US 20190255192A1 ( 19) United States (12 ) Patent Application Publication (10 ) Pub. No. : US 2019 /0255192 A1 KIRN et al. (43 ) Pub. Date : Aug . 22, 2019 ( 54 ) ADENO - ASSOCIATED VIRUS VARIANT CO7K 14 /005 ( 2006 .01 ) CAPSIDS AND METHODS OF USE THEREOF A61P 27/ 02 (2006 .01 ) (71 ) Applicant : 4D MOLECULAR THERAPEUTICS A61K 9 / 00 (2006 . 01) INC . , Emeryville, CA (US ) 2 ) U . S . CI. CPC . .. .. A61K 48 / 0016 ( 2013 .01 ) ; C12N 15 /86 (72 ) Inventors : David H . KIRN , Emeryville , CA (US ) ; (2013 .01 ) ; CO7K 14 / 005 ( 2013 .01 ) ; A61K Melissa KOTTERMAN , Emeryville , 48 / 0025 ( 2013 .01 ) ; A61K 9 /0048 (2013 . 01 ) ; CA (US ) ; David SCHAFFER , A61K 48 / 0075 ( 2013. 01 ) ; A61P 27 /02 Emeryville , CA (US ) ( 2018. 01 ) ; A61K 9 / 0019 ( 2013 .01 ) ; A61K (73 ) Assignee : 4D MOLECULAR THERAPEUTICS 48 / 0058 ( 2013 .01 ) INC . , Emeryville , CA ( US ) (21 ) Appl . No. : 16 /300 ,446 ( 57 ) ABSTRACT ( 22 ) PCT Filed : May 12 , 2017 Provided herein are variant adeno - associated virus (AAV ) ( 86 ) PCT No. : PCT/ US17 / 32542 capsid proteins having one or more modifications in amino $ 371 ( c )( 1 ) , acid sequence relative to a parental AAV capsid protein , ( 2 ) Date : Nov. 9 , 2018 which , when present in an AAV virion , confer increased infectivity of one or more types of retinal cells as compared Related U . S . Application Data to the infectivity of the retinal cells by an AA V virion (60 ) Provisional application No .62 /336 ,441 , filed on May comprising the unmodified parental capsid protein . Also 13 , 2016 , provisional application No . 62 /378 , 106 , provided are recombinant AAV virions and pharmaceutical filed on Aug. 22 , 2016 , provisional application No . compositions thereof comprising a variant AAV capsid pro 62 / 384, 590 , filed on Sep . 7 , 2016 , provisional appli tein as described herein , methods of making these rAAV cation No . 62/ 454 ,612 , filed on Feb . 3 , 2017 . capsid proteins and virions, and methods for using these TAAV capsid proteins and virions in research and in clinical Publication Classification practice, for example in , e . g . , the delivery of nucleic acid (51 ) Int . CI. sequences to one or more cells of the retina for the treatment A61K 48 / 00 ( 2006 .01 ) of retinal disorders and diseases . C12N 15 / 86 ( 2006 .01 ) Specification includes a Sequence Listing. SIRSWA 40MT Proprietary Libraries (n = 204) In Vivo Vector Selection : Vivo Vector Selection : resign . : od 200 muutos a @ob the 0 transmitimijeni Vector kits Identified Vector???? Hits ?? ???? ???? ???? ?? ?? ?? ?????Identified ??? : : ??? ?? ? 