AU2014271317A1 - Methods for production and uses of multipotent cell populations - Google Patents

Abstract

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) CORRECTED VERSION (19) World Intellectual Property Organization International Bureau (43) International Publication Date (10) International Publication Number 11 December 2008 (11.12.2008) W O 2008/150814 A8 (51) International Patent Classification: EG, ES, F, GB, GD GE, GIL GM, GT., HN, HR, H1U, ID, C12Q 1/68 (2006.01) A61K39/21 (2006.01) IL, IN, ISJP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, C12N 15/00 (2006.01) LK, LR, LS, IT, LU, LY, MA, MD, ME, MG, MK, MN, MW MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, (21) International Application Number: PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, PCT/US20081065007 SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (22) International Filing Date: 28 May 2008 (28.05.2008) (84) Designated States (unless otherwise indicated, for every (25) Filing Language: English kind of regional protection available): ARIPO (BW, GHl, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, (26) Publication Language: English ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, (30) Priority Data: FR, GB, GR, HR, HU, IE, IS, IT, IT LU, LV, MC, MT, NL, 60/932,020 29 May 2007 (29.05.2007) US NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, 60/933,133 5 June 2007 (05.06.2007) US CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). 60/933,670 8 June 2007 (08.06.2007) US 61/006,449 14 January 2008 (14.01.2008) US Published: 611064,761 25 March 2008 (2503,2008) US - with international search report - with sequence listing part of description published sepa (71) Applicant and rately in electronicform and available upon request from (72) Inventor: REID, Christopher, B. [US/US]; 880 N. the International Bureau Alameda Street, Apt. 312, Los Angeles, CA 90012 (US). (88) Date of publication of the international search report: (74) Agents: FOX, James, A. et al.; Goodwin Procter LLP, 135 5 February 2009 Commonwealth Drive, Menlo Park, CA 94025 (US). (48) Date of publication of this corrected version: (81) Designated States (unless otherwise indicated, for every 30 July 2009 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, (15) Information about Correction: CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, see Notice of 30 July 2009 (54) Title: METHODS FOR PRODUCTION AND USES OF MULTIPOTENT CELL POPULATIONS (57) Abstract: The claimed invention is directed towards the generation of pluripotent, multipotent, and/or self-renewing cells which are capable of beginning to differentiate in culture into a variety of cell types and capable of further differentiation in vivo. The claimed invention is also directed towards the generation of desirable, differentiating cell populations transplantable to patients, genetic modification of endogenous cells, and the treatment of patients suffering from diseases that may be ameliorated by these methods. This invention also provides methods for preventing, treating, or retarding disease related to immunodeficiency virus (e.g. HIV-, HIV-2, SIV, FI, etc.) infection.

Description

EDITORIAL NOTE 2014271317 - There are 101 pages of Description - The first page is not numbered I\hotb fd r rhcIon and 1AC h Of Aultpoi All PopulalN, Plunuacnt Cel lpulatis, Neunal Cc Populin d ade Pu p ul atnCl FWAGGRO1 AD OF I H F II \ N i ON 1 he uzunn ThInTse 10 mcdime io the 1 St euii n u he repkeag darmIaed. womrtp t or gnulkco imed dcc! I kt ran ion hetorN ons speciiicallv to DuA pla a ux ia ole m~ reuatngn efipres\ n It a\ the taruu complement of irnsenpflen factrs un an indivudua cceli tht~ deiniu wxhch celular programs are ae cand W hieh ar turn) off in Ilths capaci tfranfrptfn fuehos pla a d deersnve role in deterrlu and maimtainmg cellular ideniy, as well as deternm celuhar valnerabihats StMM4AR1 OF ME INVENTION he abidv so derive prohraung sel renewmj muinpotenu and pluripotent cell popuhAMtnt im otheinie norwp ripotet fonnelf renewitmg 'VW may hal e Siga 1 5 posve imcpheanos for all cids tlhiing edlLar therapies i bu WieldS melude boue nmrow ransplaau, transfusin medicine. and gene therapy 'nd nbk the prou'xedn ot' ptic-speitie em cell and other desired cell types. likeWse, thw Ai li to nmiete di elt'rentiaeion of cells in bo neural. musoe ad vaioute 01b desirable ed populations is and w il als be of iicant value to medicime and commer cial process o mg animals 20 lAnoingnly the present imxentiom prodes methods i giene producuen and uses ot mhultaplteml tel populutmns pluriotenm cell populations. ireuroinal cell populaneons, muscle cell population and other desied celi opulatons such as, fr example. 1I1 distant cell it is a propushtion of the present invention that the efficient introduction or 25 oveeepresen of seeic transnpon factors. alone or in combhation w"ih okher cell ue deternnants isuch as notch, numb and numbbikei cuenales the ierceono of w hat hav e been considered trasitol)v nr lupotenm Plrinlemh atd i selfr n'eenn( or Lived bliftbeiiated or somulk I eel'Ilay ph0eno\ pes I he ailit tol1 reliabdiducdke oheno1\ rPe eon version or ec lluhr re pngrantg al lows thbe prouction of stem eel IS replacement cells, A issues, and oryan that match n idual patient I coniutction with gene therapy tchniues and cell culture techues cell 'pe inerconmersion also proudes the WVO 2008/11=814 PCTIA1208/M5ti" productumn of disease d:esitant an geneticalv-epaircd edls that are :>uitable for ,anspintalion, it an lect of this im 0 on to rwovde a-ous mannersf eencratmi pibtert. scf renewing, mu bpotem andLor plunpotent cll pop atioms)L as well as 4N04y 'esiW cll t ~utns romo eh~ ceilsmil x;'tlaxc "w. othe ~ e d puton -mither dividing or non-dVidine itte ha the use e oneogenes itfereniaung cell popular cumpehe cells expi csing SOme, but not all markets assoaed nih special cell type c-ategorzaion, It ishis)ed herein tht aptproprlute Numb isorms cxession in conmbioaton with other trarigenes especeda1 transeripn factors enables the produton diS piptent cell puh muons or I) ditllerlating ccl populations \More0\ve7 the genelice ctors of the present invetionrtthc be used to produce genceic modidcathm (ce.g, expression at gene pieduicts deficient in die putient) a nd to trasientl\ or pernranemly induCe proliferation, scitrencual, or dcn1 oroagemtor cell belnctr 1i n endoenons cels in \vi\a, partitiar l those cells ibund in issues whIh noundi do not show ar no longer sho such behavior. [mall other genetic 15 vectors ofh present venhon ma b used to produce genetic mrw tion nd4oJ to block prohi Tzram, Ae~tenl orh stemn prowemitor cell behavior in cls abern:ly displaing such behavuor ie g cancer cells) It i' wh o air objec of the present miention to pioudie therapeutic Vc(t0re Gd cells capable 01 expte~ing synthetic ol ig~ouilctiode Seqwtences predicted to attenuate disease processes or example, the uent mention disdlss the 20 use of synthetic oligonuecmides to reduce gene ex pression ct ical DI\ arid other inainuuodehlcien cy vv a in fection, propagailor and spread. the inemnion may be used uith any suitable cel. including vertebrate cls, and including fish, mammauma. avian. amphibi m, and repilian cells BRWEF IESCURWP tON OF TIAW DRAW INC 25il'inur < Aschemati/ed eele ma-p coroespondme oth eer sequence o Example 13. rF txIU2IE DUSCRI PTION \ll paten~t~s pate-m applications and pubbications cited in tis application are hrerebs incorporated b-c reference herein in tlheireantreules s discussed hetemin "DNA" refers to deoxsKboiIce ac d aI ' refers to ribortude acid As discussed hereDin " A lith reNrs o con plementar 1 NA 'nmR \ WO 2008/15014 PCT/US2t}8:i65OM7 efes m ssenger R.NA NIRN4' rfers to sa interfering RNA hRNA\ reirs to .snmadl hairpm RNA: "mi RNA\Y refers to .micr&RNA such as singleouranded R(NA moleeuues, tYUWplV0 ithut 0 0lul~tg in lma|Lwhgh00 regulate gene expressmort 'ilecov amd "decox RN\'A^ and "[RNA decref cer to an RNA mol0eule tlaidn uildcs the natural t binding domain for a ligand A toed leldtr the maeamllngt of tW term "dml(0ltUW Nighdes ICSenIng a ellel or irducim dda'gte or Ummf the elett rU1 or acod an 3nactin Orit or huntebot, and fl des t01 esmanle, fessrssin the deleteriou0d? eerl o a di seas or condition. V ued9 hier ith the medo the te1m vc11 rlrdnc. indaled slowing ot ie0ssening the 10piras of im fl ee or aten.n and0 ml\hidts (Or LNample, lownet 4 th progress of a disease. 4s0owing the rate of ia fectio. or other\\se acting to slow ot reduce the d\ne or progress of a disase or cnidont Used liretn, an "lTning agnt is an uc-nt iha aids or isa dt' ektt cte\ to promote an ation 1:or exampleam exogenous agent that afers a promoter, e g. b m5idong or enhancing its activ ity, and so affe pression a gene under control of the o'he tu\ be emcd on MUnl y aelt. Wor ample. etrachc in ho e Used as an nducmp akent and doxyycIin ma\v he used as an indugly agent A~ nuleic acid sequlence ( eg. a nucle damd sceee ccoeedinjs a po0ypept1:1i is termed "operdNi ATuiedto another n1kic a dtquence (emg. a pi0Mottia) wen the first 20uciec acid sequence is placed in a fancaol i dt1onhip w ith die second nuelelc acid iopwNeh AMke to a.,dn~ eh sequence. IHr edaple. tI promotler prebN~e is . e~.~ .,q fnee i the promoter anects the transcriptiorn or expression of the codin" sequence s used herein, the term "drven by rcir to a gcne or coding sequene that is operably hrted to a promoter sequence. and that the promoter sequence affects the taunscription or expression Athe 25 coding sequience As used herein tnarke a mulel tat i detectable, or codes fat a detectable moecule or acts on other molecules so that the presence of the marker is detectabe A niarker proteni 0r mrker polpepride' is a protem or p0151peptide that 1iteetabe i a taboratory or Chnuial environment and, m emlbodirmemit. may he detectable lh eve. A 30 "mnuher grene." cendes a marker protein or arker palvpepide. As used herein, "111\'' refers to human immunodeicienev irus, and mecludes W0 2000 1$08 14 r 0PC 0S/2nd65M7 trluiamis Such DS.egi i it.l. 1IVK \t.)1ber mmiantfilcm v 1 russ imehude simumn rrnruuodebtetcnev vurus i V } and tel itc inmmunodeheiiieny v irus (FIV\. En/wines rvttcd to mV a bet temed '1V enIA I la,',nd me tlde. fRq cumpleirtegrase proteaseev erse transen~pta:<e and transacthvatnwe :egulatorv protein (AT), ) inteetton by ll is belie\ t d jo 'aoivC~ receptors termed 'lil\' rect'told~ here nav heimuitiple such re eptors, some of uich may be temed 'll\ eo-eeeptor. ' diuted hereint iY H\eonecptors include C'CR4 and (CR 5 A theoretwal basiq fr the embodlism of h imntion is described herein. ho \ er, ilistt diKuiljun On wo mr' wata to he considered s bindiln or nitM on the il present ventjon. Those of skdl i the art wil understand that the vfnous embodoments of the invention may e pratied rerdless of the mode used to desenhb the theoretical undlpminatt the imenftiConl in a prceremcd trxbtime~nt cells are "selec-ed^^ omm accessible, dividing or non dividing cell populaons fo the purpose of generating the desed a) pritmtig S munipoten (r ph ipoten cell populAion, diitreniatung hi populations o nteulponal cels e mu cle I ell, d) and or an oher desired cell popuhlo moreover the desied cel populen may be capable of lather di ferevunaniu in vuno. m vito, and ri ussue aproua: udteiouta ~pprop)flte di ftrenilauon inl meY sonrees of cels weed tor use in the inventiu: 20 Selected cellk mat include any cell pracucable mt the present mntein. Cells selected imy use in the present invention (hereim termed "selected eel Is1 mar oriumt as cndogenos cell of the patient -nicIdine, cells derived from other 1rga systems; or from exogenous sources imedinig thoe dem ced from cell Imecs. ervopureser eJ soures, banked sources and donsds. Cels ma al be seleceddmm cell genetkictealnmdited nith 25 synthetic or nature nucleic acid sequceas. Te tman Ackeed cesI as used erin does not incude human rmie tonic Stemu cells. in embohments of the present miventon m order that they may be isoWted without the movement ofansiv procedues selected ell wi preferaNe eastis accessible celHs periphery al bhond Ieutotes circulautng hematrpoiette stem tck epuheiin cell A eg buwed chect els (og. Michieryk vt a. 204) adipse tissue (g. (Snbeet c, .. 2007. \ et a, 20 mbiical cord blood cel MIS A g hao ei alt. 20% 1an ct al . 2{07 4 W a 0"M/ ISN4( I206: ' h7 eCI. I [1seYr, 1ie mIArow stoln cels, sermatemd e (Tan eta A 2017 prun.ovdil ge ls (Pnt') eelN isolated rom AammotiC m embruns (e g Woaklnan e W, 2007) lmu ele Od (e a e Dc tOpi et J IO al eO iel Jis ee inotated r o skin (c z, ubdL uOQA Dunwlaid ,k d1 b 2001: /udalsea dcv at A. 2007) ec. are ao \ol ed b the pesernt inwentidn. Such Ls \an he isolated fhon the issues in wAich thle" aesdk b 'n' means~ known to the art :Spermatoig0nid cells can be isolutd iusing a twn sten enl/\maue digestion lio i-ed btCO sep atin 1l can then b resuspened m mmimum essential medIum Il "Inda~io Ucvdupl vi'I ntif supplemrented with boime serum albumim to a flia concentration of t)04. mi h detadL 10 ubule Irdgments are decreased sutreiail and teased aparn pnotr to treatnmn with I mn'ml tivosim, h s-dutromi JaSe, Sad C0agtendSe, and thenf I mU mi0 hlflrtUo-lidase and -col genase- in \NI'\t contimn 0 10% sodium bicarbonate. mM i utamme, nonesseIntal amin duds, 40 rinmerifrnnru nenaavem, 1010 il to 10 n meroguanml ni emiltinstrevtnrnvm, ai 15 as!i TI PI' Spermd d 0 aella ute her' iar e ro0 'ulie irdement V) 5 eenfuation at 30 tmas '4axitv Afet iltrauon through 'mou ers n 0ith 77- ardprn 55 micron pore wes cel are Lllesied and loaded onto a disconuuuous Per-l denr prddlent I' cti ns aith ari 'it greater than 40% rogemtor stm -spermatecoma cells are \ashed and esuspended to a nCCncentron of cels$; q uivAet us 10 progemior stemnispermatogomia cely per~ ml. \fterw ards cells arc tzuttured andior stored -lw 20 an .. opw.. catibm technique known to the art, he selected Ces my he gCicY--nild Ves, espedial e that have been geneiealy mdined b any means known to the art, to encode therapeutic or commneil use (u| deoxtrihanuc Ic- acid (DNA ) or riboanuclele acid (R\ A) segn.enees In accordance wtth an aspct iathe pnkti 4u , itere. 4i pwoded a method ( 2 i pouing a denied cell popunon (eny plutlent Deumal iuS te, an Im the selected cc hs Achiei m mip ;aoten, pnotent, and/or sel renewing cl -populdtions hi order to achi ee a poputatbn ot p:rohi itranny,. selh'renewng.; plunipotent ells the selected cells) candor their progeny are tUansfected uwith nucleotide sequences) 30 inCludig those encodmg the "long? (' RR inser -t} tsotors) of the inanunalin numb aene \t abou the same toe the selected cel ma- alo he transtected with syntbetic WO 2008/150$14 PCA TUS2StM/5007 eonuclelades laurg(1n it) , W: short N umb Wiims and Numbike, then culured tnder c idtlns inch promote grow5h ot the se cd cell dO art optmal rowth rate. elected cells are mvntaued under these coDdioons tor the period of ile sufflcient to achieve the desnred ell nunlber I he cells arc ttronu an the ( optirn allre of growth uchier eJ byirmeul ann with I It, teel factor, and or equipotet ewncnaon5 ofH l6 bp v1 11h7 OncOsiftnM ard/or ardiotrtph 1; thatogr that th rate achlehed in the presence of other growth enhancmy e tokmestes those eondinons desenbed for euhri g plunpotent cells eg Guam et A. 2000 '6 . I gronth rate is determined lam the douhing times of the selected eel in said 10 grows tuh ture medium, ikisesse, cuulr o oditions such sa those desIbed i US Putemts '4327 I and 5453357 ady also be stable or the fpopagation and expdfShuLt, at yn maf growS ote. of cells tr n leted ith the I g PR 4 y Nu 1 mb 1isi)Of Ns Other appropriate proios and reference eCylUme concenatnsM have been taqi by Koshimirua L l 19Q: Skel we V 0, Pique-PeWlorce, I t4, Rose at at, 1994 Park and 1 n 000 uAan et al., 13 20(06 Dvksa ei al. , 2000 /hang et wk. 2007). i fowever e pratie it the present imcntion is not limied to the details of these tedchigs. In a. preferred embodUment the elcted ells are cltured in a standad gaob medo ini (n.iimal E ssential Medium wth r thout supplements (ecg gutme,1 and hel~tu-merdp1ftoetaol. T he medium may include basic groth1 factr (b1EGP' 20 steel facwr, lcukemia inhibitMrY taltor I WF), and/or fiatwrs with LI aetivty (e. LWE' KId receptor (LitW ciRihar eurotrophic facor jCN 1 F, oniostatin TO M receptor (SMR), eardiotrophin. iuterleukin IL) such as il- -,bper ILL o PI etc ) as well as horse seru. IUl' as wel iS other 1ictos wth Ll' actvty prevents spontaneus di terentation of the ceHs Under these condit ions selected eelIs an feted with the PRR-, umb ofern(S 3 and thh Proyeu\ die expected to y~l\eanptusjli~ee midor0 sel -ren ewal. In1 a pit fUtred embodimtent the selected -'eilt} t~ t.I ot their progeny are trdadeeled with c nueoude sc uence) s encoding the lng I PRR inseit N umb io fornt s1 as c eli s sequences encodmuc other transgenes.\ M Ya of those ra nes aresed belarat ong. wa M it 30 thern crrespondnj tdentilleatinu Ranrer tacycslnn m WnneiN) nthe NC3l setquence databaseT In anotller preferred embodimenlt, the selected ecllts) ad/dor their pi'o:11) are 6- WCO 2(M*/15tiM14 PCVTUS20t8/16607 transtbcted w ith nucdeutide sequencebsi cnccdirg a portion of the '^long ' FPR Rosen t + \umb isorms as el as sequences encodig other tranagenes. T'hu1 of those tanscenes are itied below along nh: their crrespondog identIteatin (accession) iumbens (odes) i t he Nc El sequence database. In a pweiened embed mnent, the selected cellS and/or their pro gcn arc transfeeted uith long (PRR -1 Numb isotorm encoding sequence as well :s sequences eicoding other trm/enes, mehdmt i F. In a preened embodiment, the selected cells and/br their progeny are transficted w oh long (PR R - I Numb soun encodmg sequences as welo as sequences encodg other i) transpenes, muc ndog ones wunb lA activity. in a preferred eMbodtent the selected cells and /r their progeny are r1 sfCeted w"th long (PRR #1 Numb isofruna encodig sequences as wedl as sequences encodmr other transgenes, tided thbi e i1 :11R, n a preferred embbmienr the seacted cellsmd/or ther progeny are uusjeered 5 n'ih long (PR R 2 i nmb isofkormnii enica dirg sequences as wudl as sequences encedinn cuher git M In T WPcludints oncaostatinM \ (OSM) in r preferred embodiment the seeted edls and or thenr progen) aire trasfuected Ath lonw tPR R uRmb isoinsUrm encoding sequences as well as sequence ncodng ther moangnes. idingdt oncostato I reeptor iOS MU). 2.0 In a vreterred voibudhment the sadered eIs and/er their prupenv ar tauskested wb it ong (PR R g,+) Numb js:.onnms i encodmng sequences a xwdil as sequences eneoding other transgeecs. including eca'diotrophin-) in a preferred embodiment the select ted! cells andor dithe nrenv are uranstetted with long ( PRR -l Numb isofotmhs) encoding sequences as well a sequences enreodinga other u ansenes including CN 1 m In a pretered embodiment the selected cells and/or their pmoer are transieted w ith long (PRi ) '>-umnh saorma en coding sequences as w ell as sequences eu,:odmg other transpenes, meidmg i 00*13 4,md SON In a pre/erred cembod imem, thme selected eltls and/ua their prodt;enY are trand ec red 30 w ith lon *.PR i Nim isotormpsi encoding sequences as w ell as sequences enicodone other WO 200W1 PCT/US2008 145P07 trusnes incidmg N A NO 00 OC' / 4 and SOEl Ih a pICrned enhodiment, the selected elIS and or theO progeny, are uanUsteted nx th long (P'RRA-i Numb i80h)olt0it nding SequenlCeSkaS wel as sequwees encodmne other rausitene. ineldum O 4 and ;Q\ arnd a travo ene w uh nt a1 set, 5 0i a pre ferred !.mbOdint te 1slected c-elkndo i thn pR ony arc iransfected £equience/s tIne dmlg Othekr tansgrenes, t mindg <X i34 and SON andm a transgene w ith l(Ji acta n in a preterred embodimnent the sebtected tells and/I their prog;efl arc transfcted with Vuri (lP RL) Numb Nstformt s) enceodiig :3equencoe as w all as~ segnenees encodmint other 10 transens. includog Notch I a. (iano v1 ., MNm oa preferred erbodont. the selected celS andor ibe& proenv ae nansfected \ uh ioqg i PRR '-) Numb isoformiis) encodmei sequences n~ el as sequencs encodoge other fansgene ineludiaX 0 K3'4 .SOX2 and Noch (eg notch i un 1 or notch 2 In urcferred embodiment the elected cells and/or hei progenv are 1.5 w 0ih ln I PR ' Numb ishinns) encodmg sequences as Wel as sequences enodin Onher transeenetneS chading O( 4, ',,'O ANG\' ad Notch. I a prend embodanem, the selected els and/or their progCn) arc translteted "Ath long (PRR Numb moform tio ng sequences as wel a sequnces ncoding other trausgenes inelung O)04 oN, \ANO.o and a tlanTSCene wln LUh- c 1eas.. 20 In a preee id mbodment the seleted cells and/or their proueny are ttanstected whlong I IR R \m \nt 1 roibim) encdog sequence as -wel av seences encoding other transgnes rm ludca n(' a G lh aN , and rtiln9 p.e ernsWenes ith D nl act 0 p e in a preferred embocdtrient the selected cells and/or theIr progerw are uasseted niah long ( PR ,1nm soltarms} encodog seguence asI a l s sequences erucadiru; other 25 1 anisgenes, mduding O0TD4 N otch, 110XB4 and SG0l. Over inme, other gene comtbimmons dif fermyw rom those describedt hereim mar he desctrihed or disco1'ered capake om causing cells 1o become inilt ipolent, p unpolenot. capable of s:lleneua or to becm ditleremtitnto However IhN ptnt application ever~ uch "genet reproctamnunn' of ans nud eated cell util mny nueleicacid r protem 30 electroperafion Isee (iane ct al I I Saiito et it. '00 I Yu~i '00KS I'hragI et al, 200 7/ WO 2008/150814 PCIA0T0820VO65fM7 Xia and hang. 200 ? 'enwar and Sursa 2007 saka aid hmai, 2007: 1 ucem et a .

