K Cheng
John Wiley & Sons
e druk, 2013
9781119976868
Advanced Delivery and Therapeutic Applications of RNAi
Specificaties
Gebonden, 534 blz.
|
Engels
John Wiley & Sons |
2013
ISBN13: 9781119976868
Rubricering
Verwachte levertijd ongeveer 16 werkdagen
Gratis verzonden
Samenvatting
Commonly used by researchers to develop technologies for modifying and studying genetic process, RNA interference (RNAi) has many potential uses in medicine, biotechnology, and functional genomics. This book covers all essential aspects involved in the development of RNAi therapeutics, providing detailed guidance on the challenges and opportunities of bringing RNAi technologies from bench to clinic. It explores the design and mechanism of RNAi molecules, delivery strategies, and therapeutic applications in various diseases. Preclinical, regulatory, market, and intellectual aspects of RNAi technologies are also covered.
Specificaties
Inhoudsopgave
Preface xvii
<p>Contributors xix</p>
<p>About the Editors xxiii</p>
<p>Part 1 Introduction and Basics of RNAi 1</p>
<p>1 Mechanisms and Barriers to RNAi Delivery 3<br /> Jiehua Zhou and John J. Rossi</p>
<p>1.1 Introduction 3</p>
<p>1.2 Barriers to Systemic RNAi Delivery 5</p>
<p>1.3 Rational Design to Improve RNAi Efficacy 6</p>
<p>1.4 Chemical Modifications to Enhance siRNA Stability and Reduce Immune Response 7</p>
<p>1.5 Cellular Uptake and Intracellular Release of siRNA 7</p>
<p>1.6 Combinatorial Targeting for Targeted RNAi Delivery 8</p>
<p>1.7 Cell–Specific Aptamer–Functionalized Nanocarriers for RNAi Delivery 9</p>
<p>1.8 The Clinical Development and Challenges of siRNAs Therapeutics 10</p>
<p>1.9 Conclusion and Perspectives 12</p>
<p>References 12</p>
<p>2 Analysis of siRNA Delivery Using Various Methodologies 19<br /> Yi Pei</p>
<p>2.1 Introduction 19</p>
<p>2.2 Checkpoints for Analyzing siRNA Delivery 20</p>
<p>2.3 Methods for Analysis of siRNA 26</p>
<p>2.4 Case Study for siRNA Delivery Analysis 38</p>
<p>References 39</p>
<p>3 Challenges and Opportunities in Bringing RNAi Technologies from Bench to Bed 45<br /> Sandesh Subramanya and Lance Ford</p>
<p>3.1 Introduction 45</p>
<p>3.2 RNAi Mediator (siRNA or shRNA) 45</p>
<p>3.3 Safety Issues of RNAi Mediators 50</p>
<p>3.4 Efficacy of RNAi Mediators 52</p>
<p>3.5 RNAi Mediators in Clinical Trials 53</p>
<p>3.6 Conclusion 54</p>
<p>References 55</p>
<p>Nonclinical Safety Assessments and Clinical Pharmacokinetics for Oligonucleotide Therapeutics: A Regulatory Perspective 63<br /> Shwu–Luan Lee, Paul Brown, Jian Wang and Robert T. Dorsam</p>
<p>4.1 Introduction 63</p>
<p>4.2 Unique Properties of Oligonucleotide–based Therapeutics 63</p>
<p>4.3 Regulation of Oligonucleotide–Based Therapeutics 65</p>
<p>4.4 Conclusion 79</p>
<p>Disclaimer 79<br /> <br /> Appendix 79<br /> <br /> References 80</p>
<p>Role of Promoters and MicroRNA Backbone for Efficient Gene Silencing 83<br /> Feng Li and Ram I. Mahato</p>
<p>5.1 Introduction 83</p>
<p>5.2 Promoters for shRNA Expression 84</p>
<p>5.3 miRNA–based shRNAs 96</p>
<p>5.4 Concluding Remarks 100 References 101</p>