900000 i AAV. Capsid. .4VriA 2 Variants4 . PER Patent Application Publication Aug. 22 , 2019 Sheet 1 of 38 US 2019 /0255192 A1 a124444444447%. VivoVectorSelection: Stage2 extr IdentifiedHitsVector AAVCapsidVariants M24 wwwrrrr* 'r-47. v . IdentifiedVectorHits inVivoVectorSelection: Wehill .* th ? WWW Wh WWWWWWWWW!! M34 st * 2 KOO ** 0 !! !! i 4DMTProprietaryLibraries 0 . 200 BUIENRATIA WWW 0WW 1FIGURE . Patent Application Publication Aug. 22 , 2019 Sheet 2 of 38 US 2019 / 0255192 A1 :. arcadevascular ".: 1471 T513TI 12T4T6 fovea . B 6 15 S6S5 5453 S2S1 22 LeftEye 14 16 WT. ** ' NIN3N5 N6N4N2 N5N3N4N6 WIN 16 . 14 P #UZ WW Y : 15WWW :. VI B T6T412 Afovea FIGURE2 arcadevascular A Patent Application Publication Aug. 22 , 2019 Sheet 3 of 38 US 2019 / 0255192 A1 WHE WWW 2000x outmidoutinmidoutin Ult RPEONLINL RPEONLINLGCL midinout midinout WITH GCL outmidin WWW FIGURE3 Patent Application Publication Aug. 22 , 2019 Sheet 4 of 38 US 2019 / 0255192 A1 36.00 Round4 Round6 WWW. (6) (d) NE Round3 Round5 WE96VEL (a) © S FIGURE4 Patent Application Publication Aug. 22 , 2019 Sheet 5 of 38 US 2019 / 0255192 A1 WY FIGURE5 Patent Application Publication Aug. 22 , 2019 Sheet 6 of 38 US 2019 / 0255192 A1 ko 30 30 30 30 Sets PCAPKPK NOVY TER 20 1427 WWWWW WHERE POPKKAADGYIPD?LEDNESEGIREWWALKPGW W WWW. WRITE WEMO WALEXA BAADGYEPDWLEDTESEGTROWWKEKPOPÉPEK POPKPOWLEDNESEGIRENWALKPDRKAADEY?WWW RTDGYOPDÚBIEDNISECVREWWANOPGABERX EXTRARIAHLAX UNU . h RE 10 10 VANDRUPDWDSREGERENDUPOAPXPK WWW WERTHE 7777 Wh CA SHA WIR YUPO FAIXXVXOKOAX VAADGEPOISDNB SEN PUEDE UN . Hl BE WEET27 1 * " AAV3B(SEQIDNO:4) 2 AAVS(SEQIDNO:6) AAVG(SEOIDNO:7) AAVO(SEQIDNO:10) FIGURE6 Patent Application Publication Aug. 22 , 2019 Sheet 7 of 38 US 2019 / 0255192 A1 ? WWWS !? ? * 2 . 1 AVA VRH 17 THE71A/ 4* . 60 09 60 00 WWW ???????? VA . PENGEDKE MAXXX 2 2. os H 50 39 .WWW WA PROQHODOARCUVEPEYNYNGPENGEDRGEPIN EVAMA ANOOKODDERGLVOPGÝKYLGPPNGEDEGEPIN . 24. 