510 Index. 22 rationale, 15, 16, 25 stability of complexes, 21 Atomic force microscopy (AFM), DNA conjugate particle size analysis, 41, 42

Transcription

1 Index AAV, see Adeno-associated virus Acellular dermis, see Skin substitute Adeno-associated virus (AAV), genome structure, 446, 462 herpes simplex virus amplicons for hightiter stock production, amplicon features, 448, 450 cell culture, 451 DISK-HSV-1 as helper virus, 450, 451 materials for production, 451, 453 plasmids, 451, 453, 458 purification of recombinant virus, concentrating, 457, 458 heparin affinity chromatography, 456, 457 iodixanol density gradient, 455, 456, 458 overview, 454, 455 release from infected cells, 455 rationale, 450 transfection, 453, 454, 458 herpes simplex virus-1 hybrid vector for gene transfer to neurons, advantages, 464, 465 components, 465 cosmid DNA preparation for transfection, 467, , 475, 476 gene transfer, animals, 469, neuron culture, 469, 474, 476 helper virus-free stock preparation, 467, 468, 472, 473, 476 materials for production, 465, 467, 468, 475 packaging, 463, 464, 467 plasmid construction, 469, 470 rationale, Adeno-associated virus (AAV) (cont.), herpes simplex virus-1 hybrid vector for gene transfer to neurons (cont.), safety, 464, 465 titration, 468, 473, 474 transduction efficiency, 464 protein function, 446 recombinant vector production, DNase-resistant genome quantification, 438, 439, 441, 442 materials, midscale production screen, 432, 436, 437, 441 optimization strategies, 447, 448 packaging cell lines, plasmids, 447 polymerase chain reaction assay of infectious virus, 439, 440, 442 producer plasmid generation, , 440 purification, 430, 432, 433, 437, 438, 441 requirements, , 447 titration, 430, 431, 433, transfection, 435, 436, 440 replication, 446 serotypes, 445 transduction efficiency, 427 transgene integration, 462 tropism, 427, 445 Adenovirus, advantages in gene transfer, 371, 389 first generation vectors, overview, 371 genome, helper-dependent vector system, cell culture, 377 concentration determination, 384 design of vector, 373,

2 510 Index Adenovirus (cont.), helper-dependent vector system (cont.), helper virus, 375, 376, 385 materials, 373, overview, 373 purification of vector, 376, 377, 381, 383, 384, 386 rationale, 371, 372 rescue and amplification, , 385 restriction enzyme digest analysis, Southern blotting for amplification monitoring, 380, 381, 385, 386 ovine vectors, cell culture, 420, cell lines for production, 422 design, 416, 417 genome structure, 415, 416 genomic plasmids, 417 materials for production, 417, 418, 420 purification, 423, 424 rationale for construction, 415 recombinant genome generation, direct cloning, 420, 421 recombination in Escherichia coli, 421, 422, 424 shuttle plasmids, 417, 418 titration, 424, 425 protein functions, 390, 391 replication, 391 replication-deficient vectors, 391 serotypes, 389 tumor vaccine modification, 236 two-plasmid rescue system, cell culture, 395, 402, 412 cesium chloride banding of virus, 398, 408, 409 concentration determination, 409 genomic plasmids, 393, 395 materials for production, 393, 395, monolayer cell production, 398, 407 optimization of production, 411, 412 overview, plasmid preparation, 399, 401, 412 Adenovirus (cont.) two-plasmid rescue system (cont.), replication competent virus assay, 410, 411 restriction enzyme digest analysis, 405, 409, 412 shuttle plasmids, 395 suspension cell production, 398, 407, 408 titration, 397, 406, 412 transfection, 395, 397, 402, 404, 405, 412 AFM, see Atomic force microscopy AIPcS 2a, see Photochemical transfection Asialoorosomucoid, gene therapy targeting of liver, asialoorosomucoid-lysine methyl ester- VSVG peptide conjugate synthesis, 19, 20 asialoorosomucoid-polylysine-listeriolysin O conjugate synthesis, asialoorosomucoid-polylysine conjugation, 18 DNA complex formation, 19 listeriolysin O synthesis and conjugation, 18 cell transfection and luciferase reporter assay, 21, 22 DNA compaction measurement, 21, 27 materials, 16, 17 mouse liver transfection, 22 particle size determination, 21 PEG-polysine conjugate synthesis, PEG-polysine, 20 PEG-polysine-VSV, 20 PEG-polysine-VSV/asialoorosomucoidpolylysine-DNA complexes, rationale, 15, 16, 25 stability of complexes, 21 Atomic force microscopy (AFM), DNA conjugate particle size analysis, 41, 42 B BAM, see Bioartificial muscle Bioartificial muscle (BAM), advantages and disadvantages, 220, 221

