WO 2010/105243
`
`PCT /US2010/027253
`
`614
`
`u
`560 GAGAAGCCAUUAUCUGCA
`A
`·CUAUCAUCAUAGGUCGUC
`A
`618 CAUCAUAGGUCGUCAUGC
`u
`621 CAUAGGUCGUCAUGCUUA
`u
`691 GAGAUAACCUACACACCA
`A
`735 CCUGGUACAUAACUUUGA
`A
`747 CUUUGAAGAAGGUGGUGG
`u
`775 GGGAUGUAU,Z\AUCAAGAU
`A
`811 GCACACAGUUCCUUCCAA
`A
`818 GUUCCUUCCAAAUGGCUC
`u
`844 GGUUGGCCUUUGUAUCUG
`A
`851 CUUUGUAUCUGAGCACCA
`A
`882 GAAGAAAUAUGAUGGGCG
`0
`942 GUCCCAGOUUGAAGCUCA
`A
`968 GGUAUGAGCAUAGGCUCA
`u
`998 GGCCCAAGCUAUGAAP,UC
`A
`1001 CCCAAGCUAUGAAAUCAG
`A
`1127 CAGAUGGCAAGACAGUAG
`A
`1133 GCAAGACAGUAGAAGCAG
`A
`1184 GCAUGUACCAGAAAGGAC
`A
`1214 CCAAUCCCAUUGCUUCCA
`u
`1257 CCACAGAGCAAAGCUUGA
`u
`1258 CACAGAGCAlv\GCUUGAU
`A
`1262 GAGCAAAGCUUGAUAACA
`A
`1285 GAGCUUGCCUUCUUUGCA
`A
`1296 CUUUGCAAAUGCUUUGGA
`A
`1301 CAAAUGCUUUGGAAGAAG
`0
`1307 CUUUGGAAGAAGUCUCUA
`u
`1312 GAAGAAGUCUCUAUUGAG
`A
`
`G
`68 UUGCAGAUAAUGGCUUCU
`C
`70 UGACGACCUAUGAUGAUA
`G
`72 AGCAUGACGACCUAUGAU
`G
`74 AUAAGCAUGACGACCUAU
`G
`76 UUGGUGUGUAGGUUAUCU
`C
`78 UUCAAAGUUAUGUACCAG
`G
`80 ACCACCACCUUCUUCAAA
`G
`82 UAUCUUGAUUAUACAUCC
`C
`84 UUUGGAAGGAACUGUGUG
`C
`86 AGAGCCAUUUGGAAGGAA
`C
`88 UCAGAUACAAAGGCCAAC
`C
`90 UUGGUGCUCAGAUACAAA
`G
`92 ACGCCCAUCAUAUUUCUU
`C
`94 UUGAGCUUCAAACUGGGA
`C
`96 AUGAGCCUAUGCUCAUAC
`C
`98 UGAUUUCAUAGCUUGGGC
`C
`100 UCUGAUUUCAUAGCUUGG
`G
`102 UCUACUGUCUUGCCAUCU
`G
`104 UCUGCUUCUACUGUCUUG
`C
`106 UGUCCUUUCUGGUACAUG
`C
`108 AUGGAAGCAAUGGGAUUG
`G
`110 AUCAAGCUUUGCUCUGUG
`G
`112 UAUCAAGCUUUGCUCUGU
`G
`114 UUGUUAUCAAGCUUOGCU
`C
`116 UUGCAAAGAAGGCAAGCU
`C
`118 UUCCAAAGCAUUUGCAAA
`G
`120 ACUUCUUCCAAAGCAUUU
`G
`122 AUAGAGACUUCUUCCAAA
`G
`124 UCOCAAUAGAGACUUCUU
`C
`
`- 123 -
`
`69
`
`71
`
`73
`
`75
`
`77
`
`79
`
`81
`
`83
`
`85
`
`87
`
`89
`
`91
`
`93
`
`95
`
`97
`
`99
`
`101
`
`103
`
`105
`
`107
`
`109
`
`111
`
`113
`
`115
`
`117
`
`119
`
`121
`
`123
`
`125
`
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`Page 462 of 582
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`

`

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`
`PCT /US2010/027253
`
`1315 GAAGUCUCUAUUGAGACA
`A
`1356 GGACUUGGCUGCUUGCAU
`u
`1359 CUUGGCUGCUOGCAUUAA
`A
`1371 CAUUAAAGGUUUACCCAA
`u
`1385 CCAAUGUGCAACGUUCUG
`A
`1390 GUGCAACGUUCUGACUAC
`u
`1396 CGUUCUGACUACUUGAAU
`A
`1415 CAUUUGAGUUCAUGGAUA
`A
`1422 GUUCAUGGAUAAACUUGG
`A
`1425 CAUGGAUAAACUUGGAGA
`A
`1455 CAAACUAGCUCAGGCCAA
`A
`1487 CCUGAGCUAAGAAGGAUA
`A
`1493 CUAAGAAGGAUAAUUGUC
`u
`1544 CUGUGUUACACUCAAGGA
`u
`1546 GUGUUACACUCAAGGAUA
`A
`1552 CACUCAAGGAUAAAGGCA
`A
`1581 GUAAUUUGUUUAGAAGCC
`A
`1646 GUUAUUGCCACCUUUGUG
`A
`1711 CAGCCUAGGAAUUCGGUU
`A
`1713 GCCUAGGAAUUCGGUUAG
`u
`1714 CCUAGGAAUUCGGUUAGU
`A
`1718 GGAAUUCGGUUAGUACUC
`A
`1719 GAAUUCGGUUAGUACUCA
`u
`1725 GGUUAGUACUCAUUUGUA
`u
`1730 GUACUCAUUUGOAUUCAC
`u
`1804 GGOA.AAOGAOAGCCACAG
`u
`1805 GOAAAUGAUAGCCACAGU
`A
`1816 CCACAGOAUUGCOCCCUA
`A
`1892 GGGAAGUUCUGGUGUCAU
`A
`1897 GUUCOGGUGUCAUAGAUA
`
`126 UUGUCUCAAUAGAGACUU
`C
`128 AAUGCAAGCAGCCAAGUC
`C
`130 UUUAAUGCAAGCAGCCAA
`G
`132 AOUGGGUAAACCUUUAAU
`G
`134 UCAGAACGUUGCACAUUG
`G
`136 AGUAGUCAGAACGUUGCA
`C
`138 UAUUCAAGUAGUCAGAAC
`G
`140 UUAUCCAUGAACUCAAAU
`G
`142 UCCAAGUUUAUCCAUGAA
`C
`144 UUCUCCAAGUUUAUCCAU
`G
`146 UUUGGCCUGAGCUAGUUU
`G
`148 UUAUCCUUCUUAGCUCAG
`G
`150 AGACAAUUAUCCUUCUUA
`G
`152 AUCCUUGAGUGUAACACA
`G
`154 UUAUCCUUGAGUGUAACA
`C
`156 UUGCCUUUAUCCUUGAGU
`G
`158 UGGCUUCUAAACAAAUUA
`C
`160 UCACAAAGGUGGCAAUAA
`C
`162 UAACCGAAUUCCUAGGCU
`G
`164 ACUAACCGAAUUCCUAGG
`C
`166 UACUAACCGAAUUCCUAG
`G
`168 UGAGUACUAACCGAAUUC
`C
`170 AUGAGUACUAACCGAAUU
`C
`172 AUACAAAUGAGUACUAAC
`C
`174 AGUGAAUACAPAUGAGUA
`C
`176 ACOGUGGCUAUCAUUUAC
`C
`178 UACOGOGGCUAUCAUOUA
`C
`180 UOAGGGAGCAAOACCGUG
`G
`182 UAUGACACCAGAACUUCC
`C
`184 AUAUCUAUGACACCAGAA
