Antisense Drug Technology: Principles, Strategies, and Applications

Cover......Page 1
ISBN-13: 9780824705664......Page 2
Preface......Page 4
Contents......Page 6
Contributors......Page 10
A. Polynucleotides......Page 15
B. The Antisense Concept......Page 16
C. Strategies to Induce Transcriptional Arrest......Page 17
D. Ribozymes and Oligonucleotide-Mediated RNA Cleavage......Page 18
A. Factors that May Influence Experimental Interpretations......Page 20
4. Variations in In Vitro Cellular Uptake and Distribution......Page 21
5. The Binding to and Effects of Binding to Non-Nucleic Acid Targets......Page 22
8. Kinetics of Effects......Page 23
A. Occupancy-Only-Mediated Mechanisms......Page 24
2. Inhibition of Splicing......Page 25
4. Disruption of Necessary RNA Structure......Page 26
1. 5'-Capping......Page 27
2. Inhibition of 3'-Polyadenylation......Page 28
D. Activation of RNase H......Page 29
E. Activation of Double-Strand RNase......Page 31
REFERENCES......Page 32
A. Generic Nature of Antisense Oligonucleotides......Page 43
C. General Structure-Activity/Property Relationships Pathway to Enhance Oligonucleotide Drug Properties......Page 44
D. First-Generation Antisense Oligonucleotides......Page 46
E. Oligonucleotide Modificatoins and Drug Properties that May Be Altered......Page 48
II. The Medical Chemistry Challenge......Page 49
IV. GENERAL COMMENTS ON THE VALUE OF CLASSES OF MODIFICATIONS WITHIN EACH SUBUNIT......Page 52
A. Heterocycle Modifications......Page 53
1. Introduction......Page 54
2. Cyclo-ribofuranosyl Modifications (Conformationally Constrained)......Page 56
3. C2-Ribofuranosyl Modifications......Page 58
C. Linkage Change......Page 59
D. Phosphorus Atom Modifications......Page 60
E. Oligonucleotide Motifs......Page 61
V. PERSPECTIVE......Page 63
REFERENCES......Page 65
I. INTRODUCTION......Page 71
II. HPLC ANALYSIS......Page 75
III. RADIOLABELED OLIGONUCLEOTIDES......Page 77
IV. DIRECT CELLULAR VISUALIZATION METHODS......Page 81
V. CAPILLARY ELECTROPHORESIS......Page 82
VI. FLURESCENT CAPILLARY GEL ELECTROPHORESIS......Page 85
VIII. HYBRIDIZATION......Page 86
IX. MASS SPECTROMETRY......Page 87
X. NEEDED METHODS IMPROVEMENTS......Page 90
REFERENCES......Page 91
I. INTRODUCTION......Page 97
II. TRADITIONAL APPROACHES FOR STUDYING CELL SIGNALING......Page 98
A. Overexpression Systems......Page 99
B. Gene Knockouts......Page 102
C. Small Molecules......Page 103
D. Monoclonal Antibodies......Page 104
A. The Antisense Concept......Page 105
B. Specificty and Simplicity......Page 106
C. Versatility......Page 107
D. Antisense Oligonucleotides as Drug Candidates......Page 110
E. Potential Limitations......Page 114
IV. FUTURE DIRECTIONS......Page 115
REFERENCES......Page 116
I. INTRODUCTION......Page 120
II. RNA STRUCTURE CALCULATIONS......Page 121
IV. CELL-FREE SCREENING AND COMBINATORIAL APPROACHES......Page 123
A. Target Specificity......Page 124
A. Cross-Species and Cross-Isoform Oligonucleotides......Page 125
VII. SUMMARY......Page 126
REFERENCES......Page 127
I. INTRODUCTION......Page 132
II. SEQUENCE-INDEPENDENT PHARMACOKINETICS......Page 133
A. Intravenous Administration......Page 136
B. Absorption......Page 142
1. Parenteral Administration......Page 143
2. Nonparenteral Administration......Page 144
C. Plasma Protein Binding......Page 146
IV. TISSUE DISTRIBUTION AND CLEARANCE......Page 148
V. METABOLISM AND EXCRETION......Page 152
A. Nuclease Metabolism......Page 154
B. Excretion......Page 158
VI. SECOND-GENERATION PHOSPHOROTHIOATE OLIGONUCLEOTIDES......Page 159