3 Index 511 Bioartificial muscle (BAM) (cont.), applications, 220 formation, cell culture, 222, 228 materials, Matrigel seeding and maintenance, 222, 223, 227, 230 primary mouse muscle cell isolation, 221, , 229 retroviral transduction, 222, 225, silicone tissue mold fabrication, 222, 226 implantation, 223, 227, 228, 230 principles of ex vivo therapy, 219, 220 C Cytokine-modified tumor vaccine, see Tumor vaccine D Di-n-propylsulfoxide (DPSO), solvoplex vectors, 84, 85 synthesis, 89, 90 DNA vaccine, gene gun, see Gene gun muscle, 49, 50, 58, 109 DPSO, see Di-n-propylsulfoxide E Electron microscopy, DNA conjugate particle size analysis, 4 Electroporation, muscle delivery of genes, advantages over viral vectors, 55 applications, 55, 56 duration of expression, 54, 55 efficiency of gene delivery, β galactosidase, 59, 60 interleukin-5, 59 overview, 49, 50, 59, 110 GeneSwitch system, , 119 instrumentation, 56, 57 parameters affecting efficiency, DNA concentration, 52 electrical field direction, 53 pulse length and number, 52 voltage of pulses, 51, 52 Electroporation, muscle delivery of genes (cont.), technique, 58 vector, 53, 54, 56, 60 F Feline immunodeficiency virus (FIV), gene transfer vectors, advantages, 335 cell culture, 342 concentrating, 343, 345, 348 first generation vectors, 338 harvesting, 343, 345, 348 infection, overview, 345, 346 Retronectin utilization, 346, 349, 349 spinoculation of target cells, 346 supernatant infection, 346 materials for production, 342, 343 plasmids, 342, 343 pseudotyping, 340, 341 second generation vectors, third generation vectors, 341 titration, 347, 348 transfection, calcium phosphate, , 348 FuGENE 6, , 348 tropism, 341, 342 genome, 337 replication, 337, 338 FIV, see Feline immunodeficiency virus Foamy virus, see Simian foamy virus-1 G Gaucher's disease, gene therapy clinical trials, Gene gun, advantages, 137, 144 applications, 137, 144 devices, 138, 139 discharge and transfection, 142 DNA cartridge preparation, 1441 DNA vectors and coating on gold, elemental gold particle preparation, 139