`
`- 124 -
`
`127
`
`129
`
`131
`
`133
`
`135
`
`137
`
`139
`
`141
`
`143
`
`145
`
`147
`
`149
`
`151
`
`153
`
`155
`
`157
`
`159
`
`161
`
`163
`
`165
`
`167
`
`169
`
`171
`
`173
`
`175
`
`177
`
`179
`
`181
`
`183
`
`185
`
`Rigel Exhibit 1021
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`
`PCT /US2010/027253
`
`u
`1934 GCUGUGCAUUAAACUUGC
`A
`1937 GUGCAUUAAACUUGCACA
`u
`1939 GCAUUAAACUUGCACAUG
`A
`1953 CAUGACUGGAACGAAGUA
`u
`1960 GGAACGAAGUAUGAGUGC
`A
`1961 GAACGAAGUAUGAGUGCA
`A
`1972 GAGUGCAACUCAAAUGUG
`u
`1976 GCAACUCAAAUGUGUUGA
`A
`1982 CAAAUGUGUUGAAGAUAC
`u
`1987 GUGUUGAAGAUACUGCAG
`u
`1989 GUUGAAGAUACUGCAGUC
`A
`2020 CCUUGCUGAAUGUUUCCA
`A
`2021 CUUGCUGAAUGUUUCCAA
`u
`2024 GCUGAAUGUUUCCAAUAG
`A
`2035 CCAAUAGACUAAAUACUG
`u
`2067 GAGUUUGGAAUCCGGAAU
`A
`2073 GGAAUCCGGAAUAAAUAC
`u
`2074 GAAUCCGGAAUAAAUACU
`A
`2080 GGAAUAAAUACUACCUGG
`A
`2133 GGCCUGGCCUGAAUAUUA
`u
`2134 GCCUGAAUAUUAUACUAC
`u
`2136 CUGGCCUGP.AUAUOAUAC
`u
`2166 CAUAOUUCAUCCAAGUGC
`A
`2180 GOGCAAUAAUGOAAGCUG
`A
`2182 GCAAOAAUGUAAGCUGAA
`u
`2272 CACUAUCUUAUCUUCUCC
`u
`2283 CUUCUCCUGAACUGUUGA
`u
`
`C
`186 UGCAAGUUUAAUGCACAG
`C
`188 AUGUGCAAGUUUAAUGCA
`C
`190 UCAUGUGCAAGUUUAAUG
`C
`192 AUACUUCGUUCCAGUCAU
`G
`194 UGCACUCAUACUUCGUUC
`C
`196 UUGCACUCAUACUUCGUU
`C
`198 ACACAUUUGAGUUGCACU
`C
`200 UUCAACACAUUUGAGUUG
`C
`202 AGUAUCUUCAACACAUUU
`G
`204 ACUGCAGUAUCUUCAACA
`C
`206 UGACUGCAGUAUCUUCAA
`C
`208 UUGGAAACAUUCAGCAAG
`G
`210 AUUGGAAACAUUCAGCAA
`G
`212 UCUAUUGGAAACAUUCAG
`C
`214 ACAGUAUUUAGUCUAUUG
`G
`216 UAUUCCGGAUUCCAAACU
`C
`218 AGUAUUUAUUCCGGAUUC
`C
`220 UAGUAUUUAUUCCGGAUU
`C
`222 UCCAGGUAGUAUUUAUUC
`C
`224 AUAAUAUUCAGGCCAGGC
`C
`226 AGOAGUAUAAUAUUCAGG
`C
`228 AGUAUAAUAUUCAGGCCA
`G
`230 UGCACUOGGAUGAAAOAO
`G
`232 UCAGCUUACAOUAOUGCA
`C
`234 AOUCAGCUUACAUUAUUG
`C
`236 AGGAGAAGAUAAGAUAGU
`G
`238 AUCAACAGUUCAGGAGAA
`G
`
`.187
`
`189
`
`191
`
`193
`
`195
`
`197
`
`199
`
`201
`
`203
`
`205
`
`207
`
`209
`
`211
`
`213
`
`215
`
`217
`
`219
`
`221
`
`223
`
`225
`
`227
`
`229
`
`231
`
`233
`
`235
`
`237
`
`239
`
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`

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`PCT /US2010/027253
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`· Table 8. siRNAs targeting wildtype IDHl
`SEQ
`ID
`NO:
`
`sense (5' to 3')
`
`Position
`on mRNA
`(FIG.
`218)
`611 AACCUAUCAUCAUAGGUC
`G
`612 ACCUAUCAUCAUAGGUCG
`u
`613 CCUAUCAUCAUAGGUCGU
`C
`614 CUAUCAUCAUAGGUCGUC
`A
`615 UAUCAUCAUAGGUCGUCA
`u
`616 AUCAUCAUAGGUCGUCAU
`G
`617 UCAUCAUAGGUCGUCAUG
`C
`618 CAUCAUAGGUCGOCAUGC
`u
`619 AOCAUAGGOCGUCAUGCU
`u
`620 UCAUAGGUCGUCAUGCOU
`A
`621 CAUAGGUCGUCAUGCUUA
`u
`622 AUAGGUCGOCAUGCUUAU
`G
`623 UAGGUCGUCAUGCUUAUG
`G
`624 AGGUCGUCAUGCOUAUGG
`G
`625 GGUCGUCAUGCUUAUGGG
`G
`626 GUCGUCAUGCUUAUGGGG
`A
`627 UCGUCAUGCUUAUGGGGA
`u
`
`antisense (5' to 3')
`
`240 CGACCUAUGAUGAUAGGU
`u
`242 ACGACCUAUGAUGAUAGG
`u
`244 GACGACCUAUGAUGAUAG
`G
`246 UGACGACCUAUGAUGAUA
`G
`248 AUGACGACCUAUGAUGAU
`A
`250 CAUGACGACCUAUGAUGA
`u
`252 GCAUGACGACCUAUGAUG
`A
`254 AGCAUGACGACCOAOGAU
`G
`256 AAGCAOGACGACCUAUGA
`u
`258 UAAGCAUGACGACCUAUG
`A
`260 AUAAGCAUGACGACCUAU
`G
`262 CAUAAGCAUGACGACCUA
`u
`264 CCAUAAGCAUGACGACCU
`A
`266 CCCAUAAGCAUGACGACC
`u
`268 CCCCAUAAGCAUGACGAC
`C
`270 UCCCAUAAGCAUGACGAC
`C
`272 AUCCCAUAAGCAUGACGA
`C
`
`SEQ
`ID
`NO:
`
`241
`
`243
`
`245
`
`247
`
`249
`
`251
`
`253
`
`255
`
`257
`
`259
`
`261
`
`263
`
`265
`
`267
`
`269
`
`271
`
`273
`
`Table 9. siRNAs targeting G395A mutant IDHl (SEO ID NO:5) (equivalent to
`
`G629A of SEO ID NO:9 (FIG. 21B))
`sense (5' to 3')
`Position
`on mRNA
`(FIG. 218)
`611 AACCUAUCAUCAUAGGUCA
`612 ACCUAUCAUCAUAGGUCAU
`613 CCUAOCAUCAOAGGUCAUC
`614 CUAUCAUCAUAGGUCAUCA
`615 UAUCAUCAUAGGUCAUCAO