92 PARKODSRPGYKIEGPFUGLDKGBP TARGET&PCYK BUL WALIO WAX ht ANOOKODE2 OONERS WWW 32 NEW ANQOKODDGRGIVLPGYKYILGPENGEDEGEPUN W ODBODRARGER 2007WALA LI NGORONGROPOLKUPENGER 2 22 MAS AAV2(SEQDNO:2) PAVBR(SEQIDNO:4) AAVA(SEQIDNO:5) AAVS(SEOIDNO:6) AAVO(SEQDNO:8) FIGURE6(contd) AAV10(SEQID Patent Application Publication Aug. 22 , 2019 Sheet 8 of 38 US 2019 / 0255192 A1 20 90 4 90 90 00 WWWWWWWWW WWW WWW 37277 ADABAZENDKAYDOKAGONPYURYNARDAR 4 BOKADOBRASONPADRYNRADAS AADBABLONDKAYDOOLLAGDARYERYNHADAS 30 AADAAAZSIDKAYDOOLKABRYNHADAGD 80 PADARDXBYDOORRAGDNPYEZINHADAS www SO EADÅAADERDKAYDROLDSGDNPYDKYNHADAN WHAT SADARANDKADOORKASDNPYZXYNRADAS wwwww 2 20 RADZARBADOSYNBOLAGDNPIERRADA70 70 ADBAARDRAIDODEKAGENPYRANHADAN70 W AAVBA(SEQIDNO:3) AAVI(SEQIDNO:1) AAV3B(SEQDNO:4) AAVS(SEQIDNO:6) AAV7(SEQIDNO:8) AAVS(SEQIDNO:9) AAVO(SEQIDNO:10) AAV10(SEQIDNO:11) 0233 Patent Application Publication Aug. 22 , 2019 Sheet 9 of 38 US 2019 / 0255192 A1 129 130 WW PPCD . WWW WWW 120 PAKKRESPEC120 120 120 120 re 010 601 WORKBOTSFOORBEEOAKKRUP FORREOBDTSEGREGRAZIOAKKREAPLES XX DOBROOTSIGOREGRAYSOARER MA 12. WORLOD32GBELGRZOAKKREDPLG LOORXOGOSPARAGRAFOAXKREEPEG OXBADDESIGN Harm FOLOSETSEGGKLGRASOAKER how Thomas sobooks 1)NO:IDSEQAAVI( AAV3B(SEQIDNO:4) AAV6(SEQIDNO:7) AAV7(SEQIDNO:8)23 AAVO(SEQIDNO:10) AAVIO(SEQIDNO:11) A FIGURE6(cont'd) (SEQ 1 Patent Application Publication Aug . 22 , 2019 Sheet 10 of 38 US 2019 /0255192 A1 091 159 159 139 159 160 160 *159 WA . WWW Who AXTAPIGXRIDDOPKRKLART-EDS w 149 350 150 190 150 . .. IA SA CACTEPERKRPSP-OPPSTICKYGY AKTABOXERPISOSPO-SDSSSGUGATOR WWW 77UDEPREZZO WRAAKTAPOKKRPSBOSPO-PDSSSCSKSGX 11 RAAKPAROKKCASOSPOLPDSSSGIORSOK GALAPARKROPSPORSPDSSTGGKUSO* ERAAKTAPGKKRETROSPO-EPDSSAGEGKSGA 140 A 129 140 140 . UK2 DRAAKTAPGKKRPEEPSPORSPDSSTGEGEKCO NA AAVBA(SEQIDNO:3) AAVIO(SEQIDNO:11) tir 2 AAV7(SEQIDNO:8) AAVE(SEQIDNO:9) AAVO(SEQIDNO:10) FIGURE6(cont'd) Ver YA ook Patent Application Publication Aug . 22 , 2019 Sheet 11 of 38 US 2019 /0255192 A1 mas Kok XXWh we Q.1 / FARIS WWW ost621 687 981 067 WY 061 .WWW L-ardaokaacausa909Paza I-dua2aoaga99899RONDO maWWW azadodiacansas?n WWW W SOPARKRINEGOTGDSESUPDPQPEGÖPPAAP- S-aydanozaaos20:09Nazao W 02 180 180 621 180 081 627 621 a ambalaagd$3991093128g ???????? wancein ???? ???? WWW WIRAWWW With relation was 169 post OLL 691 að At + V . 18 W12 AAVI(SEQIDNO:1) )NO:2IDSEQ(AAV2 AAVBA(SEQIDNO:3) 4):NOIDSEO(AAV3B 8):NOIDSEQ(AAV717 NO:9)SEQIDAAVE( WY Patent Application Publication Aug. 22 , 2019 Sheet 12 of 38 US 2019 /0255192 A1 72% YMAG 2.29 .Prs 228 228 228 222 218 228 229 228 229 W ASON . WS AHMINIMA WINWA UNION W VE 210 226 21.2 23% 219 612 218 219 WW EX WS SONRAGUNSSADPADASEGSITRASSGGA WWW WBXWA WWW SWEGADGAGNSSPADNESGSBELASGGGA 4 . * 22 * SGDIHGGBSRBIRADBGGAASGGOGADSDO GDWRGADGEGNASSAGGGGPGDNNODUSASAD SOMWAGADGGSSTRAAGGGAPRADACGSG 210 200 200 200 203 200 ay HEN27 Ken 22222 . 