200:; \im Alendloo. 2007; iakahashi, 200.; etQX )lposonw, 7ab501e5l atuo\autA etc. ee Golberg eg aL, 200; 71i et al . 2107n andibr another approach an hiding vial i megoo I o ther random rilterattion of the cell genomef as such means increasu safet anod ettclcy, h'cdudcd, Ot cours IYfomS the CeeotY 01 randem) uhierttOn are appreiicbes imnvoking genre ling111 and 'ildirele a nthodk designed to 10tirOdtlCC Ot renlove DN a t specdle Iikewse, this pment a covers t gleetic rcprogamrm a any nuQeated ell utlizig ucleic acid or proldht elWeltoporiitlon. H po0onlet anocapsuies, nano auts eIQ. >ild wTi another approach a5Oldina r051r1 Ilifluit inietUain 0Mo O rh radom alteruaon of the cell's genome as such ata% itJreaf&tv and e iten\, Such appmozalhes and methods inlude all known to h art and praetiale i die present mention. In a pre ferred embodiment. u atte tC d() ot proe ints) erresponding to a simgle i5 gene or p0own therof, (Prie0hir t1OhWe named heren., dicered dcc0iding tO methods doeribed heren. discered according to othr publshed methods; or knoo lo he mlttipotee plufpetency Or sfe mCa ducmg) are the on nuclear ac id ortm proteints) o\ erexpressed andi a otoduved t0 produce lmuiupotent, pbmutiem and tor self rene\ine cells from the selected ells, o.n a prelermed embodimeeo nucleic ac d"y or prItIsi corresponding a sMge gen, or pAron thereot (paricubei those named herein, discovered accorim to methods descIbed hireDn, discovered acoding, o other published rethods; or kiown 1 he nultipotnr pluti ipotency or seirenew ndcmglaree the onl nute acids) Or protem(s Oere preed and Introduced to produce ultipotet plutipo1r0t and or self ene wng els fram the selected ells ard the method utilied is eectroporation- hpsomes, nanocapsulen nanoaldt, and or another approach aitoidima retiosual/lenaital idi steuagtn or other r andom alration0 of dhe ell's aenorne In a epar ate preterred embodimenLt other nuclei acids or proteins) can he utilized in concert with the nuclei, acids or protun(5) correspondmng t0 a single gene, Or peiuiOn 30 itereoL (particultrlh thosc naimed herCen disov ered aceording to methods deserthed hevreinr disco\vered according to other published methods: or known to1 be multipotenet. 0 WO 2008/151P4 PCSO TU8/%5Mi plutipotence. or selbrenend mrduemg) so log as a population o.f multipostent phuripotent, ,nd/or self-rcne~nmg ccl Is is produced fron the selected eel I n epatnt preferred embodilent, other nucleic acid(s) o protem(s) can 1e uti/ed in coucert 0th the nucle acid') or proteins) e responding to a smgle gene, or poron 5 ritercot (pmasotlarh ithosc mued hcem. disovered aceobug to methods descrbed herein, disco\vered acs cordhng 1o other pbl ished methods; or knn to he mutnpoeno\, phnpotenev, or seL-tenewal iducing ) o nhg as a ptpuknou 1 rnhltipotent pluipoent aod/or sel-renewmg eelsY is produced rom A he selected cells and the mehod utilhed a lrotfporation.hose, itp0uCaUnocapsuales. nanulb anitior other approah RGda Idretu\ouiale(cntal iru tegrathon or other ran d alat of the cell s genoine. In a praerred emnhodiuent. nuclee au to p pro0tem ornespomdng to Nanog arc the only nuclei acids) or prot1i0o0 et\pr en\d adr0d irduced to produce ntwpotent. purip0tent. andier sellrencw up cel from tie selected cells crnd the method utilied is clectroporation. lesomes, nanadjpule nwnovaults and/or another approach 15 avoidmg rero\im vleontiv irai nuegraton or other random alteration of the cell' genome in a separate preferred embodiment other nucleie acid(s) o protein(s) ca he W Obred in conrt wilh the nuclei acids) or protein(s) correspondingfto Nandg so long as a population of mulpotet. plutpoten. and/or sIfrenewing cells is produced from the selected eels and the method utilied is electroporaton hi posoets nanceapsues. 20 nano\aubs, andor anther approach nviding Vita imtgranon or other random aberailo of ie eil 's aenome. hn a separate prekerred embodinent nudlei0acidcuy or prolete curespndig 1 Oct4 and S\2 we the on nuceic acidu or ptotebo 0vrvxprcned Sond/r moduced to proulce nuu li iteL pluipolent. andor self-rc'in ) cells trut the selected celsn And the 25 illthod utl 0d is elatroporbatin, ti n narocetpsules nnal and/or another approach aoidi ng viral integrallon or other rindy ou'ralien of the cell's cenorne. in d fa . eserred e r'nldi L ul' nlcl ed or protebobw ore utilized i conert with the nucleic acids) or protein(s) eol dig to Oet4/Sox2 so ong as a population fuipotent. p1 ccipoel. and/o se - nn cc Ifs, is produced fro the 30 selected cells and the method luilired is electroporation liposuons, nanoeapsules nanovauh, and/or another apptach avoiding vral integration or other random alerton of 1R WO 290811 4 PCT/S2UO0 In a separate preferred cinhodinemi nlucleic aeldt si or0 proteimts corespon0di0g. to Ioon ) \Zurit isoorns are the od, I e aci rr s) Srerceopressed and/or produced to produed nltulutien plri potent andor s~elenwin ceIls I' fron 0 the elete 5 cel and the method utb/ed is electropoath, brlisoies. naucapsukd nanov tuhs andr unther proh aoidg iral im rn o hrdm e action o the celL's genome in a separate' prtred ellib diment. uher nlece acid(s ) or protenur} are tili/ed in concert with the nueleic acid(s) or prcneis) corresponding to LoUng PR R \urnimb iso0b0rms so long as a popubrti1ri 01 muttipotirL pliporo t and/o el ene wing coIy is produced t rn the selected ells and the method is electrq rati iposomes, nanocapule. I~t.nOdriVl(5s anrd/or another appioach avodig vral integratI or other random almertlrU of, the cel7s genrrme In a prelerred mcl acidt 1or protcius) coresondig to\ang are tie on> nucleic I rit~d(s) or protein) oNreal ~resed d;an intr o duced o1 produce multirdipotent, pluripotent dnnd GTsel-reneWng cells horm the elected cells. In a pre d ertodimernt uesei nCidt 1or protein s corresponding to anog are the unlv U leic ac d(s) or proteint s) i erespr ewd and /o introdecd to produce mulipotentL pirpotentL andor sel-renewmg eells rn the selected celz: and the method AN bed is eleerroprat100 iposotrt nanocapsuleas nanauts and/or another approach M a dirw retrial r entivi ral mieganon or other random atetion Wo the cell> gnoe in a separate oreerred enhat, ther nuclei aidos) or proteus t e utlized in coaert witl the nucleic acids or proeis correspond. L. to 0s l0na as. a ponul soon otniul trooent. plu spoten t ad/or s0el f-enewn1ng ell ia pro01duced front the selecled eellIs 2. In a separate preferred embodint. other nuclei acids) or proteo) van n iiized in concert ih the nuclei acids) or protemn) corresponig to Nanoso long asi a wwpulattn o f multipotentL pluripotent and/or sel f-ewre cellts is produced from the .iecued cells and the. method utili/ed is eletroporauton, hposomes nanocapsules nanov aults ando an other approach bi aidmng r etroviral/lens iral iteaaion or other 30 random alteration ol the els genome I a pr eferred embomient .uien c aciden or prolinm correspondmg to a gene 6ith I i WO 2(008/151$4 PCTJ/118200IO%507 oI actiit are the od 2 nuclei audSt ( protem s o\eexpressed and or introduced to pr-odu c maopotent phtripotetl d sellreneumg cell from the elelc d C b A pkrTrd emblhOdimem mUakne ciss) or proheiit) corresponding t0 a gene with LIV detii &r e o no iucter cd i or protem' i ovcrtpresscd silor introded to produce rmulHipoteti Qplun I it o sell- Wreus COl\ h n the selected celk and the mocthed utilireJ is electruprain. 1 ip Osomel naiuOapul5 es nanoruuhs. andior another appnuch ,naigtro\-it .etumi m0gaile othe .jodem therttLon of' the tcelPS qcnomie. in a separate preterted embodiuro other nucleic adts) or protens) van he WOi1id ncert. ntllh the nIuleic <a'd(s) or proteinl(s) wolrsponh to 10 <o gnce Wth IF acdi slorg as a p puatilon of Muitpotent. lItPOtet, :Md/or seWTrenewi C ells is produced Lom the seected cells in a separate preferred ehOndment other nuclic acido or potemins can be united in ert with the nuclei oaedtk0 o p eint() correspoimng to a gene ub io y:I ac\'rv 1, I lg as a population 01 of' foUltpeet, piuripotent. and/or se w-renen cemi l MIS produced rnt the selected 2clls and the ncthod utiltd is clc0totori, liAi om to netpstlerti namo\n ults. anwod another approach aTilding reuuuralemturl imteration or other random alteration of the cel's geome. in a preferred embodiment. nuclec acd(s) or prt:0s) c0rresponding to On 4 are t.h-ic only uclec acd(s) or proteints) on-respressed and or intrduced to produce tilti potent pluripotelnt andor slf-renwing cells front the seleed cells In a prtekrre d emodumeUt, ncleic acid(s) or proteinrs) correspoindig lo uA are the only nu1leic acid U or protetal) o\ re prcssed an/e f rtod uced to Prudce mu ilpoteill, pjhripotent and/o scWl-vcei clls 10 Irathe elea180cted J ad the method 25 u tilired is eletroporation liposomes nanocapsuies nanonaua, and/or another approach ao luidm -il i l l oetti rz3i integralton or mother rando01 alteration of thet C IIS (iflOmYt in d eparate preferred embodimnent, other n1uclec add) or 0 pr~tn( sif can betlb/ed in tened sv Uh the nucleic acidt s) or proterUf.s correspundyo O lit so luti dt a popula~nto0 0n'n19l1m0ent. pilurpotent, and/or sc Ieewn . eclsx is prodatdd hr the 30 sdeted cell In d separate pregerred embodiment other icleie Aid(s) or protcims) can e util. ed l 2 WO 2008/150814 PCT/US208/6%5007 in emeert with the mon eS, Qid~s) t protcints} corresponding to Oct4 so bon as a popuhaiot ( nutipokruL pitipotelt dior selfrenen cells 1s produced i TIn t he selected ce! sad the method Uil iaed is electroporatlionlb hpisnlesA wnntc~pst Ido I2OIOS ItsmadOr anlother approach >IVOldiIL cttitkell10i)( iitt)eg2rat<Th or othes ) radom salton of the celps genorne i a fered embodiment nuteIe ctd(:>l or protemn s) correspoThling to %\2 are the only nuclecatS eSxrpessed ando mtlredwed to proIte multpotent, plunripoten. andor selrcneing eelb fronm thselected cell In a preferred enodiment, nelec acidt s) or protein) coresponding to SoAW are i he onl nucleic dc id(s ) a pteiril o\ereiprsed and/or intrtduced to prude nulipotena pluipoterti andr selfencwig m yels e the selected cells and the methcd ut lil/ed isN edtrOpOrdtionlliposomnes, tianoctpsuik'. naaoltunits anid/or ittothea approach ,vi ding retro\viral/bentn ial integrdti011 or <oter 'abdotn ahcleio $01of llte eel ls eninle. in a separate preferred cmbodimemt, thot nucleic acids) or pin(s can be utih/ed I > in concert \Vith the nuclei. ~ded}Or yotins) re pding to SA W lOng a a populatun t tmuluortent plalpjoient and or settiening cells is produced trom the selected c ells in a separate preerred em odament, other uleic acidts) or protei(s) can be uired in concert wlith the PUt b~lR fields) or ptotcm(\ 5 toespondsinga to Sox solng as a (1 populaton of inuil[ipoltn phlurtuent anda NC elt-ienewinlg eelse is produced font the selected cells ad the nethod utlihzed is electroporatinr hiposomes namncapldes anOautlts and/or onihnr 'ppromAs) oidm" retrovuym ent i rcd integiOn mo Other random alteralin o vthe cells nuotrAice Un a pretbrred emnbodinlent nucleic altdo p 2> the onry nucleic acidis) or prorteiros oeernesd ado introduced to produce noitipotent, piuripotert, and or seli-rerne'us en&ells horn the selected cells. In a pret'erred embohment, nuelic acido ofrprotcios) cesmesponding to Iir 2 are the on!y nucle aciden) or protemt 1OVletexpreswed ador introduce to pr oduce multipotent, pluripotenh .ad/or s'elf r enenn cells t110o1 dhe selected els and the nietnod 31 utdhted ts dectroporatuon hiposeme mmh capsdes nanovtdtus, and/or aothe approach a\ oldinretu 0rainr vrald n1 Stegradiion or other rdadom altercation of the ell's genune.