VII. PHARMACODYNAMICS CORRELATE WITH TISSUE PHARMACOKINETICS......Page 160
REFERENCES......Page 162
I. INTRODUCTION......Page 168
A. Autoradiography......Page 169
C. Localization by Subcellular Fractionation......Page 172
D. Summary of Literature Survey of Suborgan Distribution......Page 173
A. Histochemical Techniques......Page 175
1. Rat......Page 178
2. Mouse, Rat, and Monkey Comparison of Suborgan Distribution......Page 184
3. Subcellular Distribution in Human Hepatocytes......Page 186
IV. CONCLUSIONS......Page 187
REFERENCES......Page 191
I. INTRODUCTION......Page 196
A. General Pharmacokinetic Behavior in Humans......Page 197
1. Maximum Plasma Concentrations (Cmax)......Page 199
2. AUC......Page 200
3. Plasma Half-Life and Clearance......Page 201
1. Metabolism......Page 202
2. Excretion......Page 203
D. Plasma Protein Binding......Page 205
F. Route of Administration......Page 207
G. Sequence Independence......Page 209
H. Interspecies Scaling: From Mouse to Human......Page 210
REFERENCES......Page 211
I. INTRODUCTION......Page 214
A. Too Much of a Good Thing- Exaggerated Pharmacology......Page 216
B. The Luck of the Draw: Toxicity Resulting from Hybridization to Unintended mRNA Targets......Page 217
C. Bits and Pieces: Metabolism and Toxicity......Page 219
D. Nonspecific Protein Binding: Class-Related Toxicities......Page 223
1. Hemostasis: Acute and Transient Toxicities......Page 224
Complement Activation......Page 225
Inhibition of the Coagulation Cascade......Page 233
Immune System......Page 237
Hematopoietic......Page 249
Hepatic......Page 250
Gastrointestinal......Page 251
Renal......Page 252
Cardiopulmonary......Page 254
Integumentary......Page 255
Nervous System......Page 258
E. Reproductive......Page 260
1. Salmonella-Escherichia coli/Microsome Plate Incorporation Assay......Page 265
2. In Vitro Mammalian Cytogenetic Assay......Page 266
5. Micronucleus Cytogenetic Assay in Mice......Page 267
6. Exposure of Cells to Oligonucleotides in Genotoxicity Assays......Page 268
G. Research Applications of Antisense Oligonucleotides in Toxicology......Page 270
REFERENCES......Page 272
I. INTRODUCTION......Page 281
B. ISIS 2302- An Inhibitor of Intercellular Adhesion Molecule 1......Page 283
III. DOSE- AND SCHEDULE-RELATED SAFETY PROFILE OF OLIGONUCLEOTIDES......Page 285
A. Complement......Page 286
B. Coagulation......Page 290
C. Thrombocytopenia......Page 291
D. Renal and Hepatic Toxicity......Page 294
E. Constitutional Effects of Oligonucleotide Administration......Page 297
IV. SUBCUTANEOUS DELIVERY......Page 298
V. SUMMARY......Page 299
REFERENCES......Page 300
II. THE RECEPTOR......Page 303
A. Antisense Mechanism of Action......Page 306
B. Proof of Antisense Mechanisms......Page 307
C. Nonantisense Effects of Phosphorothioate Oligodeoxynucleotides......Page 309
IV. IN VITRO PHARMACOLOGICAL EFFECTS OF PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDES......Page 312
A. Neuropharmacological Studies......Page 314
C. Cardiovascular Pharmacology......Page 315
D. Cancer Pharmacology......Page 316
REFERENCES......Page 317
I. INTRODUCTION......Page 331
II. PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDES......Page 332
III. CARBOHYDRATE 2'-MODIFIED OLIGONUCLEOTIDES......Page 333
IV. 2'-O-(2-METHOXY)ETHYL CHEMISTRY AND BIOPHYSICAL CHARACTERISTICS......Page 334
V. 2'-O-(2-METHOXY)ETHYL-MODIFIED OLIGONUCLEOTIDES: IN VITRO PHARMACOLOGY......Page 336
A. RNase H-Dependent mRNA Cleavage......Page 337
B. Modulation of Pre-mRNA Splicing......Page 338
C. Inhibition of Translation......Page 340
D. Decapping Strategies......Page 341
