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The Two Tort Dit U Moltoto Umuhimu THETWO TORT DIT UUS MOLTOTO20180044662A1 UMUHIMU ( 19) United States (12 ) Patent Application Publication (10 ) Pub. No. : US 2018/ 0044662 A1 PLATT et al. (43 ) Pub . Date : Feb . 15 , 2018 (54 ) USE AND PRODUCTION OF CHD8 + / C12N 15/ 86 (2006 .01 ) TRANSGENIC ANIMALS WITH C12N 15/ 85 (2006 . 01) BEHAVIORAL PHENOTYPES ( 52 ) U . S . CI. CHARACTERISTIC OF AUTISM SPECTRUM CPC .. .. C12N 15 / 1024 ( 2013 .01 ) ; C12N 15 /8509 DISORDER (2013 . 01 ) ; C12N 15 / 1082 ( 2013 . 01 ) ; C12N 15 / 907 ( 2013 .01 ) ; C12N 15 /86 ( 2013 . 01 ) ; (71 ) Applicants : The Broad Institute Inc ., Cambridge , C12N 2830 /008 (2013 .01 ) ; C12N 2015 /8536 MA (US ) ; Massachusetts Institute of ( 2013 .01 ) ; CI2N 2310 /3519 (2013 .01 ) ; C12N Technology , Cambridge, MA (US ) 2310 /531 ( 2013 . 01 ) (72 ) Inventors: Randall Jeffrey PLATT , Basel (CH ) ; Feng ZHANG , Cambridge , MA (US ) ; ( 57 ) ABSTRACT Ian SLAYMAKER , Cambridge, MA (US ) The invention involves inducing a plurality e . g ., 3 - 50 or more mutations ( e . g ., any whole number between 3 and 50 (21 ) Appl. No. : 15 /467 , 949 or more of mutations, with it noted that in some embodi ments there can be up to 16 different RNA ( s ), e . g ., SgRNAs (22 ) Filed : Mar . 23, 2017 each having its own a promoter, in a vector, such an AAV vector or a lentiviral vector and that when each sgRNA does Related U . S . Application Data not have its own promoter, there can be twice to thrice that (63 ) Continuation - in -part of application No . PCT /US15 / amount of different RNA ( s ) , e . g ., sgRNAs, e . g . , 32 or even 48 different guides delivered by one vector ) in transgenic 51691 , filed on Sep . 23, 2015 . Cas9 eukaryotes to model a neuronal disease or disorder. ( 60 ) Provisional application No . 62/ 181, 002 , filed on Jun . The invention comprehends testing putative treatments with 17 , 2015 , provisional application No . 62 / 054, 675 , such models , e . g . , testing putative chemical compounds that filed on Sep . 24 , 2014 , provisional application No . may be pharmaceutically relevant for treatment or gene 62 / 180 ,859 , filed on Jun . 17 , 2015 . therapy that may be relevant for treatment, or combinations thereof. The invention allows for the study of genetic Publication Classification diseases and putative treatments to better understand and (51 ) Int. CI. alleviate a genetic disease or a condition , e . g . , autism , C12N 15 / 10 ( 2006 .01 ) autism - spectrum disease or disorder , obsessive compulsive C12N 15 / 90 ( 2006 .01 ) disorder , or psychiatric disorders . O * goesofou WITH ** * * * * ** ** * * 45 Patent Application Publication Feb . 15 , 2018 Sheet 1 of 10 US 2018 / 0044662 A1 > xrotypes m 0 2 WICX Step4wWeek1?karo Characterizestanekunders ahaviat,&culas 13) FIG.1E synapkolevelsand RosakEXO ales:01 FIG.1D NO.277 fiutatWtMom 20 CH00 geun GAPDA 3 140 576ERAG7X MAGIA 63 OXIAGRA .8976:41 TIETAISAXTIGT.6476:2042 WE.60& MIXKGKTAACC7667. ATEXTERCATATACOX.-TALAGA AOUIRAKA MORAESIXMAOO7.MUMMYAG WATERMA56:48%.-6677827086 IG*XXC.2667676080M3 MUME&AUTIXC%2X70G.MPYAO MANAGINWAGENKTISGK.15AAGOGIKUR* CrossmosacxS transionofmutantalake Y*SEX:48.