WO 2008/1501 14 PCTUS200/%5027 In a sepm'ate preferred embtlodimeet, other nucleic auidos or proteinms) can he utilized in coe t \xith the mtieet: acid() 0o pf0tsi(s) yortespondhng to l0n28 so 101r as a Population to mulotem, Arip(tent. rnd/or seinenen wig cells is produced Non the selected ced I ad the method ui 1iied 5 eltrtoporattion, hiposomcs nanocapsulesx > nuanoeudHis and/or another approach a\oidiun e iral l1tm ird integranon or other random aitertion 0f the cell \ genomne IQ a p Sef enred ?Inh~dilent nulee.i dste( or protein(s) corresonding to e-Aile are the on\ nuctle aeAdis) or pote h in s) erexprissed and/ao intrdhu ed to produce multiptent. plunrmter, and/or sel rencump ell hrom the selected cclk { i a prfeiried emodiment nuc le i idts or proten( s) cresponding to ec are the onlb nuclei acid(s) or protem(s) oerexpesed andAto introduced to produce nuupotnt pluripotent. and or sed frenei. cells hon the sOlewted cebs and the method utihwed i electroprn on, Isomes nmOeApsues nauIt, and/or anther approach at oidlmg rerirarllentir itaimegratnon or other ra dom alteratin of the cell's grenunme, 5 in a sepiarate prefebrred embodiment mother nuclei acidts) or pr oteinits) can he utilized in Concert uith the nucleic acid(s) or problems) corresponding to c-nyc so long as a poplaton of mul1t0o te, pltripolalet ld/or sefwing Clls is Produced from the selected cells. in a separate Piowtrd emhodinent other nece aetdsI or protemnhs ct he utih/ed 20 in concert with the nucleic acid(s or proteins) corresponding to enc so long as a population of muin potent, puipotent. and/or seW renewnmcells S produced finsm the elected cola and th method wt bred is dectroponition, hipsme, nanoennaules, nano\vau)ls andOY another approach avoidincretroiritenirhai integnrom or other random alpeca3 ton ot the cell s* enon0,eC 2 ha a parati p dried elhdimeL nuc leic acd(s) or proteins) correspoding 0ledt and !01 are the only nuc leic a 4d(s or pACeM M \erckprtssec iNd t introduced to produce multpotent puripoent. and/or sel renenmg cells rom the selected ellsa In a separate prefer ed emnbod inent nucleic acidt s) or pr owemns ean cresponding to Get <nd Soxd are the onl) nucleic acid(s) or proteil(s) 0 oer eviretshed andior introduced to 30 prydte ntultipotent pinripotent, and/o 9selfrenew ing ccdls ho0n the Selected cells and the method uticd is eeAropration. liposome nanocapes. s, nano\aus ,nd or another 14 VO 2(M8/150814 PCT!IiS200S/065007 appah avoiding. retroviral/ientn ital imegrat in or other random aiterat n of the es genine, In a separate pretered embodiment. ther nuclei acidtS or proteinS) are utilized in concert with the nucleic acids or potein(s) coiespotiding to Oct4 and Sox so loin as a populaton tf multpotent, pluripotent and/or ad Prenewing cells iS produced ITom thie sekCVOted cols. h a separate preferred embodimN nt other nucele acidts) or protein() are utilized in concert w1h the nucleic or protein: correspodmng io ani 3d Ss' Wi long as a population of mudipeut p and'or sell tenn ing ccllt i5 pt0dtced tIof the 2 e lcted cells and the method utlzed is electroporation, iposomtes nanocapsules nano\ axlts. antd/or Ja~ bepproach avoiding ret ro\ traileniO v il tnieygradion ox otber randin alteration of the cols genome in a separe pref erred inboddimnt. nucleic acids) O r protein) are pidirag it Loe (PfRt) Numb iusormns are the onl\ nucleic aidf s i or proteinH t uo erexpressed 15 and or introduced to produce notipotent, piuripotem,. andior self-renew ing cells frorm the in a separate preferred emhodiment, nucleic aeidi or protemis corresponding to hogne ifplRRi Numlb isotornis ar e only nu1 aCleic acidts) or proteins) trvcrexpmesSed anJ hr introduced to produce im uipotent pur potent, and or sef-renen eng es trom the 29 selected cells and the edhod Udili /ed is eleetrop fanon hIosoles, nanoeuptuie& niinovtiults and or another approach avoidimg retroviral lemdi iral integration or other random alteratIOn of the 6Cels cenotne, in a separate prefer red embodiment, other nuclei. acids i or protemii are utili/ed m~ conern wth the nucleic acids) or proteist eonrespondmii to lag PR 1 um tsofke ma 25 so long as a population of noipoxteme. pl uripotent arnd/or self-reuen ing coils is produced from the seated eelkV I a a scpraic preferred embodhiment> other nuclei, acids or proteints)i are uti tied in concert w ith the nucleic acids o1er proteinos corresponding to ILong { PR R l Numb Loforms so long as a population of muolupotenit piun ipotent andhor sel f-renewmng ells is produced 3t0 from the selected cells and the method utilized is eleetropomanon. liposomes. nanocapsuiles WO 2008/151P 4 PCTUS2ItS/065007 a nt y pro la votdrng ttvrlIitrvd i Iutegratton rtao\>ults, kind/or another approach amdn ersta etsfa tgainor cther random abernon of the cea itenome. in a seprate preterted ebodiment nuc lei 11 protein(s) corresiphdin (to Ct t So , mid NanoM are the only nuclei acid(s) oa prtcM(s) o ereapressed t and/or introduced to produce iniupthen puipotent. andor S ht enewi ma cei foom the Acered ellsA in a separate prefer einodimeut nuc leic acids) or prote) coresponding to 0c14. 50\. arnd Namoc are the .tl aclR 1idts or 0 pro leinisM\ eetls and'or otroduced to produce inultipotent. pinipot and or se'tenmAirg cebs firom tlhe 1 sAlected cels and the method utilized is ectroporarlo, Hposonmes. namocapsuis, nrians.307 iind/or Uaoter approach JNadvoitg retrovirdiflernivirai interat ion or other random alteradio~n oftIhc ccl irenorie It a separdte preredtt embodiment , other nucleic aeld(ai or ploemiil ar util/ed n concert with the nucleic acids) or proteinist corresponding to 0ct4. cRX2. and Nanos No 15 long as a poplati on of mutpoLent plur lltnt. And /or .e .wog w is produced from the select ed cel ls. in a separate preferred embodient other nuelee acid(s) or proteins)ar tited Min cer w ith the nuclei ac ids or proteints) corresponding to 4 eN, S 2 fmd n long as a population o' IutipoteIt ptipoteti and' seI f-renew g cell s proditced t'rm 20 the selected ells and the method utired is eleetioporatwn, Iiposomes nnoapsules 1XahO\uaults. and/or another approach a\ iding rora i/ lenti Vral tutiou or other r andom alteraton of the eW' aetinme. In a separae premerred eiedimnlt nucleIc acidts) or prde.Mts) cirntspnndtn to I. onc IJRR ) \utrmb isotrs rc fthe onl ucleic acids) to proteins) overexpressed and/br o introduced to produCe nitipotelt itpte tit ando sc n .lenew c ells frun the elCeted ln a separate let'krred embhodinemnt nuclei acids or prteims conrespondmng to Lon iPRR,) Numb isoforms are ihe onl nuclei acids) or proteins) ot'erexpressed and/or inroduced to produce mulhpoent. pluripotent, anchor slfrenenmg cells from the selected .) cels nd ie method is eletoportion, Uposomne nanoepsuies nanoauits ador other WO 2908/15184 PCT/UST28/065(M7 approach av oidmg rletov r adedivul lteration or other random dleratlon ot the cell's giohne. ht a separate preferred ebodAiment. other taucleic aida) or protan arc utdzed mw covc t with the ntl aidi or prin ) coTrespondmg to i ang VPR R ) Numb isolims so long as a population . on ltipotent, ptunpotenL didr i -tenewmg Cll6 is produced irorn the selected ed In a separate preered embodthmenit, other nucdei aie tpoein(si are utthed ni concert with then ucice acids) or protein(S) wrrespondinp to Uong (.WR&) Numb isoioroi 5o long as a populaton oX tmuhipotent pbmnpotent, and/or selfrenewing eelN is proahtced tr om1 dhe eeted cells and tl: nethod is eietroporattort ilposonfeet fanocapso Ies. nonaults. andor ano thc approach avoiont lcryu alenti ial itglatvon or other rodem alteraion o3 the ci 1 eome B is to be understood that. ar> combination of nneletc acid or protemn sequence described heein can he mihied y Icludng thoe corresponding to Numm oulr umbhke so long as the desired cel population or bchavOr is achieved. Si arU , fi shoul be understood that the methods described herein for initialtog dhk itiman are applcable to any induced ornon-uduced umtipotent plunpotent or sell> aenawfing 'Item kdi eiter progenitor cdlls. or other selected ells. not cnN those obtained i the. sa) nner JLMenbed herein. 20 1is to be uruders tod that anm combmInaion of nucleic acid or protem sequences described heren cn be moditied by excluding nuce acid sequences or proteins corresponding to Numb and or Numblike so long as the desired eel population is achie ed In another crmbodimen: the arous nucleic acid or paotei conhioatimo deribed herein are employed with the exclusion of the nUClecl acid or protei corrspondig to the z) nmbtie and or Numb isolorms W a pretR'ited embodiment, the selected cells andor Their proaenv arc ells ta hav e been rrcnedtial) i-rnoddtId betowbed.d in a pretred embodiment the transbetion steps desenbed herein represet transient transtect ion 30 In a further preferred enmbodinent such transient iransetion s accomplished using 17 WVO 2008/150814 PCTW$1s/65mr vial \etW tAtl do nineitiah trio the host unorc In another pret0rred emtothnenl such traisent trantectionis aouplin shed using skandart transteetior; technjtj es cdectTopoHit10n. chemncdd vmcddi ed tr ans teetion, ()oCapstt wi o es.s tc) 1A tlcor an uOtnem ii0po1ome9, limn-m ue. aO~h l \ ttune, Qther gene edoain dllfermgr un0m those. dees heAei % p he deceri bed or discovered Iapble t'edtN~Dg ecC! to hycenimltipetfi~ut pteent tcralle of sI ttoewal or t begi doierentiating lin e\er this ptentappenion als cmees the pleni reprogranmn g 4 'inV md heated cll u/mlu nude icJaid or prtlein eleetropuraUion m example methods e e evntrt ci ai., 191, Saito i 4., 2A. 01; Yuan, 2001 i hunig et a, 2) 7 AXa atd /bag '2007: Cemaar and ersa 2007: laka and lmai 2007; I iembiura A W \an I endion. 2007; DMEaha. 2007; etc )eeimprion. ipsomes Th00n490 in ads anol va Its, and another appach d\voidg \i ial integration or other random alteruen o the cells enToIne as such means incrase safet and cenC) III amvn(Ph)r pre dmhodimcnt uransetUmn With Yag MR' -) numb h IN ormn 15 eneding eguenes and't er fltfdhenei nchpmeltidc\ targrunu numhble and sbort .numh ISOoltemlS} iS aiceoumpatmd 01l eatid b drnienlt o; pur 1ein, t anAlection n\ ih other sequences including hone l sceed t0u those encodo human I II re u tod , i tO onic~tstau ttN caidiotr;ophinI .11 -1 L. 11 A6 hpAW !! A 11 R R pp , 30. tX F A K 4t ano "SOX". andior Gi-4. Simrldanus Irnstci ion with any subse ol these distnct tYnspeene seqences can be acoimplisheJ b many means known to the att including th une of Angl eenetie ctor, ultitple genee \ ectOry. sexual {ranseiofln and selec'on based oi diict marker' or10emn ,rnd.?omanuthiotic risaunces In another n erred embodiment. eClI transteted Sit 0na {PR+ nutit 25 Wis-1a ) ita C cUitied iMt a &di cultue pr"onOM an Optimn growth r at sIht a described jbn e, and that iudes LhUb h. oncdsant in, i D 10 iU and SI, 196NY steel ator, 11 achdotrophin- I 114 hyNper-IL"6 4 Th, -nd or soluble apr 30 Assessme of Poteny and an i terentation Puripotencs and muNipotenY can be assesd bh any means known tohe at 30 i ncdg I naUspkraoniw .a s2)da. e undtler cIlnltljols pso0mtia cdrnhrood bod) tonin 3) iectito of Aels ilo anmal biastocst stage embqis wh sunSNeuent 1 8 WO 20 l$MI0814 PC14I820i 1m? development and :4 R.NA \ region asss egS RaV IPCR and mironaA based anAses ior gene erpessim asow jated w i dIi'erendaon. oul diptencx ph iteaI ec (see G(un e at,. Nuh) SI cobny toriatom as w el as hv x ke mor phoAog One approach disclosed her etn lot dctimt. plrtripoteney in selected cel nd mor their progeuv invoktes Itansteet ion with a repoler constrUct N011 g the ig poter ope ably eied t a Soreseent prot gene ha allo s identiiention and tnnehment f Namo exessmfg celk using F luorescence Activated C'elI Soing (:'\Uz4 etc. la a oteferred mbodinvrt endyo'llouS cCls (0eg cells surrounding a bom il0 Ro sue) re transietted n v1t n wit gerea it Sector\ encoding the log (P1", } nmb ioiokrl(s) 10 ale Am e W uninm n ith other ranseenes named heren to transiently promte renewed O neitased cell prolietGoThIs approach can also be nh h/ed clinical) in the seltina ilt h popastuc tissues diwrders where stem progenitor cels arc anrrmalb depietee and other disoriers where the approach can be show n to be beeial Whicp Im Itt ct:ie aug I'll Aolmnus 15 a order to achew b! neural c musle a~ and other cell populatinS eapabe of i u0h r am runnta l o reuidted difelrntiatein v \ivo. selecied cells) an ib heir progenv aire Aop ll lilateAted w ith 1(PRO-) MunihltOm SqutJliee) dbt S:UitSt ohgcnucleowide sequences and expanded by growth for sufficient time to achieve the desirable anohber ofteNl imtgert m \j l) (Js deseibed iibo0), owing ti optional sep, the selected celh and or their progeny are washed ree of the ctokimes and agents comrisn the expansion optimal growth media. and are optionall\ transifeted with ithe aueleotide sequaeneecs) encodirw the Numriblike gene andior "shor{' (PklvI Numb isotormis} and of1thtie 1liponaleottldes targeting the long I PlI isciins etc Ic g /aehres et aL , 200n then euured under ndiftums whih promote 5 dilticeuniation of the selected cels into the Jesired ell lx pcest i'n most llnsuees the cels are then caused in the nprsere of 5N > eu ime setumin and agentst 3 promnolng diherentit tion of the selected cells and/or their pinnen\ into a desired cell population The presence of the fMtal bovine and of the agn(s) provides i growth or po hiteration at a rat that i less than the opmhial {or expansin) growth rate> and 30 !I ors dit erendiation of the elhs into a desired cell popular tion The agents and precise cihure condmntxns arn sected accordmg to the desied cell population as described below 19 \VO 2008/1501 4 PC T/US20081065007 Achieving Neuronal or '\erM i Popvdadons When the dewred cl popukain in a neural c ,l p0uiofl. the 9essiull ransfetcd ells are oltured under Lihtonsy that proote grow th at a rate whbleh is less than the opumial trate and in dhiree 0'ign( r)porinei different iation of the c ells nto neur ce!ls (ondtions proniot u d1il'ecnntdoA intlo ne ons have been desefibed ii numerous public ath in dn e user etdi, 2003 C'ihun et a n 2001 ldrha> c 01 , 200 ikeda et at, 2004 lied orA 'd W0 O \ nti at. 200:rd \erig et al. 200) [urthecmnte. comtbiomg TImOOWIC :Ild ep tl su with the presence (d additonal e tOkieS \ttvrsafipeclies nenronat eU ivpe. dd1~leRt ati 1\ viro (e S0ndiarjdiic t. 20 0fx JO Soundardaan ci ni / ill', l N l tc61 4i ti a pTefired embod owen x n ar din ufAeremi o1 neurons or neural cell occurs i the presence of A 1 50 ni nen e gYowib fattr NUl In a prectl'ed enildment, when a nevulmni popidation is the desired eel [huniL 1ransfetio s11b $Ug 4entx eneoitdlg shlt wUm? oto0i0 s ( andt ri Rhliike) is dee0 aedor ePn 1e 'htnsiet or perm,1nent tlorlo u t oher "eloets ineludina ones selected fon. Os eroding urr .l RI N urosenn I, Ndurogen1n2 Nearogenm. \lash 1. Phw2b, Phom d Iand. Gana Shh. 108. 1 ixl N\2 Pet, ULsI and o1 Rnxa in another preferred embodiment, when dopaminerie neuons are the desired 20 eurondl pouad10. iranDletion wit s1 equellces eneodl~ in0 s1Uort nuni0 hsobri (and/,r wih6ke) is.0c pdnied or replaed ytansient or permainent trsfeti with other sequences neludin, ones selected ron te cruncdinw \IddlIl, NW. N' ur. pin l in 00a hU another prcmerced emibodimellcth tvh00 sero10oelcr tl eurons are the desired neturonal 25 transecuon with sejuenes encoding short nu1m isolorms ad nuiajli ke} i dat.hnint ed or replaced by trarklent Or peritanent Iran sleet0 on withter secjberves inCttdng ones selected ifrm those e'ncodhmin MlIMS. ..\nix I K N\A 1i32.d (iat3 alldor Aell, In tuother pre:fered embusdiment. uwlen dh10inergie w71trotis are the dk ltrrd tiCU00itd 30 p*oudat91, transfetion with .yequences at shodrt 500 numb I1sitl0smaddor BIIII iet is atNtpqaned or replaced by tnsient ox permanentl transe010 mith other squenees

A)

WO 2008/150814 PCIT/US208/q6507? neudgKXlS selected irsmi those efleodutg \ilS i PhOX2tI and/or IU/f 4 i another pre. 'ered em bodinrenL when C\AB \A7r1de eurrons are dhe desired neur0ontd n0ptlaon, itrnsietln w ith sequences. enleod m shortll nib s~ds'sms (and/o ntittlike I is acuompttlie et Orephaced by trasient or IermanhQ' tttabteettfn with Other sequences 5 inehidingL ones selected froni these enethiuc MAS\Y11, Phozaa and 05.Ra il f'o11ed. Optiovdis b culare ut media suppleniemknd nikh IK Neutolphin 3 ON\ I vd'or nere growth JAactor{ Mli. another prekrred cmbodint twhen nor g neuons ae the deed DeuRolad popuattio. tAtnsNetilon with seutene t'niodwti short null 'O1Urns imd/doT I5 nummke} is accp un1id or teplaced h\ lnslienlt or pernuent transfectit with other sequence including ones selected (torn those e ncodig \ash1, di/lald, Pbs,1 Ptl Cata dad/or Gatad. in another wielered embodidnt, when1 (I Bergic ecurons are thle desired nieuronid popul d httio. evnsied tun With S eqtnesl encodil short numb io srms (ando num n blike) is 15 accompamed or replaced h) irnsient or permanent trasetion \t'h other sequences ncluiln oes selected li'm thOse coding PV X itlx\. N , dNtisense hln RN/\ ndo l Wther wVFS o tare 'tm s\Mnthetic ohgthuclelsdN hi another p lTed emodiment when a neuronal or neUral cell population is the desired population, cells transtctd with short (PRI& nuimb isoforms' (and/or numbhhle) are culttued n a Cell cultie nmedum e promoting dilferetiadon such as described above and that includes one or more of the ibiloniin agents; etimoic aciud, N lL 4a iclinme dem ecd P31r att Kammr (A)\ I and interfron gamma MinNneNnma \chteu ng~ \1 usee (Cel Ppuhaiurn. \\hen the desired cell population is a muscde popukaion, the successfuldvtransfrczed 2> cells ar e cuaured mn the pr esencet an agent promoting didttiennau of the ellin to muscl cells and growth at a raze 1 c) thin the optimal rate. (I'ud io018 promotln differendation wits nuscle cells hav e alsi been deseribed pri ly Makamura ct al. 2103 Pal and Khanla, 2th5/Pipes ct a ,O k0 lhiie i i. 2006 Pa d ekhanna. 2007: lehiir ci a. NE. 20 \ Patent 6127 i1) I uiernere. exposure of selected cels and/or their 30 Iprogenls to heaVe\leile I s-avr)cide or Int I h W A'Nlde in the premece to additional cvtomnes lihvrs the Inittion ol muscle ty pe diUrelntiaion lt vitro \VO 2008/15014 PC/S0200S/065007 In a pretbrred embhodinent when a cardiac muscle cell population is the desired en t 1PRI(- innmb jotorms rndi or numbhkel are eovtured in a cell eultre mediwun promoting difaleiatioti in c-rihny) vies {neot "a] MO; (uan al., 200t. e or that Unc specI c emns at coneentdaos pronmng N (oza 47e"W didere1imlou te a , Ch J .rn ul fide (DMUMl 2p% nord bnme serms\ (nD. A 1h 7) mM retmoic acid {RAd and '0% cardornmocy tes ondjianed medium d ud at atL 2 , In am>ther preibeued ermbodiment,. when a ealdtectmuscle cll population is the desired population, the cells are lso transfeeted wilh maclecoode sequence let tIding one s o0 seieered from those seguences encoding Gat 4, (iato 5 and (aud 0 In a peterted embodimem, when a muscle cel popuaton-is the deAired cel popu aboaL ra"sfeenlon wth sequMens cuodma shor umb isoirms (and/or numblike is e nnipdmed or replaced by rasient or penmnanerI trance n wi dter seque11ces ncdudiog ones selected from those encoding muscle type speCific N Alvencodmng 15 sequences. \hvoD ,\togenin Nti MvW Ma l. \lyvo arint f'rJ an:d/or other muscde aa scniption actors. In a preferRd embodinent when a smooth mUscle ceI populdtoi i the desired CeI popul auna tanstectron w ithb sequences encoding shot mnm soioimw (and/or numbhket is deeonrpted Or replaced he transient or nermanet trdnsetlit with other sequences 20 inelc tkbng ones selecred ifron those enuodingthle m~uscle type speelme Mvoerdin mhue ilde In a preferred embodiment whben a skeletal muscle eel populruion is the desired cell populIation, transtecuon w ith seque nen encodm g short. nmb isoanus (aud/o numbh kel is accompamned or replied by transent, or penanent ranstection with other seuenees 25 mdudre.nes selected from those encoding the amusece ty pe >pecific. \ts ol and mn vogen nucleoride sequences. In a preferred embodiment, when an oligodensroc~yte ell population is the desired el popr nen tanstection with sequence encoding short mumb iso orms (and/or numnb ke) is accompanied or replaced bV trnsentho per manent transiection with other 30 sequeness meludiog ones selected from those encoding the ohgoedoeyspechie (1 GL 01 A2. arid /fp 4 88 uudeonide saqus AND 20 M/150814 PCT/S200W06=7 Sirmdtaneous transIeton With nyov subset o these distinct transgene Sen abo'e can be accomphihed by any mnczs known to the art ineIding the use of multiple g enetic veers, senal iransfection as uedi a seledism based on distinct nrurke:r protemns and or antic resiStanCe