B. Pharmacokinetics......Page 342
VII. CONCLUSIONS AND PERSPECTIVES......Page 345
REFERENCES......Page 347
I. INTRODUCTION......Page 351
A. Synthesis of LNA......Page 352
B. Synthesis of 2'-Thio-LNA and 2'-Amino-LNA......Page 356
C. Synthesis of a-L-LNA (a-L-Ribo-Configured LNA)......Page 357
D. Synthesis of Abasic LNA......Page 359
A. 9-Mer Mixed Sequences......Page 361
B. 10- and 14-Mer Homothymine Sequence......Page 363
A. NMR and CD Studies......Page 364
B. Hybridization Properties of Abasic LNA......Page 365
V. LNA AS DIAGNOSTIC PROBE OR ANTISENSE AGENT......Page 366
ACKNOWLEDGMENTS......Page 367
REFERENCES......Page 368
II. CHEMISTRY......Page 370
III. MODIFIED NUCLEOBASES......Page 371
IV. ANTISENSE AND ANTIGENE EFFECT OF PNA IN VITRO......Page 373
V. CELLULAR UPTAKE OF PNA......Page 374
VI. EX VIVO AND IN VIVO EFFECTS OF PNA......Page 378
VII. ANTIMICROBIAL PNAs......Page 380
REFERENCES......Page 381
B. Chemistry......Page 386
A. Biological Advantages......Page 388
1. Hybrid Arrest of Trasiation/Interference with Ribosomal Assembly......Page 389
C. Specificity Studies......Page 390
1. Mouse In Vivo Efficacy Studies; Lewis Lung Carcinoma......Page 391
2. Mouse In Vivo Efficacy Studies in a Polycystic Kidney Disease Model......Page 392
4. Rabbit In Vivo Efficacy Studies; Endoluminal Delivery......Page 393
B. Multidose Toxicity Study in Rats......Page 394
C. Single-Dose Toxicity Study in Primates......Page 395
IV. PHARMACOKINETIC STUDIES......Page 396
V. SUMMARY......Page 397
REFERENCES......Page 399
I. INTRODUCTION......Page 401
1. Rationale: Conjugates to Improve Cellular Uptake and Alter Pharmacokinetics......Page 403
3. Cholesterol-Conjugated ICAM-1 Antisense Oligonucleotides......Page 405
4. Pharmacokinetics of Cholesterol Conjugates and Other Lipophilic Conjugates......Page 409
5. In Vivo Therapeutic Efficacy of Cholesterol-Conjugated ICAM-1 Oligonucleotides......Page 411
6. Evaluation of Cholesterol-Conjugated Antisense Oligonucleotides in Other Biological Targets......Page 412
7. Effect of Cholesterol Conjugation: Reports from Other Laboratories......Page 414
9. Toxicity of Cholesterol Conjugates and Other Lipophilic Conjugates......Page 417
10. Antisense Applications of Other Lipophilic Molecules......Page 418
11. Complexes Derived from Lipophilic Molecule-Conjugated Oligonucleotides and Liposomes and Other Oligonucleotide Carriers......Page 421
12. Novel Polyaminolipids Based on Cholesterol......Page 422
B. Vitamin Conjugates......Page 423
1. Folic Acid Conjugates......Page 424
2. Folate-Conjugated Oligonucleotide Carriers......Page 426
2. Cationic and Zwitterionic Oligonucleotides......Page 427
1. Biological Activity......Page 432
2. PEG Copolymers......Page 434
2. Conjugates of YEE(ahGaINAc)3......Page 435
4. Cholane-Based Galactose Clusters......Page 437
2. Peptides as Polycations......Page 438
3. Peptides for Endosomal Release: Fusogenic Peptide Conjugation......Page 440
4. Cell Permeation Peptides: Ant Peptide and Tat Peptide......Page 441
5. Reports on Synthesis and Purification of Peptide Conjugates......Page 445
2. Lanthanide Complexes......Page 446
3. Peptides as RNA Cleavers......Page 448
1. Psoralens......Page 450
2. Mitomycin C and Analogs......Page 452
1. Porphyrin Conjugates as Cleaving Agents......Page 453
3. Other Applications of Porphyrin Conjugates......Page 456
J. Minor Groove Binder Conjugates......Page 457
K. Polymer Conjugates......Page 458
L. Antibody Conjugates......Page 459
M. Protein Conjugates......Page 460
N. Miscelleneous Conjugates......Page 461
