Genome Editing: The Next Step in Gene Therapy (Advances in Experimental Medicine and Biology Book 895)

This comprehensive volume explores human genetic engineering its pre-clinical and clinical applications, current developments, and as treatment for hereditary diseases. It presents and evaluates the most recent advances in the understanding of mammalian host DNA repair mechanisms, such as double-strand break induced gene targeting and mutagenesis, the development of zinc-finger nucleases, genome editing for neuromuscular diseases, phase integrases, triplex forming oligonucleotides and peptide nucleic acids, aptamer-guided gene targeting, AAV gene editing via DSB repair, engineered nucleases and trinucleotide repeat diseases, and creation of HIV-resistant cells. The expertly authored chapters contextualize current developments within the history of genome editing while also discussing the current and potential safety concerns of this rapidly growing field. __Genome Editing: The Next Step in Gene Therapy__, the latest volume in the __American Society of Gene and Cell Therapy__ series, deftly illuminates the potential of genetic engineering technology to eradicate today’s deadliest and most prolific diseases. It is ideal reading for clinicians and researchers in genetics and immunology. Dedication 6 Preface 8 Contents 10 About the Editors 16 Gene Editing 20 Years Later 18 Introduction 18 Facile Gene Modification in Yeast but Impediments in Mammalian Cells 19 DSBs as Initiators of Homologous Recombination 19 Groundbreaking Experiments: DSBs in the Genome Induce Gene Targeting Orders of Magnitude 20 DSBs in the Genome Induce Mutagenesis 21 Two DSBs Induce Genomic Rearrangements 22 HR Studies Using I-SceI Endonuclease 22 Collaboration and Competition Between HR and NHEJ 25 Genome Editing with an Emerging Range of Nucleases 25 Conclusions 27 References 27 The Development and Use of Zinc-Finger Nucleases 32 Introduction 32 Origins of ZFNs 34 Characterization of ZFNs 34 Designing ZFNs 35 The Utility of ZFNs in Gene Targeting 36 ZFN Contributions 38 References 39 The Use and Development of TAL Effector Nucleases 46 Transcription Activator-Like Effectors 46 DNA Binding Domain 47 N-Terminal Domain 50 C-Terminal Domain 50 Assembly of TALE Arrays 52 Standard Cloning Assembly 52 ‘Golden Gate’ Assembly 56 Solid Phase Assembly 57 Ligation Independent Cloning (LIC) 57 Designer TALEs and Their Use 58 Specificity of Tailored TALE-Based Effectors 60 Conclusions 61 References 62 Genome Editing for Neuromuscular Diseases 68 Introduction 69 Overview of Common Neuromuscular Disorders 69 Duchenne and Becker Muscular Dystrophies 70 Limb-Girdle Muscular Dystrophy Type 2B 71 Myotonic Dystrophy 71 Fascioscapulohumeral Dystrophy 72 Spinal Muscular Atrophy 73 Huntington’s Disease 73 Genome Editing Technologies 74 Introduction to Genome Editing Strategies 74 Tools for Targeted Gene Modification 75 Meganucleases 75 Chimeric Nucleases Based on the FokI Domain 76 CRISPR/Cas Systems 76 Integrases, Recombinases, and Transposases 77 Adeno-Associated Virus-Mediated Gene Targeting 78 Genetic Modification with Oligonucleotide Complexes 78 Introduction of Genetic Corrections In Vivo 79 Cell-Based Therapies to Introduce Genetically Corrected Cells In Vivo 79 In Vivo Correction by Direct Delivery of Genome Editing Tools 80 Genome Editing Methods Applied to Neuromuscular Diseases 81 Correction of a Native Gene In Situ 81 Reading Frame Restoration by Targeted Genomic Modifications 81 Complete Gene Restoration by Knock-In of Essential Gene Sequences Absent in Mutant Genes 