4 512 Index Gene gun (cont.), gene expression assays, cytokines, 141, 143 β galactosidase, 140, 143 haat, 140, 143 luciferase, 140, 143 tissue extraction, 142, 143 materials, skin preparation, 140, 142 GeneSwitch system, electroporation, , 119 injection, 115 materials, mifepristone induction, 112, 113, 115, 117 nonsecreted gene product assay, 118 plasmids and formulation, 111, 112, 114, 115, 116, 118, 119 principle, 111, 112 promoters, 113, 114 secreted gene product assay, Genetically modified skin substitute, see Skin substitute H Hemagglutinating virus of Japan (HVJ), liposome gene transfer, animal studies, 70 artificial viral envelope liposomes, cationic lipoids, 65 cultured cell studies, 70 efficiency, 63, 65, 66 preparation of conjugates, DNA complex preparation, 69, 70 flow chart, 68 lipid mixture preparation, 69 materials, 66, 67 virus preparation from eggs, 68, 69 repeated injection, 63, 64 safety, 65 Hematopoietic stem cell (HSC), abundance, 188 CD34+ enrichment, antibody selection, 187 Ceprate system, 195 requirement for gene transfer, 188 expansion ex vivo, 188, 189 Hematopoietic stem cell (HSC) (cont.), functional assays, 187, 188 markers, 188 retroviral transduction ex vivo, clinical grade amphotropic vector generation, , 197 clinical trials, materials, 192, 193 multidrug resistance engineering, 190 optimization, 189, 192 prestimulation of cells, 196 transduction, efficiency, 190 protocol, 196, 197 Herpes simplex virus-1 (HSV-1), adeno-associated virus hybrid vector for gene transfer to neurons, advantages, 464, 465 components, 465 cosmid DNA preparation for transfection, 467, , 475, 476 gene transfer, animals, 469, neuron cultre, 469, 474, 476 helper virus-free stock preparation, 467, 468, 472, 473, 476 materials for production, 465, 467, 468, 475 packaging, 463, 464, 467 plasmid construction, 469, 470 rationale, safety, 464, 465 titration, 468, 473, 474 transduction efficiency, 464 advantages in gene therapy, 481, 483 amplicons for high-titer adeno-associated virus stock production, amplicon features, 448, 450, 462 cell culture, 451 DISK-HSV-1 as helper virus, 450, 451 materials for production, 451, 453 plasmids, 451, 453, 458 purification of recombinant virus, concentrating, 457, 458 heparin affinity chromatography, 456, 457

5 Index 513 Herpes simplex virus-1 (HSV-1) (cont.), amplicons for high-titer adeno-associated virus stock production (cont.), purification of recombinant virus (cont.), iodixanol density gradient, 455, 456, 458 overview, 454, 455 release from infected cells, 455 rationale, 450 transfection, 453, 454, 458 genome, 483 protein functions, 483, 485 replication, 461, 462, 483, 485 replication-defective vectors for gene transfer, cytotoxicity of recombinan vectors, 490, 491 DNA isolation for transfection, , 498, 499 double mutant virus lacking ICP4 and ICP22, 488, 490 engineering goals, 485 first generation vectors, 487, 488 immediate early gene targeting for cytotoxicity reduction, 486, 487 limiting dilution for β galactosidase expressing recombinants, 496, 500 materials for production, 491, 492 second generation vectors, 488, 490 stock preparation, 497, 498, 500 titration, 497 transfection, 494, 495, 499, 500 structure, 461 HIV, see Human immunodeficiency virus HSC, see Hematopoietic stem cell HSV-1, see Herpes simplex virus-1 Human immunodeficiency virus (HIV), constitutive transport element in simple retroviruses, 280, 281 function of proteins, Env, 260, 277 Gag, 260, 277 Nef, 260, 277, 279, 280 Pol, 260, 277 Rev, 260, 279, 305 Human immunodeficiency virus (HIV) (cont.), function of proteins (cont.), structural proteins, 277 Tat, 260, 278, 279, 304, 305 Vif, 260, 280 Vpr, 260, 277, 280, 305 Vpu, 260, 280 gene transfer vectors, advantages and limitations, 264, 265, 275, 286, 287, 319 applications, 308 cell culture, 265, 266, 270, 289, 293, 309, 310, 315 central DNA flap creation, 285 concentrating, 266, 268, 288, 290, 294, 295, 309, 310, 312, 313, 315 design and performance, 263, 264 first-generation vectors, 284 β galactosidase reporter gene assay, 310, green-fluorescent protein-encoding vector detection, 291, 292, 295 harvesting, 312, 315 materials for production, 265, 266, , p24 assay, 266, 269, 270 plasmids, 265, 266, 270 replication-competent retrovirus assays, 266, 269, 288, 289, 292, 293, 295, 307 Rev-independent vectors, 284 safety in production, 307 self-inactivating vectors, 284, 285, 306 stability, 308 Tat-independent vectors, 284 titration, 266, 269, 270, 288, , 310, toxicity, 308 transfection using calcium phosphate precipitation, 268, 270, , 294, , 315 vesicular stomatitis virus G glycoprotein hybrids, 261, 285, 306, 308, 319