V\

t hej the desed Mel MUM Ippukhi is a honatopotetic cell population, the di(Therentiatlon medium mechides spedi ie agems ut coneentra nous prmotin dill'erent im ion mi) aCpluc progcemtor' cell s ?Le auseumr endothielial growth Vactor (VE\ Mil thtonW)opOctm ine. (1m (hiaono, 1997: \\ ngt S 2005, Srivastav c JL 2007: upta ct aL 2007) or dfferenaed henumtoposene cell vyows C acoiding to methods known to the Val aM rmng di lerecntiatt'd bematopolic n cl tvpcM from indi(tlrentuaed o pluaipotent ehs V \\Wheu the desired~t edl p0opU 1nioI ii t germ ell population, the differenta o medium includes specitle agents at cuncentradions promroutng dieretotiation into aerm cells mg Naunemia ta 'AtMA Y 1$hen the de\ired eel noplation ism endoderm md pancreanc islet cell populaon. the ddi erenthuio n media includes specie agents at conentrations promoting differentanon into crdsederm nd r p eanrle iset els (e. 01 t alAJ. 20I6' Dennor et it, 2007: Shim d a, 2007: Jiang wt 2007). hi t preferred embodimemnt di Ufreemdion of selected cells and/or the i progeny nj'i 20 occur in the ddferenuation medin in the absence of transition With numblike, short N Iob wdotormtr other transgenes. shhough the difftereiation medimn ny be In obodoteuts a smnle vetor ul be ulied which comrols the expression of nuceode eqencs ieneodag thie "long" (PR i-1 ic'thrnn of the moktnmahian numb 5 gene and/or symheic oligonudceolides targeting iumbike or the shown numb iofrna under one regulable promoter (e~g a ietracv clinec-reguluted promioter), while the Numoblike and short Numb i storms (and or synthetic ohgonuoleondes largeting t lorg PR- K isoforms) are expressed under the control or another, distinct, but ac reauhbie piomoer hus, the long IPR 1 numb isoornm) can he expresed (and or Wort iinomu pressed 30 w hen ax pnslo n to t1 selected acs is desired ln an inducing ngent e.v, etracyli i added to the rouTh medium; biter nonmlike and the short isofnrms n he xpresd und- 01 2I3 WCO 2008/150114 PC T/US20081065007 boe (PRR , numb iaronlts repressed I uhen dei renfitoaa n is des red. \lernatiely Proema and peptides corresponasding to Numb iooorm \'otch, 001 4, t 3 and other D\A sequences hated herein may be apphed mn nalogous fashion to Elected Mlin and or their pr ogelny a electroporaton ( I oten ci at , 1994 Ri te and 5 ito,199h8t un nat particles. eatiui lipids tusogem hposomries eg. Yoiaa et W, 200. 20070 n) m in le 0uA of in combinatlom with Agenetic transition. Generad jy, etroporation allois fomr hbhi~nfeln feee gdelent productionl of the desieed VL widwnittelnie ntet:'t in of the tamspelle and ls therefre sisocited with increased safeic. Ie MA or RNA enodin protin(s) or pa\ ppid) pirnt in proI ferion mittrattenit W, phmripotent ality or diurerentian on of the selected eellIs rman be isated in aceordance with standard greneuis enymneermg techniques f'or esaimpte, by isolaimg such DN \ from a WDNA lbaNry of the pecei cell hnue and placing it io an appropriate exprteknion x teeOr, w bich then is tranafecied io the c wte t s 15 n another preferred embodiment, endoder and pancrc idet cells are the desired popuaidoit and Itrdesttion with sequences eneodn Shon numb i4uorns (and ar nntlbke) iaccomnpanied or replaced ha transent or perunanent11riustection w ith other sequencees includmgvq ones selected Irom those encodlm l'osal Soy 1i L N and/or Pdx! i another prere odd bodnsent hepatocy les axe the desied pulMadOn, aid rdsfeton wih sequences enoding short n ibseso rm (and/r nuntliket Is crntpanied or replaced by tsient or permanent tuantbetion witlh other sequences meludin ones selected from thoe wenodrig hepatic nucea (tictor in- N1 INP 1\-4 t NJNIdand ereh-bimding patein, 1n another preferred enshmb nm henatopoec e ellIs are the desired population, and 2$ tnansfecoon unth sequceS enleodmg t>Short ntumth isotorms tinidior nutmbhike) isa accompanied or repla cd by transient or peirmanent trnseco u wth ot her sequences mrehudine ones seleelted Lin those encodhng Run' \ \ML!J and \0\'fQ t'('\A anr cell cuure in the presence oF colony stiimulam WacS specilie or the desired ce popunaors. I' be Run I . a isoform is iroduced when engratne is desired and the I voform (1 whben differentingtion ta desired (Creemer< t al 06 In another preferred embodiment. chondrocytes are the desired ppul en, and 241 WO 2OWWONW PC/U S201W M907 transhon uilh seguenes <tenio Shor n o orWnh ans W Jm rn mbhkell i cc0opanied or replied by transi'm or permanen; transfeetion of ther .sequences inet din ones encoding ot CRltbindmgo poein, GA., andior RX2 Un. another ptcferred emodinot ine cells (cespeciaUV osteobitsts) are thie desired 5 pIlain, and tranlsection with sequences encoding short numb isotbrms ndior umnblkik) isaccomlpumed or repl~aced hx tr;msienrt or permanent iransfecti on of other SqusOCDO meMudingo Runx in a pefered emlbOdimenft the QCenectors eoding the long Numb isofbrns (suet as those described here in Sre introduced i0Tanty or under W Control ofi ra gubbe 10 promoter. into endoacenus cells m in ur in ordferto caSeS those cells proiiferate urnuntlyr, In a pi terred emboimen endOgenous cells (am cpendvnud zonc CellS o'the central nervou!Ss sstr) are transiceted in \l vo with rgenellc \ectors encodilg either the shortest nm isotorm or the numbnhike prcdemnts} alone or mn tonjtiton uith other iruus'eres n aed hcem, n. order to lransiently or permanenten promote rnewed or 5 increased differentiatton iespee'iallyi nurana different ion) ansd migratiol) of progentorpedymt cells nM tm !he cman nrrv as nltm). FI renc d or increase K nmsnred in rztms A1 the number of cells show in new-onset expreSsion o marofkers assoiateJ w ith di fferemiation, I his ma1 be accomphshe~d bx int'odu ti n h eetic vectors io the organ system usmg' methods suitable r that prpoe (See e~tnpkLe In ai preFrred emnboaient endogcnus cl s e~g0 ependyal m one cells of the central nervous sstem) are rmsfectcd in via 1 Aeneue vwsto> uncoding the long numb isforis andi or other transpres named herem, in order to traniiemly promote renewed or inc.reased stein cell proliemion (w ith subsequent difrcntvaan of pwn cy ellns hn. renewal or inirase is measured in tis of the nundber cellshbowina new-onset e5 xpression ot nmarlers associated with disviding pronenitors 'lhis may be accomplished It irodhction of the genetic kvetors into the organ syenm using methods suible for thai purpose (ee u.mples) likewise this approach issalso be suitable for mnducing renewed or mecreased ditecnttat0n frfm other len cl popilaions in other Ntsues (such as the sin, ete) rhis 0appnno can be utilized. (or example, clinical F in the setting o' central nerous ystem iu~lirv disorders of' other tissues wher e normal diilkrentiation or nuoration arc mnadequate, WLI 2in10 1$ 4 PC/US2Q00/95U'f dv spl te th orden~ and other dsordeta whee the approach is beneficial In a pretIt emboent nueic anid(s) or poeIn(s) e npndg to a ng ie a genc, 0or nliti theueof, (p tthularbl those named hereto, deseocred accorim to muedhods described herein, dise ce ac. tcoriog te oter pubhshed methods anchor known to be a capable o imnating the deHired nmu of diler-entiathon) are the only nuclec acid S) or protein. overeaprsed ad h introduced it Kindate differentation i the selected celk in a prne erredl em bodment nueleic ad proteins eorespondng to a single gene, or portion dherco t onrucularlv thse naned herein, diseo\ci ad accordiz tmedhds described herein, disevered accrding to oter pubbshed rnethd anmd or know n to he 1(4 capable of initiating the desred manner of derentianon1 are the om) nucleic acid' or prein(s) oerepressed nd/or itrduce to imte doentuition n the selected cell and the method utilied is electropo ration, liposomes nanocapsules nanov aults and 0w another ppudch a\>m r raItiral micfation or other rando aiteratin * thle ] genome. 15 hx a separate preferred cinhodiunent, otter ucleic acids) 1 rotemit) ean be un i in1 encert w ith the nuclei acidtk 01 palt frespondig to a sinlet' ene, or pUnsn thercof I iii en lat d those nanlmedi llrunf disecm1ered ocecorditin to nethiods described herein idc to other ptbllshed xethodx, and/ o kun\wn to be cadbie of mntlato. the' desrahe manner af dificlentiauitm so tong :is a population 01 dmferentiatmnu cells is 230 proved from the selected ce.& In a separate :pret'brred embodiment other nucdei. *ud, cif roe(s) can beuI /d in coneti th the .nuceeteacidtsi or groteints) ceYtsponding to a single gene, or .fl0't10if theeot, tpurtieodarty those named here in disco sld aecording to methods dleseri ed hiere inL discN trled acce cding to etr pUbikbed methods, aan Vii known to be capable of iniQiatinN the desirable manner of dilerentiatin) so lone as a ppudaion differeniathai ee] as produced Woem the seleted celh and the method utd ized is .electroporthn, hiposomies nanocipsuilea non luts and ra ntothter approach avoiding retro iriu lenxral imegarationr or other random altertinl o0 the cell' a genome. I is ie hr uderstood that any combinaUil of nucleteecd or protein sequences 3( Joenedherein can be miodited by excluding those corresponding to \tnb and/or >hxmblikso long us athe desired cell population oT behi a is ac hie\ved WO 200115014 PUT/USTMN/6og 7 Nmd~ii Should be understood that the metods desclibed herein (or elsewhere) fs9 Itiati g di firenti Inu are applieable to ani) induceed or non-I ducred inuhtipotenL. plunpoieznt or self renenmne stmr tells or other seleeled cells not only those obunamed 1n the manner desorbed heini ) Soures o[ heleelwd eels Ushe popuhauion ol seleeed cells ray deRome from arous stem eels.k piegetor cells and somatic ceI Avever snaie cells lackirm ucet e . "Maie, htDiun red blood eys are speci hcaly eluded. Selected stem cells mays he dem\ 'd frm existing ell lhnes or slated rom sored, banked. or eupreserved sources I ril scores of stem ells inlude 10 bone marrm peripheral bNond plaeenaul blood, amn iote ithiid (e g. De C oppi et. aI. 2007R umbiial cord Nood tey, Ao et K 2,06, lin et a , 2007 adipose tissue ie.g. imbNe et aL 2007; elt al . 2007 mon-human embros, arnd others. teuAt'Ang leukoevtes and other non-stem cels max ikevse be selected and subjected 10 thy same cuare eondaiens as described above elleoe\ r thm they acquire mnhlipotenex> plaripatLene) and/or seltrenewal as 5 a result. Kampls of other acseible somate ells useful in this mventon include lymphocy tes and epAheha (e-g bucua chee cells, Isolain mad colMec 1on of cl ls selected Or use wuhm the present iention may be perIImed by am; netod known to the art. In embodirments inmolhin annuals. stem eels isolated frnm prostate..1ests 20 embryonic BoanL and intestine are also disclosed ts being preferred soues of seleted ells In a prelenred emubodimren, the selected, cells and/or their proemn are euhurea in a three-dimsensio nal homniut A fOther su of the present mnenton i to pvdc els o i& uei the producion of' panent-comopabe and paiet-specific Isues and orgais fOr transplantation to parents 2$ deemed to be recnn such organs or issues It is disclosed herein iha~t tK rilipotent. mtlipoet andor u itrentiatn g eel s povided by the methods desribed herein (or timusr methdl n miil/ed in corunetion w ec lhQuges unled at the produdton cl such eyans andor us'ues e g oAW cn tI a . N00. Nu c aL. l 2006, Campbd m Is 7. Such utilization is spseificalh covered by the pi esent to cntion, 30 Fo i mstance, pluripotent nmutipotent and 01 differrnthttng eellIs produced or treated actwidoint the methods describe herein( e lr publishd irrethods} tray be crown to V0 2051150814 PCTUS2QO8/Q65007 ass0nA0 n wth thvIee-dimesnionaI or iwo-dimendjnal scalliatngs emeerd h 'pbeate normaM fissu strelure atd/or organ s'trtures (es Yarlagsda Dd 2 : A ci alo 1908; \\ )/20>01 tro4 I P14: : L\\00100084, 1 Patents 2395691 7207540; 6995011; 680075 isenberg WI A. 200L. )Smuln \ ,k Sa dhildings to be teUpted my the pinripk dent mtipotenL ander diflereniung CelHs may he derived rom cadaemrie rgans or fasuct a Ster the cadavcr orgdns i f$s9Ie (e g. bone, Ian kint llu, \i ) any a he treated in Ahl awaty that the host irmmUnre cells resident hi ibha iissue. arnd o~her undesirable or aneillnry host cells are eliminated (cx yi by tonizin raditon eeili tion te p. \boz et al , 2000 and/or smous 10 1wthods oF deeeluhu ton ii alled tes Patens 67340 18: 692814 6479064: 676244: S i- u P. /s 5032508; 4902508 495617 T 5281022. 54389; N09956. and 626911, 4361552 and 657618, 75318 L SM application 5cr 1 .11 16.2 i WO 2001 048153 \\ Of 4 W4 \0003002 165: WO 200'{49211, \\O!20t7}0233R European Patento FP! AI82871 IS .46901V 12443% FP098799: hP! 24436: P1111870 Riedec ea, 15 2004; Ou e a A. 200$; l r a eiAel i 99)S) liken se., n is ntiuped A the plubpoau gen. ukiw- anor dilqrentimy cels o the present ienti may be uoed in applications lol/mg. inict-style pimng for issue enginecrmny (eg. Bond ci , 2006 X4 v! e , 2L006; Campbell eo A, 20M iheret'ore such use et the celb ipiduved or treated accordmg to the mebods desihed 20 heren is cornered in another preferred embodiment, the selected clls and/o eir~e progeny) arc utudt mi hanging drops. hi accordance with atnuther aspect of the present *mventot, selected cells may be mddied cencical beinehand. 25 In accordance with another aspect ol the present invention, selected vels InaW te meuditited nwot DNA or RNA ens.n poet .. wet ppidc ) pronmeag djfletation of the cell ito a de1sred cel popukniion. secreig ('l1 .Popuiations in one embodiment. the methods oit ibs imvenuti nimriSe screening cells fronm cell 0 lines donor sources umbilical cord blood, and aumnogns er donor hone marno, blood. spetr8ofma, prnoridial gn ermeIs bueal cheek eis or anl aiher ell source efieui e 28 NVO 200O/1 P$814 P /It12n0i 65UM? in the cure n t Iion >c d ReAW t can he screened to 0nhm successful tranfectiori nwit be beeicial sequecees) or therapeutic Vettor( as well as succesful mnitatin ut Jileemiai~m b any metod kunwn to thei (Guain ct A. 200J6 t S Phient M A, 7 n mne emnbodovets, the cells are screened usime standud PCR and nmue acd ) hyhbridihzn-based methods ni' usmt rapd tU ipmg methods. Inpreferred emodiments the CeC <1 e increCenedt according to e>;pressjom of reporter genes ln some embhod m tivm cells axe screened K expreswe Ohs marker pene en tded the tranagene espressmA aesoor(sn such as. an 4mtibiouc resstance gene or a fluoresent protein (e g. GP) gene. \ercening bci Uhetape ntl \Vectors and ienefial NequeleC$ 0 cl lsm can be screened er the presence o hene deia sequences and therapeutic ul itort usm fn mcethodi known to the art .tr detection ofpei sequencesV 1ach cel simp Qn be screned fmr n iety ot seguecs smh mun ot rls A \eriatis .hi, onltiple sbampl"n e st be screened amnutaneously. ( Il di tberition tha) he troniOted h) se\veral means: inchidnigt t i) orphologica 5 assessed, @ nuinm rewrse transnptase polmouse chain reaction (R ACom), MAbern blot, or m ira teclhmiqueA s to momtr chances m gene expression (ni assaying ethda epe f of speci be mrkers ch us beta ubulm .1 (fr neurCas) ete (0/awa, et ,.aL 1t08) I sumea omboimenis the cells ar e screened tor successful inmtation ot dit iekentiation using FA\GS soring based on cell type speedic markers or transcernic marker 20 cxpressim i (eg. antibiotne resistance or t11 noesent prin eXlot5sFon) under the conmIrob of cell ty pe speiIC promoters suieh as the mi osni promoter inm iuscle cells; the human cardiac tidtit promnt'el- in cadiomvoces:e the insulin pr0tuoti ni insuhl n prodcing cells; the neuronalaspecd ie enola e iNSI )promoter for netronal daierentmation, or neurainsitev rehited promoters such as thes tine hydroxytase promoter in doprnmnere i neurons< et .1 25n me erbodnitt the cel. arc screened usi standard PCI and nucleic adId hybridizationwhrsed mnethod. In a rae ub ary piecfzired cembodimieni, the cells are screened us]n rapid ti 2. methods Screedag for human leukoevie antigen uiI. Aj ty pe in certam embodimens, the selected cels are selected with respect to compaublW 31 1L\ typing The .1 A genotype can he determmed by any means knon to those of skill the art.

WO 2008/150$14 PCA/0T200/0650M7 !he ell used or screerinm ma csati of cells taken dicty in a donom or Wom cell LN esNished from donor celIN or other praetible cell sourcesT 'he cellsecn he screened lA boeneletat sequences <ndlor therapeuic vectors) and I A type at once, or separately. Those cells svccessllN trsmt'ected with a hentieil sequence and showIng an r5 apoprnate lA\ genoty pe can be prepared for transplanttma to a patient Iln ceri cmh~dtitlts the transfected cells tire tranplated without i A tv1pmn. ia other embodiment the celIs ar eIL\ { pcd for oimpatibhit 'Neeening lor atgents pronoting a cellular phenotype I he present icnlet ion als ro 0ides t A a methods o sereenimn protemis ard agenh I to f their abihti to induce phenowpc ehanes or ditllerrearion of the selected cells anJd/ their propen nto desired cell popuitions Bre x sectors enudorm comp00lm etarDNAs tlN\s) fromt rppr-apriaue eDNA brt es ar e traastected into the select Pe edik N d or their promgenv, Once a speci tec eDA \u ihtnduces de dercenhtionm or other phenotx pit. cne.ms dnuled, such e D\A thertna; be isolated -id clmed ino anpproprite expressron vector I 1 o protein produchon in approprite cells (t2. CUS eelIs.) in \tro later the protein eonucnm supernatat cart be applied to the selected ell culhures to deternmmncif any created Proteins from such cells induce ddirentitiort \lternatwel. , ndidate agents an be nAphed to te selected cell ctes to detemine if any seceted proteins frm such cells mnduce dillerentiat ton Ise US9 Patent 6432 ii. 20 i ht present nventien also pmides for methods of screen nucleic acids t0r their abiit to i al duce nuluipotemut itIii paariptnhtiis. and/or sell-enew at or to intate diler enuatlon 01 selected eld, and/or their progent . I these methodt Vector enceoni eeted eAs (or eDN \s tomn approprute )N & libraries, or Other sguences) are itrduced 0io the selected cel diand or tNei pregenv usain cectr0p0r3tior, narnocapsules 25 naino aults, hi vom.3 ret viruses. ienti\ruses and/o any other practicable meas A, .transteetmon. Once a spec(I i )N\ that induces a phenhic change, i}tigkpl1alt, pitrripotenmi1hr and or selfrsetewath is idemi tied, such eDN \bt thnmay he isolated arnd cloned into an appropTnae expresson "ecor Assays for deteimmmg such change> melude those described el sew here herein. 30 L ikewise the proteint caesponding to the identiie eDN\ may he produced in appropriate cells (esg Ct)S cells) i sitr to deterne whether thme proein coutmno W 2008h150814 PCT/AS2008/065}07 yupemwatt owm be,, apphed in rho selcted Mel euures and induce the deshed Aunqes. 0Hna'ly. protems may be produced into the seleced celkand Or thenr progeny usm el ettroporda e.naRnoedapsules nano\ tJuIts, liposoines. ret ro\ iru5es, leti\iruse. daid/or ans tAhr prtettitce meSi of tmsteetion, and the resuliun cells passed as descnbed here s for' nuapotent iY plunipatetait set-enewva or the imtiation of dillcrcnuion. i rainrdantahon of Cell mto Patients A fter screening. selected el andow their proenly may he crvopreserved, maiinedt as ell Ines in culture, or ny be admimtered to the put ent Selected celS enn be cn'opresenxed or maintained M uhure by 'ay roots kn\nn to the arc and preserved (r 0 kre transplantation proceduws P:rlervibkg the cells to be screened are obtimned tromi accessble scturtes idhowung eass collectumn \'Virh regxard to podouemy I II' resistant cells targeted soatic ells and stemn cels of this inmetion can he - f n ~ e capable otdddTereuatlma into celli that c an be mneed hr hof any opc S-\ i that can sustain the trnursption and'or repleataon of IV, that can alter the I IV inmnC response, or that can retard prgeinto A IDS. Such stem cells mdcude, hut are not limited to punpotent cels dered from spermatogorua, prmordial germ cels hematopotetic stem cells peripheral blood cels placental blood cell amniic (luid ells. umbilical cord blood cels buccal cheek ells, adipose tissue cels (includimg stem ells 20 dern od from those tisse, epwgrammed cells, induced muhipotent celk induced pluipotent cel O ee. non-human enrvnos. and/o any other el tnpc that can torr blood and ininune eI I hR idret cedl and other cells. !hearapeutIe v etortsa exprew "berneticial sequence intended to render tansfected or elected CeM less napIble o suainmi' II\ replication and ransei'pior he genec 2 v ector ecxpessmg enelia ( seuCne'C S" as nel as aun' rus dcri\ eJ.Irom such genetic vector arc herein earned herapeuti c vecor' A er sreeening, ells iknseeed "Ith the desired thempeud uu etog ( and expressing beneticial sequence a hh or xxuthoeompatihle ill A eeno tr pe) may be expanded eN rivo (in viro uing sitndad methods to cuhure druding cels and maintained 30 pa N e cell line msPaenst 042711 d J533 erein m rpe itod by reference \ternatiel, these ccl Is can~ he admmtered to the pati ant dexpmded in vo.