`
`antisense (5' to 3')
`
`SEQ
`ID
`NO:
`274 UGACCUAUGAUGAUAGGUU
`276 AUGACCUAUGAUGAUAGGO
`278 GAUGACCUAOGAOGAUAGG
`280 UGAOGACCUAUGAUGAOAG
`282 AUGAUGACCUAUGAUGAUA
`
`SEQ
`ID
`NO:
`275
`277
`279
`281
`283
`
`- 126 -
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`616 AUCAUCAUAGGUCAUCAUG
`617 UCAUCAUAGGUCAUCAUGC
`618 CAUCAUAGGUCAUCAUGCU
`619 AUCAUAGGUCAUCAUGCUU
`620 UCAUAGGUCAUCAUGCUUA
`621 CAUAGGUCAUCAUGCUUAU
`622 AUAGGUCAUCAUGCUUAUG
`623 UAGGUCAUCAUGCUUAUGG
`624 AGGUCAUCAUGCUUAUGGG
`625 GGUCAUCAUGCUUAUGGGG
`626 GUCAUCAUGCUUAUGGGGA
`627 UCAUCAUGCUUAUGGGGAU
`
`284 CAUGAUGACCUAU.GAUGAU
`286 GCAUGAUGACCUAUGAUGA
`288 AGCAUGAUGACCUAUGAUG
`290 AAGCAUGAUGACCUAUGAU
`292 UAAGCAUGAUGACCUAUGA
`294 AUAAGCAUGAUGACCUAUG
`296 CAUAAGCAUGAUGACCUAU
`298 CCAUAAGCAUGAUGACCUA
`300 CCCAUAAGCAUGAUGACCU
`302 CCCCAUAAGCAUGAUGACC
`304 UCCCCAUAAGCAUGAUGAC
`306 AUCCCCAUAAGCAUGAUGA
`
`285
`287
`289
`291
`293
`295
`297
`299
`301
`303
`305
`307
`
`Table 10. siRNAs targeting C394A mutant IDHl (SEO ID N0:5) (equivalent to
`C628A of SEO ID N0:9 (FIG. 21B)) (Arg132Ser (SEO ID N0:8))
`SEQ
`antisense (5' to 3')
`SEQ
`sense (5' to 3')
`Position
`ID
`ID
`on mRNA
`NO:
`NO:
`(FIG. 21B)
`309
`308 CUACCUAUGAUGAUAGGUU
`611 AACCUAUCAUCAUAGGUAG
`311
`310 ACUACCUAUGAUGAUAGGU
`612 ACCUAUCAUCAUAGGUAGU
`313
`312 GACUACCUAUGAUGAUAGG
`613 CCUAUCAUCAUAGGUAGUC
`315
`314 UGACUACCUAUGAUGAUAG
`614 CUAUCAUCAUAGGUAGUCA
`317
`316 AUGACUACCUAUGAUGAUA
`615 UAUCAUCAUAGGUAGUCAU
`318 CAUGACUACCUAUGAUGAU
`319
`616 AUCAUCAUAGGUAGUCAUG
`321
`320 GCAUGACUACCUAUGAUGA
`617 UCAUCAUAGGUAGUCAUGC
`323
`322 AGCAUGACUACCUAUGAUG
`618 CAUCAUAGGUAGUCAUGCU
`325
`324 AAGCAUGACUACCUAUGAU
`619 AUCAUAGGUAGUCAUGCUU
`327
`326 UAAGCAUGACUACCUAUGA
`620 UCAUAGGUAGUCAUGCUUA
`329
`328 AUAAGCAUGACUACCUAUG
`621 CAUAGGUAGUCAUGCUUAU
`331
`330 CAUAAGCAUGACUACCUAU
`.AUAGGUAGUCAUGCUUAUG
`622
`333
`332 CCAUAAGCAUGACUACCUA
`623 UAGGUAGUCAUGCUUAUGG
`335
`334 CCCAUAAGCAUGACUACCU
`624 AGGUAGUCAUGCUUAUGGG
`337
`336 CCCCAUAAGCAUGACUACC
`625 GGUAGUCAUGCUUAUGGGG
`338 UCCCCAUAAGCAUGACUAC
`339
`626 GUAGUCAUGCUUAUGGGGA
`341
`340 AUCCCCAUAAGCAUGACUA
`627 UAGUCAUGCUUAUGGGGAU
`
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`

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`
`PCT /US2010/027253
`
`(SEO ID N0:5) (equivalent to
`Table 11. siRNAs targeting C394U mutant IDHl
`C628U of SEO ID N0:9 {FIG. 21B)) {Arg132Cys {SEOID.N0:8))
`· antisense (5' to 3')
`SEQ
`sense {5' to 3')
`Position
`ID
`on mRNA
`NO:
`(FIG. 218)
`342 CAACCUAUGAUGAUAGGUU
`611 AACCUAUCAUCAUAGGUUG
`344 ACAACCUAUGAUGAUAGGU
`612 ACCUAUCAUCAUAGGUUGU
`346 GACAACCUAUGAUGAUAGG
`613 CCUAUCAUCAUAGGUUGUC
`348 UGACAACCUAUGAUGAUAG
`614 CUAUCAUCAUAGGUUGUCA
`350 AUGACAACCUAUGAUGAUA
`615 UAUCAUCAUAGGUUGUCAU
`352 CAUGACAACCUAUGAUGAU
`616 AUCAUCAUAGGUUGUCAUG
`354 GCAUGACAACCUAUGAUGA
`617 UCAUCAUAGGUUGUCAUGC
`356 AGCAUGACAACCUAUGAUG
`618 CAUCAUAGGUUGUCAUGCU
`358 AAGCAUGACAACCUAUGAU
`619 AUCAUAGGUUGUCAUGCUO
`360 UAAGCAUGACAACCUAUGA
`620 UCAUAGGUUGUCAUGCUUA
`362 AUAAGCAUGACAACCUAOG
`621 CAUAGGUUGUCAUGCUUAU
`364 CAUAAGCAUGACAACCUAU
`622 AUAGGUUGUCAUGCUUAUG
`366 CCAUAAGCAUGACAACCUA
`623 UAGGUUGUCAUGCUUAUGG
`368 CCCAUAAGCAUGACAACCU
`624 AGGUUGUCAUGCUUAUGGG
`370 CCCCAUAAGCAUGACAACC
`625 GGUUGUCAOGCUUAUGGGG
`372 UCCCCAUAAGCAUGACAAC
`626 GUUGUCAUGCUUAUGGGGA
`374 AUCCCCAUAAGCAUGACAA
`627 UUGUCAUGCUUAUGGGGAU
`
`SEQ
`ID
`NO:
`343
`345
`347
`349
`351
`353
`355
`357
`359
`361
`363
`365
`367
`369
`371
`373
`375
`
`SEQ
`ID
`NO:
`
`Table 12. siRNAs targeting C394G mutant IDHl {SEO ID N0:5) (equivalent to
`C628G of SEO ID N0:9 (FIG. 21B)) (Arg132Gly (SEO ID N0:8))
`antisense {5' to 3')
`SEQ
`sense (5' to 3')
`Position
`ID
`on mRNA
`NO:
`(FIG.