6 514 Index Human immunodeficiency virus (HIV) (cont.), gene transfer vectors (cont.), woodchuck hepatitis virus posttranscriptional regulatory element modification, 285 genome, , 276, 304 packaging cell system, components, 281, 306 plasmids, elimination of Vif, Vpr, and Vpu, 282 first generation plasmids, 281, 282 preparation, 309, 314, 315 ptv, 306 pty, 306, 307 Rev-independent plasmids, 283 segregation of Gag/Gag-Pol and Rev functions, 282 segregation of Gag and Pol coding regions, titer, 307, 308 types, 261, 263, 285, 286 replication, 277, 278, structure, 259 HVJ, see Hemagglutinating virus of Japan L, K Keratinocyte, see Skin substitute Lentiviral vectors, see Feline immunodeficiency virus; Human immunodeficiency virus; Simian immunodeficiency virus Light-induced gene delivery, see Photochemical transfection Liposome, liposome-polycation-dna, see Liposome-polycation-DNA viral liposomes, see Hemagglutinating virus of Japan Liposome-polycation-DNA (LPD), formulations, 73 gene transfer efficiency, 74, 75 preparation, 73 incubation conditions, 76, 77, 80 liposome preparation by thin-film hydration and extrusion, materials, 76, 79 Liposome-polycation-DNA (LPD) (cont.), size, 74 storage, 74 toxicity, 75 Listeriolysin O, synthesis and asialoorosomucoid-polylysinelisteriolysin O conjugation, 18 Liver, gene therapy targeting with protein delivery systems, asialoorosomucoid-lysine methyl ester- VSVG peptide conjugate synthesis, 19, 20 asialoorosomucoid-polylysine-listeriolysin O conjugate synthesis, asialoorosomucoid-polylysine conjugation, 18 DNA complex formation, 19 listeriolysin O synthesis and conjugation, 18 cell transfection and luciferase reporter assay, 21, 22 DNA compaction measurement, 21, 27 materials, 16, 17 mouse liver transfection, 22 particle size determination, 21 PEG-polysine conjugate synthesis, PEG-polysine, 20 PEG-polysine-VSV, 20 PEG-polysine-VSV/ asialoorosomucoid-polylysine- DNA complexes, rationale, 15, 16, 25 stability of complexes, 21 Low-density lipoprotein receptor, gene delivery mediation, 33 LPD, see Liposome-polycation-DNA Lung, applications of gene delivery, 83 polymeric immunoglobulin receptor, gene delivery mediation, 34 solvoplex gene delivery, advantages, 83, 84 di-n-propylsulfoxide synthesis, 89, 90 formulations and efficiencies, 84, 85 intratracheal injection, mouse, rat, 91