<p>Part 2 RNAi Delivery Strategies 109<br /> <br /> 6 Bioconjugation of siRNA for Site–specific Delivery 111<br /> Bin Qin, Wei Jin and Kun Cheng</p>
<p>6.1 Introduction 111</p>
<p>6.2 Conjugation Strategy 112</p>
<p>6.3 Bioconjugates for Site–specific Delivery 120</p>
<p>6.4 Conclusion 129</p>
<p>References 129</p>
<p>7 Multifunctional RNAi Delivery Systems 137<br /> China Malakondaiah Kummitha, Anthony S. Malamas and Zheng–Rong Lu</p>
<p>7.1 Introduction 137 7.1.1 Chapter Objectives 139</p>
<p>7.2 Lipid–Based Delivery Systems 139</p>
<p>7.3 Polymeric Multifunctional siRNA Delivery Systems 150</p>
<p>7.4 Conclusion 157</p>
<p>References 157</p>
<p>8 Dendrimers in RNAi Delivery 163<br /> Jose Luis Jimenez Fuentes, Paula Ortega, Sara Ferrando–Martýnez, Rafael Gomez, Manuel Leal, Javier de la Mata and MaAngeles Munoz–Fernandez</p>
<p>8.1 Introduction 163</p>
<p>8.2 Challenges in RNAi Delivery 164</p>
<p>8.3 Dendrimers as Non Viral Vectors 166</p>
<p>9 Development of Pharmaceutically Adapted Mesoporous Silica Nanoparticles for siRNA Delivery 187<br /> Wilson X. Mai, Tian Xia and Huan Meng</p>
<p>9.1 Introduction 187</p>
<p>9.2 Mesoporous Silica Nanoparticles as Novel Inorganic Nanocarriers for siRNA Delivery 188</p>
<p>9.3 Safety Assessment of Nanocarrier and Design of Safe MSNP Carrier 199</p>
<p>References 179</p>
<p>9.4 Summary References 202</p>
<p>10 Environmentally–Responsive Nanogels for siRNA Delivery 207<br /> Atsushi Tamura and Yukio Nagasaki</p>
<p>10.1 Introduction 207</p>
<p>10.2 Reductive Environment–Responsive Disulfide Crosslinked Nanogels 209</p>
<p>10.3 Temperature–Responsive Nanogels 211</p>
<p>10.4 pH–Responsive Nanogels 212</p>
<p>10.5 PEGylated and Partially Quaternized Polyamine Nanogels 216</p>
<p>10.6 Conclusions 220</p>
<p>References 220</p>
<p>11 Viral–Mediated Delivery of shRNA and miRNA 225<br /> Fredric P. Manfredsson</p>
<p>11.1 Introduction 225</p>
<p>11.2 RNAi A Brief Overview 226</p>
<p>11.3 shRNA or miRNA? 226</p>
<p>11.4 Rational Design 227</p>
<p>11.5 Viral Vectors 227</p>
<p>11.6 Tissue–specific Transduction 233</p>
<p>11.7 Applications of Virally Expressed shRNAs 241</p>
<p>11.8 Viral Gene Therapy in the Clinic 241</p>
<p>11.9 Conclusion 242</p>
<p>References 242</p>
<p>12 The Control of RNA Interference with Light 255<br /> Simon H. Friedman</p>
<p>12.1 Introduction 255 12.2 The Importance of Gene Expression 255</p>
<p>12.3 Light Control of Gene Expression 257</p>
<p>12.4 Why Use RNA Interference as a Basis for Light Control of Gene Expression? 258</p>
<p>12.5 Light Activated RNA Interference (LARI), the work of Friedman and Co–Workers 259</p>
<p>12.6 Work of McMaster and Co–Workers, 50 Antisense Phosphate Block 262</p>
<p>12.7 Work of Heckel and Co–Workers, Nucleobase Block 263</p>
<p>12.8 Use of 20 FsiRNA, work of Monroe and Co–Workers 264</p>
<p>12.9 Photochemical Internalization 265</p>
<p>12.10 Future Directions and Conclusions 266</p>
<p>Acknowledgments 267</p>
<p>References 267</p>
<p>Part 3 Applications of RNAi in Various Diseases 269</p>
<p>13 RNAi in Cancer Therapy 271<br /> Cristian Rodriguez–Aguayo, Arturo Chavez–Reyes, Gabriel Lopez–Berestein and Anil K. Sood</p>