- WookStep3( wwidtypeanmeisforcorino ISACACAXACA6764I.MAAX GAROTAMIGIKA-66767KOAGUA N MEE460479&*:44CT997.180GEWIMMIGAS MAGITATOAMIGO8PXSET-SGIGTAKAS AKAZIGRAWATANSerumSSWW467 A&74:KAIKHA?ACACC78K.SAATORSTW14 KAANIAWATOGRXACT-. ALAS.20367766X7XXI- AAAAMGO* MAYASAVISO:-*I7797XROXIMA29A MAGAMISR:C FIG.10 PRAW XXX WA* W mica aird alleles mutant -113 WW Step2(Wook3Mainmogenyafter buitadiyger16otecRossioKonies * *** A64A6668600* *oncm. * EL ACSATSGATACACC766TOSAKAS66S.?! * 7000WWWww CHADEK 184414H ** * 16TTACCTATGISGACCAGCIT1000 5 0000 W FIG.1A ster1-Woak01 CasoaBWAANA120 gritzedzolas FIG.1B mostro 0806oxotein Cidagene UWAANA Patent Application Publication Feb . 15 , 2018 Sheet 2 of 10 US 2018 / 0044662 A1 * febrer Vico L a IM - 5 * --- FIG.2E - no * 4 OM - - URU - » info to *** * 3 ( 9 ) Ourogator UK our 2 - FIG.21 1 -> (235)eu C com OIM { s } YOK 09 10 „FIG.2D LS & hueso aos dun Out - 17} 83X10 - 14 O- SM $33-23}{ sitting 3500- med Alo8ÁLBU004 WOIMMUM ; is center in me FIG.2C - il01 - einu jaxou uw waox0 . FIG.2H, onge:2 IN 39 - Cx" (9 o confins - = FIG.2B » , - Stwo: + + + + + + + + + + W mitand {01«)(75 - 19) Aug Juaris eum FIG.2G ha * Ons?W irsi urte * Exty E$1 2-0 - Qla. (012) fimifmati O E L- ?W Karina S=id; OOOOOOOOOOOOX IM artis 66 $ u 51&e: (91) FIG.2A - 1.15€ FIG.2F L009 lounodem oda 90 gt Srce Patent Application Publication Feb . 15 , 2018 Sheet 3 of 10 US 2018 / 0044662 A1 OR1:33) WTOU* 13) * prosessoring In:25)1720 W( FIG.3G 2.0- } { { d sp KOARINA2AN7728GLORIPEXCINNBIAOIBGAF0:1 dosahrumog CXB FIG.3F W 35319 FIG.3B FIG.3E IM L1JC mbrie e sept 054 0. watatatatatatatatatatatatatatatatatatatatatatata cs \ } ?{ { } z {} ?? * ( 0)????sue ????? .040 m:19 . WT(1-5 de123 * IM FIG.3D * * * (nu: se 28 KWdebe 504 09 : 25 . III . CINNOSGARDAGLORIFTENACTE#28 . B Cixg * OYNSEEDLI00Sne:334 d - * prising NOAAINEVA WT FIG.3A 0.* FIG.30 2011 wootor: 0.2390 1. Sisag mod pozjeu3 ! 013 100A Patent Application Publication Feb . 15 , 2018 Sheet 4 of 10 US 2018 /0044662 A1 FIG . 4A man synapsio 1 {HSyn ) promotor I EGFP - KASHODO DemogaITR SOANA FIG . 4B *** * * (week 1 (weck 2 ) (WEEK 4 otwarta ) VIRS Proxduk { }} xtion xHVOX FIG . 4C nocleus acuXXIS (NAC Boxsai sukan (DS ) NO FIG . 4D SCHWA NAc single FIG . 4E 100 genotypes 990 NO * H intactedcellsinNAO 30 0190103 mm ( 1039 ) 080 16 ustant W ON alelas : 0 2 2 3 5 6 (14 } na17) ( 10 ) Patent Application Publication Feb . 15 , 2018 Sheet 5 of 10 US 2018 / 0044662 A1 nu 2 180=upovo Q 2 ouou:* (22Log 0 A se siewe ueru Sesong SYNWS wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww S8! NSÖR wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww 1NUÔS wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww 6300 doa Solo Patent Application Publication Feb . 15 , 2018 Sheet 6 of 10 US 2018 / 0044662 A1 FIG . 6A FIG . 6B 357 mm (6)jucam e 000 a hrownegooooooooooooooooo W Oxig W Oce (1338 ) n = 18 ) FIG . 6C Moses * Suodantjuodsun ou Ol .. .po cosec 01 junction W COX * W Choo WT CIX * ( n = 10 ) (n = 10 ) = 10 ) { 1} = { 0 } ( Elo 1 0 } FIG . 6D FIG . 6E Oro * * distancewaveled(m) (SiyoyesofBull W * : 0 } wannequences. agaansa com wwwwwwwwwwwwwwwwwwwwwwwwwwwani. www w W One 3 2 3 4 5 6 ( 0 : 0 ) (8 :: 8 ) Patent Application Publication Feb . 