`218)
`611 AACCUAUCAUCAUAGGUG
`G
`612 ACCUAUCAUCAUAGGUGG
`u
`613 CCUAUCAUCAUAGGUGGU
`C
`614 CUAUCAUCAUAGGUGGUC
`A
`615 UAUCAOCAUAGGUGGUCA
`u
`616 AUCAUCAUAGGUGGUCAU
`G
`617 UCAUCAUAGGUGGUCAUG
`C
`618 CAUCAUAGGUGGUCAUGC
`u
`619 AUCAOAGGUGGUCAUGCU
`
`376 CCACCUAUGAUGAUAGGU
`u
`378 ACCACCUAUGAUGAUAGG
`u
`380 GACCACCUAUGAOGAUAG
`G
`382 UGACCACCUAUGAUGAUA
`G
`384 AUGACCACCUAUGAUGAU
`A
`386 CAUGACCACCUAUGAUGA
`u
`388 GCAUGACCACCUAUGAUG
`A
`390 AGCAUGACCACCUAUGAU
`G
`392 AAGCAUGACCACCUAUGA
`
`- 128 -
`
`377
`
`379
`
`381
`
`383
`
`385
`
`387
`
`389
`
`391
`
`393
`
`Rigel Exhibit 1021
`Page 467 of 582
`
`

`

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`
`PCT /US2010/027253
`
`u
`620 UCAUAGGUGGUCAUGCUU
`A
`621 CAUAGGUGGUCAUGCUUA
`u
`622 AUAGGUGGUCAUGCUUAU
`G
`623 UAGGUGGUCAUGCUUAUG
`G
`624 AGGUUGUCAUGCUUAUGG
`G
`625 GGUUGUCAUGCUUAUGGG
`G
`626 GUUGUCAUGCUUAUGGGG
`A
`627 UUGUCAUGCUUAUGGGGA
`u
`
`u
`394 UAAGCAUGACCACCUAUG
`A
`396 AUAAGCAUGACCACCUAU
`G
`398 CAUAAGCAUGACCACCUA
`u
`400 CCAUAAGCAUGACCACCU
`A
`402 CCCAUAAGCAUGACCACC
`u
`404 CCCCAUAAGCAUGACCAC
`C
`406 UCCCCAUAAGCAUGACCA
`C
`408 AUCCCCAUAAGCAUGACC
`A
`
`395
`
`397
`
`399
`
`401
`
`403
`
`405
`
`407
`
`409
`
`Table 13. siRNAs targeting G395C mutant IDHl (SEO ID N0:5) (equivalent to
`G629C of SEO ID N0:9 (FIG. 21B)) (Arg132Pro (SEO ID N0:8))
`antisense (5' to 3')
`SEQ
`sense (5' to 3')
`Position
`ID
`on mRNA
`NO:
`(FIG.
`218)
`611 AACCUAUCAUCAUAGGUC
`G
`612 ACCUAUCAUCAUAGGUCG
`u
`613 CCUAUCAUCAUAGGUCGU .
`C
`614 CUAUCAUCAUAGGUCGUC
`A
`615 UAUCAUCAUAGGUCGUCA
`u
`616 AUCAUCAUAGGUCGUCAU
`G
`617 UCAUCAUAGGUCGUCAUG
`C
`618 CAUCAUAGGUCGUCAUGC
`u
`619 AUCAUAGGUCGUCAUGCU
`u
`620 UCAUAGGUCGUCAUGCUU
`A
`621 CAUAGGUCGUCAUGCUUA
`u
`622 AUAGGUCGUCAUGCUUAU
`G
`623 UAGGUCGUCAUGCUUAUG
`G
`624 AGGUCGUCAUGCUUAUGG
`G
`625 GGUCGUCAUGCUUAUGGG
`G
`
`410 CGACCUAUGAUGAUAGGU
`u
`412 ACGACCUAUGAUGAUAGG
`u
`414 GACGACCUAUGAUGAUAG
`G
`416 UGACGACCUAUGAUGAUA
`G
`418 AUGACGACCUAUGAUGAU
`A
`42Q CAUGACGACCUAUGAUGA
`u
`422 GCAUGACGACCUAUGAUG
`A
`424 AGCAUGACGACCUAUGAU
`G
`426 AAGCAUGACGACCUAUGA
`u
`428 UAAGCAUGACGACCUAUG
`A
`430 AUAAGCAUGACGACCUAU
`G
`432 CAUAAGCAUGACGACCUA
`u
`434 CCAUAAGCAUGACGACCU
`A
`436 CCCAUAAGCAUGACGACC
`u
`438 CCCCAUAAGCAUGACGAC
`C
`
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`SEQ
`ID
`NO:
`
`411
`
`413
`
`415
`
`417
`
`419
`
`421
`
`423
`
`425
`
`427
`
`429
`
`431
`
`433
`
`435
`
`437
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`439
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`626 GUCGUCAUGCUUAUGGGG
`A
`627 UCGUCAUGCUUAUGGGGA
`u
`
`440 UCCCCAUAAGCAOGACGA
`C
`442 AUCCCCAOAAGCAUGACG
`A
`
`441
`
`443
`
`Table 14. siRNAs targeting G395U mutant IDHl (SEO ID NO:5) (equivalent to
`G629U of SEO ID NO:9 (FIG. 21B)) (Arg132Leu (SEO ID NO:8))
`antisense (5' to 3')
`sense (5' to 3')
`SEQ
`Position
`ID
`on mRNA
`NO:
`(FIG.
`21B)
`611 AACCUAUCAUCAUAGGUC
`u
`612 ACCUAUCAUCAUAGGUCU
`u
`613 CCUAUCAUCAUAGGUCUU
`C
`614 CUAUCAUCAUAGGUCUUC
`A
`615 UAUCAUCAUAGGUCUUCA
`0
`616 AUCAUCAUAGGUCUUCAU
`G
`617 UCAUCAUAGGUCUUCAUG
`C
`618 CAUCAUAGGUCUUCAUGC
`u
`619 AUCAUAGGUCUUCAUGCU
`u
`620 UCAUAGGUCUUCAUGCUU
`A
`621 CAUAGGUCUUCAUGCUUA
`u
`622 AUAGGOCUUCAUGCUUAU
`G
`623 UAGGUCUUCAUGCUUAUG
`G
`624 AGGUCUUCAUGCUUAUGG
`G
`625 GGUCOUCAUGCUUAOGGG
`G
`626 GUCUOCAUGCUUAUGGGG
`A
`
`444 AGACCUAUGAUGAUAGGU
`u
`446 AAGACCUAUGAUGAUAGG
`u
`448 GAAGACCUAUGAUGAUAG
`G
`450 UGAAGACCUAUGAUGAUA
`G
`452 AUGAAGACCUAOGAUGAU
`A
`454 CAUGAAGACCUAUGAUGA
`u
`456 GCAUGAAGACCUAUGAUG
`A
`458 AGCAUGAAGACCUAUGAU
`G
`4 60 AAGCAUGAAGACCUAUGA
`u
`462 UAAGCAUGAAGACCUAUG
`A
`464 AUAAGCAUGAAGACCUAU
`G
`4 66 CAOAAGCAUGAAGACCUA
`u
`4 68 CCAUAAGCAUGAAGACCU
`A
`470 CCCAUAAGCAUGAAGACC
`0
`472 CCCCAUAAGCAUGPAGAC
`C
`474 UCCCCAUAAGCAUGPAGA
`C
`
`- 130 -
`
`SEQ
`ID
`NO:
`
`445
`
`447
`
`449
`
`451
`
`453
`
`455
`
`457
`
`459
`
`461
`
`463
`
`465
`
`4 67
`
`469
`
`471
`
`473
`
`475
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`627 UCUUCAUGCUUAUGGGGA
`u
`
`476 AUCCCCAUAAGCAUGAAG
`A
`
`477
`
`IDH2
`
`Exemplary siRNAs are presented in the following tables. Art-known methods
`can be used to select other siRNAs. siRNAs can be evaluated, e.g., by determining
`the ability of an siRNA to silence an e.g., IDH2, e.g., in an in vitro system, e.g., in
`cultured cells, e.g., HeLa cells or cultured glioma cells. e.g.,
`The siRNAs in Table 15 were generated using the siRNA selection tool
`available on the worldwide web at jura.wi.mit.edu/bioc/siRNAext/. (Yuan et al. Nucl.