<p>13.1 Introduction 271</p>
<p>13.2 Therapeutic Opportunities for Noncoding RNAs 274</p>
<p>13.3 RNAs as Drugs 277</p>
<p>13.4 Overcoming Anatomical and Physiologic Barriers 278</p>
<p>13.5 Advanced Delivery 283</p>
<p>13.6 Clinical Experience 294</p>
<p>13.7 The Next Steps 298</p>
<p>Acknowledgments 298</p>
<p>References 298</p>
<p>14 Adenovirus–mediated siRNA Delivery to Cancer 309<br /> Chae–Ok Yun</p>
<p>14.1 Introduction 309</p>
<p>14.2 shRNA–expressing Adenoviruses: Cancer Biological Studies and Therapeutic Implications 312</p>
<p>14.3 Exploiting Oncolytic Adenovirus for siRNA Expression 315</p>
<p>14.4 Current Limitations of Adenovirus–mediated siRNA Therapy and Future Directions: Smart Adenovirus Nanocomplexes Expressing siRNA for Systemic Administration 318</p>
<p>14.5 Conclusion 320</p>
<p>References 321</p>
<p>15 RNAi in Liver Diseases 327<br /> Jiang Li, Jianqin Lu, Yifei Zhang, Mohammed Ghazwani, Peng Zhang, Xiang Gao and Song Li</p>
<p>15.1 Introduction 327</p>
<p>15.2 RNAi in Viral Hepatitis 327</p>
<p>15.3 RNAi in Hepatocellular Carcinoma 336</p>
<p>15.4 RNAi in Liver Fibrosis 340</p>
<p>15.5 Delivery Systems in RNAi 345</p>
<p>15.6 Conclusion 352</p>
<p>Acknowledgments 353</p>
<p>References 353</p>
<p>16 Approaches to Delivering RNAi Therapeutics that Target Hepatitis B Virus 367<br /> Carol Crowther, Mohube Betty Mowa, Abdullah Ely and Patrick Arbuthnot</p>
<p>16.1 Introduction 367</p>
<p>16.2 Vectors Suitable for Hepatic Delivery of HBV Gene Silencers 369</p>
<p>16.3 Conclusions 381</p>
<p>Acknowledgments 382</p>
<p>References 382</p>
<p>17 RNAi in Respiratory Diseases 391<br /> Ciara Kelly, Awadh B. Yadav, Paul J. McKiernan, Catherine M. Greene and Sally–Ann Cryan</p>
<p>17.1 Introduction 391</p>
<p>17.2 Respiratory Disease and RNA Interference 392</p>
<p>17.3 Delivery and Development of RNAi Therapies for Respiratory Disease 397</p>
<p>17.4 Conclusions 408</p>
<p>Acknowledgements 408 References 408</p>
<p>18 RNAi in Ocular Diseases 417<br /> Andrey Turchinovich, Georg Zoidl and Rolf Dermietzel</p>
<p>18.1 Introduction 417</p>
<p>18.2 The Principle of RNAi 418</p>
<p>18.3 In vivo Delivery of siRNA 419</p>
<p>18.4 Delivery of siRNA into the Eye 420</p>
<p>18.5 Conclusions 431</p>
<p>Abbreviations 432<br /> <br /> References 432</p>
<p>19 micro RNAs as Therapeutic Agents and Targets 439<br /> D.S. Karolina and K. Jeyaseelan</p>
<p>19.1 Introduction 439</p>
<p>19.2 miRNA Therapeutics 440</p>
<p>19.3 MicroRNAs and Cancer 447</p>
<p>19.4 MicroRNAs in Stroke 450</p>
<p>19.5 MicroRNAs in Heart Diseases 452</p>
<p>19.6 MicroRNAs in Diabetes Mellitus 454</p>
<p>19.7 MicroRNAs in Liver Diseases 457</p>
<p>19.8 MicroRNAs and Ocular Diseases 461</p>
<p>19.9 MicroRNAs and Respiratory Diseases 462</p>
<p>19.10 MicroRNAs and Stem Cell Research 465</p>
<p>19.11 Conclusion 468</p>
<p>References 469</p>
<p>20 Delivery of Micro RNA Sponges for Interrogation of MicroRNA Function In Vitro and In Vivo 483<br /> Jiakai Lin and Shu Wang</p>
<p>20.1 MicroRNA Loss–of–Function Studies 483</p>