2 . 73 * an * wWS al R w ef. WS 29 HE 198 198 293 WWE 3.30 URUMEA 139 24 24 FIGURE6(cont'd) . AAV10(SEQIDNO:11) 1)NO:IDSEQAAVI( )NO:2IDSEQ(AAV2 4)NO:SEQ,IDAAV3B( AAVA(SEQIDNO:5) )NO6:IDSEQAAVS( AAVX(SEQIDNO:9) pour 3 Patent Application Publication Aug . 22 , 2019 Sheet 13 of 38 US 2019 /0255192 A1 WA 36 * NO 11 wa MW 259 258 252 W 240 259 259 258 1 EYROISSW USB C 00 4. WWXXXXX : 270 SSKOBRYTYNNPSTRTWALCDSTNEGDRETT 15 WWWW 249 248 242 ARNE 238 248 ZAKOISSPTYNNEDIAPRCOSTNEGORIOTTS NA NUPTINNEXOS3IRCORTASCoSon 22:13 23 ti UPLYNNYKORSSSTRINACOSINECORETT STRADOSNRE WA 239 240 gez 230 238 232 228STRTIPSYNCHOYBLIKSCOSTUMODRATX 236 WIB WWW * w UA2 ure 1 12 AU 3. AAVI(SEQIDNO:1) )NO:5IDSEQ(AAV4 )NO:8DSEO(AAV7 )NO:9DSEOAAVO( (SEQ Patent Application Publication Aug . 22 , 2019 Sheet 14 of 38 US 2019 /0255192 A1 3P WWW 11102 290 266 286 286 277 287 200 289 . BRFBCBPSP 280 276 276 276 267 277 279 SPPOPNROACHSZOSRTYNGESTPROY- YFGYSTISNANA YA mSDXBLOGY 02 GYFDFNRPRCRESPBESYSTPRSNDSTGA4- ho GYSTPAGYROPORRCROSPCASNODAYI- HOW 268 PWGYFDFNRFHCHFSPA-STGÅSNDNHYFGYST266 266 266 267 268 269 SNDNHYFGY?TPWGYFDFNRFHCHTSPSGA-0 VOX TNDNTYFCYSTPNGYFDFNRFECHPSPETSOGA SGGSTRDOTYPGYSTPRGYEDENRI 1 AAVI(SEQIDNO:1) NO2):IDSEQAAV2( )NO:3SEQIDAAVBA( )NO:6IDSEQAAVS( 8)NO:SEQIDAAV7(H ):9NOID(SEQAAV8 FIGURE6(cont'd) to *74 hoood 1 SLOW Patent Application Publication Aug . 22, 2019 Sheet 15 of 38 US 2019 /0255192 A1 329 330 326 3 WOMAN DOBRO 316 301 313 319 342 306 306 305 297 NDERZORROTTNGERPKRDROREN 308 BREAPRODITAPRPGRORORO309 302 GOPOR GUTONQERRDVORIINNNAGERPKKESFKEFNI):3NO KROSFKELÉERDVOEDIENNAGERP 292 296 28 287 267ZGPRERSERKOPALO2030 RONORED 297 298 NORORORDNINGERPEREREKBERO2)NO:IDSEQAAV2( SZKÖPROGRESSIONGEROKKUPOHOREN)NO:4IDSEQAAV3B( DROBNOZRPRESE at DORORDNINGERPRENORDENLOSEDN 11)NO:IDSEOAAV10( )NO:3IDSEQ(AAVBA NO5):SEODAAVA( 7)NO:IDSEQAAVO( 8)NO:IDSEQAAVO( NO9):IDSEQAAVE( 10)NO:IDSEQAAVO( S )'dcont(6FIGURE2 Patent Application Publication Aug . 22 , 2019 Sheet 16 of 38 US 2019 /0255192 A1 . A 323 358 358 359 356 . 22222222 GOEDÅYUHYELPFADSS YDLEGŠARO IAC 350 346 346 346 349 . WETDŠEYO?PYVEGŠAHO TVOVFTDŠEYOUPYVEGŠAHO. With ELGSASXPSDSSTEPODERATED WHA HOTÉTIANNÉTSTIONFTDŠEYONPYKTIGSAHQ w WA