83 Correction of Point Mutations 84 Wholesale Replacement of Mutated Genes 84 Promoters to Drive Transgene Expression 85 “Safe Harbor” Sites for Integration 85 Disease Modulation by Modification of Compensatory Gene Targets 86 Discussion and Conclusion 86 References 87 Phage Integrases for Genome Editing 97 Utility of Phage Integrases 97 Integration into Pseudo Sites Versus into attP Sites 99 Use of phiC31 Integrase for Constructing Transgenic Organisms 100 Gene Therapy Studies Utilizing Integration into Pseudo Sites 100 Utilizing phiC31 Integrase for Reprogramming Mammalian Cells 101 Site-Specific Integration of a Therapeutic Gene at a Pre-­integrated attP Site 102 The DICE System: Combining Homologous Recombination with phiC31 and Bxb1 Integrases for Cassette Exchange 104 Reversal of the Integration Reaction with phiC31 Excisionase 106 References 106 Precise Genome Modification Using Triplex Forming Oligonucleotides and Peptide Nucleic Acids 108 Introduction 108 Triplex Forming Oligonucleotides 109 Binding Code 109 Chemical Modifications to Improve TFO Binding Affinity and Stability 109 Triplex Mediated Genome Modification 111 Targeted Mutagenesis via Triplex Formation 111 Plasmid Based Assays for Detecting Mutagenesis in Mammalian Cells 111 TFO Induced Mutagenesis at Chromosomal Sites in Mammalian Cells 112 TFOs in Homologous Recombination 112 Intramolecular Recombination 112 Intermolecular Recombination 114 Peptide Nucleic Acids (PNAs) 114 Use of PNA in Repair and Recombination 116 Summary 120 References 120 Genome Editing by Aptamer-Guided Gene Targeting (AGT) 126 Introduction 126 AGT Origins 129 Aptamers 129 The Site-Specific Endonuclease I-SceI 131 Single-Stranded DNA Oligos as Donor DNA 131 Characterization of AGT 132 The Utility of AGT for Gene Targeting 134 Future Directions for AGT 136 References 137 Stimulation of AAV Gene Editing via DSB Repair 140 The Vectorization of Adeno-Associated Virus 141 AAV as a Clinical Gene Therapy Vector 142 AAV Vectors and Gene Editing 144 Stimulation of AAV Gene Editing via Site-Specific DNA Damage 145 Challenges for AAV Gene Editing via DSB Repair 147 References 147 Engineered Nucleases and Trinucleotide Repeat Diseases 153 Introduction 154 Microsatellite Repeats and TNR Instability 155 TNRs and Human Disease 156 Engineered Nucleases and TNR Contraction 160 Challenges and Limitations of DNA-Directed Therapy 165 Summary and Perspectives 166 References 168 Using Engineered Nucleases to Create HIV-Resistant Cells 174 Introduction 175 Disruption of the CCR5 Co-Receptor by ZFNs 175 Engineered Nucleases and DNA Repair 175 Rationale for CCR5 Disruption as an Anti-HIV Therapy 178 First to Clinic: CCR5 Knockout in T Cells Using ZFNs 178 CCR5 Disruption in HSC Using ZFNs 182 CCR5 Disruption Using Other Engineered Nucleases 183 CXCR4 is an Additional Target for Gene Knockout 185 Beyond Gene Knockout: Site-Specific Gene Addition and Combination Anti-HIV Therapies 185 Disrupting CCR5 in iPSC 188 Disrupting HIV-1 Genomes with Engineered Nucleases 188 Progress to Developing HIV-Specific Nucleases 188 Challenges for the Use of Anti-HIV Nucleases 190 Considerations for the Clinical Use of Engineered Nucleases 190 Strategies to Deliver Engineered Nucleases to Human Cells 190 Off-Target Effects and Toxicity 192 Summary 194 References 194 Strategies to Determine Off-Target Effects of Engineered Nucleases 200 Introduction 201 Factors Influencing Off-Target Activity 202 Zinc Finger Nucleases 203 Protein-DNA Interactions 203 Poly-Zinc Finger Arrays 204 Protein Linker Domains 204 Transcription Activator-Like