<p>20.2 Considerations in MicroRNA Sponge Design 486</p>
<p>20.3 Advantages and Limitations of MicroRNA Sponge over Other MicroRNA Loss–of–Function Strategies 489</p>
<p>20.4 Interrogating MicroRNA Function via Transient MicroRNA Sponge Expression 493</p>
<p>20.5 Interrogating MicroRNA Function via Stable MicroRNA Sponge Expression 494</p>
<p>20.6 Utility of MicroRNA Sponge in Living Organisms 496</p>
<p>20.7 Future Perspectives 498</p>
<p>References 499</p>
<p>Index 505</p>
<p>Contributors xix</p>
<p>About the Editors xxiii</p>
<p>Part 1 Introduction and Basics of RNAi 1</p>
<p>1 Mechanisms and Barriers to RNAi Delivery 3<br /> Jiehua Zhou and John J. Rossi</p>
<p>1.1 Introduction 3</p>
<p>1.2 Barriers to Systemic RNAi Delivery 5</p>
<p>1.3 Rational Design to Improve RNAi Efficacy 6</p>
<p>1.4 Chemical Modifications to Enhance siRNA Stability and Reduce Immune Response 7</p>
<p>1.5 Cellular Uptake and Intracellular Release of siRNA 7</p>
<p>1.6 Combinatorial Targeting for Targeted RNAi Delivery 8</p>
<p>1.7 Cell–Specific Aptamer–Functionalized Nanocarriers for RNAi Delivery 9</p>
<p>1.8 The Clinical Development and Challenges of siRNAs Therapeutics 10</p>
<p>1.9 Conclusion and Perspectives 12</p>
<p>References 12</p>
<p>2 Analysis of siRNA Delivery Using Various Methodologies 19<br /> Yi Pei</p>
<p>2.1 Introduction 19</p>
<p>2.2 Checkpoints for Analyzing siRNA Delivery 20</p>
<p>2.3 Methods for Analysis of siRNA 26</p>
<p>2.4 Case Study for siRNA Delivery Analysis 38</p>
<p>References 39</p>
<p>3 Challenges and Opportunities in Bringing RNAi Technologies from Bench to Bed 45<br /> Sandesh Subramanya and Lance Ford</p>
<p>3.1 Introduction 45</p>
<p>3.2 RNAi Mediator (siRNA or shRNA) 45</p>
<p>3.3 Safety Issues of RNAi Mediators 50</p>
<p>3.4 Efficacy of RNAi Mediators 52</p>
<p>3.5 RNAi Mediators in Clinical Trials 53</p>
<p>3.6 Conclusion 54</p>
<p>References 55</p>
<p>Nonclinical Safety Assessments and Clinical Pharmacokinetics for Oligonucleotide Therapeutics: A Regulatory Perspective 63<br /> Shwu–Luan Lee, Paul Brown, Jian Wang and Robert T. Dorsam</p>
<p>4.1 Introduction 63</p>
<p>4.2 Unique Properties of Oligonucleotide–based Therapeutics 63</p>
<p>4.3 Regulation of Oligonucleotide–Based Therapeutics 65</p>
<p>4.4 Conclusion 79</p>
<p>Disclaimer 79<br /> <br /> Appendix 79<br /> <br /> References 80</p>
<p>Role of Promoters and MicroRNA Backbone for Efficient Gene Silencing 83<br /> Feng Li and Ram I. Mahato</p>
<p>5.1 Introduction 83</p>
<p>5.2 Promoters for shRNA Expression 84</p>
<p>5.3 miRNA–based shRNAs 96</p>
<p>5.4 Concluding Remarks 100 References 101</p>
<p>Part 2 RNAi Delivery Strategies 109<br /> <br /> 6 Bioconjugation of siRNA for Site–specific Delivery 111<br /> Bin Qin, Wei Jin and Kun Cheng</p>
<p>6.1 Introduction 111</p>
<p>6.2 Conjugation Strategy 112</p>
<p>6.3 Bioconjugates for Site–specific Delivery 120</p>
<p>6.4 Conclusion 129</p>
<p>References 129</p>
<p>7 Multifunctional RNAi Delivery Systems 137<br /> China Malakondaiah Kummitha, Anthony S. Malamas and Zheng–Rong Lu</p>
<p>7.1 Introduction 137 7.1.1 Chapter Objectives 139</p>