7 Index 515 Lung (cont.), solvoplex gene delivery (cont.), mechanism of transfection, 87 plasmid preparation, 88, 89 preparation, 90, 92 readministration, 87 reporter gene studies, β galactosidase, green fluorescent protein, 92, 93 luciferase, 91, 93 overview, 86 rodents for study, 88, 92 solvents, 88 storage, 86 toxicity, 87 M Mannose receptor, gene delivery mediation, 34 Mifepristone, see GeneSwitch system MMLV, see Moloney murine leukemia virus Moloney murine leukemia virus (MMLV), see Retrovirus Muscle, bioartificial muscle, see Bioartificial muscle DNA vaccine, 49, 50, 58, 109 electroporation, advantages over viral vectors, 55 applications, 55, 56 duration of expression, 54, 55 efficiency of gene delivery, β galactosidase, 59, 60 interleukin-5, 59 overview, 49, 50, 59, 110 instrumentation, 56, 57 parameters affecting efficiency, DNA concentration, 52 electrical field direction, 53 pulse length and number, 52 voltage of pulses, 51, 52 technique, 58 vector, 53, 54, 56, 60 regulated gene therapy, GeneSwitch system, electroporation, , 119 injection, 115 Muscle (cont.), regulated gene therapy (cont.), GeneSwitch system (cont.), materials, mifepristone induction, 112, 113, 115, 117 nonsecreted gene product assay, 118 plasmids and formulation, 111, 112, 114, 115, 116, 118, 119 principle, 111, 112 promoters, 113, 114 secreted gene product assay, ligand-dependent systems, overview, 110, 111 rationale, 110 N Neuron gene transfer, see Herpes simplex virus-1 P PBMC, see Peripheral blood mononuclear cell Peripheral blood mononuclear cell (PBMC), replication-competent retrovirus assay, 292, 293 Photochemical transfection, advantages and limitations, 126, 127 cell culture, , 132 DNA/poly-L-lysine complex preparation, , 132 in vivo potential, 127 light sources and dosing, 128, materials, 127, 128 photosensitizer, AIPcS 2a solution preparation, 129, 132 cytotoxicity and cancer photodynamic therapy, 125, 126 definition, 123, 124 intracellular localization, 124, 125 reactions, 124 structures, 125 principle of photochemical internalization, 123 transfection reaction, 130, 132, 133

8 516 Index PLL, see Poly-L-lysine Poly-L-lysine (PLL), binding assay using fluorescence quenching, 9 charge ratio determination, 9 copolymer conjugate synthesis, calculation based on composition, 9 materials, 4, 5 nuclear magnetic resonance, 8 PL26k-graft-(ε NH-CH2CO-NH- PEG ε trilactose ligand)2.5, 6, 8 poly-l-lysine-graft-r1-graft-r2-- graft-r3, 5, 6 graft, attachment, 3, 4 structure, 2 ligand, 3 luciferase gene expression study, 10, 11 nucleic acid delivery rationale, 1, 2, 15 particle size analysis, 10 PEG-polysine conjugate synthesis for liver targeting, PEG-polysine, 20 PEG-polysine-VSV, 20 PEG-polysine-VSV/ asialoorosomucoid-polylysine- DNA complexes, photochemical transfection, DNA/poly-Llysine complex preparation, , 132 plasmid DNA preparation, 8 polyplex formation, 9, 10 Polymeric immunoglobulin receptor, gene delivery mediation, 34 R RDO, see RNA-DNA oligonucleotide hybrid Receptor-mediated gene delivery, see also Asialoorosomucoid; SEC-R, DNA compaction importance, 27, 28 endosomal escape and nuclear uptake, 29, 30 ligands, 26 mechanism, 26, 27 polycations in DNA condensation, 28, 29 rationale, 15, 16, 25 Receptor-mediated gene delivery (cont.), receptor types and ligands, asialoglycoprotein receptor, 33 low-density lipoprotein receptor, 33 mannose receptor, 34 polymeric immunoglobulin receptor, 34 SEC-R, 34, 35 table, 31, 32 transferrin receptor, 33, 34 Retrovirus, see also Feline immunodeficiency virus; Human immunodeficiency virus; Simian foamy virus-1; Simian immunodeficiency virus, advantages for gene transfer, 173 alphavirus for cytoplasmic vector construction, see Semliki forest virus, bioartificial muscle gene transfer, see Bioartificial muscle hematopoietic stem cell gene transfer, see Hematopoietic stem cell keratinocyte ex vivo retroviral gene transfer, see Skin substitute titer assays, active virus per cell determination, 165, 169 cell culture, fibroblasts from human foreskin, 167, 168 materials, 166, 167 cell surface area determination by fluorescence microscopy, factors affecting titer, 161, 162 β galactosidase assay, 167, 168, 171 multiplicity of infection determination, 165 recombinant retrovirus harvesting, 168 serial dilution, 161 start of infection, active virus concentration, , transduction efficiency of different cell types, determination, 165, 166, 170, 171