`Acids. Res. 2004 32:Wl30-Wl34.) Other selection tools can be used as well. Entry
`1356 was adapted from Silencing of cytosolic NADP+ dependent isoccitrate
`dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells
`toward stauropine, Lee et al., 2009, Free Radical Research, 43: 165-173.
`
`The siRNAs in Tables 16-23 represent candidates spanning the IDH2 mRNA
`at nucleotide positions 600, 601, and 602 according to the rnRNA sequence presented
`at GenBank Accession No. NM 002168.2 (Record dated August 16, 2009;
`0128178831) (SEQ ID NO12, FIG. 22B; equivalent to nucleotide positions 514, 515,
`and 516 of the cDNA sequence represented by SEQ ID NO:11, FIG. Fig. 22A).
`
`The RNAs in the tables can be modified, e.g., as described herein.
`Modifications include chemical modifications to enhance properties, e.g., resistance
`to degradation, or the use of overhangs. For example, either one or both of the sense
`and antisense strands in the tables can include an additional dinucleotide at the 3' end,
`e.g., TT, UU, dTdT.
`
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`Table 15. siRNAs targeting wildtype IDH2
`sense
`(5' to 3')
`
`Position
`on mRNA
`(FIG.
`22B)
`
`SEQ ID
`NO:
`
`antisense
`(5' tO 3 I)
`
`SEQ ID
`NO:
`
`250 GUGAUGAGAUGACCCGUAU
`252 GAUGAGAUGACCCGUAUUA
`264 CGUAUUAUCUGGCAGUUCA
`274 GGCAGUUCAUCAAGGAGAA
`451 GUGUGGAAGAGUUCAAGCU
`453 GUGGAAGAGUUCAAGCUGA
`456 GAAGAGUUCAAGCUGAAGA
`795 CAGUAUGCCAUCCAGAAGA
`822 CUGUACAUGAGCACCAAGA
`832 GCACCAAGAACACCAUACU
`844 CCAUACUGAAAGCCUACGA
`845 CAUACUGAAAGCCUACGAU
`868 GUUUCAAGGACAUCUUCCA
`913 CCGACUUCGACAAGAAUAA
`915 GACUUCGACAAGAAUAAGA
`921 GACAAGAAUAAGAUCUGGU
`949 GGCUCAUUGAUGACAUGGU
`1009 GCAAGAACUAUGACGGAGA
`1010 CAAGAACUAUGACGGAGAU
`1024 GAGAUGUGCAGUCAGACAU
`1096 CUGAUGGGAAGACGAUUGA
`1354 GCAAUGUGAAGCUGAACGA
`1668 CUGUAAUUUAUAUUGCCCU
`1694 CAUGGUGCCAUAUUUAGCU
`1697 GGUGCCAUAUUUAGCUACU
`1698 GUGCCAUAUUUAGCUACUA
`1700 GCCAUAUUUAGCUACUAAA
`
`478 AUACGGGUCAUCUCAUCAC
`480 UAAUACGGGUCAUCUCAUC
`482 UGAACUGCCAGAUAAUACG
`484 UUCUCCUUGAUGAACUGCC
`486 AGCUUGAACUCUUCCACAC
`488 UCAGCUUGAACUCUUCCAC
`490 UCUUCAGCUUGAACUCUUC
`492 UCUUCUGGAUGGCAUACUG
`4 94 UCUUGGUGCUCAUGUACAG
`496 AGUAUGGUGUUCUUGGUGC
`498 UCGUAGGCUUUCAGUAUGG
`500 AUCGUAGGCUUUCAGUAUG
`502 UGGAAGAUGUCCUUGAAAC
`504 UUAUUCUUGUCGAAGUCGG
`506 UCUUAUUCUUGUCGAAGUC
`508 ACCAGAUCUUAUUCUUGUC
`510 ACCAUGUCAUCAAUGAGCC
`512 UCUCCGUCAUAGUUCUUGC
`514 AUCUCCGUCAUAGUUCUUG
`516 AUGOCUGACUGCACAUCUC
`518 UCAAUCGUCUUCCCAUCAG
`520 UCGUUCAGCUUCACAUUGC
`522 AGGGCAAUAUAAAUUACAG
`524 AGCUAAAUAUGGCACCAUG
`526 AGUAGCUAAAUAUGGCACC
`528 UAGUAGCUAAAUAUGGCAC
`530 UUUAGUAGCUA.AAUAUGGC
`
`479
`481
`483
`485
`487
`489
`491
`4 93
`4 95
`497
`499
`501
`503
`505
`507
`509
`511
`513
`515
`517
`519
`521
`523
`525
`527
`529
`531
`
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`anti sense
`tO 3 I)
`(5 I
`
`SEQ ID
`NO:
`
`Table 16. siRNAs targeting wildtype IDH2
`SEQ ID
`sense
`Position
`{5 J to. 3 I)
`NO:
`on m.RNA
`(FIG.
`22B)
`584 GCCCAUCACCAUUGGCAGG
`585 CCCAUCACCAUUGGCAGGC
`58 6 CCAUCACCAUUGGCAGGCA
`587 CAUCACCAUUGGCAGGCAC
`588 AUCACCAUUGGCAGGCACG
`589 UCACCAUUGGCAGGCACGC
`590 CACCAUUGGCAGGCACGCC
`591 ACCAUUGGCAGGCACGCCC
`592 CCAUUGGCAGGCACGCCCA
`593 CAUUGGCAGGCACGCCCAU
`594 AUUGGCAGGCACGCCCAUG
`595 UUGGCAGGCACGCCCAUGG
`596 UGGCAGGCACGCCCAUGGC
`597 GGCAGGCACGCCCAUGGCG
`598 GCAGGCACGCCCAUGGCGA
`599 CAGGCACGCCCAUGGCGAC
`600 AGGCACGCCCAUGGCGACC
`
`532 CCUGCCAAUGGUGAUGGGC
`534 GCCUGCCAAUGGUGAUGGG
`536 UGCCUGCCAAUGGUGAUGG
`538 GUGCCUGCCAAUGGUGAUG
`540 CGUGCCUGCCAAUGGUGAU
`542 GCGUGCCUGCCAAUGGUGA
`544 GGCGUGCCUGCCAAUGGUG
`546 GGGCGUGCCUGCCAAUGGU
`548 UGGGCGUGCCUGCCAAUGG
`550 AUGGGCGUGCCUGCCAAUG
`552 CAUGGGCGUGCCUGCCAAU
`554 CCAUGGGCGUGCCUGCCAA
`556 GCCAUGGGCGUGCCUGCCA
`558 CGCCAUGGGCGUGCCUGCC
`560 UCGCCAUGGGCGUGCCUGC
`562 GUCGCCAUGGGCGUGCCUG
`564 GGUCGCCAUGGGCGOGCCU
`
`533
`535
`537
`539
`541
`543
`545
`547
`549
`551
`553
`555
`557
`559
`561
`563
`565
`
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`Table 17. siRNAs targeting A514G mutant IDH2 (equivalent to A600G of SEO ID
`
`N0:12, (FIG. 22B)
`
`Position
`on mRNA
`(FIG.