<p>7.2 Lipid–Based Delivery Systems 139</p>
<p>7.3 Polymeric Multifunctional siRNA Delivery Systems 150</p>
<p>7.4 Conclusion 157</p>
<p>References 157</p>
<p>8 Dendrimers in RNAi Delivery 163<br /> Jose Luis Jimenez Fuentes, Paula Ortega, Sara Ferrando–Martýnez, Rafael Gomez, Manuel Leal, Javier de la Mata and MaAngeles Munoz–Fernandez</p>
<p>8.1 Introduction 163</p>
<p>8.2 Challenges in RNAi Delivery 164</p>
<p>8.3 Dendrimers as Non Viral Vectors 166</p>
<p>9 Development of Pharmaceutically Adapted Mesoporous Silica Nanoparticles for siRNA Delivery 187<br /> Wilson X. Mai, Tian Xia and Huan Meng</p>
<p>9.1 Introduction 187</p>
<p>9.2 Mesoporous Silica Nanoparticles as Novel Inorganic Nanocarriers for siRNA Delivery 188</p>
<p>9.3 Safety Assessment of Nanocarrier and Design of Safe MSNP Carrier 199</p>
<p>References 179</p>
<p>9.4 Summary References 202</p>
<p>10 Environmentally–Responsive Nanogels for siRNA Delivery 207<br /> Atsushi Tamura and Yukio Nagasaki</p>
<p>10.1 Introduction 207</p>
<p>10.2 Reductive Environment–Responsive Disulfide Crosslinked Nanogels 209</p>
<p>10.3 Temperature–Responsive Nanogels 211</p>
<p>10.4 pH–Responsive Nanogels 212</p>
<p>10.5 PEGylated and Partially Quaternized Polyamine Nanogels 216</p>
<p>10.6 Conclusions 220</p>
<p>References 220</p>
<p>11 Viral–Mediated Delivery of shRNA and miRNA 225<br /> Fredric P. Manfredsson</p>
<p>11.1 Introduction 225</p>
<p>11.2 RNAi A Brief Overview 226</p>
<p>11.3 shRNA or miRNA? 226</p>
<p>11.4 Rational Design 227</p>
<p>11.5 Viral Vectors 227</p>
<p>11.6 Tissue–specific Transduction 233</p>
<p>11.7 Applications of Virally Expressed shRNAs 241</p>
<p>11.8 Viral Gene Therapy in the Clinic 241</p>
<p>11.9 Conclusion 242</p>
<p>References 242</p>
<p>12 The Control of RNA Interference with Light 255<br /> Simon H. Friedman</p>
<p>12.1 Introduction 255 12.2 The Importance of Gene Expression 255</p>
<p>12.3 Light Control of Gene Expression 257</p>
<p>12.4 Why Use RNA Interference as a Basis for Light Control of Gene Expression? 258</p>
<p>12.5 Light Activated RNA Interference (LARI), the work of Friedman and Co–Workers 259</p>
<p>12.6 Work of McMaster and Co–Workers, 50 Antisense Phosphate Block 262</p>
<p>12.7 Work of Heckel and Co–Workers, Nucleobase Block 263</p>
<p>12.8 Use of 20 FsiRNA, work of Monroe and Co–Workers 264</p>
<p>12.9 Photochemical Internalization 265</p>
<p>12.10 Future Directions and Conclusions 266</p>
<p>Acknowledgments 267</p>
<p>References 267</p>
<p>Part 3 Applications of RNAi in Various Diseases 269</p>
<p>13 RNAi in Cancer Therapy 271<br /> Cristian Rodriguez–Aguayo, Arturo Chavez–Reyes, Gabriel Lopez–Berestein and Anil K. Sood</p>
<p>13.1 Introduction 271</p>
<p>13.2 Therapeutic Opportunities for Noncoding RNAs 274</p>
<p>13.3 RNAs as Drugs 277</p>
<p>13.4 Overcoming Anatomical and Physiologic Barriers 278</p>
<p>13.5 Advanced Delivery 283</p>
<p>13.6 Clinical Experience 294</p>
<p>13.7 The Next Steps 298</p>
<p>Acknowledgments 298</p>
<p>References 298</p>
<p>14 Adenovirus–mediated siRNA Delivery to Cancer 309<br /> Chae–Ok Yun</p>