1. Rationale......Page 463
IV. CONCLUSIONS AND PERSPECTIVES......Page 464
ACKNOWLEDGMENTS......Page 466
REFERENCES......Page 467
I. INTRODUCTION......Page 480
A. Immune Stimulation by Palindromes and Antisense Oligodeoxynucleotides......Page 481
B. Identification of the CpG Motif......Page 482
C. Immune Recognition of CpG Motifs as a Defense Mechanism......Page 485
A. B Cells......Page 486
B. Monocytes, Macrophages, and Dendritic Cells......Page 489
C. Natural Killer Cells......Page 491
D. T Cells......Page 492
B. Cellular Uptake......Page 493
D. Activation of the Mitogen-Activated Protein Kinase Pathways......Page 494
E. Activation of Nuclear Factor kB......Page 495
F. Activation of Transcription and Translation......Page 496
G. Influence of the ODN Backbone on the Immune Effects of CpG DNA......Page 497
A. Activation of Innate Immune Defenses Against Infection......Page 498
B. Role of CpG DNA as a Vaccine Adjuvant and in DNA Vaccines......Page 500
1. CpG DNA as an Adjuvant for Cancer Vaccines......Page 503
2. Activation of NK Cells with CpG DNA for Immunotherapy of NK-sensitive Tumors......Page 504
3. Activation of Antibody-Dependent Cellular Cytotoxicity with CpG DNA for Immunotherapy of NK-Resistant Tumors......Page 505
D. Immunotherapy of Allergic Diseases with CpG DNA......Page 506
VI. CpG-N MOTIFS COMPRISE ANOTHER CLASS OF IMMUNE-REGULATORY SEQUENCES......Page 509
VII. CONCLUSIONS......Page 510
REFERENCES......Page 511
II. PRE-mRNA SPLICING......Page 525
III. ALTERNATIVE SPLICING OF PRE-mRNA......Page 527
IV. MODIFICATION OF PRE-mRNA SPLICING PATTERNS BY ANTISENSE OLIGONUCLEOTIDES......Page 528
V. PROPERTIES OF OLIGONUCLEOTIDES FOR MODIFICATION OF SPLICING......Page 530
VI. RESTORATION OF CORRECT SPLICING IN MUTANT PRE-mRNAs IN GENETIC DISORDERS......Page 531
VII. MODIFICATION OF SPLICING OF mdx DYSTROPHIN PRE-mRNA......Page 535
VIII. OLIGONUCLEOTIDE-INDUCED MODIFICATION OF ALTERNATIVE SPLICING IN CANCER......Page 537
IX. TARGETING OF ANTISENSE OLIGONUCLEOTIDES TO SPLICE SITES......Page 538
REFERENCES......Page 541
I. INTRODUCTION......Page 548
A. Phosphorothioate Oligonucleotides......Page 549
B. End-Protected Phosphodlesters......Page 550
D. Antisense Mechanisms in the Brain......Page 551
E. Other Oligonucleotide Chemistries......Page 553
B. Intraventricular Administration......Page 554
D. Intrathecal Administration......Page 555
F. Cellular Localization......Page 556
G. Crossing the Blood-Brain Barrier......Page 558
IV. TARGET VALIDATION TOOL......Page 559
A. Alzheimer's Disease......Page 561
B. Pain......Page 562
C. Affective Disorders......Page 564
D. Obesity......Page 565
V. CONCLUSIONS......Page 566
REFERENCES......Page 567
I. OVERVIEW......Page 572
III. EXPLOITING THE HIGH SPECIFICITY OF ANTISENSE OLIGONUCLEOTIDES......Page 573
IV. CURRENT AChE PHARMACOLOGY......Page 574
A. Specificity......Page 575
B. Feedback Overexpression of AChE Alters the Balance and Levels of AChE Isoforms......Page 577
C. Noncatalytic Activities of AChE......Page 579
VI. ANTI-AChE ANTISENSE OLIGONUCLEOTIDES- THE STATE OF THE ART......Page 581
B. Neuromuscular Disease......Page 584
C. Head Trauma......Page 585
D. Neurodegenerative Disease......Page 587
ACKNOWLEDGMENTS......Page 588
REFERENCES......Page 589
I. INTRODUCTION......Page 594
II. THE SERINE/THREONINE PPase FAMILY......Page 596
A. Cantharidin......Page 600
B. Fostriecin......Page 601
A. Identification of Antisense Oligonucleotides that Inhibit the Expression of PP5......Page 602
1. Specificity of PP5 Antisense Inhibition......Page 605