WO 2000 /$0814 PCf/S2nt$/:065O!)? Selected eeis ton be cnepeserved b, an; means known t0 die at and preserved i Ittre turaplantation1 procedures IJ'OnSplaIaO Lt 01Of de>ile CCII Popttftions 11V0 Padients in cer tai embhodinlets. eel poptla 111 arc aiiicbed for stem ccl1 Ipr or 10 t v~v~i.jpid10 \aicusictbods n selet lIT stem cells arne well nown in the dri or cmplCe cel Samphis can he em iced by .luIresemiIV hibeled mon oCW MAti bodie< rteCgniAng eelb.uface nmkers U OunderentlyNed henmpoieue sem cells e g(0X3,' T I . inhiL CD 103 OW N.t hn- Om r torting ia fureseence-Jemialed Cel sording l ACSL In other embodbmeuts a sample oi the eted ell a in tansplaracd, without enlorent. In sonc nbimlenis, the cndE enhm Q steell a of the borie maml are ehm"ndled or r edA ned 01i0t to rnalplatton10 0i tle there dpeute stem cells. Ihelrdpelltici er cell are delIued as til we Ow ineells contaunn, he-netidl se4Ue-1I r e 6 15 In some iAlodirments. the tra1iplantrinon prOceSS may iinrole 00 ling phaes: it } enditionmu () Stem cell mfuStow (3 neutropeni phase. (4) engrtmen thliase, and () potengntiltmet. peu td. In NOmelC WmnodIQnelS, thec endo genous 4ter eellS ibl na ll15 pr fIoduthe deWSredJC ells e g bone marrwstem cu-cl l r eWluindinned or reduced prior to maspanaltli. 20 hemothenmy. radiaior. etc ando methodsinalouus to those desecnbed mn Patent 21867 Dni) he ised 1 eondit h the bone marw i0r appropoate ofaraitent ci the transplant. insdht l- lIlcrpeticttz'rn1 cells mdx' be transpldnted htto the patient tletm adi\ nmethod known to the ort De4han of Numb \/ umble and othe' tran ee- e'ne ng veetors In IWW emI11000tii t(4a$.kvta0 'd I t 8Itel.- JcId Mjt-uke(N e.uN nunmbl hke/u tumb iso[0rm01s is ace-o0ptis9d0v0i Nra trans01ion. Ihe terre 'unm Nuabhlike eoding vct.ol(5 " rcf&.r to Whe \ctors incorprinsdaihe ucle c acid ee s encodmn g numblike nub i-'urmis) and/or sy ntheic oh elcodndes uro tt numNike or unnb asorms, as well as any aditioaii tran-senesegucnccs synthetic limunuc.eories, et. and anly associaed vjradi uperati ior led n ii0ht' vecor 12 NM 208215081 4 PCiTIS28/%MM07 equence. tit Numb ;Nuunhl iT Cncding veetors) nay co\poe an expresion I \e \ pporiate exptes1on vctor ae those that may he employed A'n tra teting I M or RN \ into cukarvotc ce-lA Such \ eCtors include, hut are not1 innied to, prokarvotic vectors > <uelh :. f0or \exmple, battetz- Vectors; eukawtr1 wectors. such as 4\ examnpteyeust 0e"Ars and Jurg ets, mod viral sectors, such as. hu not ultced to adenovital ctors Menassociates W d url vector, and retro nil vecors. Lxamplcs of retoviad actors %vbch oial he empled uretUde. blit are no hinted ton those derived fron Molone\ Murine Ie nia Virus Motonev Mrao Sarcoma irhs and ItouSarcoma irus, N Id 1-11W, \ md ai b brid \vectors ft is disclsed that the Numb Nunike eneaodin \etors) may be used U; transet cells 10 vuro ando in i\ I ransfeceon can he arried out by amv means kuwn to the art, e-speuU t ug i produced from \virl packaging cels. uch \irus1 a he encapsidated so as to e capable to infeang a vrsty of cell types. Nver esS <mn euapdanou technique ahaing incton of selected cell types and/or their prooy s practteabe \within the context ot' the pr esent im ention. Desgn of human imnRuodehaIency virus (11W) (}cne ther apy Vecorfs. I he 'Aherapeute vector(s' may icoqmuratc an epession vecor Appropate expression vectors ar those that may be employed R transftelng 1 -\ or RNA it; 210 'eukarvouc sb. Suh siectrs inicde biu are not mnuted ho, W r)oote ectors c such as. K example, bacterial vetors: eukar oare vcetors, such us, 10w example s-east \vectors ad lunga1 detours: and iral vectors, such a hut not0 W W uted to adeno\u'al \c10M iatleno-asseiaed ral vecor, and retrovral vectrs xrples of ietrmral vectors which ma be emphed inlhude but arc rnot hmihed to, those denied ftom \1hmev NIhaine Leukemia Virus, 23 M oney \ ne coma Vrus and ieo-s Sarcoma irus. tW necl onRodetieue \Iu (LIV ), ill\ , smitan mmDunodelcencv \lirtis ('Y) and hl\brid \ectors 11 i disposed herem tha the: therapeutc weo ) ma he used to trasect target e 1t in i andhor in vio lanswteon cin be carried out b any mena kno\w o the an. espCeial through virus produced horn iral p\achaging cells Such \us Nto,% h, Sencapsidaned so as to he capable of iDe3i a cells and/hr Q ta els 0no erh r some imtancs other cel types are trans-'eeed by means not nonhm the CDJ4 or (Ds '.3- W) 2008/15 14 PC /S0200/065007 pisel ms, Nierertheless, anlVCi eapsidtatloi tehalqueC jajlk £nt iti on t sudh el 1 tpes ma) 1therelbre be Alued in the diclosure ot/he preit i onni in Pseudot) ping with diffintt t\ lope ptolems expalds he ralqze of hst colN tvansduceabke h A Veosc > and iberpette \ ectos and allos the virus to be t cmcntrated to hvh iters esaplcumk , hen pseudo wyped Wvid the veseiultr stomaltus vivs glOpe uto YOtin V S V l 1 jt A , 199X; Reiset cl a L. 2(00), 'eetuir Costruetion rd \ vecto1 utliz eA in thS tuve1n01 tt be Of NyirnSlOpus it lAding h;rid actors. Vetors oar ,lforinstance, he thiid-gcnesatolet iral11J v ehnm wib ch include only D r v c mail tirac'ion ' " the native genone {/ufferer e L 99) Production of Iausgene .PodeJ \Cotor{ manyn oing andr etOrs 1 /uffreW tv C, W 199$ N is nht wi at , 199$) i eating the production of til-llenth vector RINM after inibion M laceo cell Vi! vetors mar be tli/ed which are replieathm-mnotnpetem dhte o hl'ahre to 1.5 e'pre certain viral protem ne ssary tir replicati. However the posi e t' cxAs Isd hee uirus maw enable therapenue rims repheaton. t his likelihood can he reduced bs the use of sde imaerih aig \tttors. In a preirred embodunent raiscene sqietences are driv en by a uiguni promoter it) prooit. I' HNalpha promoter, CiN promoter, arguable promoters and 01 desired eel 20 typ spee1ic t)J0n10ters. \'rl lopim 'in u prelerrd embodiment virus dcet fre jom the Nrmb isofrmr NAmhke encodUwg \ectprr(N Ltlcrapentle \ eCtOr(s) and/or other transtgenet e \seclnr( st of this In\ventioti pt'h ta ixjd 1k \ vesicua iah00( is vrus eD\velope gitcproteln to enalde conentraton 21 ore i vtrs to lih oters and io facditate ini06etO of CD oells. Nr eence SeOet on s o seqence wIth 7as r iretleT or AcDnpoleenarn i) t an d\ 9egunae retenreJt as a \N 'M1 or Numnbhde suence {swarehabte ushng the 'nircx P/ubmned database) is covered hr the ienion i f uthai d in the manner desenied mn the 31) present inmention WO 2008/150114 PCI IS20M5007 I he currceni invention also rates in par to a genete eta that in cides sequences eapabie otfmarkedly reduemy i the suscepti biht of' mamnmahanu cells to ikcuin b\ 11 B\ 1 and 111\2 v irses ihoth together rclened to herein as 11\ Vi I he current ins ntion discloses the ruoel conhmanon 01 sythette ohynueoudes tthe eSpIo of enes erPea to the l.\ \ DS disease proenss Ihe desirabi Ut c Qoinbiog svnhet oplmenelcotdes to efect c.0<ceeptar "knock soW" with epreion of TAR and RRI' deo> souenes ises rom the propson, expressed her .m,.i nitt ombiu muhiple tiene U ietarv approuches simultaneouiss l imtrme S111\' mfection, 2) 1117 \casimnon tin idual cellH is hkely io 10 produce superioriherapeune. benie iit in tny of' 'hese appruches n WSOklo llhentpeue \c Vector(s) cx press Theneticial seqttcncet si tended to r endertranusfected or mneted cells less capable of'sustainig 111\ repliedtion arud tralncetiohn 1W Lgeie \ector expressng '%enehicia sequeneets)a as w ell as an' irus dern ed front such gnet \etor. sare her ein termed "Nherapeurni et r awte lc r%'3jlOK 1 , ~ t~~lk~ il~~l' It Ihe present m eloit in k3rold in am! to gen nthdlion A celk susceptible to intecn by I\ or capable of popiganig 111 Such cells are heein terned 'itagt cellh' Ihe present iventon provides a compostion und method for using therapetic viral vectors to reduce the susceptibluA of mature or immature tuget ceA Iseukoetes, blood 20 cells any stem/progenitor cels and/or their pogem to inc n by [11l\ It 1 lis that the present invmenion ahs no pnid a myo siou and m iehud tor usgthap irat vectoar to reduce the susceptditq of reproammed ceik induced nmultpotent cells, induced plunpotent celk and or thcir progent to inkeni t' 111\ h is a Ibriher objective ol this nvention to reduce the abls of mature or inuature 25 targe cels stenmonimor cels. ticda rewrme ellmeed <ntpott induced piuripotent ells) und or their progeny to sustatmi~t irtudetueietW) S YillM replicato~n and traSCmetiuun, is anther objective of this inventing to achele effIent. longyteroexpre ssiorn of the therapeutic sequence in mature or ifmatec target ells, other quiesent els. 3 stem proaentolr cells, and/br their pRogengh WO 2008/1SO1 4 PCIT/US2u/%5607 in one spect, th ismennen provides a method for prevention or treatmg i Y mufeuon 1 he method im olves u~asralantnm stem cels transtected with therapeutic veeno~s Or sequeneci ito patients nith U.] Y ittmarn iennfied \equeneeyd mag be ones that reduce thre ability of f11\' to imkt a ellt tansibe h tral I N A, o reph ate tin affected cell, or which enhances the ability o a cell to neutrah xe ' l\ infection. la certain embodiments the Ibe11la wsetect s) repiresent sv nthetue oligtonuefeatittciil WIde ineltele nth 10 V entry. unelkdin silA, shR . antiense RN or mRA directed agnitst jn ofh i h v co-recepwors tc eluding. b Iu noi limted tO (' R4, (( AT A CAT amd UR In a preferred embodument, the therapeutic v ectorts) incudes synthetic hgompouiendcs tar getmg one orImNe 111\ eowreveptors indudng CMA u4 (CR, (CR (C(R2.CN \i.'.R6:md/or [R013 in another preferred embodtment the theiarenu ti vectorm)inmcludes synthetic ohgonuclcntides targetng the n jo ll cureceptors CXUC 4 and ((KS In a firther pieterred embodnient the therapeutic veons meudes s nheue oligonucdeohdoes tattttil ti iAC more Ill\V eniy)3e esuc0h as Il|| rev erse lransceriptase. rtlegrilse anhd protetse. \ppropriate sequences for the syntheti oh rhgnucleotides are those 1) prietcable b >1 computer algornthms to be enietelne in red ucing' isrgeeo eunead2aal 1 successhully 1educe the amount o a tngeted enzyme by ' '7" in standard quauntitwve RNA assay Sand in assay a en ynatic aci\ty or to a lesser hut theapeutic degree. the phrase "wr geted sequence" mtniates that a paricular sequence has a nueleode base sequence that has at least 711 ideny to a vr u enulonn'c nucleotide sequence r is em imet t . (cg, is the same as or colaplementary to such vital genotim epec fl correspondhm; RN/ sequence. in particular embhoimnems of'the present ienun the term ind tes that. the sequence is at least In odentc ai to a viral genemae sequence of the particular virus aamst nwhieh the olioeleoide is directed, or to its corpementary sequence. Any of he various 1types o1 synthe ohiuonuckeo ides mar: he expressed vija WVO 29*811S$1 4 PCVIROMO600O tevtie \~y~f' tnsfe I in nd the current ivvemin iq d'eevc ton A possile. cnbaosid ch&hmceids in a pMAMferreetbodAQen. the qsahti obgoNueA~j Ie xq e eecs arc dven hby target~~~ ecU ipc hepoi~4 apf a ;en a In aher peferr o the. sc O u~lC by suns dN, dv~~~ap~uuttcna vecoti Wayh le&R 6l"o. lx\'\I. H In alfre rnohe.Ie~ opiesn Itpzdeoly Inacte pc1rde0bdm tedcyR Al- sequce are 1R NAo TAR. 20 in1 anote prefIard embodiment the ve nes nldePieemn eo ln~~~~~~~~~~~~~ a7rfre moiettedcysqene r ahdie yaft rmtr AW0 210& 1011 PCT pLS 200S,!0650io In WAwtthr pCeh.rid embnocmct the vactoW&C in dhA ol i gou Iotde Ii noie pre"itited chodhunt. the vector inclde iR lg.elo d In a Nohw preterred enhdtinent, heor cludes: Rnywv onto nconcde a In noherpritred ciboonei.theec i po iclue jmape.n hiinin f eniet v hisoisclcoude ckes. (Iina pirthrw, Von;inet. the gxr enuclodeA y seuec targetI IY excen ue as CCR5. d &XCR4, al etcW!.Cu in a frushe em bodbu Aen thed sytetcnolcoie cn. A .e IL u, a e sInt rae ortae.rvrs rfleitts.T .ec Ina hrhe eibduen te i oain squncs aretfl OWeecnOOssc ISk~ CCS{XC4 ec.oriV mrme uca merAs.oroee reesetaserpae A.

