Jurnal Stem Cell Thalasemia

8/16/2019 Jurnal Stem Cell Thalasemia

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SAGE-Hindawi Access to ResearchStem Cells InternationalVolume 2011, Article ID !"!0, 10 #a$esdoi%10&'0(1)2011)!"!0

Review Article

The Ongoing Challenge of HematopoieticStem Cell-BasedGene Therapy for β-ThalassemiaEkati Drakopoulou,, ! Eleni "apanikolaou,, ! and #icholas "$%nagnou, !1 Laboratory of Cell and Gene Therapy, Centre for Basic Research, Biomedical Research Foundationof the Academy of Athens BRFAA!,11" #$ Athens, Greece# Laboratory of Biolo%y, &niversity of Athens 'chool of (edicine, 11" #$ Athens, GreeceCorres#ondence should *e addressed to +icholas & Ana$nou, ana$noumed&uoa&$rRecei.ed 2 /a 2011 Acce#ted ' Au$ust 2011Academic Editor% R& eith Hum#hries

Co#ri$ht 3 2011 E4ati Dra4o#oulou et al& 5his is an o#en access article distri*uted under theCreati.e Commons Attri*ution6icense, which #ermits unrestricted use, distri*ution, and re#roduction in an medium, #ro.idedthe ori$inal wor4 is #ro#erlcited&)-thalassemia is characteri7ed * reduced or a*sence o8 )-$lo*in #roduction, resultin$ in anemia&Current thera#ies include *loodtrans8usion com*ined with iron chelation& 9/ trans#lantation, althou$h curati.e, is restricted * thematched donor limitation&Gene thera#, on the other hand, is #romisin$, and its success lies #rimaril on desi$nin$ e:cient$lo*in .ectors that can e;ecti.eland sta*l transduce HSCs& 5he maed HSCs& 5his #a#er#resents the current status o8 $ene thera# 8or )-thalassemia, its success and limitations, and theno.el #romisin$ strate$iesa.aila*le in.ol.in$ the thera#eutic role o8 HSCs&

$ &ntroduction 5he )-thalassemias re#resent inherited, mono$enic anemias,arisin$ 8rom autosomal recessi.e mutations, a;ectin$ thesnthesis o8 the )-chain o8 hemo$lo*in ?1@& 5he arecharacteri7ed * reduction or a*sence o8 )-chain snthesis,

resultin$ in ecess o8 +-chain molecules, which #reci#itatein red *lood #recursors, leadin$ to im#aired erthroctematuration, mechanical dama$e, and ultimatel to a#o#tosis?2@& 5halassemias are caused * more than 200 mutationsa;ectin$ the human )-$lo*in $ene and are most #re.alentin the /editerranean re$ion, the /iddle East, India, andSouth East Asia, re#resentin$ a serious health #ro*lem&6atel, due to #o#ulation mi$ration, )-thalassemia #resentsa clinical #ro*lem also in B, BS, and Australia& Glo*all, itis estimated that there are !0 million carriers ?@&)-thalassemic #henot#e is .er hetero$eneous anddirectl lin4ed to the $enot#e& In hetero7$otic state, theoutcome is consistent with clinicall normal indi.iduals, who

are lar$el unaware o8 their $enetic condition& Inheritanceo8 two co#ies o8 )-thalassemia $enes causes thalassemia


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macantl delaed the onset o8 iron-related or$an8ailure, treatment noncom#liance is common, leadin$ to cardiac,he#atic, or endocrine 8ailure ?'@& Allo$eneic hemato#oieticstem cell HSC trans#lantation o8 human leu4octeanti$en- H6A- matched si*lin$ donors can *e curati.e,reachin$ cure rates u# to 0 in #atients oun$er than 1"ears o8 a$e ?@& Howe.er, it is associated with a num*er o8 draw*ac4s, such as the limited matched related donors andthe need 8or lon$-term immunosu##ression to #re.ent, treator dela $ra8t-.ersus-host disease GVHD, o8ten associated

2 Stem Cells Internationalwith allo$eneic HSC trans#lantation& 5here8ore, an alternati.emolecular strate$ *ased on $ene thera# is undou*tedla radical a##roach that o.ercomes all the a*o.e limitations&