<p>14.1 Introduction 309</p>
<p>14.2 shRNA–expressing Adenoviruses: Cancer Biological Studies and Therapeutic Implications 312</p>
<p>14.3 Exploiting Oncolytic Adenovirus for siRNA Expression 315</p>
<p>14.4 Current Limitations of Adenovirus–mediated siRNA Therapy and Future Directions: Smart Adenovirus Nanocomplexes Expressing siRNA for Systemic Administration 318</p>
<p>14.5 Conclusion 320</p>
<p>References 321</p>
<p>15 RNAi in Liver Diseases 327<br /> Jiang Li, Jianqin Lu, Yifei Zhang, Mohammed Ghazwani, Peng Zhang, Xiang Gao and Song Li</p>
<p>15.1 Introduction 327</p>
<p>15.2 RNAi in Viral Hepatitis 327</p>
<p>15.3 RNAi in Hepatocellular Carcinoma 336</p>
<p>15.4 RNAi in Liver Fibrosis 340</p>
<p>15.5 Delivery Systems in RNAi 345</p>
<p>15.6 Conclusion 352</p>
<p>Acknowledgments 353</p>
<p>References 353</p>
<p>16 Approaches to Delivering RNAi Therapeutics that Target Hepatitis B Virus 367<br /> Carol Crowther, Mohube Betty Mowa, Abdullah Ely and Patrick Arbuthnot</p>
<p>16.1 Introduction 367</p>
<p>16.2 Vectors Suitable for Hepatic Delivery of HBV Gene Silencers 369</p>
<p>16.3 Conclusions 381</p>
<p>Acknowledgments 382</p>
<p>References 382</p>
<p>17 RNAi in Respiratory Diseases 391<br /> Ciara Kelly, Awadh B. Yadav, Paul J. McKiernan, Catherine M. Greene and Sally–Ann Cryan</p>
<p>17.1 Introduction 391</p>
<p>17.2 Respiratory Disease and RNA Interference 392</p>
<p>17.3 Delivery and Development of RNAi Therapies for Respiratory Disease 397</p>
<p>17.4 Conclusions 408</p>
<p>Acknowledgements 408 References 408</p>
<p>18 RNAi in Ocular Diseases 417<br /> Andrey Turchinovich, Georg Zoidl and Rolf Dermietzel</p>
<p>18.1 Introduction 417</p>
<p>18.2 The Principle of RNAi 418</p>
<p>18.3 In vivo Delivery of siRNA 419</p>
<p>18.4 Delivery of siRNA into the Eye 420</p>
<p>18.5 Conclusions 431</p>
<p>Abbreviations 432<br /> <br /> References 432</p>
<p>19 micro RNAs as Therapeutic Agents and Targets 439<br /> D.S. Karolina and K. Jeyaseelan</p>
<p>19.1 Introduction 439</p>
<p>19.2 miRNA Therapeutics 440</p>
<p>19.3 MicroRNAs and Cancer 447</p>
<p>19.4 MicroRNAs in Stroke 450</p>
<p>19.5 MicroRNAs in Heart Diseases 452</p>
<p>19.6 MicroRNAs in Diabetes Mellitus 454</p>
<p>19.7 MicroRNAs in Liver Diseases 457</p>
<p>19.8 MicroRNAs and Ocular Diseases 461</p>
<p>19.9 MicroRNAs and Respiratory Diseases 462</p>
<p>19.10 MicroRNAs and Stem Cell Research 465</p>
<p>19.11 Conclusion 468</p>
<p>References 469</p>
<p>20 Delivery of Micro RNA Sponges for Interrogation of MicroRNA Function In Vitro and In Vivo 483<br /> Jiakai Lin and Shu Wang</p>
<p>20.1 MicroRNA Loss–of–Function Studies 483</p>
<p>20.2 Considerations in MicroRNA Sponge Design 486</p>
<p>20.3 Advantages and Limitations of MicroRNA Sponge over Other MicroRNA Loss–of–Function Strategies 489</p>
<p>20.4 Interrogating MicroRNA Function via Transient MicroRNA Sponge Expression 493</p>
<p>20.5 Interrogating MicroRNA Function via Stable MicroRNA Sponge Expression 494</p>
<p>20.6 Utility of MicroRNA Sponge in Living Organisms 496</p>
<p>20.7 Future Perspectives 498</p>
<p>References 499</p>
<p>Index 505</p>