NVO 29O81114 PCvIASIs5007 o other theru<ut in U d 15 t~~ rnurt (%t ia ddrO)rftlpetltrs \ I a di r h rRRY opplaoflte r - I AnMleo sequencerpesITo weis jte VbN cthtifha.Uf ot d it) alphas ugand ti efde t othermbidite~i appte rpeuU prmoer Inea pretrrfed 00 e ethirwp\ 11 1 ilha) adudS butN exresno whgill the Cdlle rm onter drep h A dedo es Or 10M e n te Ike mapling0 v v v Ft hv adhv prlere embroiet CMPs W lh rvssRAsqec xrsinw h a206,~~~~~~~~~~~ HVbR 190(ud1 nidclt rhnd, pechetal ",'lO~ 3D d upiB h 25 6 p he prest Rni de o Cesesi 1ontra.hectio of Du i SeCd prolterds Apdecoy e preni D t m l \ In autreerred edb\dim e , he hee m 004 veo includs n tife d Cl ietiRN10 sequcrpae-ouweted aai t C wh n u c U sepee di erected ce thIe2R . cane he seuenU as in Im w o n{decoy, see! anwod th vter iosai thatn hare n dd te do oul etlen combiatie t h ths t her emtl metl WO 2908!1501 4 PCT/US2U08/060 By hc. sarnc token, self-lrnewto. i mdpctcni ,nd/or pituitpotcnt 5tei0 cels inchithng repro ad and 4i htekd pluripotent cci s represent a&1rthut ogea Lar et w tr It gene herap\ , and Tir o is speci fectil co\ red b) the preseD in\ennon. in ene entodiemt 0f this prwew selected cel Peg heinatopoleic stem ceBk im Stem cels uiiWa cord el ls pimird l germ eels (PO( s pematogona, any accessibe Sonmalyt c01l Oi 1 ec arc pIoptialcd in culture lrig re cv t) nes such as sce:l Ow, lettki'a m ibior bitoi I IF) , cardhroni n- s I -1 1 ThiL -6 p- I )430 CNS F 1it. bPGl and or vnesahm\l n ?\1rtnsfeced nith the therpeune veetorun or be Oelid yequ oci c(s) pitwr to di nffltori sOn any methods n n to d thle art, smh si those deseitned m I Patent 5677 139 hMi incorporated by reference, or bA methods Judlocous toL Paw adt~ 5{>73l w ith respect to mother target c eds In separate 001bodimnet. it may be desirable to perform the arious, steps rior to trntioeln. 15 In separate emobdinento the purpose of gcneratog pluriporent lem cell poptillions It may be desirable to perform only the incubation steps above e appropriateoe conc11liti1 o i)' and K t0 1at0tl f stVmIpro enjior Cell Propti~aiIuprlhderauo t wel as: other calf culture conditions have been desenbed prevouivl) (cog, 1i lptent' 6412711 dad 5453357 here iK'orporated by crencei ( iter 20 ppropri-Ae prtcosad rt eccy neconceTratins have been tam by Khimbru ci aL, l99; keller 01 Yl , 199' glqueteilorce. 1991 Rose c& cil , 994: Park nd Ihan 29it (luadn t. 3%I DIt sta et. at., 200%), The popu t ion of taret e1s may idude somatic ccll stem cells and promenior ells sern cets in e died om extin cell lines or isolated foni stored, honked, or 25: crvpeed tSouwk s. Iypl soures of stem ells include nsmxv. periphmlA blood, pQlcental blood, donioc AU thd ibillCJ cord Nood. adipse ioue. noi-unan embryos Sonmaticellsd, especially circultig Icuk ocytcs and other nonf Ngcil itemstn cels moa likewise be subIecnd to the same cullute condiion as described abve Ir: in piogcntur cells cffbcti e that they acq re stemipgenitor cel properites asult T)1 WO 201W/i14 PCT/ 12tf 28465P07 '<he mflmsiI also dsloses the prodtion (exg US Patent \ppbeatton 2003009621 Y of tar et clls trm stemprogentr cell that maal be made re lativc resistant to 1 i ceOn andor 1 B\ rupeation. it understood, hover that any method Al di ttfrentraing preenoush propagated s 5 temr f~prfemtm leUk0 4e cells nt the desied tanet cetsamay be unpl oed uvim the wo 1 a jttorI4mo So tOng tt hme~ftio.ld r/el ec el el renti tsistant to .1 I\ intectiu amid'or I [1 rcphcations andior I ll\V triseription are produced. an a preIbered embodiment the therapeutevi ral vector is packaged with one or more envelope proteins ronm native e Il V mirses sKtC'enmg upon the theropettte irms the calucenl to in et any ell that a e I strais ar apale of' iCOecinit. ( Is selected lot use i thS iventan wll be sonme instances accessible ce w vanbibeal cord ste celS. bone marrow stem cell spermatopmia and pronordial germ els ol thx toa stem cells isolated norn armuothe lmd. stem cells isolated fron the sN. etc L Such cesb can be Smoltd tMai the tassues in xheh they resie by any means knon to the Other selected cell M a oDm rise r eprogrammed cclkl ndutzed na0l tipore1 cel mnduced pluiripotent cells. ec In accordance wh an aspect of the present mention there is prodded a method prodeminy a desired el line, cell ty pe, o e I ltw from the selected cell, ttteudlv, the 20 method ctomnpnses cultmring the selected eCh andI or their pr ogeny under condinons which promote growth o1 the selected ell at an opnmai powth rnte, The result ed population is then cultured under conditions which p-ntote Al growiT at a laite whi )ien 'l)mnl less than the optinal rate, and in the presence o an agent promAang dlerentiaton o c the cells into the desired Cel line cell ty pe, or cel eass (e g. 4 1 cells) 23 boe present of mention alo Jieloses the popaeauon ol the selected cells and or their progeny Ut etth ie helre or itel transtectonl wid thertapeuic \vecor, by any means know n to the art e~g IS Patent \nnpheatton 2t060000)1 77). Such methods also include incubation with IN steel Iuetor, 14*6 I 113 oucostatimM\ and or cnrdiuoivn-4 and other r1 SIM'th enlaining cytok 0e06 etc. Ihe present inention Inther discloses the directed ITfeenation of cels tats=ted wi h the tetapti vCos) i nto desired -Ce \ pes by Etnher incubat i media 4 1 W O 2=)8/15"t814 PcTft i H2§8/065M7 coviamine the appop~riate ctokine. aid amih futors such as COa w m triaating ntoo such as NCSK i US ( \ S ), G M S Ii ant eo tokmAw promoing. 'D4' c ell differentiatiom, etce r[ans leo uO Sentic mdi eton of seleed ceW and jaret cells whether (he e eXQignons (: (1 elndok:Cnous relh can be nel sH ed aeemdmg to onl PubWAlS or tnpublvhd method known to the a g, it 25 pat, 43171 1, t .059375,' S () '8; . US 521041 t S A '.Y2454~ e JT or b other gena auc ed means. Suitahe methodN ib tnsdimmyni host Cels tan h l ound if baarook ct al 0 N (olecular Cloning A I ahoratot \lanuiu, 2nd H ion, Cold Sp4ring iadbor l.aboatorl pes M$N anod thr laboratory tetbsks Successfulls transeted ce'N can be idenutied b sceleiun protoc(os im oini markers such as anuthiotic leaistance cenes oi addition to RN \ expression assays and mo-phological analses, 'KCS mes fromt suesss y iansieted cells. express the C. tpprognte eSOgenou\ D)\' a t appropriate levels, can he present ed as ell ines by ztvepteserviauon iutdL'ing an)v appropl ite mtlod 04 crx opizservaditm know n to the art>t Slectable X-LIkeR (Ce P. atblotcs resistance genesi may include 1hose which Confer ssane to dmnes sa h as 04 N i& n remym. rmpicillini anJd Nlasuadini, tc Cells cortamning thet gere of interest c-un be wiel. Red yJru selectnon where cels that base 20 incor pirated the selectabl market tgene sunvv e, and &ahcs die. A theoretiedl basis loi thei emibean ents of he in ventjon is described her ein, how ever, t iw do>, eu-0i is not n ains uY to be voenidered as bmndmg~ or hittig or t present Ientdon I hose of skill in he art wil undersand <hat the varnous embodimenhs 04 the imventuon nay be practiced r eeuxdle.ss of the model utsed to desceibe the theoretal 25 underpionings oft he invention. T he ivention w il now he deseied arid illusiratedl with respect to the following csaniphs- however, the serpe of' the pr eenti invention is not mended un he lioned thereby liumple ; C'onstruelion &f the mmatngemec v ectors suable for use in the presen movnnri is apup AWDNA4a 26 0$115N14 PCT/TS2M/65007 Sitabl ucnN1umb and IM >0 % imhike. and TFVX lentiviral vectors wheree S mny Iransgene described m the present buenunul can be produced by elomg mto an apprspnatviral vecior {e. the Ivvo-y-ene ! NAGP lT SA vector (iteser et aW ,WHdi O Adapter primers (", he selected tot P-R ampiication o! Numbhike and Numb ifdrm ) LI DN \ and cloning mate a geneti vctor, in preparanoln for nlonn. the gene v ector as dgested nith en,:Q v w Snthsevqulv the eDNA icI Wach tnsaene i msered into the net coding region preOons occupied by the i 1k < N \ C I P anged peen fluot esen protein) id a wel populationppropite promoter (e NI M o r V t alpha) ha ig been prcvoui i msened mio tihe a r edm~~fg epn eniccsru m euda eo 1) bxackboue, and rnsacNvator "hich ren Wles a pHomter OperaV imked to heter\o0hus nuice acid sequences. fampiWs 0l'0enmval vetOs which may be employed clude but aie not hmited to, those derived eJ io \ttohev A burine Leukemia Virus. \hialoue \urtme Sarcoma Nus and Rous saronm \ nusV IV. and it V Appropr iate ex pression vectors are those that may 15 he emphyed on tramsfe tm' D\' \ iu NL\ tmo cukt L Such vectors include, hut are mnt honed to, pwar ne 0 edto s such a for eimne, bacterial vectorts eukarotic vectors, such as fnr temple \'cast vector and fumgal ctos and ural vectors, such as but nt liMed to, entiis irhee tr. zdenovind vectors denoasomted viral ectorsm d rctMroidal vectors 20 >hereplicaton mompetent peDN 2!h'rtWP~RsdV\ PI'SI ector is n camnle of an appr operate expression vector (lmvitropein and allow sof spreuin of Sownthetic oltignucteodes (e. , mi RN\si }tismsferred irom the peDNA6 \\nu iR etot have the capO v t dem targeted Iequences These aetors mcude fknmg and loop setjnenes (trom endopenous in' R \ \ io direct the excison of the engineered in R.N\A temr a loner PW 23 Il imaascipt (pi -mtR\ \ X C o 'mibmnu t y miultipl tmi RNA, sequences duietted against specific eudogemous R&N\ spcaes mrrass the hfebhood of success i ra sequence extprcM on. mi \nc\ "Y Vrevuk!Lked e or tiss uec t segnenc es may be opermtNv hoked to reul.e0 asu pelie pYomloters. By ubh ring Nenaa 1 ectors tor gene expresion, the resultmgi Ninnmh Ntnmbhke S eneod mg vectr} and o eth er transgoena aexor n s) of this imention, becomes capable of stal 6 tdlsuemp both di\idhnga anhd non-dividin cell t5pes. 43 S2O MI8/15ON 14 PCTM/USIMTA65OO7 in a prckerred embodmen. thc resuitung Ntimb 'Numblke enc emg orevs). and/o other {rmse i ectort 5) ofdi th n mitnon ontain muluijpie svnthetc; oiigomUiel 1 tid seqTiences' dri en bX tle! or more pwomotcrs so as to reduce e\prescsn o seci(le nm isofornS aidt U LIh im e.e samnple 2: \other example 01 a sotahble etor is a retro\ tiWl vector. Reirovirstos are RNA uusw ta ontai an RNA aenomi'. he gnq polemJ em genes are CMlad b lor teninual repeat ili sequence. he and P L Rl sequences promote tansenptbon md poilvadem lanton of mRN A\. the retrovrwa vector may provide a reptublae trnus tisn element. an mtimni 10 rib~ome 'geintv ysite ( R LW a seleeion marker, and a taret heieroioouIs cne operated b \jai man i e w mutupie sequences nu ot be pw under fiomoters ionudi the rcirovital vector may contamnc acmten se'teiies necessary for C kTC trause'ilp n and inLgrariiun. 1 pu ietnt dK RN \ is reNer$e treansribed iw I5 DEs \ that iegrates elieientv into the host genome. I he recombinant rettovris of tis mvenrion is geneucad'y moditied in such a wayh onic of the rctrur. ifetns geCnes 01 the name virus have oeen remove d andin certain insttaies replaced Mstead th a 12ra nuc kIie aid sequence 6 genetic modiicaton of the CCi The sequences may he ewtenOus MA or RNA. in narlnd or altered S'm 20 FI rampl : anple methods for generation of Numb/ Numbke ending v ectont. antdo or er Iern usienm vectorts) of this mwentio Ibc mncthods h h0 genemon ofthe resultig Num 3 \u0hhk. teding xelottm and Br other iransgenic. vectoriN 01 this imnnoin mdu tde these -uIJit in Ilouuingen's \ iral Pow er I entiai expression Svstcnm :\anual 2007^ ILhiet thE l b-m~d'P-hsd cassetc is 25 lhned s a PmklpA S mOs ino the pLnte R4R' DIN Iector while the miR-Ion (PR nin isoforn or m-Rhort numb isoomtnumblike cassctes are umudanen l transiened by HP reaction :nto pDO'NR22 I1'hen the regohib le pmuoterNs and mniI isO' non catawes are Muhlisnie It c rossed in the mnodhtied pl enPnre Fm P -bsdd(4R> 30 Muhtiple vectors can he generated in this manner compning di tberert combmianions of syntbenie 01ligonuckcotues and transgene cassettes. r r j' C. 44 WO 20)8/I50Q14 PcT/Us208:%3t5m7 CiX 'N :C AidflVs PC Ai aP:Ik <dcayid ad jw y h agdit''. d aUl y ':0 C4 09 10 q qw g Z s. ,N s."0 s Olt ,w ,A ,s , s goas s d l ta-gl~ c t.3'btA!.. a'ilS - .: ,i ksti .* '.",'fa i l","' 'a 'pi agP i'd0 i a ac ;5 .z.Ci~tCC3y ago OMIT~t< A: a?4. 05=004 1 u AI.l:nd ! 4$4"$S? c- " .. 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CA,,N fi t Z1t'Nt '- h h s ad 4-'4'Z.tak 4-'"ac""""JtZ ieN R"''e K i hxcnc 'd5&td U ll kC tCa' t " 1- ie IIACA$5C1$W5- 5a 5~~$ utd-4-dd yd R" '<d** t~ita'a cfr5iiiiltE* caagfri e a gegci44 C$ ttgusitLgdtige 3 e5iddg ga$ t t eg..)den u i~-dCQa eNl~dt ad1sedXiiti$I dil c ll5tpa d$5g idn a $5akt.:-tft k dek$5$h$cc01a t! $54ut*ga.i a..'a $5R A casere engha; ega 30a e:aa:: R egees -are a- .- '.4 k 'Z 'ZN e:gu o sau asg I 0 iC~ce!$5ta k sd tdtdtildat~idg g:. $5ct.,gtcdr4z1gemna t Nalcti llddtcletskand3Cetagrgetuaadetta si$53p e cLt-.i::sidac alee et:e 'uguag au./a uae etMaica 0nd3-41 .NZ-4 -4' Uiifr5 tct~c2glti dc~ct\tthh nit hd g fg;3ttir ts;dd'tu ag i. the Idpeuintd ti{$5dimid~d$C$Cd$dtf WO 2108/150814 PCT/1 nSt)o6 ? Rios- e i is" demved fro ahumm ndO a amm ibrohkwt eel i s result frin tranfbecinu. or itetme normal Oe lines wth viral gag. pel ad en Sttural genes On thother hand, packaing c! ines produce RN \ det old of the rsi sequence o tha the vbi partiules produce on 't&UngIng cel doe eotai the gag pM, or reCnc's Once 5 the therapeDtA etr Dl \ ontanmng the psi negancl Awt .rm wt e therapeutic gene) iS mirdcld mio the puckuiog e b means of tran\eek ton or infecton, the pahaging l may produce uras capable o transmitting the hermpeutic RN\ to the ial target cell (eg. a i04 cceim I he "QCe i'Se'ang' o1 the thertpette Vtectot' ) i deetaied h\ydte package it ell 10 he A nunbe of pakagig cci lines are avadable for producoen of virus suitable for inlciisishbrd ranap of Slun W k IS inteciing a bra a~t futs telIlI CC'S. these ( tkXtelt m cell lines are lievertheless generally capable1 Vofl'en4upd&Ifng \tral Veti's der ived frinm uruses that in nature usal mIet JiTeremi anna1 species o e'canmple, vectors de ed frm Il\' or MML6\' can be 10kaged by OP 10 enc'apadmy cell lines 15 An example protocol for producing a therapeutic \ii'al supernatamt s provudedlas l I'ntv rnerogl'ams of retrours vector arc mixed uh ib mierogri'ms of vial DNA \ontaing the seketaE rnarke Yene (0 a. aniblodi resistance gene) by gentle tapping in (Lf I nniltlible. of licpec buftered 94l md (pllt'.QS*) im I i n i plastic tube, Seven t iemhiterns o i2 Catl' are addMd to the no \'n hr created gentle tap png. 3' When a blue precipitate irst begins to appear within the tube,. the product should be gently apphed to a 30% conMuem laye ol packagno g cells tom any number of ommi~eal vendors T!he D\A inntre should be apphed only afler first rems mg the ) miedhumn fromt the packaidmi c ells 4. he packagmy cell pre set to incubate for ttt3 minutes at room temperature art degrees (esis) before dansierriug them back to an feubator at 3 1 dlerees Ceiu tor \ \ddi 3 S4 milhhters of i lenes buered sahne contm Ng v l elcerol bir 3 301iniutes then wash cc nI With 1i lhcccok Modi led e~gf \IedMP tur fl)M\P' fI 1 (%H0 X02008X/ 1501 4 P( .. IAU$- 2 i10 A650i1 W. Add tack DMLM "0 144S. a1W i nocu Mg cOs r 20 I hAmr wi 37 deusees Rem~ oe ar!d fiter mehum outaiung the apunie en par s, .sIs ind supratant in m atcy ord a comeated und stored at -80 ves~ 1. elMiI, erndayt s)w stred \\h 5- netcgrmtus [f pohriene to Kowefcdae the eU2tJKenc of tagCet cell infeeion. khermise polvhrenwe m he cludor added st bebre T LthE pveducer lines can estitsbed b\ N30UiS1 cueNgy cell ies I to 20 or 1 to 40 and sub quemtv meubahnm these upls ir up" 10 d <ayS twhangng 13dom eCrC IU thee da) in m0Aedium coidiuning selocu\ o dynegh Ie crfuun 5u 1 io 5N coYrespondil4g to rransfeied resisanme genes)I j vs isolated. c&Ole 5re nicd waj 9 \l ier 10 dag orsledclr u oep ed nn-up aliquoted and frozen fot storage \Asd ol' Rctwm irus I elul\bc ityi ataton is ahexved h\ apphcauon of a defined 1 \gotlme ot viral supernatant to d la e~r ot cordluent "test' edls such as \IH 3113 cells plated at 20 confluence Afler y cell di\sion imes (236 hours or N1 3111 cell) cohimes of es t cells incubaed d! 3 direes in anabtiecnrtainui mediumi cr eumned, T he supernaiant S 1cr arc e stated hmn these coha) COunts lo tev n ibmub! (ohm rout I m Pml etdomiesidenlbed X Ws sph la tor) volumre of u> u's 20 wnh1 t he acura ol this tumat. inc reused b testingiN a udumes 4tanpcruutant oer mam plates ell "100" celk AppQUao oflh thorapeute und supemt to target cell may be accomplished bs \:flat(~; iT'UmeaM a irs ne to the c ncel itouai he 5ir\Wib Medium iI ( cele es Selected cells can he expanded p icon in hubecoI modified Minima ssental Medimm I spplemented with [vim netnmereaproethan) 10 vounme) hofme ~ertnm and human recombinamt I etemia lhhitumr Facto 41 la " replaeeS the .rneed lb roantining leseleed CellS on IeKder kIwi> cells. (u h ma\ Jlso be employed) JId h 30 cssent i nto muaiani selected cellS in an undileeniated, muhipotet or pharten 48 WO 2008/1501 14 PCT V/0200s/065067 ste. such cells can he nisimained ln Dulbeco'S modhle d flnotd isential \edium (DIMEM) supplemented nith chtamine, beiaemeraptoethanoL 10% (by volume) ho se serum, and hunarm recoban Y eukema i nibor actor kLI 1he LW replaces the nced for maremainmn eik on fMeder laiksS oicelk as hilh max also be cemploved and is essntial 5 oh manduin Cel in an uSrditl' nRit eer t iS Pita 4l21l In order mtoiate the dskit in Uaon f the seated cells Mt neuronal cels the cells arc trvpsinued and wahed free of 1 !. nd placed in DML supplemeted wut 10% fatal "ne e serum (MS). Aller resuspenson in DMLM and 10% U S, I \'Ce eIs ire plated in S m M M \, I BSlrc* } " mer echoic acud in a 60 mm Eisher bateriologeal grade 1 Petri dishes. Were the cel V:are expced to frm smal uregtes Agregation aids proper cell dulicientiataion lhtghI .. eW . n-nsiec tin n~ di pnr~ptate neuronal triscripuon factors can tcur before or Aler plaim mi D ll M, IP. and retmoie acid. NSec JA. Paenus 6432711 and a 3 45357 or AMonal dcAs) Fxxample e& 11l\ matchmn, Selected cells g, rmbilieal cord blood or cels f rom 15 anv otai \tutabie source and or their pogenv u, o screened gencucall-moditied d and duc to beg dfrenal io the desned cell tpe (opini I 'Ide. ceha are then tratsplaed according to simdar iem ll transpldaan pnovtool In certau instances, cels may be tnsplaned io paiens without il A matching. 2fxample 7 in some rare nstane i may be appropiate to produce trnene eneodmna eeetors into lpaicens in order to suimrlate or inhibit cellular division or cellular dalerentation, i u lxaample 8' teneti Modification of Selected Celb In vitro tgenetie mod ieation of exogenous cells or patent's endogenous cells ean be petmed wterondyg to any pubblled or unpubbsd ilhod .knrn to the art tee. Pm, E at Nvs 6 4 71i1, ti5A938'7S U ( Th %AUS ,w .. X . W M4, UtO.511,488, (>8. S4> etc.) or by other generally accepted means Suiable method< fu transorming bost cells can he And in Sambrook ci al (Molecular Clonig A I aborator Manual 2nd I'du rt ( ld Sprwing arbor L aboratory press ( 198% and other labortor textbooks SSueenuil trnnsecled cells are identied by selection protocols imolving markers such as anibiotic resisumae genes in addiion to R A expression asays and murphulogeal .4.k" WCO 2008/1514 PCO 1TS200/657 analle VS i lones rrom suceessli lv transrtted cells expressimg the appropoate ewgen1ious5 DN at appropriate lvAels can be preserved a cel lines bn ervoresenaion (utih/mg ant appropriate method of0 epreserv ati on know n to the art> ~eieetable Dmalkers iea, autlhioties resistane genes) nrav inc lude those conferring > 0 stadnee oiD s d Lr S h i\ (W I . hygroInmo and rucihoreaHte, Cels coutsuimn the aene t intent can be idemnoed y dru seectimn where cells that hav e incorporated the Wecable marker gene vunae, and others die. I he ennent ienion dincloses the select on of genecad lyoditied eetls as 'eected el' of the im ennn. Ihe term Mn i mIod insaton refers to ateraon of the I0 celudar venotv pe hy itrodmuig natural or sy hectic nucle c acids ino selected cells andhor their pigen or immortaled cel lines anod r their progeny by arn menus known to the art Alernatie culture cniditons that mdnc ermancnt changes in gene expression Patrns are consieted heemn to represen geneic mOci Anon, Mddiianun et stem cel, whether they be derived from the host brin, endowenous donor sources exofenous donor souces, or 5 cell bnes, represems a t'asie rprac to the treaAment of cenumn humi diseses espeuiallh those of the human ner\ ou w ~vtemi Genenc modiications com LrId hy this discloure include, hut arc not limited to, nete modiicationx performed in o: medi ations that aler the actit or amount 01 mnetablhe eixnes expressed by' endopenous or exogenoUs seeted cells and/er thei progen: tnodititctons which aher the actv . autit or antihemiits of cliulhr proteins: mxodiicantins which alter the active or amount of proteas imobed i anal trasducton pathwvas: mdhiiation w hieh abler i M \ype; modiicnomo whi aler elhua differentiation n, oditcanons which ate neoplasnic potential Mnodicaons whtic- alter eluldta i'erentj at om: modilicatious whieh ater the amomt or acvt)t of sxrctunil 25 proteins: modifiations which alte r the aimni or n \v 01 membrane associated proteins (tuctual or en/i made ; moihtikdtsop\ which ate th1e actin i or amnouni ot protemns i h l m di D\A reipair and chromosome intmenaince: moditiedtions w hich alter the u th i armount ci'pro teams involved in eelhului trnspot: niddinions which alter the IL li\ Id\ or amount ofen/1ymes: modh tications t ich alter the aetitt o r amlout u'proteis 31) iii ulbed in s\ napse fornation and ntamteitnle iodi ixcations which alter t(he acttvitv or amount oft proteins involved in neurrte outgrowth or ax on outgronwth and tormuatnon, rnodtIncauons altering the anmounrr or acdi\1it olfltto~idaint odcne'\es Ulth VO 20& 50$ PCtS2ONQ5tl eil fl10d iations ulneh blead It0 watered post-rtraiittd mtodi hea 01Of eelIu hdt proteit1, mo etiuns which alter the actt or amount of proteius vmolved on other spects ot cellular repar and 'terations which minceae the iikspani of the edl (such as production of telemerasel Such protens a those mennened above may he encoded r b) DNA or K\ derivcd Aon the human genome or oTer animal plat, iAl, or bautendl genoms. W.s iention also cGxeis sequences designed de no\vo in addman, this imwention rlues to the in sta genetic modiiauan oF selected eels dnd~ theirr iprogen ce l lor the ixeatnrem ofdisease. l'dogenotN stem cells mJy be u'toded in shtu divc't opetion or applteaon n of DN \o RNA v sector meludina vrues 1 retroiruses hposoms. ce, ito the substance of the Nssue or woo the appropriate pomon A the \ en1neular sytem th einn Sice h ave il odited thousands of s~etemprozenltor ells and in 0) boos msd prootn\s Cells in this manner. tur data shows at, tho' manne r of mnodiimg pre nnr eeMs ieoh in a tremendous uriety o mOdtitel edl t\vpes lhrnuihout the ter\ o IS stem aind Iha e\et rested i adverse efeld 1.5 1' sampic 9s lnm1dueuun 01' Genetie \Veero intmo the host In a preet te mbodtrnen, endxgenous cells are uansteeted with vector such as those deserted herein in ivo b\ irodueion of the therapeutic \vetorts) into the host blood tisuCke nervous qStent bone arrow, ,e the gretet beneit ma3\ be achieved by mDodi t u a largc nmeI'l h iaCofdIe&lO\ tarCt cells. ihis m c Ke a0omphte ON usine Bt tn appin atelyweb~d, catheter hke dt e etneIc to mnlect lthe thiapetic \ectorls ino the v enous o arterial circulaion, ine a speci ie tissue. such as mue tissue, a ino the nvow stem In a pdueied emboduneet the \virus is pseudotvv pdvn h \'s\'-G env elope nrlvprotem and natis bc Il\ i em proteins. 00 11 pe ie lncion imto the Nervous m 2'S Ttabphmtalion of select ted scl ('omi either the growth or ddieienanaion media) mnto the ietl oet waso spstem ortigenti mroddlcauin 01' endogenious letal cells ubiiimy 'et \cclors td) heaccol isned n the 1ol0t ig manne o Uder sterile conddtions the .terus and futuses drc \isaabed o) lhs mUd or other radiologtea guidance. \Aterative the uterus max he exposed surtgicadh mu order jo facliate direct ideriiniobn of fetal skul 3(dI ladars. Selected cells canu then he itUodtuced byini ectioa (using tit adprloprttei\ siA'd catheter or needle) into the \enmtrkc isirC tem nerrminai 0on 5s or t te substance of the MO 08 /150814 PCT/MSW8/W506M7 eANus sstem. 111h104 may be performed m Ceriam msiances, dhrough the mtherns abdor natal, the iterine w all ,md fetal m bones boo the fens The accuracy of the tin .euun N nioord hr direc t observ ation, ultrasoumd. contrast or radiologicl mitoe based methods or bV Jny utter means 0f radiiczpeal guidate know n to the ant. : nder aippyopriate steille eondhtions direct identilation of fetal skull landmxurks i accomished 1isually as well as CIIIh Iea ispeetur n d pal pation ecoUpled with stcr eolanic and radtu logic guidance. bal movit cell e uh ure, appro prende amounts K the elected or dtferenaung cells can then be introduced by iectm or other means io the c entrica svstem, terminal ores or into the substance of Ike nemavi sstem i he awcuratcy ot the. imes tiun may be mnonured by direct obsen atin udtras'oumdor other radiouaigeal guuin n ceten m. an lgieal d diseases uf the adul nerous sstwm such as Flumngtnonis dl1 ae 25 and I aikmson dusesecso a spec ti punion of the brain are seleei. v afecd, in the case on Iakhtson Pdeas. t i the d mmergk cells it the subostala Mahn m r in aueh retnonal ix -3peciti diseases ato.edlag JdndbsloalizLed ltransnlanedhc of cr'lIs mnay It atennpished b\ 'udiulowe ally'-"tuded trIidpantotUn of difremntitg ce lk under sterle conduions. .abdiogicp andante malk 0n1ude th use of t' and/or \R L and may take adamage contrast o isotoe based techniques to motor ijeewd materials GnrefllO neui'ologie dsosees such as some mejabixK storage disorder. eels are aIteted aciros da vremins of the nenvous sy stem, and the greatest benefit may be ac hiev ed b geneticall v medi NOmy endogenotis cs or etteduem di<elected elks of the present mwemui i 25 either from the gruthl cultue media or the difitremiating medium) into the tin large numbers inoa dittus mnanner. bi the nervous s'tem, these diseases maty he best approached by intra\ ent, uIllar mjecions us ion an appfrnpilahe i-sied, eatheter-like de\vic, o needle tespecodll averhsae tdreloprment) x nhich adlowsa di fuse endonenous cell mrobhkalun or diffuse enrat'unen of selected cells isoated triom the epowth antdror 30 shi fibrentiation media. Nonetheless, injecsuon of the ells io the circulatory SvSem toi the same purriose is als eudercod o0w ev er with .egad to Any disorder atkcflnig m utiiple organs or Abe body dilhusely (e g h1 ysomal storaric disorders hermuogh1.inpathies. muscular dv struphv t the ell6 isolated f romt the g:"ux'th and/b d Jiterenoun media amalsuo be WO 210/I5Q C114 PCT U s2%i 65ta7 pre'eremiially ontrodiced directly ito the crreu li/n and/o 0iceral ordan, such 4s the i erh kidney, gut. spleen. adrenal gladus. increase WD ql, and th00 usnig endoscopi gwhidre and mny apprOpr:,te.s, edJ. uateter-h1c dcCe, aliow n. diftie engamntia of the cells thiughou the bodj a-' nell a Speific m Uetlt <md mitr1tion 01 the cells into the Selected orizms. Fi tnplei 11 leh\vey of Lefls b, Injchon n to he irculatory Stream and Orgins lIDiseases of one organ Sytem may be tedatKble nith geneieahy modified cells tirn a separate oryan sytem, Also m wme instaneet ay become apparent that the sele eted eds ma\ integrate and dterenuate un tncir W n n ino. in sulteeent numfben they are S injected into bloo siream either aerial. venous or hepatic. ater cuhming in the grow th atidor dif lereniation media, 'i approach is co ered hh present iention. 'he ttiei I 01 didI fuemuscle (e~g. muscular d vstroj~ e1 or'gan. hisse, or blood disorders (e~g1 iedavv Spherocyosis, Sickle cell anemia other hemogiobinopAies ec.) maY. 10r instance, is oh ijectio 01 .eW t" )le ITft hot th iot med5i dioifrentiatdI 5 media tto the parent, especially the ontud sa eirculanon l'hi alpracih is also beefed tO drnehlrare .e hemne mdica esSuch its m) ocaduled ltrel ion, Stroke, etcn d1 well 'a 'Jamnauc mitunes to biain~ and othe issues huestionx 01 such elL produx ed b)' the current iion~ti. direl imto the uirculation, by needle or aihtedy so that the s eLe are nked to "home ' to the bone marrirnitusele. kidne s. huozs and/o an ; other other orgom system, as well as 2.0 iestion dirct v ihe hone marrow ace ts sutabe fo the prace of the present omvention, Likenwise snicior of the wels direct in lesion site with or without radoowie utHrasome or Twuoroscopic miance is aso snUable Or the pcie of the present Nlehods of isolting selCeed ells us l in the present innon or lad c hose 2 described 1) ZOilo et atd 2(1(16 In a prelerred msbodient genetic \vector encoding numblhke and/or numb tSof ormi comprise regulable Promoters ope'rably linked to the Numb or nUtbtie (fl'5taVt', in another preferred embodiment the mrde of transtetion may be selected on'm those modes of tansteetto that pr0\svde IbAr unplsierl rather itn pernarwnt expression of the Rnubike and numb Koorms .irnmpe iiI ample ienet \v Xwledificat'ins M) OMN P(304I Ttummi U. is bhe\v eda thttrudtelh: N detseds and dml nicOl coolditton, wa able ta be treaed and s an horteJ A the nthods of he tpreent imvvntof meludjg, but in no wa lmbed to G. rta\ atl - dikeat' Vi G I tv-\tielks d1c9'se0 Ill' MA I t rEli Nth 4 s\ dtpoll Ui 1K I I umiwgton dinee mill 1I) vfS rtidrome, it p A and l 'p B \cmutn Pick disea,1 s SanhOI S JIwase ' 111\Nin: FabrS diSaased 0 At py Ie ( emann PIeKtI ds4PCI I: aucher\ diseae alli Parkisois disemseftRMl et \ on Hitpl LiuN disa\e. Siekk cel aneia ImB) and other thdaw07mias as well as similar doeas I'hese trasgenes may' represent the o inri or porions of the coo ng reg am of the nmwud to be ~imdlerstood, leWCta lb the upe o the Qtrescmi ifVCTis not w W imed to the speeeCmbodimwts and e!ples dested above t he imemi na be prncced OnbeI than a pONeuh& described ad sid be 0thin the scope o. the aucopanus in claims \awI 3 n eample semleuee hor a \vecto caphie of renderng cell 15 pluripotem and expressing a lng~ stmb ifra. 0cP4. Sox2' and 1:mOFP nuelce acid sequences under the cmnrol of tetk einensimte promote is daggIsa ive;ti1Q itt ica'aIg e ue iac'r2ht'a tgg likactlcuaitaage~tgagagtanlgtltItxgtgets34 rI tytl amd t a o ltag g aa 10 ~ ~ ~ ~~~ l~w aC.! avolowl~ sty.r iy iig zc1 C It Ci i1.Cc Ctaewe ty~ ~~iy 23 a'Mdgutngpaeaegeugetus LPy gUsa dogueggjgdlgldd costl 5 wilisL3dfgeg3Cg CdgugtEagatgtcungk kZeaia tettaa CgsCIgCegat' ta'tve'' «'Y$<' daIt e~giitya ce ist t ggedda d >EIaald Itditaan C I iS n344 tc44 eg aagt iZg iiagstttkiI E t c i t de1341 est a da at 5 talaCC t derCdeatggazIatad'vi P l >alaggt aa hgatt\agta tat.egtgttsg~aa * itsa agaaalt - g.4 .(q1g'$d<tadiYa3 d" 3 -cfl'' fia sa4 e atagi u at eca't e'Xali<a'"'v as i tg Sn atgd etueggag gladgstag .tltg ~.adeius ak tl ca~ 1CC it-ta gla OICI tatlgCtga e 2iteld.a atltgCe '1' 5 l' t lQtl&1litggatitggiu IliggyCCC llU tiglet gaat~ueile C a ZU'tigiIC aecC t biet 3e aaccegnav t .satJaA gczgaataEdla pt li dgli2C arCga lCttIaatrcaatt t;-i tat:Ita S~rtsakatgagtagt tR tu oat4..tti.4'dlat1' fi'b< i'"1 <'41 3 'a<ltrilgdiatatlta'2ttnC~agtslatta d.e.&, daaawk ia Aacardfalatccai1taaldaj&acga ci 1aaAg idat SCCC Matddedttelat~gataaiacttgal setb dat a d ;a kd worm a N gO t Ntc a tg NO 24I50$14 PCT/ S8;$65Q47 Oam tQoo; i y 1 rng g vnC xM go t i Yt~tCt: it3 :Cmm myZC~~ dVP: iC t:V zit tR1 , - s- lId t C CCL C Cv u" x 2flCC. tirp1 .. iC uC - .-... g....t ~ ;ay illC gta1 a~t CitCt ;VagCagIu CjCidst ka~ Conn )n t ,;gigCTgb rtqt 'mit gCca asgt :. 0 dCO ig tig L M I a 00 1 nod'mdod.. wytyna a 1 B 36 4 oC mv~ , .. ,n a, omc O CI CCCiX C ~ CaCC C C ail efltuCCuC C tCt hti ~ t Cat /CNCgC gNCC C/ Ig aCC~ acxn~ s s, - - , . s Y- ee e k . 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CdQaaV csca d~a~~~ C3.gIdtC. dC;U Ju t 2 d iJ R du V ' < te4~ .4S " " .3tgetg4waCdsfheC I2ons.3~CCg tc'ntdl (> ,.3th u4 hV de' not>a' o ee oh o np .A u hICZ' h d og ofi~l dIC N3 lb Stet~~i!3 andl ~ tIZ ettud~a Icequnced2.t 32m4 the p osm Con zit A0 2108150114 PC /US2nu}5U ccupwed by joet i the in virogen pe)N\4tode/:Z eetor, Samdativ the tetR gel ' tomd mn the Iviuogen Ie,) \ApcR Iecoto Cod ing sequences of genes referenced are dso rppte tfl 6r 1m m10 1,: the pc DN\4Iae/ veor. Altematively, the tetR gene ma, he transt:ezed io tarc"t cells sparateY utiW/p z the peDA 6A\ 'R v ector in combmnaion wilh a ector comprsng the Sequena hlee moi thectet.Rgenre amd 119 PC M\ promoter. I Akew e, am ile v ec tors may be emphe d so hi as ekment sWma rio the lenmms mduided n he abow sequence are present, Es may redce the hkelbeod of promoter compettin. It is to be underNtod that tither condmannl promoter eents 1Id)I b e stubstuted hot the tetr aevetine sensi isC e f oflOter elements. Example 14 1t I expected ilt ima\enous and other administation of phuupOtent jiem cclls produeed dccordin to (he methods described herein (01 Other published inebods) one or more tumes con pro\ide replacement cells to the bodnht such udmidrior nml ser\e to e Qtend the it or improve the health olhie patnieni suffe ing agei:elted 5 sentescence. I \dnple I 5. l'rouuchuon of tler c evil. Ihe current mnventin co\ era the derivanon of germ eel l'rom muldpoent, plr ipotenL t d 0ano se it' n ig m d pC r ted c ording to tie methods des cbed heam m aiccordum Li O the irbbshed metodwi I he producon of such Leii ells may be 20 Suldbe 101 {tetCot oetil th and prodtuiy embi iOs in vitr (exg. I ilbr e i ,at 200K; Lehlrt at - 20h 'Naverma et NOW - 0ta N' Lir o t .A '00T: lrusenhennier eo aL . 700: \'l<ore et ~i 200' ek 1 Eimple 1t, -( eneratont tfn'aeenc animals. 11e present inention c\vers th1e genleratil of ni (tigenic animals. As w ith other 2A p1 WfiPOttfl gel js, the plaripotent ed u produced Wv the mellhods derbed hereinl (or mother puhsheod methods ay be Uib5ired to produce transgeme anna b anv method krono to the art. l'\amplc 17 Ibherapeune \vector (i0liSiruenOn Examples OR reirUViraI vectors \hich ma be employed INlude, but are noa ilmired 30 to, those derived trom \mlone\ Muame L eukemia virus . Molone ,\arime Sarcoma Virus WVO 23/150814 PC TfS2081Q0047 adl Rous 8iuona \Virus, I I\, and Hi A\ Appropglde esprcs:,in \echo are that limay b emnplod ltr (fuitfetm DN A or .RA 1 mo eukarvoti clls. eh eettos mdclude, but de not boimtted ao pmrot\ yot c vectors sutch ast for e\Jmnple, lacterts Lvl n ers, eukaryotie l ectors. WN o \ a \ dt \ector ad tbal ect, and \lT ectott such a. anot 1 icntel J. lel\ n ee *hrr,4,.d\ a tear .dll mt~ 4 vail0 vCtors, >nd retrovial vedors. Vhbe r'epitaliN inR1nipelent pel) \. 62 GWrnil(R ind pcdNA 62/B'latils4d5 VLS v1ctors 4atre e\xmpie' ol an uppropritci exprcsston\ v eto0 (invithogen ) Jrd alo\\ e\n s00 of s\yldhetic oliportide (iler miRN \s) that la\e dhe cdnacltv to cleave (1 rlfged 5 4~uenQtL.ee vectors t10lute tltmktn >nloop00 seoucec drI'm endoceno11s :niRNA \to direct the excision of the ce need niil{fr ll a lontger Pol II Utercrpt (pre6 ,wterat ivel. inclusion 0f the 111\' ps sequene allows the theaupeutecutor to compete wrt. naiv\e il Vl gennne fir packaging into viral pariceles, also ihblm I 1l\ ( S trasm.Ission. Coinirtg. muli pie mDiRNA sequenes dirtected agdas a singe larger incea uses the hkehhxood ol success un reduc liget sequece erpresn . miRNA \eguences may be opctaht hkfld to tisue speethe promoters such as the H - alpha plomufe, any 1 cell Teci ic poloter or macrophage sprben promoter to ensure Cxpression i the desired cell 20 tye F 'hrmg i topen s lentiira dest maton lWIT) \vectors fot gene e xpresson, the re llun .terapeutic \ector{s) becOmes capalS e OfI d r \rdneducn bot d\ iii no and 000 diu dog ksel ty pes. In a pr eered embhodimnent the thcepennec \vector~s otinas muhmple synthetic: 2 g0nukledcidequences diven bM one or more promoters so s to reduce expresion of C lN ( (I> anQd/o row tter10 t'efltidd protein IU0OV110 toeat as a eSi-eept10Y for Ill\ infeceion in hrc wel st In one iherapetce v ec1r ( constructed in %L6} four nRNA sequeceD txtigetil CXCR4 and (UR S coueceptors were cmed into the peDNA 0.2 G\W R c etor along with 30) decoy RN\ sequences targetmg ii1 c2 I A R and R RP Genei ontructs tmay ic lde a vetor backbone, rI a tnseti\ vator w\hich 61 NY0 2t1015Pt4 PC /US2OLPN0i65001 reguhiles a promoter Qpertr3 huh ined ho hetentlogou~s nucleic ae.d sequences \nothermamde a a sWrable ichr is a retvrual ector, Retroyiruses are RN\ uiruses hgh conta n, .PA4\ gomeC The ga. poA and Cn gnsr long teonimmal repeat 0 i.R) .equene Ut atid Y 1 R seqienes parmote t ranseriptaon and potyadenY LtD00 of rnRNA\s Ihe .rettr I ector ei a0 poiue ieuabltrans 15s1tng element an internal ribosomne rtst se (IRWA. a selecton marker and a tar get heterologous gene opeated by a regUlable prolintef Aliti No\ , nmdltipie seguettes$ ir) he expressed Ufldet thae ontrol of multilis2 H) Wiomote. l ,fwav. the i tviu l vecor may Contam enactof seqences necessary ihr rev erase atsertnon <mdl inteton, U'poin mieoa the RNA is reverse trmscribed to D'\, which itegrates etiieiendy linto tKl host geriome, T he reombinat retrM truDs 0f this inemtion IN genenkalIy nr adild m nilch a way than some ot the relo\iral, irdleuioaa genes of the odlr\ye vjirns arremov10\ed and in embostdiments replaced inteadl witlh a trOct DflticI 15 acid sequeiwe toa geetic modidkationt of the cell. The sequences may beeo rms DA or P \ i, w as aof o altered tFrin\ F ample it lixample methbods for generation ot the therapeunec vector I hermeibods for 'eneration of the therapeute i ce&urailadde those taunt in lnvdrlger's Vrt) P'ower I enui\ id E preh mat bSt~env \Ianu~ (teoTporated by rei erenee 20 bewein) Briefly, thel o't' Ilt d casette i\ c'loneCa a Pl tpltfranmtatmo Ihe pl entifl R2.C/\ 1S1 vector while the maR dco eassetle ga 'imultaneously transferredt ba HP metton io pRI0\dC2 I hen the o i promote and nub.dceco ar" \ltiAue iR cromsed into the moahiied p enti 6/ m n P-hsd R4R / V A etor, p i\ k t or seque nace.