9 Index 517 Retrovirus (cont.), transduction efficiency, 149 transgene cassette stability, 173, 174 tumor vaccine, see Tumor vaccine vesicular stomatitis virus G glycoprotein pseudotyping of murine leukemia retrovirus, cell culture, 153 concentrating, 156 env protein replacement, 150 materials, 153, 154 packaging cell, 150, 151 plasmids, 153 producer cell line generation, 155, 156 pseudotransduction, 152 rationale, 149, 150 titration, 156 transduction efficiency, 151, 152, 156, 157 transduction protocol, 154 transfection, , 157 RNA-DNA oligonucleotide hybrid (RDO), gene conversion, assays, bacterial transformation assay for gene correction in eukaryotic cells, CC106 competent cell preparation, 103, 104 P90C competent cell preparation, 102, 103 principle, 102 transformaton and plating, 104, 105 cell culture, 99 materials, 99, 100, 105 mutant β galactosidase plasmid reactions, 101, 102, 105 nuclear extract isolation, , 105 overview, 96, 102 comparison of different cell types, point mutation correction, 95 S SEC-R, cell distribution, 35 ligand, 34, 35 SEC-R (cont.), receptor-mediated gene delivery, conjugate analysis, 36, 39, 43 conjugate construction, 35 39, 42, 43 DNA condensation, 36, 37, DNA plasmid preparation, 36 materials, 35 37, 42 particle size analysis, atomic force microscopy, 41, 42 electron microscopy, 41 overview, 37, 41 Semliki forest virus (SFV), replication, 174 retrovirus cytoplasmic vector construction, analysis of vectors, 182, 183, 185 cell culture, 176, 183, 184 minigene incorporation, 175, 176 packaging cell line, 182, 185 plasmids, 176, 178, 179, 184 principles, 174, 175, 183 RNA synthesis, 178, 179, 181, 184 titration, 178, 184 virus production, 178, 181, 182, 184, 185 Sendai virus (SeV), gene transfer vector, cell culture, 365, 366 F-defective vector design, 362, 363 green fluorescent protein as reporter, 364, 365, inducible expression of F protein, 366, 367 infection, 368 materials for production, 365, 366, 368 overview of generation, packaging cell line construction, 363 photoinactivation of recombinant vaccinia virus, 366 propagation, 368 titration, 368 genome, 361, 362 liposome generation, see Hemagglutinating virus of Japan protein functions, 362, 363 replication, 361