15 , 2018 Sheet 7 of 10 US 2018 / 0044662 A1 GAPDHE GAPOH ????? 910V W . anygal(AM PTEN KOLO PIEN GLUAI EZUN 4 OLURI . NR2A NMDARI NMDAA1 FIG.7B L02 FIG.7D 209 SABUBOX pohjausje Sonosoki pozeludu 3) 100 WT( GAPDHY GAPDH ACTE ACTE sobnxiouG2- PTEN (0) PTEN GLURI 189 003 90od EIIN4177 NA2 VEHN NRAA 1NMDAR .071 FIG.7A .0 e pop u lations are on FIG.7C HomepageNMDARI S avoxoki pozg @ glou ?? {{ { ????? ????{ { { } Patent Application Publication Feb . 15, 2018 Sheet 8 of 10 US 2018 / 0044662 A1 CB CX OS HPF HPE N828 GLURI STAM STAN NAO 2.0 FIG.7F 20 ??????0. Som u oxioozequiou FIG.7H genottooxipezielou 08 X0 PE 90 1 M AM TITIEUTILEW WRZA NMDARI 87 NAC WN . .. FIG.7E 00 FIG.76 201 Sana vigold panelou GOA11000 poxyewou Patent Application Publication Feb . 15 , 2018 Sheet 9 of 10 US 2018 / 0044662 A1 )5.E18 (brainadulti (wholeNAQ WOOZ ORFCHDÉ N2a FIG.8B OH08Westem 00.000 Nza adult FO x00 . : : : .: : : : : E19 : :: 3: : i E1. 12 E17 ES? 6$E+ QAPI 3. Chd8 Gepon 30010 ou 41 *** 19 05w. FIG.8C W FIG.8A " CHD8QHD8 C-08 exposure GAPOH hite Patent Application Publication Feb . 15 , 2018 Sheet 10 of 10 US 2018 /0044662 A1 FIG . 9A GFP ( 200m ) 17. : SOL CZ : : : : : :: : : : : : :: : : : NAC : : : : : 1 : : :13. : : 1:. 7: NAO : : : : : : : ..SzChds : : FIG . 9B FIG . 9C LAXWE . ($}voivupnouje jaundi)omauuen * * maramamayana maneno OL {- sguacz soci8 sou A LACZ sgcus SCHON NAC NAC OS NAC NAC OS {} } - } 5 } 215( ) ( 11) ( n = 15 ) ( 13 : 15 } # { 1 } FIG . 9D FIG . 9E 90 dancetraveled(m) (3)ladovicu it online find * framfarafen homoporoscom * erere voorop 06 .. lacz XC sgcn8 sglacZ sgchu8 NAO NAC OS NAC NAC 08 353 } {f } = { 5 } = : { }} 815( ) ( n = 15 ) ( 11) US 2018 /0044662 A1 Feb . 15 , 2018 USE AND PRODUCTION OF CHD8 + / ability to catalog and map genetic factors associated with a TRANSGENIC ANIMALS WITH diverse range of biological functions and diseases . Precise BEHAVIORAL PHENOTYPES genome targeting technologies are needed to enable system CHARACTERISTIC OF AUTISM SPECTRUM atic reverse engineering of causal genetic variations by DISORDER allowing selective perturbation of individual genetic ele ments , as well as to advance synthetic biology , biotechno RELATED APPLICATIONS AND /OR logical , and medical applications . INCORPORATION BY REFERENCE [ 0007 ] CRISPR -Cas9 is a powerful technology for genome editing and is being widely adopted due to its [0001 ] This application is a Continuation - In - Part of Inter efficiency and versatility . While Cas9 -mediated genome national Application Number PCT /US2015 /051691 filed on editing applications are compelling , applying them in vivo Sep . 23 , 2015 , which published as WO2016 /049163 on Mar. and ex vivo is challenging , since commonly used delivery 31 , 2016 and claims benefit of and priority to U . S . provi systems are inefficient and limit accessible cell types . More sional patent application Ser . No . 62 /054 ,675 , filed Sep . 24 , over , certain cell types, e . g . , neuronal cells present chal 2014 , and U . S . provisional patent application Ser . Nos . lenges due to their particular sensitivity to perturbations, and 62 / 181 , 002 and 62 / 180 , 859 , both filed Jun . 17 , 2015 . are often
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