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SeLv-nego d tnaap c (ht xtatn e horwe senm and htiannrainbinxlta lae geeru WA imtor a Ho W t Me I it r'epaau Tce taL ad for wiatc g .e gu g wcaandr t ietat ce lgon ateeder asrtgs t-lsa \eg ndaa so be m noyewd) and is e sntia i wua tiri0ping pronattrsn cl 3g an Mn MM A t atct. vap ntle i2t0: stetn Cel are eaecOW ng n inyt viinIak the;cellsg u irnaee gti 21 h 'Ua qgc etors, hem prpared t rPdlp iabi tsadrmtos iho ~tl', cip w11 iltt aIi .rnawhnaw "; i in Lxnmpe l1 tinibhds cord brlaoo g m ble atCobied froge mb isic 10( cor haL ini el o rde hn o trbanLu etmer slf te raec vhectar orelanefily equemt eso then01 npreihon yo ip Yed standrd netds it oranYerthoid roLA tem and mtchin 23 jtmie L ta mpe.:brncpesaoSht %ic olueotide seume smd htanl r embma in in ekerS e utc vector p~xrgesitor meel oaneecas 7t'cl is covere mw thoe ineponehd.sesea iiame te edlnue seouenced theluat tdeesM. vtheuc~he abiantecidtwrieh 30 ells t Ir:ain I eTo! ton p y r > 7e d f r to a esskeri l te ru d agree or d wilb :r Ii It 1 a bstilicpeu dgreare lob c red bdihis beoo d Larpsy (0htd(c slgncetd egences uuceudell rniAqrethdried Kh prote c~f~s1U 1sadeedSt{enes' 9 l C~ee b te reet n\eno XVO 2TS15014 pC/gSt4f60 aon thmnt }m fropen)H iOJRN\A Seqtient> ts(ignyth 4gne~ 10lode top strnnd: 5E CTGIATAtAMU(CA(IOAKAAG WCAGG rUrWcACUAfC.TC fTCT A AL I A Q\kG AUG A I\ \ WCiCAGI t ' CA CAAA CA 3 , A\ I'RC CC it A,\ C-sAndb'tu -lan bafn ,r d CCTG (IA '\ C \G \ \I C '\GTTGIJC\TCAG\lTh CCA \A\AC' A' \C C Al '1 ACAn aIC 'n t l m top htrandS> i (i \ NC \ti\GI\CAC\CirIAlAa TiGCAC \)O\ T\ACI\ACA'\Kt A IA AId 0 NtM 'ahd i Kword: n CC Ci s(r FC\tid \ \ I t CCAC'AG' C i \bl (i&AAA Cl'.AAC iAO5tittC i C O(( 6At 20 vector ilAny lcy San3 RN\ Prgu ne s ugftu reethe ablagene mdudet cehll) tossl HC I G'e con by T A lesser but h'i GreCAC IW Iia AeG\01 \ * A '0 or to\ a tre iG t hn \ x UtG(iC d i<e ac I coee by(ibi inventions, f fAC ii AC V(6A J:C as nld as IlOp sthMb S1 GUl, W \1k A M l ONWCA"'' AMlAG I T]' 6(0 IcfuAIG\ X\G C A ample A1 N: dapv iece Is e catige sequengeag tact pxgonfulseuctet he a ht f ase dh t li e N phemin hy ' ^3044 tow a eWar but theapeube destec oT HI\ s AdnM witi bIns 25 C~tt~\nt eCtmCC i (Broeev i)UrtC ct P1tea goyctggugg1ag ap C C q eaggeltC to i C agwnecieluaa gige itO n Ynu myw gceggt ndCnegu gyw est go en f 1 so g;q yqau K 3(5 aaauec~ccaggeztag Ucc~' Iflsc c~i-aaame ;eeI Cgct awaatet q tg (DillonWh wi A,. g at g i a i \VO 208/150814 PCVV02U0S/065007 E example 24: ranking sequences pioridmn stathtv fr RN \ decoys xainpes of appopriA clanking sequences K R\ \ decoys are as toikvs GiU'C.'CGCJJ CiUAGCA C:tiUMAC -TI.R UFCOY SliX i \AUA AC: ACI(GMUCCGAi' U S 1;(U1C(,Cl t'(t1 0(0'A\CCl.CAC -RRE DE(J OY SE'Q 'C1.,\nA\AGCiit l'(' (iUC1C t ' ' Preous nly, it w as demnonsoated that deC sequences flanked by hairns neth side, 19 nieleotides (nds of the 116 RN \in te side as w ell as a V stem iomnediatels preccdinp a poly L terlunator ior Pi al II alth\ ed' greater sLabjhtv. Ihis aril neement ts 10 expected to protect agamint 3'. exounclcas attack, and to reduce the chances of the 3 tmadICr minicferingn with the insert INA iblding, Since only the thrst 3/4 of the tiRN\ sequence is present, the 5' end of the insa should be protected and export from the nucleus should be prevented i(Good et aL 1997. example 25 ntdidtuou of drerapeuic Veesu to the 110o1 15 In a prefened embodimem, blood stem'proeemtor els. and mtcl cells arc transeCted "ith the therapeutic \>t"s) (Or assoiaed ther apeuti 1ius) in Vio be mnWdu uon of the thereune vecton Into the host blood, tissues, or bone martron ei She greatest benefit emay he achieved by modi ma a Are number of endogenous target and sitn4m~ rockniior QCIIS I lhis n ia) be ac.-lsedb sn appropriately v-iz/cd, catheter 20 ic devie' or needle to mnicet the therapeuc v ector ilto the enous or arterial ciclation. in a preyed embodiment, the virus s pseudo) ped with vs- emvelope glyecprotem and native 111 l 1 mn protemns, i example 26: introdueiOn of geneteallymeditied celY io the Ilst flood ells such as maure peripheral blood I' lymphoe tcs, nmonocytes 2 mnarcphage.. i eON prosenbrs .mcrophage-mon oete prenitor cells an or pluri potet hematopotetic stem wli (such as those tRund in umbilial cord blood and occupying bone marrow spa& stNe l as otbeW stem oipgen bor cells can be transtycted 1ing the t herapeutic te Ctuit s in vitr A00ppr opriate conc ent rations of the ther apeutic vetoirts) may be those constent oth Wownimng et aL. 1999. Shsequeritly ,cellb are expanded ipropagated) 30 in vitro, and are then transfTrred to the host i a inmodution of the cells 10 the enous or