!$ Designing E'ecti(e )ectors for β-Thalassemia Gene Therapy/ost research e;orts towards $ene thera# 8or )-thalassemiaha.e 8ocused on em#loin$ retro.iral .ectors as a means o8 $ene deli.er, since these are ca#a*le to inte$rate into the tar$etcell $enome, resultin$ in sta*le and lon$-terme#ression&Howe.er, the inte$ration has to *e tar$eted and should occurin a s#eci>c manner in order to a.oid #oor $ene e#ression or

e.en silencin$& 5here8ore, 8or $ene thera# 8or )-thalassemiato *ecome an e;ecti.e and realistic thera#eutic a##roach, the8ollowin$ .er im#ortant criteria need to *e met%I the thera#eutic .ector should ehi*it sta*ilit, hi$htiter and erthroid-linea$e s#eci>cit, .ia the utili7ationo8 res#ecti.e re$ulator elements,II the trans$ene must *e e#ressed in thera#eutic andsustained le.els,III the thera# itsel8 should *e sa8e and e:cient in termso8 .iral transduction&Earl attem#ts *ac4 in the 1!0s and 10s utili7ed $ammaretro.iral.ectors to achie.e sta*le and hi$h-le.el trans$enee#ression, howe.er, with no success& /ore s#eci>call,

illiams et al& ?(@ mana$ed to introduce a mar4er $eneinto murine HSCs, usin$ a .ector, deri.ed 8rom murineleu4emia .irus /6V a8ter re#lacin$ %a%, pol, and env $enewith the trans$ene o8 interest, while later on, in.esti$atorsutili7ed these .ectors to dri.e e#ression o8 )-$lo*in $enesinto murine HSCs ?"J@& 5he outcome, howe.er, wasunsatis8actor, as #oor $ene-trans8er e:cienc was o*tained,with le.els o8 )-$lo*in reachin$ 0J2 o8 the endo$enousR+A le.els& In an attem#t to increase )-$lo*in e#ression,+o.a4 et al& ?10@ incor#orated into/6V the newl identi>ed#ower8ul D+A-enhancer elements 8rom the )-$lo*in locuscontrol re$ion 6CR, which is 8ound to *e essential 8orhi$h-le.el e#ression o8 $lo*in $enes ?11, 12@& Bn8ortunatel,#oor .ector #roduction and $enetic insta*ilit o8 the .iral.ector $enome were o*ser.ed under these e#erimental


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conditions, #rimaril due to the lar$e si7e o8 the 8ra$mentsincor#orated into the .ector& Etensi.e muta$enesis studieso8 the transduced )-$lo*in $ene * 6e*oulch et al& ?1@identi>ed a "2 *# intronic se$ment and multi#le re.erse#oladenlation and s#licin$ e.ents, which were res#onsi*le8or low .iral titers and insta*ilit o8 #ro.iral transmission,

u#on in8ection& Around the same time, Sadelain et al&?1'@ mana$ed to $enerate a hi$h-titer retro.iral .ector thate#ressed hi$h le.els o8 )-$lo*in in an erthroid-s#eci>cmanner, * com*inin$ the human )-$lo*in $ene and the6CR core h#ersensiti.e sites HS 2, , and ' howe.er, the$rou# 8ailed to reduce #ositional .aria*ilit o8 e#ression& 5a4en to$ether, the a*o.e >ndin$s indicated that .ectorinsta*ilit mi$ht *e caused * s#licin$ o8 the retro.iralR+A $enome, a conseKuence o8 cr#tic s#lice sites withinthe $enomic seKuences ?1, 1'@& A wa to circum.entthe a*o.e was to turn towards lenti.iral .ectors 6Vs, asthe latter, in addition to the common Ga$, ol, and En.#roteins, also encode 5at and Re.& A ma


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com#romise #ractical use the mechanism underlin$ thisdecrease has *een recentl elucidated 8or the >rst time ?21@&Howe.er, when Hanawa et al& ?22@, included onl the 0&2 4*core element o8 cHS', it was shown that the s#eci>c elementcould rescue .ector titer * alle.iatin$ a #ostentr *loc4to re.erse transcri#tion associated with the 1&2 4* element&