3. ISIS 1534- Mediated Inhibition of Cell Growth Correlates with the Induction of p21WAF/Cip1......Page 606
4. PP5 and Glucocorticoid Receptor Signaling Cascades Leading to the Induction of p21 WAF/Cip1......Page 609
5. Effect of PP5 on the Binding of Dexamethasone to the GR......Page 612
B. Identification of Antisense Oligonucleotides that Inhibit the Expression of PP1 y1......Page 615
REFERENCES......Page 617
A. Inflammatory Processes......Page 622
1. Major Histocompatibility Complex......Page 626
Interleukin (IL)-10......Page 629
2. T-Cell Antigen Receptor (TCR) and TCR-Mediated Signal Transduction......Page 630
Protein Tyrosine Kinases (PTKs)......Page 631
Transcription Factors......Page 632
B. T-Cell Costimulation......Page 633
1. CD28......Page 634
Tumor Necrosis Factor Alpha (TNF-a)......Page 635
Interleukin 4 (IL-4) and 5 (IL-5)......Page 637
Interleukin 6 (IL-6)......Page 638
3. Cytokine Receptors......Page 639
Interleukin Receptor 1, Type 1 (IL-1R1)......Page 640
CD14......Page 641
Ha-Ras......Page 642
IL-1 Receptor Associated Kinase 2 (IRAK-2)......Page 643
Tyrosine Kinase 2 (Tyk2) and Janus Kinase 2 (Jak2)......Page 644
Protein Kinase C......Page 645
c-Raf......Page 646
Signal Transducer and Activator of Transcription 6 (STAT6)......Page 647
Nuclear Factor Kappa B (NFkB)......Page 648
Inducible Nitric Oxide Synthase (iNOS)......Page 651
Phospholipase A2 (PLA2)......Page 653
E. Apoptosis and Its Role in Inflammatory Disease......Page 655
2. c-myc......Page 656
4. Lyn Kinase......Page 657
ICAM-1......Page 658
Allograft Survival......Page 659
Ischemia/Reperfusion Injury......Page 660
Lung Inflammation......Page 661
B2 Inegrins......Page 662
2. Chemokines and Chemokine Receptors......Page 663
Macrophage Inflammatory Protein (MIP)-1a, B......Page 664
IFN-y-Inducible Protein (IP)-10/crg-2......Page 665
A. Application of Antisense Oligonucleotides to Inflammatory Diseases- Novel Strategies for the Use of Antisense Oligonucleotides......Page 666
B. Antisense Oligonucleotide Delivery and Distribution......Page 667
REFERENCES......Page 669
II. THE POTENTIAL ROLE OF PULMONARY SURFACTANT IN RASON UPTAKE AND DELIVERY......Page 686
IV. DELIVERY DEVICES......Page 687
V. EPI 2010......Page 688
VII. TOXICOLOGY......Page 689
A. desAdensosine RASONs......Page 690
REFERENCES......Page 691
I. INTRODUCTION......Page 693
II. ANTISENSE c-myc......Page 694
IV. ANTISENSE PROTEIN KINASE A TYPE I......Page 695
V. CHRONIC LYMPHATIC LEUKEMIA......Page 697
VI. Bcl-2......Page 699
VII. MDM2......Page 701
X. c-RAF KINASE......Page 702
XI. c-JUN N-TERMINAL KINASE......Page 703
XII. SUMMARY......Page 707
REFERENCES......Page 708
I. INTRODUCTION......Page 714
II. CHEMICAL MODIFICATION OF ODN......Page 715
III. SPECIFICITY......Page 716
IV. TARGETING......Page 721
V. TRANSFER......Page 722
VI. PERSPECTIVES......Page 724
REFERENCES......Page 725
I. INTRODUCTION......Page 729
II. THE TARGET......Page 730
III. ANTISENSE OLIGONUCLEOTIDES AS ANTIVIRAL AGENTS......Page 732
IV. HUMAN PHARMACOKINETICS OF FOMIVIRSEN......Page 734
V. CLINICAL EFFICACY OF FOMIVIRSEN......Page 736
VI. CONCLUSION......Page 739
REFERENCES......Page 740
I. INTRODUCTION......Page 743
A. ISIS 3521 (Isis Pharmaceuticals)......Page 744
B. ISIS 2503 (Isis Pharmaceuticals)......Page 753
C. ISIS 5132 (Isis Pharmaceuticals)......Page 754
D. G3139 (Genta)......Page 756
G. MG 98 (MethylGene)......Page 758
III. OUTLOOK AND FUTURE DIRECTIONS......Page 759
REFERENCES......Page 760
I. INTRODUCTION......Page 764
A. In Vivo Treatment Modeling......Page 766
B. Pharmacokinetic/Dynamic Studies......Page 767