10 518 Index SeV, see Sendai virus SFV, see Semliki forest virus SFV-1, see Simian foamy virus-1 Simian foamy virus-1 (SFV-1), advantages in gene transfer, 319, 320 gene transfer vectors, cell culture, 325 development, 322, 323 infection of nonadherent cells, 326, 328 materials for production, 325, 326 packaging cell lines, plasmids, 325 titration using β galactosidase, transfection using Lipofectamine, variations, 323, 324 genome, 321 replication, 321, 322 safety, 320 Simian immunodeficiency virus (SIV), gene transfer vectors, components, 353, 354 concentrating, 356, 358, 359 envelope protein selection, 354 Gag-Pol expression plasmid optimization, 355, 356 materials for production, 356 minimization of construct, 354, 355 plasmids, 356, 358 titration, transfection, genome structure, 351, 353 replication, 353 tropism and safety, 351 SIV, see Simian immunodeficiency virus Skin substitute, demand, 203 keratinocyte ex vivo retroviral gene transfer, acellular dermis preparation, 205, 206, 209, 210, 215 advantages of vector, 204 applications, 203, 204 cell culture, 204, 205, 208, 215 Skin substitute (cont.), keratinocyte ex vivo retroviral gene transfer (cont.), genetically modified skin substitute preparation, 206, 207, , 215 genetic modification of cells, 208, 209, 215 materials, , 214, 215 transplantation, 207, Solvoplex, advantages of gene delivery, 83, 84 di-n-propylsulfoxide synthesis, 89, 90 formulations and efficiencies of lung gene transfer, 84, 85 intratracheal injection, mouse, rat, 91 mechanism of transfection, 87 plasmid preparation, 88, 89 preparation, 90, 92 readministration, 87 reporter gene studies, β galactosidase, green fluorescent protein, 92, 93 luciferase, 91, 93 overview, 86 rodents for study, 88, 92 solvents, 88 storage, 86 toxicity, 87 T Titration, adeno-associated virus, herpes simplex virus-1 hybrid vectors, 468, 473, 474 recombinant vectors, 430, 431, 433, adenovirus, helper-dependent vector system, 397, 406, 412 ovine vectors, 424, 425 feline immunodeficiency virus vectors, 347, 348, 347, 348 herpes simplex virus-1 vectors, 497

11 Index 519 Titration (cont.), human immunodeficiency virus vectors, 266, 269, 270, 288, , retrovirus vectors, active virus per cell determination, 165, 169 cell culture, fibroblasts from human foreskin, 167, 168 materials, 166, 167 cell surface area determination by fluorescence microscopy, factors affecting titer, 161, 162 β galactosidase assay, 167, 168, 171 multiplicity of infection determination, 165 recombinant retrovirus harvesting, 168 serial dilution, 161 start of infection, active virus concentration, , transduction efficiency of different cell types, determination, 165, 166, 170, 171 Semliki forest virus vectors, 178, 184 Sendai virus vectors, 368 simian foamy virus-1 vectors, simian immunodeficiency virus vectors, vesicular stomatitis virus G glycoproteinpseudotyped murine leukemia retrovirus, 156 Transferrin receptor, gene delivery mediation, 33, 34 Tumor vaccine, antigens, 233 gene transfer, cytokine genes, allogeneic tumor vaccine clinical trials, 241, 242 animal studies, autologous tumor vaccine clinical trials, 239, 240 mechanisms of antitumor immunity, 239 Tumor vaccine (cont.), gene transfer (cont.), cytokine genes (cont.), rationale, 235 selection of cytokine type, 237, 238 physical transfection methods, 235 retrovirus transduction, advantages and disadvantages, 235, 236 cytokine product assays, 244, gene transfer, 244, materials, 242, 243 mouse challenge assay, 244, 245, 251, 252 producer cell maintenance, 243, 245 supernatant preparation, tumor cell line maintenance, 244, 246, 247 viral vectors, 235, 236 T-cell antitumor immune response, 234, 235 V Vesicular stomatitis virus G glycoprotein (VSVG), asialoorosomucoid-lysine methyl ester- VSVG peptide conjugate synthesis, 19, 20 feline immunodeficiency virus pseudotyping, 340, 341 human immunodeficiency virus pseudotyping, 261, 285, 306, 308, 319 murine leukemia retrovirus pseudotyping, cell culture, 153 concentrating, 156 env protein replacement, 150 materials, 153, 154 packaging cell, 150, 151 plasmids, 153 producer cell line generation, 155, 156 pseudotransduction, 152 rationale, 149, 150

12 520 Index Vesicular stomatitis virus G glycoprotein (VSVG) (cont.), murine leukemia retrovirus pseudotyping (cont.), titration, 156 transduction efficiency, 151, 152, 156, 157 transduction protocol, 154 transfection, , 157 Viral liposomes, see Hemagglutinating virus of Japan VSVG, see Vesicular stomatitis virus G glycoprotein W Woodchuck hepatitis virus, posttranscriptional regulatory element modification of HIV, 285