of molecular diagnostics in safety of gene therapy 357
• Quality control of vectors 357
• Testing for retroviruses 357
• Adenoviral vectors 358
• Replication competent viruses 358
• Genetic characteristics of viral vectors 358
• Concluding remarks about safety of viral vectors 358
• Ethical aspects of gene therapy 359
• The lay consumer' s view of somatic gene therapy ethics 359
• Ethical aspects of clinical trials 360
• Ethical aspects of germline gene therapy 360
• Germline gene therapy for genetic enhancement 361
• Athletic enhancement by genetic engineering 361
• Efficacy of genetic enhancement in athletics 361
• Adverse effect of genetic engineering 362
• Problems in detecting genetic manipulations in athletes 363
• Ethical dilemma 363

11. Markets for Gene Therapy 365

• Introduction 365
• Gene therapy markets in various regions of the world 365
• Gene therapy markets according to therapeutic areas 366
• Cancer gene therapy market 366
• Markets for gene therapy of genetic disorders 366
• Markets for DNA vaccines 367
• DNA vaccines markets according to technologies 367
• DNA vaccines markets according to therapeutic indications 367
• DNA vaccines markets according to geographical areas 368
• Competing treatments 368
• Antisense 369
• RNAi 369
• Cell therapy 369
• Strategies for developing gene therapy markets 369
• Collaboration with pharmaceutical companies 370
• Collaboration with companies developing cell-based therapies 370
• Overcoming obstructions to the development of gene therapy 370
• Collaboration with academic gene therapy centers 370
• Developing safer and cost-effective gene medicines 370
• Unmet needs in gene therapy 371
• Promising areas for the development of gene therapy 371