`22B)
`
`sense
`tO 3 I )
`(5 I
`
`SEQ ID
`NO:
`
`antisense
`(5 I to 3')
`
`SEQ ID
`NO:
`
`584 GCCCAUCACCAUUGGCGGG
`585 CCCAUCACCAUUGGCGGGC
`586 CCAUCACCAUUGGCGGGCA
`587 CAUCACCAUUGGCGGGCAC
`588 AUCACCAUUGGCGGGCACG
`589 UCACCAUUGGCGGGCACGC
`590 CACCAUUGGCGGGCACGCC
`591 ACCAUUGGCGGGCACGCCC
`592 CCAUUGGCGGGCACGCCCA
`593 CAUUGGCGGGCACGCCCAU
`594 AUUGGCGGGCACGCCCAUG
`595 UUGGCGGGCACGCCCAUGG
`596 UGGCGGGCACGCCCAUGGC
`597 GGCGGGCACGCCCAUGGCG
`598 GCGGGCACGCCCAUGGCGA
`599 CGGGCACGCCCAUGGCGAC
`600 GGGCACGCCCAUGGCGACC
`
`566 CCCGCCAAUGGUGAUGGGC
`568 GCCCGCCAAUGGUGAUGGG
`570 UGCCCGCCAAUGGUGAUGG
`572 GOGCCCGCCAAUGGUGAUG
`574 CGUGCCCGCCAAUGGUGAU
`576 GCGUGCCCGCCAAUGGUGA
`578 GGCGUGCCCGCCAAUGGUG
`580 GGGCGUGCCCGCCAAUGGU
`582 UGGGCGUGCCCGCCAAUGG
`584 AUGGGCGUGCCCGCCAAUG
`586 CAUGGGCGUGCCCGCCAAU
`588 CCAUGGGCGUGCCCGCCAA
`590 GCCAUGGGCGUGCCCGCCA
`592 CGCCAUGGGCGUGCCCGCC
`594 UCGCCAUGGGCGUGCCCGC
`596 GUCGCCAUGGGCGUGCCCG
`598 GGUCGCCAUGGGCGUGCCC
`
`567
`569
`571
`573
`575
`577
`579
`581
`583
`585
`587
`589
`591
`593
`595
`597
`599
`
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`Table 18. siRNAs targeting A514U mutant IDH2 (equivalent to A600U of SEO ID
`N0:12, (FIG. 22B)
`
`sense
`(5' tO 3 I)
`
`Position
`on mRNA
`(FIG.
`22B)
`584 GCCCAUCACCAUUGGCUGG
`585 CCCAUCACCAUUGGCUGGC
`586 CCAUCACCAUUGGCUGGCA
`587 CAUCACCAUUGGCUGGCAC
`588 AUCACCAUUGGCUGGCACG
`589 UCACCAUUGGCUGGCACGC
`590 CACCAUUGGCUGGCACGCC
`591 ACCAUUGGCUGGCACGCCC
`592 CCAUUGGCUGGCACGCCCA
`593 CAUUGGCUGGCACGCCCAU
`594 AUUGGCUGGCACGCCCAUG
`595 UUGGCUGGCACGCCCAUGG
`596 UGGCUGGCACGCCCAUGGC
`597 GGCUGGCACGCCCAUGGCG
`598 GCUGGCACGCCCAUGGCGA
`599 CUGGCACGCCCAUGGCGAC
`600 UGGCACGCCCAUGGCGACC
`
`SEQ ID
`NO:
`
`anti sense
`(5' to 3')
`
`SEQ ID
`NO:
`
`600 CCAGCCAAUGGUGAUGGGC
`602 GCCAGCCAAUGGUGAUGGG
`604 UGCCAGCCAAUGGUGAUGG
`606 GUGCCAGCCAAUGGUGAUG
`608 CGUGCCAGCCAAUGGUGAU
`610 GCGUGCCAGCCAAUGGUGA
`612 GGCGUGCCAGCCAAUGGUG
`614 GGGCGUGCCAGCCAAUGGU
`616 UGGGCGUGCCAGCCAAUGG
`618 AUGGGCGUGCCAGCCAAUG
`620 CAUGGGCGUGCCAGCCAAU
`622 CCAUGGGCGUGCCAGCCAA
`624 GCCAUGGGCGUGCCAGCCA
`626 CGCCAUGGGCGUGCCAGCC
`628 UCGCCAUGGGCGUGCCAGC
`630 GUCGCCAUGGGCGUGCCAG
`632 GGUCGCCAUGGGCGUGCCA
`
`601
`603
`605
`607
`609
`611
`613
`615
`617
`619
`621
`623
`625
`627
`629
`631
`633
`
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`Table 19. siRNAs targeting G515A mutant IDH2 (equivalent to G601A of SEO ID
`
`N0:12, (FIG. 22B)
`
`sense
`(5' to 3')
`
`Position
`on mRNA
`(FIG.
`22B)
`584 GCCCAUCACCAUUGGCAAG
`585 CCCAUCACCAUUGGCAAGC
`586 CCAUCACCAUUGGCAAGCA
`587 CAUCACCAUUGGCAAGCAC
`588 AUCACCAUUGGCAAGCACG
`589 UCACCAUUGGCAAGCACGC
`590 CACCAUUGGCAAGCACGCC
`591 ACCAUUGGCAAGCACGCCC
`592 CCAUUGGCAAGCACGCCCA
`593 CAUUGGCAAGCACGCCCAU
`594 AUUGGCAAGCACGCCCAUG
`595 UUGGCAAGCACGCCCAUGG
`596 UGGCAAGCACGCCCAUGGC
`597 GGCAAGCACGCCCAUGGCG
`598 GCAAGCACGCCCAUGGCGA
`599 CAAGCACGCCCAUGGCGAC
`600 AAGCACGCCCAUGGCGACC
`
`SEQ ID
`NO:
`
`anti sense
`(5' to 3')
`
`SEQ ID
`NO:
`
`634 CUUGCCAAUGGUGAUGGGC
`636 GCUUGCCAAUGGUGAUGGG
`638 UGCUUGCCAAUGGUGAUGG
`640 GUGCUUGCCAAUGGUGAUG
`642 CGUGCUUGCCAAUGGUGAU
`644 GCGUGCUUGCCAAUGGUGA
`646 GGCGUGCUUGCCAAUGGUG
`648 GGGCGUGCUUGCCAAUGGU
`650 UGGGCGUGCUUGCCAAUGG
`652 AUGGGCGUGCUUGCCAAUG
`654 CAUGGGCGUGCUUGCCAAU
`656 CCAUGGGCGUGCUUGCCAA
`658 GCCAUGGGCGUGCUUGCCA
`660 CGCCAUGGGCGUGCUUGCC
`662 UCGCCAUGGGCGUGCUUGC
`664 GUCGCCAUGGGCGUGCUUG
`666 GGUCGCCAUGGGCGUGCUU
`
`635
`637
`639
`641
`643
`645
`647
`649
`651
`653
`655
`657
`659
`661
`663
`665
`667
`
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`Table 20. siRNAs targeting G515C mutant IDH2 (equivalent to G601C of SEO ID
`N0:12, (FIG. 22B)
`
`)
`
`sense
`(5' to 3 1
`
`Position
`on mRNA
`(FIG.