Also, in an orientation-de#endent manner, the 0&2 4*core element si$ni>cantl increased trans$ene e#ression8rom an internal #romoter due to im#ro.ed transcri#tionaltermination& 5his element also demonstrated *arrier acti.it,reducin$ .aria*ilit o8 e#ression due to #osition e;ects&Similarl, 6isows4i and Sadelain ?2@ showed that the incor#orationo8 HS1 element enhances the thera#eutic e:cac o8 the $lo*in $ene trans8er in murine )-thalassemia, com#aredto HS2-HS-HS' alone and, there8ore, can lead to e.enhi$her $lo*in e#ression with lower .ector co# num*ers&Stem Cells International Also, 8or sa8et reasons and in order to a.oid insertionalmuta$enesis com#lications as in the SCID clinical trial ?2'@,

all research $rou#s ha.e em#loed a sel8 inacti.atin$ SI+con>$uration in their .ectors, * deletin$ lar$e 8ra$mentso8 the B re$ion within the .ectorsF lon$ terminal re#eat65R&

*$ Hematopoietic StemCells +HSCs andenti(iral Glo.in )ectorsA schematic re#resentation o8 the HSC-*ased $ene thera#8or )-thalassemia is shown in =i$ure 1& HSCs re#resent aminor #o#ulation o8 the adult *one marrow, accountin$ 8or1 in 2,00 to 1 in 10,000 cells in the adult mouse ?2, 2(@& 5hese cells remain relati.el Kuiescent most o8 their li.es,with murine HSCs enterin$ cell ccle e.er 1-2 months ?2"@,

while in #rimates and humans, the turno.er rate is e.enslower reachin$ 1-2 ears ?2!@& Due to the a*o.e 8eatures,$ene thera# 8or hemo$lo*ino#athies 8ocused on em#loin$such .ectors 8or $ene deli.er, as the can e:cientl in8ectnondi.idin$ cells, and thus mana$e to deli.er the trans$eneo8 interest ?2@& Howe.er, it should *e noted that althou$h6Vs ma *e a*le to in8ect HSCs in Go #hase, it has *eenclearl shown that HSCs eitin$ Go and enterin$ G1* #hasearemore readil transduced ?0@, #ossi*l due to the 8act thatre.erse transcri#tion occurs at this sta$e ?1@&-.1. Correction of (urine )/Thalassemia. As mentioneda*o.e, the ma


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In their >rst attem#t, usin$ a noninsulated .ector, themana$ed to correct thalassemia intermedia in the murinemodel& Howe.er, the #henot#ic correction .aried due tochromosomal #ositionin$ e;ects and .ector co# num*er?20@& In an attem#t to address these issues, Arumu$am et al&?21@ incor#orated the cHS' insulator in the D'2)-' .ector

and succeeded in increasin$ the trans$eneFs e#ression, inthe e#ense o8 .iral titers thou$h& Similarl, Hanawa et al&?'@ demonstrated that animals recei.in$ trans#lants o8 )-thalassemic stem cells transduced with a new 6V, containin$&2 4* o8 6CR seKuences, e#ressed hi$h le.els o8 8etalhemo$lo*in, ran$in$ 8rom 1" to , with an a.era$e.ector co# num*er o8 1&& 5he a*o.e strate$ led to a meanincrease in hemo$lo*in concentration 2( $)6 and enhancedamelioration o8 other hematolo$ic #arameters ?'@&6astl, Ohao et al& 8rom the ersons $rou# went 8urtherand incor#orated a dru$-resistance $ene, methl$uaninemethltrans8erase /G/5, in the -$lo*in .ector ?@,demonstratin$ amelioration o8 murine )-thalassemia and

enrichment o8 the corrected HSC com#artment, em#loin$in vitro and in vivo selection, 8ollowin$ dru$ treatment&Howe.er, des#ite the success8ul dru$-induced enrichmento8 the HSC #ool in the a*o.e stud, >ndin$s 8rom otherin.esti$ators tend to su$$est that /G/5 selection a##roachma not alwas result in the desired outcome and Kuite o8tenis accom#anied * dru$-related toicit& /ore s#eci>call,6arochelle et al& ?(@ 8ailed to induce selection o8 lon$termre#o#ulatin$ HSCs in rhesus macaKues and showedthat the al4latin$ a$ent *is-chloroethl nitrosourea 9C+Bcan result in si$ni>cant nonhemato#oietic toicit, such as#ulmonar con$estion)edema and necrohemorrha$ic colitis&In addition, recent wor4 * Giordano et al& ?"@ usin$ amurine serial trans#lant model demonstrated that /G/5selection a##roach can also lead to insertional muta$enesisand clonal dominance& 5a4en to$ether, the a*o.e dataindicate that althou$h /G/5 selection a##roach can *ean e;ecti.e strate$ 8or enrichment o8 the corrected HSCcom#artment, its dru$-related toicit and insertionalmuta$enesis#otential re#resent considera*le ris4 8actors 8or use inhuman clinical trials&-.#. 0n itro 'tudies with 2uman 2'Cs. 9oth )-$lo*in and -$lo*in .ectors ha.e *een used in correctin$ humanerthro#oiesis in erthroid cultures and immunode>cientmice& In a detailed stud, uthen.eetil et al& ?!@ tested a