12. References 373

Tables

• Table 1 1: Landmarks in development of gene therapy 21
• Table 2 1: Classification of methods of gene therapy 31
• Table 2 2: A comparison of various physical methods of gene transfer 33
• Table 2 3: Experimental applications of gene transfer by electroporation 35
• Table 2 4: An overview of characteristics of commonly used viral vectors 45
• Table 2 5: Companies using viral vectors 57
• Table 2 6: Companies using non-viral vectors 72
• Table 2 7: Target organs for non-viral gene therapy methods 74
• Table 2 8: Potential routes for administration of DNA 81
• Table 2 9: Companies with gene delivery devices/ technologies 83
• Table 2 10: Strategies for targeted gene therapy 84
• Table 2 11: In vivo animal experimental studies of gene delivery with polymeric systems 85
• Table 2 12: Approaches to controlling gene expression in gene therapy 86
• Table 2 13: Companies with regulated / targeted gene therapy and special techniques 88
• Table 2 14: Potential applications of human germline genome modification 90
• Table 2 15: Applications of molecular diagnostics in gene therapy 102
• Table 2 16: Advantages of gene therapy compared with protein therapy 104
• Table 3 1: Experimental approaches to gene therapy of rheumatoid arthritis 108
• Table 3 2: Gene therapy strategies for osteoarthritis 110
• Table 3 3: Cell and gene therapy approaches for type 1 diabetes mellitus 114
• Table 3 4: Indications for gastrointestinal gene therapy 121
• Table 3 5: Hematological disorders that can be potentially treated by gene therapy 123
• Table 3 6: Companies involved in gene therapy of hematological disorders 132
• Table 3 7: Techniques of gene transfer to the kidneys 137
• Table 3 8: Gene therapy in animal experimental models of renal disease 139
• Table 3 9: Applications of gene therapy in ophthalmological disorders 147
• Table 3 10: Strategies for gene delivery to the lungs 159
• Table 3 11: Companies developing gene therapy for pulmonary disorders 167
• Table 4 1: Genetic disorders that are being investigated for gene therapy 179
• Table 4 2: X-linked immunodeficiency disorders 181
• Table 4 3: Examples of inherited metabolic disorders amenable to gene therapy 185
• Table 4 4: Gene therapy approaches to Duchenne muscular dystrophy 197
• Table 4 5: Companies involved in gene therapy of genetic/metabolic disorders 204
• Table 5 1: Strategies for cancer gene therapy 205
• Table 5 2: Cell-based gene therapy for cancer 209
• Table 5 3: Companies with nucleic acids/genetically modified cell cancer vaccines 218
• Table 5 4: Enzyme/prodrug combinations employed in suicide gene therapy 221
• Table 5 5: Mutation compensation strategies used clinically 222
• Table 5 6: Companies developing oncolytic viruses 234
• Table 5 7: Strategies for gene therapy of malignant brain tumors 238
• Table 5 8: Clinical trials of gene therapy in ovarian cancer 245
• Table 5 9: Gene therapy for malignant melanoma 246
• Table 5 10: Clinical trials in gene therapy for prostate cancer 250
• Table 5 11: Companies involved in cancer gene therapy 252
• Table 6 1: Example of potential indications for gene therapy of neurologic disorder 255
• Table 6 2: Methods of gene transfer as applied to neurologic disorders 256
• Table 6 3: Gene therapy techniques applicable to Parkinson disease 261
• Table 6 4: Companies developing gene therapy for Parkinson' s disease 268
• Table 6 5: Gene transfer in animal models of carotid artery restenosis 275
• Table 6 6: Gene therapy strategies for vasospasm 276
• Table 6 7: Neuroprotective gene therapy in animal stroke models 277
• Table 6 8: Experimental gene therapy approaches for relief of pain 284
• Table 6 9: Companies involved in gene therapy of neurological disorders 291
• Table 7 1: Cardiovascular disorders for which gene therapy is being considered 293
• Table 7 2: Catheter-based systems for vector delivery to the cardiovascular system 294
• Table 7 3: Companies involved in gene therapy of cardiovascular diseases 315
• Table 8 1: Strategies for gene therapy of AIDS 318
• Table 8 2: Companies involved in developing gene therapy for HIV/AIDS 327
• Table 8 3: Companies developing genetic vaccines for infections other than AIDS 333
• Table 9 1: Clinical trials of gene therapy in the US according to applications 339
• Table 9 2: Potential future applications of gene therapy in disorders of the nervous system 342
• Table 11 1: Gene therapy market according to regions/countries - 2007 to 2017 365
• Table 11 2: Gene therapy markets according to therapeutic areas s - 2007 to 2017 366
• Table 11 3: Cancer gene therapy market according to type of cancer - 2007 to 2017 366
• Table 11 4: Gene therapy market for selected genetic disorders - 2007 to 2017 367
• Table 11 5: DNA vaccines markets according to technologies - 2007 to 2017 367
• Table 11 6: DNA vaccines markets according to therapeutic indications - 2007 to 2017 368
• Table 11 7: DNA vaccines markets according to geographical areas - 2007 to 2017 368

Figures

• Figure 1 1: Relation of gene therapy to other biotechnologies 23
• Figure 1 2: Relationship of DNA, RNA and protein in the cell 27
• Figure 2 1: Ex vivo and in vivo techniques of gene therapy 32
• Figure 2 2: Structure of the Helios gene gun 37
• Figure 2 3: Cochleate-mediated gene therapy 65
• Figure 2 4: Bacteria plus nanoparticles for drug delivery into cells 70
• Figure 2 5: Schematic of suppression of gene expression by RNAi 100
• Figure 6 1: Effect of tyrosine hydroxylase gene delivery on dopamine levels 263
• Figure 6 2: Role of cell and gene therapy in stroke according to pathology and stage 279
• Figure 9 1: Product development cycle in gene therapy 335
• Figure 9 2: Proportions of therapeutic areas in clinical trials of gene therapy in the US 340
• Figure 9 4: Proportions of various vectors used in gene therapy trials 340
• Figure 11 1: Unmet needs in gene therapy 371