Y

WO 2(0015St14 PCT/S208/Q65007 arterial circulaon usmqe a irbl ravenus needle or catheter Subsequently, s el tdas ['eted \\ih the ltherapeulic \vecors ar'e sible to TlhomC toi the bone marlrow sand other issues. it is tnderstood that the examples vand embodiment dcibled herein m Iliubtmatv ~tt1'005$ ot.lI aird that Sd i he 1s w4 ilons or changes in Q;nk thereot wil e sapows' led t s 1 le i the a r and artb included to thin111 the spiri nd puriew o' Ihis applltion and sc"pe , l the appended claims. All puica'tiowns pates, and a!pant appht eocited i r hrbin ar m- . troratd by refbrense in their entieny t0 al prploss I Amic 27 ,\dxaxn s oI Cte escwd or targeted tt'anagenes utdizled in the present 1 i invention \ al) i1mostxcnc sequenlei Olefitte inl fulfillg tlle presentinvt ioitsl suitab'le l0r use- in ilh' pa NCt a a senh0n NllitdIlc mntletilde seque11ces mald be branel no0 Ghv speies -so lone s iln lelN1 cellsl or behavior i ahdi ed, lkewise the lethod of ning s'ch sequences. eit in lower ease or pper Case letters hereinh doe not mlpl y T htilrM 1 9sec01i !h10 olwlnig sequences stored in th ON! dtabse (isd by dtcknSIO nu 1nrI V*l0i05et ex'amples at srijutsces rieferenced above mn the prc'1 applicaton. ty aoe also example of speciIC trnsenc enodin g sequences d itable r use in the prcsen inetio0n but do n0t in eau Na html the re- 1iee of the inenDthon oudietrupll mit: kl3030galggegeggggaggd agancutagcrglt Actp 4ggcagecgsge o agtgtgtegzysesgccggegaancetggi4 -gseg t~gcgct's'a ggagcge;ccg. 4 .mUtg' rues CetInesk >Cag5Ztg< in'grge ege>ew > u< rtcaeeagtcrg esgaec aeipyeo tgsz enact 1 t 1- ml 1964 notu s;intcAcni4Rcatmf.i. c< <K etagtgegt pacemci va etetiltglgetag'dcig gtlv3a t'O yk ite (55 th cateri et: MZifteae'etagaetdtJul g agag 2dae ldeg 30 ats age ovaAgatecir .. m nriaa tnegieriiu gagad1mnaggtreg citxtri gs pa cal t ki cd te aI' c htiti t l eact ;egvl'ca 1rgdeXcathtat lts kt ;lacalrlaliw vi 600 W5<4 14 On NM 0 .1S4i mt~ 13: N \ \1 '16 :aulev ttwaasea Ca g aleisC itittcftecacac aslac ggtactteitgactetsstumtai Aagregitn~aitgegadhmaathtipaptgey 4id. aatatsa talmmrclgwustgcatatavl1g>,sasa X40 POWWOW~$I PCTJUS20MSIHI4)G . tgcvgowz nc toos.~ atc atcac Kaa ~ o tw ZU ztg tuc~ot, gAR a a cc:3 1 .- l :2 [ d 442lg " O A 6 'S 1.' apolog i d . 11.6 now S5) r~~n yawtaui 0" c1cu c C goc s , s .. , - .. s . , , Cat''ti3%h t iHhdlt 11001 R flsic c t 2tild! ad~b i?c. Wl~cgata.WiJ ,a- d.c<tch%&dta . b.t 09gti,3c.<2 d"'k id1..ddP i A scaagat v I C" di K d dit c di 30 >%lctgd th'lb'n at3 Mop ;'1=&Q'. S1.. M At& W 'N . (324 SAL& " iAb t'n Yitc a "i.c ,C SONgat 0 S& ci< cCit i ag ,Ka c d 4tt nd 01 PC RI 3 tzt & G c VoClv ' 1Q.1 CM.gwmn C. ati 1fl> 1( -13>..3( < g CM - ic. c c c i i.';,za x . C32czic(Cc ti.\cc : M k1'Ot c tt t1 rc3. i t b acC uidau d;,C tuttt , aE agaQ4t~p~cC~iki~'~.~...~i.L~v3t>'c'a V g1cti4:cli4. f~J'itt3c3.CVUii, u g a~icds itg accczg g -. d Ci >cC dcid zcW .gi E"da2 .s ( a'. . dis. a Bmin 4c ~ . ;cc..geascc.t.sctaatc s flL ;c pgWtcgug:CC ccar cattcizud cic ccacrkeaM .3iitc (i Gmt333c Acittc~c~g~sic~mz~icdyccact~g ,gcga~aacdi~~tugacag~agcact ..0 s . .c -cc s~c3ck* \cucgc~ ~ c3zzct~dcit~ s3 sj , . s s3t s c~ttct u333k1 :dC(333Ck tll3.3' 0 0t3C1U(1a Ez$il t C C P~t NWI IQ <20:0(20 11. Il:N > I-, S s1i0 33 3t..>t c C C 30,3tta t~t~~4~ gztdi C tcictsi c c s e CGCRula ag d C ad 2 1 0 ikstau e~69i \V) T OJ)@ O$I4 PC''010 (50$85147 I a w <deK cttc tzalz 4 qty'a m1 Cant da 141 005 1 M 1050 41egy:10.0p3aCd~R6C0.!;Oi djC2bt d b CctR i c at w twdC d CLdadl4 2ew C ugo! 2(2 ad CtXCI. u CdCCha. ltCCigCC i hdcs C Ci MI& Q d Rt A CCS R C C a> ddCt) tCCidt di EI (C 3 It 3 t 31t0 C'.t( 'te C". ~ ~ ~ ~ - -. Sa Yn a on un w wow"-3t ' a Cjalt ua~~kt tNW vsvAt gw wg;To oJA W w dl ToMaa"d C Co ci2c ",;l CAD~a 1211331C ca3.dt3 C C C bl wlt3Ct:3 113n1C. dada2C3 .. C .. Ci'Caig ( 3 dh td: IU3 .VC d C tIC CIC 32121 UCIg332C CC&C C nu 3Ces nutus3aCsc~~addaC213$C3 3CZCititla 3 211C I 33tC C2C'fl aCP %21 i b~aailtasaI( iCg AC ItilItCb,'taCduZgli C C C L 111ChC 21TC32131 ~i3333fC~a2.2 .Ctig~a%~2CIC~iWt' 2: UtC2tC2131C tC333sh3 3bli'. CI Add121dd3 hC'la3233 2idCg3CCi3Cd CC!10'.1 2 13 dd2 da1331.ICI 1342t C'ICJ3Ct dC d -hg gI C2glcCtaZCCRAmQgag gCaIiIaa T ltt itgad( t 131.131C2SCt3CCCIi C D d'EgU-td' bI 6'tAd b12Ct ai d< [ ~ f' s <:. tu h'C1122:aaC3.CQCIt 3122 CC (2I~t 22 lbC L:.22132 aCtCC3i'Cd1Cg :.I.C d22U c- giIt - gdg i2 t3gtllC i 3l~~ C gthiCCICCIagn 2 du3dig tpaaa ut I1CCII3 u nuCC cyl -I313Cilatt 3 231 c i L332 31 I'i2 {ltdalalt~r .12 ddiI al tdIC C 43 cy 92c tjQR.. edtaClRgagg<:Gdk d & 34 'U C31a-2 932 d d3 3 2d3 -(AC s3 . -92 2 C 4~aag 41 ~ ~ lgtC g2 g 31 332 1 2C 23 1133 3922213 23 WCIIC12CCCU2- \gCgCZV h- lR ~ N:d CIt1 aIt23IC CCC:1 ICCIult 13C19d "d91 'dal 12 I2C d CIZCY1412 at.ciCC 3223ad.232131d 1CC2e3332CC<.a.CC22 d2243Cg23i2t:I g a t:itd 1dtt'2U>4di.d

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Claims (13)

1. A method for production of differentiating cells by cell reprogramming comprising the steps of: - selecting non-pluripotent, somatic cells; - overexpressing or introducing nucleic acid(s) or protein(s) corresponding to one or more transcription factors normally present in the desired cells.

2. The method of claim 1, characterized in that said cells and/or their progeny further overexpress transcription factor proteins or nucleic acids selected from the group consisting of: a. Mash 1 (Ascll), or a portion thereof; b. MyoD, myogenin, Myf5, Myf6, Mef2, myocardin, Ifrdl, a muscle transcription factor and/or a muscle type specific bHLH-encoding sequence, or a portion thereof; c. Foxa2, Sox17, HLXB9, and/or Pdx1, or a portion thereof; d. hepatic nuclear factor (HNF)-1, HNF-3, HNF-4, HNF-6, and/or CREB-binding protein, or a portion thereof; e. Runx1/AML1 and/or NOV (CCN3), or a portion thereof; f. Sox9, CREB-binding protein, Gata6 and/or Runx2, or a portion thereof; g. OLIGI, OLIG2, and/or Zfp488, or a portion thereof; h. MyoD, myogenin, myocardin, Myf5, Myf6, Mef2A, Gata4, Gata5, and Gata6, or portions thereof; i. Gata4, Gata5 and/or Gata6, or a portion thereof; j. Myocardin, or a portion thereof; k. Myogenin and MyoD, or portions thereof; 1. Runx2, or a portion thereof; m. MashI (Ascl1), Ngn2, Nurrl, Lmx1b, and/or Ptx-3, or a portion thereof; n. Mash1 (Ascll), Phox2b, Lmx1b, Nkx2.2, Petl, Gata2, Gata3, and/or Petl or a portion thereof; o. Mash 1 (Ascl1), Phox2a and/or REST4, or a portion thereof; p. Mash1 (Ascll), PITX2, Dlx2, Dlx5, HES1 targeting synthetic oligonucleotides, Phox2a and/or REST4, or portions thereof; q. MashI (Ascl1), Phox2a, Phox2b, dHand, Gata2 and/or Gata3, or portions thereof; r. Mash1 (Ascll), Nurrl, REN, neurogenin1, neurogenin2, neurogenin3, Mash1, anti-sense Hes 1 RNA or portions thereof; s. PRR- Numb or a portion thereof; or t. Numblike or a portion thereof

3. The method of claim 1, characterized in that said cells or their progeny. a. overexpress one or more of Oct4, Sox2, PRR+ Numb, Nanog, c-myc, lin 28, a gene with Notch activity, HoxB4, FGF4, and a gene with LIF activity; or b. are incubated in a differentiation medium.

4. The method of claim 1, characterized in that said cells and/or their progeny are incubated in a differentiation medium comprising one or more agents selected from the group comprising: a. retinoic acid, neurotrophin 3 (NT3), nerve growth factor (NGF), glial cell-line derived growth factor (GDNF), and/or interferon-y (IFN-y), hexamethylene bis acrylamide, dimethylsulfoxide (DMSO), fetal bovine serum (FBS), normal bovine serum (NBS), retinoic acid, cardiomyocyte conditioned medium, vascular endothelial growth factor (VEGF), LIF, thrombopoietin, a colony stimulating factor, M-CSF (CSF-1), GMCSF, IL-7, a cytokine promoting CD4+ T cell differentiation, sodium butyrate, activin A, epidermal growth factor, basic fibroblast growth factor, noggin, insulin-like growth factor and nicotinamide; b. retinoic acid, neurotrophin 3 (NT3), nerve growth factor (NGF), glial cell-line derived growth factor (GDNF) and/or interferon-y (IFNy); c. hexamethylene bis acrylamide or dimethylsulfoxide; d. fetal bovine serum (FBS); e. normal bovine serum (NBS), dimethylsulfoxide, retinoic acid and/or cardiomyocyte conditioned medium; f. vascular endothelial growth factor (VEGF), thrombopoietin, and colony stimulating factors appropriate to the desired cell type; g. LIF, neurotrophin 3 (NT3), and/or nerve growth factor (NGF); h. a colony stimulating factor, optionally M-CSF (CSF-1), GM-CSF, IL-7, or a cytokine promoting CD4+ T cell differentiation; and i. sodium butyrate, activin A, retinoic acid, epidermal growth factor, basic fibroblast

5. The method of claim 1, characterized in that the selected cell is modified to express: a. telomerase altering the cell's life span; b. a gene product deficient in a patient; c. a nucleic acid sequence encoding a protein selected from aspartoacyclase (ASP), HRPT, HTT, NPC1, GBA, PARK2, VHL, beta-glucuronidase, hexosammidase A (HEXA), hexosaminidase B (HEXB), HRPT, HTT, galactosidase alpha (GLA), glucosidase beta acid (GBA), von Hippel Lindau tumor suppressor (VHL), beta globin (HBB), NPC1, PARK2.

6. The method of claims 1, characterized in that the selected cell or its progeny is modified to expresses: a. a beneficial nucleic acid sequence; b. a synthetic oligonucleotide, complementary RNA (siRNA), microRNA, forming pegs (shRNA), interfering microRNA, antisense RNA; c. a nucleic acid sequence or protein retarding infection by a virus; d. a nucleic acid sequence or protein rendering said cells and/or their progeny less capable of sustaining viral replication; e. a nucleic acid sequence or protein rendering said cells and/or their progeny less capable of sustaining viral transcription; f. a synthetic oligonucleotide directed against an immunodeficiency virus co-receptor; g. a decoy RNA; h. an HIV psi sequence; i. a. nucleic acid or protein sequence rendering said cells and/or their progeny disease resistant.

7. The method of claim 1, characterized in that wherein a protein, nucleic acid or agent is present in order to screen for its ability to induce phenotypic changes or differentiation of the selected cells into desired cell populations.

8. The method of claim 1, characterized in that electroporation, liposomes, nanocapsules, nanovaults, a vector that does not integrate, and/or another approach avoiding retroviral/lentiviral integration or other random alteration of said cell's genome is utilized, including site- directed mutagenesis.

9. The method of any of claims 1 to 8, characterized in that said cell and/or its progeny are cultured in a three-dimensional or two-dimensional scaffolding, said cells and/or its progeny are grown in a de-cellularized organ or on de-cellularized tissue, or said cells or its progeny are utilized in conjunction with inkjet printing technology for tissue engineering.

10. The method of claim 1, characterized in that is performed in vivo.

11. The differentiating cell population, derived from the patient, cell line, or a donor, for use in fields utilizing cellular therapies, gene therapy, or animal husbandry produced by the recombinant methods of claims 1 to 10.

12. Use of the cell population of claim 11 which is screened based on cell type specific markers; transgenic marker expression under the control of cell type specific promoters; or transgenic marker expression under the control of neurotransmitter related promoters.

13. A method of treating a patient with a condition or disease a. ameliorated by correction of a genetic deficiency; b. dependent upon expression of genes producing susceptibility; c. ameliorated by renewed cell proliferation; or d. characterized by dysplasia, cancer or aberrant cell behavior, comprising introducing into said patient a therapeutically effective amount of the cells of claim 11.