lenti.iral .ector carrin$ the human )-$lo*in e#ressioncassette, Lan4ed * a chromatin insulator in trans8usionde#endenthuman thalassemia macantl reduced& 5hese $ene-correctedhuman )-thalassemia #ro$enitor cells were then trans#lantedinto immunode>cient mice, where the were ca#a*le o8 esta*lishin$ normal erthro#oiesis& In addition, Roselli et al&?@, usin$ the G6N9E .ector, re#orted success8ul correctiono8 thalassemia ma


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$lo*in .ectors and an in vitro model o8 human erthro#oiesis,showed recentl that *oth lenti.iral-mediated -$lo*in $ene addition and $enetic reacti.ation o8 endo$enous -$lo*in $enes ha.e the #otential to #ro.ide thera#eutic H*=le.els to #atients with )-$lo*in de>cienc ?'0, '1@& =inall,the $rou# o8 Ana$nou, to address the issues o8 low titer,

.aria*le e#ression, and $ene silencin$, a;ectin$ the $enethera# .ectors 8or hemo$lo*ino#athies, has success8ullused the H=H-2 enhancer in a series o8 oncoretro.iral.ectors ?'2@& 9ased on these data, the same $rou# ?'@$enerated a no.el insulated SI+ 6V desi$nated as GGHI,' Stem Cells Internationalcontainin$ the A -$lo*in $ene with the 311" H=H #ointmutation and the H=H-2 enhancer& 5his .ector mana$edto #roduce e:cient amounts o8 H*= resultin$ in #henot#iccorrection in erthroid cultures o8 CD'P HSCs isolated8rom #eri#heral *lood 9 or *one marrow 9/ ?'@&In this s#eci>c .ector desi$n, the incor#oration o8 the 8ulllen$th1&2 4* cHS' in the B re$ion and the SI+ con>$uration

had no a##arent e;ect on .iral titer, since it reached2 4 10! 5B)m6& E;orts ha.e also *een made in the a*ilito8 -$lo*in .ectors #seudot#ed with di;erent en.elo#e$lco#roteins to transduce human HSCs ?''@ that resultedin the notion that the .esicular stomatitis .irus $lco#roteinVSVG is more e;ecti.e in transducin$ en$ra8tin$ cells thanother $ammaretro.iral $lco#roteins, su##ortin$ thus its usein clinical-$rade .ectors ?''@&-.-. The First Clinical Trial. 5he >rst human clinical trialusin$ a )-$lo*in 6V commenced in =rance on Qune ", 200",where 6e*oulch and collea$ues selected two )-thalassemia#atients who underwent trans#lantation o8 6V-transducedHSCs ?', '(@& 5he >rst one, a 2!-ear-old #atient e#erienceda #eriod o8 #rolon$ed a#lasia li4el due to thetechnical handlin$ o8 the cells, without relation to the $enethera# .ector, and des#ite the a*sence o8 an ad.erse e;ects,reKuired the administration o8 untransduced cells 4e#t as a*ac4u#, in order to a.oid #utati.e in8ections& As a result, thelenti.iral-modi>ed cells did not reach a si$ni>cant le.el in9, neither did the thera#eutic hemo$lo*in, leadin$ to noconclusions re$ardin$ the s#eci>c #atient& 5he second #atient in the stud was a 1!-ear-old malesu;erin$ 8rom H*E))5 thalassemia, a 8orm o8 the disorder inwhich hemo$lo*in #roduction is se.erel com#romised& Hewas trans8usion-de#endent since the a$e o8 three, reKuirin$