`22B)
`584 GCCCAUCACCAUUGGCACG
`585 CCCAUCACCAUUGGCACGC
`586 CCAUCACCAUUGGCACGCA
`587 CAUCACCAUUGGCACGCAC
`588 AUCACCAUUGGCACGCACG
`589 UCACCAUUGGCACGCACGC
`590 CACCAUUGGCACGCACGCC
`591 ACCAUUGGCACGCACGCCC
`592 CCAUUGGCACGCACGCCCA
`593 CAUUGGCACGCACGCCCAU
`594 AUUGGCACGCACGCCCAUG
`595 UUGGCACGCACGCCCAUGG
`596 UGGCACGCACGCCCAUGGC
`597 GGCACGCACGCCCAUGGCG
`598 GCACGCACGCCCAUGGCGA
`599 CACGCACGCCCAUGGCGAC
`600 ACGCACGCCCAUGGCGACC
`
`SEQ ID
`NO:
`
`anti sense
`(5' to 3')
`
`SEQ ID
`NO:
`
`668 CGUGCCAAUGGUGAUGGGC
`670 GCGUGCCAAUGGUGAUGGG
`672 UGCGUGCCAAUGGUGAUGG
`674 GUGCGUGCCAAUGGUGAUG
`676 CGUGCGUGCCAAUGGUGAU
`678 GCGUGCGUGCCAAUGGUGA
`680 GGCGUGCGUGCCAAUGGUG
`682 GGGCGUGCGUGCCAAUGGU
`684 UGGGCGUGCGUGCCAAUGG
`686 AUGGGCGUGCGUGCCAAUG
`688 CAUGGGCGUGCGUGCCAAU
`690 CCAUGGGCGUGCGUGCCAA
`692 GCCAUGGGCGUGCGUGCCA
`694 CGCCAUGGGCGUGCGUGCC
`696 UCGCCAUGGGCGOGCGUGC
`698 GUCGCCAOGGGCGUGCGUG
`700 GGUCGCCAUGGGCGUGCGU
`
`669
`671
`673
`675
`677
`67 9
`681
`683
`685
`687
`689
`691
`693
`695
`697
`
`699
`701
`
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`Table 21. siRNAs targeting G515U mutant IDH2 (equivalent to G601U of SEO ID
`N0:12, (FIG. 22B)
`
`sense
`(5' to 3 •)
`
`Position
`on mRNA
`(FIG.
`22B)
`584 GCCCAUCACCAUUGGCAUG
`585 CCCAUCACCAUUGGCAUGC
`586 CCAUCACCAUUGGCAUGCA
`587 CAUCACCAUUGGCAUGCAC
`588 AUCACCAUUGGCAUGCACG
`589 UCACCAUUGGCAUGCACGC
`590 CACCAUUGGCAUGCACGCC
`591 ACCAUUGGCAUGCACGCCC
`592 CCAUUGGCAUGCACGCCCA
`593 CAUUGGCAUGCACGCCCAU
`594 AUUGGCAUGCACGCCCAUG
`595 UUGGCAUGCACGCCCAUGG
`596 UGGCAUGCACGCCCAUGGC
`597 GGCAUGCACGCCCAUGGCG
`598 GCAUGCACGCCCAUGGCGA
`599 CAUGCACGCCCAUGGCGAC
`600 AUGCACGCCCAUGGCGACC
`
`SEQ ID
`NO:
`
`anti sense
`(5' to 3')
`
`SEQ ID
`NO:
`
`702 CAUGCCAAUGGUGAUGGGC
`704 GCAUGCCAAUGGUGAUGGG
`706 UGCAUGCCAAUGGUGAUGG
`708 GUGCAUGCCAAUGGUGAUG
`710 CGUGCAUGCCAAUGGUGAU
`712 GCGUGCAUGCCAAUGGUGA
`714 GGCGUGCAUGCCAAUGGUG
`716 GGGCGUGCAUGCCAAUGGU
`718 UGGGCGUGCAUGCCAAUGG
`720 AUGGGCGUGCAUGCCAAUG
`722 CAUGGGCGUGCAUGCCAAU
`724 CCAUGGGCGUGCAUGCCAA
`726 GCCAUGGGCGUGCAUGCCA
`728 CGCCAUGGGCGUGCAUGCC
`730 UCGCCAUGGGCGUGCAUGC
`732 GUCGCCAUGGGCGUGCAUG
`734 GGUCGCCAUGGGCGUGCAU
`
`703
`705
`707
`709
`711
`713
`715
`717
`719
`721
`723
`725
`727
`729
`731
`733
`735
`
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`Table 22. siRNAs targeting G516C mutant IDH2 (equivalent to G602C of SEO ID
`N0:12. (FIG. 22B)
`
`sense
`(5 I to 3 I)
`
`Position
`on mRNA
`(FIG.
`22B)
`584 GCCCAOCACCAUUGGCAGC
`585 CCCAUCACCAOUGGCAGCC
`586 CCAUCACCAUUGGCAGCCA
`587 CAUCACCAUOGGCAGCCAC
`588 AUCACCAUUGGCAGCCACG
`589 UCACCAUUGGCAGCCACGC
`590 CACCAUUGGCAGCCACGCC
`591 ACCAUUGGCAGCCACGCCC
`592 CCAUUGGCAGCCACGCCCA
`593 CAUUGGCAGCCACGCCCAU
`594 AUUGGCAGCCACGCCCAUG
`595 UUGGCAGCCACGCCCAUGG
`596 UGGCAGCCACGCCCAUGGC
`597 GGCAGCCACGCCCAUGGCG
`598 GCAGCCACGCCCAUGGCGA
`599 CAGCCACGCCCAUGGCGAC
`600 AGCCACGCCCAUGGCGACC
`
`SEQ ID
`NO:
`
`anti sense
`(5 I to 3 I)
`
`SEQ ID
`NO:
`
`736 GCUGCCAAOGGUGAUGGGC
`738 GGCUGCCAAUGGUGAUGGG
`740 OGGCUGCCAAUGGOGAUGG
`742 GUGGCUGCCAAUGGUGAUG
`744 CGUGGCOGCCAAUGGUGAU
`746 GCGUGGCUGCCAAUGGUGA
`748 GGCGUGGCUGCCAAUGGUG
`750 GGGCGUGGCUGCCAAUGGU
`752 UGGGCGUGGCUGCCAAUGG
`754 AUGGGCGUGGCUGCCAAUG
`756 CAUGGGCGUGGCUGCCAAU
`758 CCAUGGGCGUGGCUGCCAA
`760 GCCAUGGGCGUGGCUGCCA
`762 CGCCAUGGGCGUGGCUGCC
`764 UCGCCAUGGGCGUGGCUGC
`766 GOCGCCAUGGGCGUGGCUG
`7 68 GGUCGCCAUGGGCGUGGCU
`
`737
`739
`741
`743
`745
`747
`749
`751
`753
`755
`757
`759
`761
`763
`765
`767
`769
`
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`Table 23. siRNAs targeting G516U mutant IDH2 (equivalent to G602U of SEO ID
`
`NO:12, (FIG. 22B)
`
`sense
`(5' to 3')
`
`Position
`on mRNA
`(FIG.