C. Uptake and Intracellular Trafficking Studies......Page 768
A. Bone Marrow Purging with Antisense ODN- Illustrative Examples......Page 769
C. Pharmcokinetic Data......Page 770
D. Toxicity Studies......Page 771
IV. CONCLUSIONS......Page 772
REFERENCES......Page 773
A. Intercellular Adhesion Molecule 1 (ICAM-1)......Page 775
1. Pharmacology......Page 776
3. Pharmacokinetics, Metabolism, and Toxicology......Page 777
A. Phase 1......Page 778
B. Phase 2a Trials......Page 779
1. Crohn's Disease......Page 780
2. Rheumatoid Arthritis......Page 781
3. Psoriasis......Page 782
4. Renal Transplantation......Page 783
5. Ulcerative Colitis......Page 784
C. Phase 2b Crohn's Disease......Page 785
III. SAFETY AND TOLERABILITY......Page 788
IV. CONCLUSION......Page 789
REFERENCES......Page 790
I. INTRODUCTION......Page 797
II. PHYSICAL-CHEMICAL PROPERTIES......Page 798
A. Brain......Page 799
1. Formulation Considerations......Page 800
2. Deposition and Uptake......Page 802
IV. ADVANCED PARENTERAL DELIVERY......Page 804
2. Anionic or Charge-Neutral Liposomes......Page 805
3. Long-Circulating Liposomes......Page 807
4. pH-Sensitive Liposomes......Page 808
5. Cell-Surface-Targeted Liposomes......Page 810
B. Sustained-Release Subcutaneous Formulations......Page 811
B. Electrical Field for Alteration of Skin Permeability......Page 812
C. Formulations for the Alteration of Skin Permeability......Page 813
D. Pharmacology by the Topical Route......Page 816
VI. ORAL AND GASTROINTESTINAL DELIVERY......Page 818
A. Presystemic Metabolism......Page 819
B. Permeability......Page 820
C. Systemic Bioavailability......Page 822
D. Local GI Uptake......Page 826
VII. CONCLUSIONS......Page 828
REFERENCES......Page 829
I. INTRODUCTION......Page 835
II. DNA-BINDING PROTEINS......Page 838
III. TRIPLE-HELIX-FORMING OLIGONUCLEOTIDES......Page 840
V. SMALL DNA-BINDING MOLECULES......Page 841
1. Introduction......Page 843
2. Pairing Rules......Page 845
4. Binding-Site Size......Page 847
5. Inhibition of Protein/DNA Interaction......Page 850
6. Inhibition of Gene Expression......Page 851
VI. CONCLUSIONS AND OUTLOOK......Page 852
REFERENCES......Page 853
A. Specificity......Page 860
B. Stability......Page 862
A. The Transcription Inhibition Approach......Page 863
1. Mutagenesis of an Extrachromosomal SupF Gene Using TFOs Linked to Psoralen......Page 864
2. Repair of TFO-Induced DNA Damage: Influence of the Third Strand on Repair......Page 866
3. Triplex Formation Itself Stimulates Repair and Mutagenesis......Page 867
4. TFO-Induced Mutagenesis at Chromosomal Sites......Page 868
5. Site-Specific Mutagenesis by Peptide Nucleic Acids......Page 870
A. Transcriptional Activation by Triplex Formation......Page 871
1. Intramolecular Recomination Between Two Mutant Genes Carried as Direct Repeats......Page 872
2. Intermolecular Recombination Using a TFO Tethered to a Donor Fragment......Page 875
V. PHYSICAL DETECTION OF TRIPLEX FORMATION AT CHROMOSOMAL SITES IN CELLS......Page 876
B. Oligonucleotide Delivery......Page 878
REFERENCES......Page 879
II. RNA CATALYTIC MOTIFS......Page 888
B. RNase P......Page 889
C. Hammerhead and Hairpin Ribozymes......Page 890
A. Sequence Specificity......Page 892
C. Determining Accessible Sites for Ribozyme Pairing in Target RNAs......Page 893
D. Ribozyme Design and Expression......Page 894
IV. RIBOZYME TRAFFICKING AND INTRACELLULAR LOCALIZATION......Page 897
A. Viral Vectors......Page 898
VI. DETERMINING IN VIVO RIBOZYME FUNCTION......Page 899
VIII. CONCLUDING REMARKS......Page 900
REFERENCES......Page 901