Effector Nucleases 204 Protein-DNA Interactions 205 Protein Linker Domains 205 Modifications to the FokI Domain of ZFNs and TALENs 206 Obligate Heterodimers 206 Sharkey Enhancement 207 Nick Only 207 CRISPR/Cas9 Systems 207 gRNA-DNA and PAM Interactions 208 Modifications to Cas9 208 Bulk Assays of Off-Target Activity 209 γH2AX Foci 209 Cell Cycle Disregulation 209 Apoptosis and Cell Viability 210 Loss of Fluorescence 210 Experimental-Based Off-Target Site Prediction Methods 210 SELEX 210 Bacterial One-Hybrid 212 In Vitro Cleavage 212 IDLV LAM-PCR 214 ChIP-Seq 215 Additional Genome-Wide Tools for CRISPR Off-Target Site Identification 216 In Silico Off-Target Site Prediction Methods 218 In Silico Tools for ZFN Off-Target Prediction 220 PROGNOS 220 ZFN-Site 221 In Silico Tools for TALEN Off-Target Prediction 221 PROGNOS 221 TALENoffer 222 In Silico Tools for CRISPR Off-Target Prediction 222 Ranking 223 Exhaustive Searches to Find All Sites 224 Search Features 224 Off-Target Activity Detection Methods 226 Mismatch Detection Enzymes 226 TOPO Sequencing 227 SMRT Sequencing 228 Illumina Sequencing 228 Conclusion 229 References 229 Cellular Engineering and Disease Modeling with Gene-Editing Nucleases 236 Meganucleases 238 Zinc Finger Nucleases 238 Rodents 239 Swine 240 Cattle 241 Humans 241 TALEN 247 Rodents 248 Swine 250 Cattle 251 Non-Human Primates (NHP) 251 Humans 251 CRISPR/Cas9 254 Rodents 254 Non-Human Primates (NHP) 257 Humans 257 Summary 259 References 263 Index 272 This comprehensive volume explores human genetic engineering its pre-clinical and clinical applications, current developments, and as treatment for hereditary diseases. It presents and evaluates the most recent advances in the understanding of mammalian host DNA repair mechanisms, such as double-strand break induced gene targeting and mutagenesis, the development of zinc-finger nucleases, genome editing for neuromuscular diseases, phase integrases, triplex forming oligonucleotides and peptide nucleic acids, aptamer-guided gene targeting, AAV gene editing via DSB repair, engineered nucleases and trinucleotide repeat diseases, and creation of HIV-resistant cells. The expertly authored chapters contextualize current developments within the history of genome editing while also discussing the current and potential safety concerns of this rapidly growing field. Genome Editing: The Next Step in Gene Therapy, the latest volume in the American Society of Gene and Cell Therapy series, deftly illuminates the potential of genetic engineering technology to eradicate today{u2019}s deadliest and most prolific diseases. It is ideal reading for clinicians and researchers in genetics and immunology. Front Matter....Pages i-xvi Gene Editing 20 Years Later....Pages 1-14 The Development and Use of Zinc-Finger Nucleases....Pages 15-28 The Use and Development of TAL Effector Nucleases....Pages 29-50 Genome Editing for Neuromuscular Diseases....Pages 51-79 Phage Integrases for Genome Editing....Pages 81-91 Precise Genome Modification Using Triplex Forming Oligonucleotides and Peptide Nucleic Acids....Pages 93-110 Genome Editing by Aptamer-Guided Gene Targeting (AGT)....Pages 111-124 Stimulation of AAV Gene Editing via DSB Repair....Pages 125-137 Engineered Nucleases and Trinucleotide Repeat Diseases....Pages 139-159 Using Engineered Nucleases to Create HIV-Resistant Cells....Pages 161-186 Strategies to Determine Off-Target Effects of Engineered Nucleases....Pages 187-222 Cellular Engineering and Disease Modeling with Gene-Editing Nucleases....Pages 223-258 Back Matter....Pages 259-263