Gene Therapy - technologies, markets and companies

SUMMARY

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
• Nanobiotechnology for gene transfer 63
• Nanoparticles as non-viral vectors for gene therapy 64
• Dendrimers 64
• Cochleates 64
• 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 66
• 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 69
• Chromosome-based vectors for gene therapy 70
• Companies using non-viral vectors 72
• Concluding remarks about vectors 73
• Cell-mediated gene therapy 74
• Fibroblasts 74
• Skeletal muscle cells 75
• Vascular smooth muscle cells 76
• Keratinocytes 76
• Hepatocytes 76
• 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 78
• 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 79
• 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 81
• Intramuscular injection 82
• Intravenous DNA injection 82
• Intraarterial delivery 82
• Companies with gene delivery devices/ technologies 82
• Targeted gene therapy 83
• 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 89
• Potential applications of human germline genome modification 90
• Pros and cons of human germline genome modification 90
• Role of gene transfer in antibody therapy 91
• Genetically engineered vaccines 92
• DNA vaccines 92
• DNA inoculation technology 93
• Methods for enhancing the potency of DNA vaccines 93
• Advantages of DNA vaccines 93
• 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 96
• 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 99
• RNAi mechanisms 100
• Inhibition of gene expression by antigene RNA 101
• RNAi gene therapy 101
• Application of molecular diagnostic methods in gene therapy 101
• 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 103
• 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
• Veterinary gene therapy 173
• Gene therapy for mucopolysaccharidosis VII in dogs 173
• Gene therapy to increase disease resistance 173
• 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 175
• 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