`22B)
`584 GCCCAUCACCAUUGGCAGU
`585 CCCAUCACCAUUGGCAGUC
`586 CCAUCACCAUUGGCAGUCA
`587 CAUCACCAUUGGCAGUCAC
`588 AUCACCAUUGGCAGUCACG
`589 UCACCAUUGGCAGUCACGC
`590 CACCAUUGGCAGOCACGCC
`591 ACCAUUGGCAGUCACGCCC
`592 CCAOUGGCAGUCACGCCCA
`593 CAUUGGCAGUCACGCCCAU
`594 AUUGGCAGUCACGCCCAUG
`595 OUGGCAGUCACGCCCAUGG
`596 UGGCAGUCACGCCCAUGGC
`597 GGCAGUCACGCCCAOGGCG
`598 GCAGUCACGCCCAOGGCGA
`599 CAGUCACGCCCAUGGCGAC
`600 AGUCACGCCCAUGGCGACC
`
`SEQ ID
`NO:
`
`antisense
`(5' to 3')
`
`SEQ ID
`NO:
`
`770 ACUGCCAAUGGUGAUGGGC
`772 GACUGCCAAUGGUGAUGGG
`774 UGACUGCCAAUGGUGAUGG
`776 GUGACUGCCAF,UGGUGAUG
`778 CGUGACUGCCAAUGGUGAU
`780 GCGUGACUGCCAAUGGUGA
`782 GGCGUGACUGCCAAUGGUG
`784 GGGCGUGACUGCCAAUGGU
`786 UGGGCGUGACUGCCAAUGG
`788 AUGGGCGUGACUGCCAAUG
`790 CAUGGGCGUGACOGCCA_qU
`792 CCAUGGGCGUGACUGCCAA
`794 GCCAOGGGCGUGACUGCCA
`796 CGCCAUGGGCGUGACOGCC
`798 UCGCCAUGGGCGUGACOGC
`800 GUCGCCAUGGGCGUGACUG
`802 GGUCGCCAUGGGCGUGACU
`
`771
`773
`775
`777
`779
`781
`783
`785
`787
`789
`791
`793
`795
`797
`799
`801
`803
`
`EXAMPLE 6 STRUCTURAL ANALYSIS OF R132H MUTANT IDHl
`To define how R132 mutations alter the enzymatic properties ofIDHl, the
`crystal structure of R132H mutant IDHl bound to aKG, NADPH, and Ca2
`solved at 2.1 A resolution.
`The overall quaternary structure of the homodimeric R132H mutant enzyme
`adopts the same closed catalytically competent conformation (shown as a monomer in
`
`+ was
`
`FIG. 29A) that has been previously described for the wild-type enzyme (Xu, X. et al.
`
`J Biol Chem 279, 33946-57 (2004)). NADPH is positioned as expected for hydride
`
`transfer to aKG in an orientation that would produce R(-)-2HG, consistent with our
`
`chiral determination of the 2HG product.
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`Two important features were noted by the change ofR132 to histidine: the
`effect on catalytic conformation equilibrium and the reorganization of the active-site.
`Locating atop a f3-sheet in the relatively rigid small domain, R132 acts as a gate(cid:173)
`keeper residue and appears to orchestrate the hinge movement between the open and
`closed conformations. The guanidinium moiety ofR132 swings from the open to the
`closed conformation with a distance of nearly 8 A. Substitution of histidine for
`arginine is likely to change the equilibrium in favor of the closed conformation that
`forms the catalytic cleft for cofactor and substrate to bind efficiently, which partly
`explains the high-affinity for NADPH exhibited by the R132H mutant enzyme. This
`feature may be advantageous for the NADPH-dependent reduction of aKG to R(-)-
`2HG in an environment where NADPH concentrations are low. Secondly, closer
`examination of the catalytic pocket of the mutant IDHl structure in comparison to the
`wild-type enzyme showed not only the expected loss of key salt-bridge interactions
`between the guanidinium ofR132 and the a/[3 carboxylates of isocitrate, as well as
`changes in the network that coordinates the metal ion, but also an unexpected
`reorganization of the active-site. Mutation to histidine resulted in a significant shift
`in position of the highly conserved residues Y139 from the A subunit and K212' from
`the B subunit (FIG. 29B), both of which are thought to be critical for catalysis of this
`In
`enzyme family (Aktas, D. F. & Cook, P. F. Biochemistry 48, 3565-77 (2009)).
`particular, the hydroxyl moiety ofY139 now occupies the space of the ~-carboxylate
`of isocitrate. In addition, a significant repositioning of aKG compared to isocitrate
`where the distal carboxylate of aKG now points upward to make new contacts with
`N96 and S94 was observed. Overall, this single R132 mutation results in formation of
`a distinct active site compared to wild-type IDH 1.
`
`EXAMPLE 7 MATERIALS AND METHODS
`
`Summary
`R132H, Rl32C, R132L and R132S mutations were introduced into human
`IDHl by standard molecular biology techniques. 293T and the human glioblastoma
`cell lines U87MG and LN-18 were cultured in DMEM, 10% fetal bovine serum.
`Cells were transfected and selected using standard techniques. Protein expression
`levels were determined by vVestem blot analysis using IDHc antibody (Santa Cruz
`Biotechnology), IDHl antibody (proteintech), MYC tag antibody (Cell Signaling
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`Technology), and IDH2 antibody (Abeam). Metabolites were extracted from cultured
`cells and from tissue samples according to close variants of a previously reported
`method (Lu, W., Kimball, E. & Rabinowitz, J. D. J Am Soc Mass Spectrom 17, 37-50
`(2006)), using 80% aqueous methanol (-80 °C) and either tissue scraping or
`homogenization to disrupt cells. Enzymatic activity in cell lysates was assessed by
`following a change in NADPH fluorescence over time in the presence of isocitrate
`and NADP, or aKG and NADPH. For enzyme assays using recombinant IDHl
`enzyme, proteins were produced in E. coli and purified using Ni affinity
`chromatography followed by Sephacryl S-200 size-exclusion chromatography.
`Enzymatic activity for recombinant IDHl protein was assessed by following a change
`in NADPH UV absorbance at 340 nm using a stop-flow spectrophotometer in the
`presence ofisocitrate and NADP or aKG and NADPH. Chirality of2HG was
`determined as described previously (Struys, E. A., Jansen, E. E., Verhoeven, N. M. &
`Jakobs, C. Clin Chem 50, 1391-5 (2004)). For crystallography studies, purified
`recombinant IDHl (R132H) at 10 rng/rnL in 20 mM Tris pH 7.4, 100 mM NaCl was
`pre-incubated for 60 min with 10 rnM NADPH, 10 mM calcium chloride, and 75 mM
`aKG. Crystals were obtained at 20°C by vapor diffusion equilibration using 3 µL
`drops mixed 2:1 (protein:precipitant) against a well-solution of 100 mM MES pH 6.5,
`20% PEG 6000. Patient tumor samples were obtained after informed consent as part
`of a UCLA IRB-approved research protocol. Brain tumor samples were obtained
`after surgical resection, snap frozen in isopentane cooled by liquid nitrogen and stored
`at -80 C. The IDHl mutation status of each sample was detennined using standard
`molecular biology techniques as described previously (Yan, H. et al. N Engl J Med
`360, 765-73 (2009)). Metabolites were extracted and analyzed by LC-MS/MS as
`described above. Full m