1(0m6 o8 #ac4ed erthroctes)4$)ear& He recei.ed ' 410( CD'P cells)4$ ?'(@& 5he le.els o8 $eneticall modi>edcells increased 8rom 2 in the >rst 8ew months to 11 at months #osttrans#lant& 5he a*o.e rise was also o*ser.edin the le.els o8 normal )-$lo*in #rotein, with 10-20 o8 HSCs *ein$ $eneticall modi>ed, leadin$ thus to im#ro.ed#roduction and Kualit o8 red *lood cells& Remar4a*l, a eara8ter the treatment, the #atient no lon$er reKuired *loodtrans8usions&He was last trans8used on Qune (, 200!, and 8ourears a8ter trans#lantation, des#ite *ein$ sli$htl anemic andunder$oin$ re#eated #hle*otomies 8or the decrease o8 irono.erload, the #atient does not reKuire *lood trans8usions,which means that this sin$le case can *e .iewed as a clinical

success&Howe.er, des#ite the success8ul outcome o8 the second


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#atient, Ca.a77ana-Cal.o et al& re#orted that one hemato#oieticclone, har*orin$ a .ector insertion into the H/GA2$ene showed clonal dominance ?'(@& At 20 months #osttrans#lantation, the s#eci>c clone accounted 8or 0 o8 the $eneticall modi>ed HSCs its contri*ution, howe.er, tothe circulatin$ red *lood cellsF #ool remains at around &

5he #otential clinical rele.ance o8 the alteration o8 2(GA#e#ression * the .ector inte$ration is hi$hli$hted * the8act that this $ene ma 8unction as a #otential onco$ene in.arious t#es o8 cancer& Howe.er, it remains unclear whetherthe .ector insertion into this $ene has actuall resulted in therelati.el hi$h contri*ution o8 this clone to hemato#oiesis,since it is concei.a*le that the a*o.e o*ser.ation ma sim#lreLect the conseKuences o8 en$ra8tment 8roma small num*ero8 transduced HSCs&In summar, the a*o.e #atient re#resents the #roo8 o8 #rinci#le that $ene thera# can *e a success8ul thera#eutica##roach& 5his success8ul clinical trial demonstrated thatlar$e amounts o8 a thera#eutic #rotein can *e #roduced in

vivo in a linea$e-s#eci>c manner and .alidated somatic $enetrans8er usin$ a lenti.iral SI+ .ector 8or transducin$ lon$termre#o#ulatin$ HSCs& It also demonstrated that somatic$ene trans8er e* vivo can #ro.ide trans8usion inde#endence8or #atients with se.ere 8orms o8 thalassemia&

/$ Enhancing enti(iral GeneTransfer E0ciency in Human and#onhuman "rimate HSCsAs discussed a*o.e, studies 8rom man la*oratories ha.eshown that murine HSCs can *e $eneticall modi>ed usin$*oth )-$lo*in and -$lo*in 6Vs and en$ra8t, resultin$ inamelioration o8 )-thalassemia& Howe.er, this is not the case

8or *oth human and nonhuman #rimate HSCs& 5hese t#esare more resistant to lenti.iral $ene trans8er and so 8ar,no more than 10J1 o8 $eneticall modi>ed #eri#heral*lood cells ha.e *een achie.ed& Recent wor4 on rhesusmacaKues ?'"@ usin$ G= 6V demonstrated an a.era$e o8 " 6V-*earin$ #eri#heral *lood cells, a >ndin$ that comesinto a$reement with #re.ious studies on #i$tail macaKues?'!, '@& Similarl, the 1!-ear-old #atient in the >rst clinicaltrial discussed a*o.e showed an a.era$e o8 1 $eneticallmodi>ed HSCs ?'(@, su##ortin$ e.en 8urther the notion thathi$her #ercenta$es o8 $eneticall modi>ed HSCs in the caseo8 human and nonhuman #rimates is not an eas tas4& 5hemost e;ecti.e strate$ies shown to increase HSC ield are

descri*ed *elow and are also shown in =i$ure 1&6.1. 2'Cs and Cellular Factors. 5he resistance o8 #rimiti.ehuman and nonhuman #rimate HSCs to in8ection * HIV*ased.ectors has drawn a lot o8 attention in the #ast decade&Recent wor4 * the +aldini $rou# ?0@ demonstrated thatlenti.iral $ene trans8er is limited * the #roteasome throu$hthe re$ulation o8 one or more o8 the cellular #ostentr ste#so8 the .ector #article& E#eriments in.ol.in$ the #roteasomeinhi*itor /G12 durin$ 6V transduction demonstrated a'-8old increase in $ene trans8er into human CD'P cells?0@& In this stud, the transient and re.ersi*le inhi*itiono8 #roteasome 8unction did not lead to an o*.ious HSC

de8ects& Howe.er, as #roteasome is the ma


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in cells, resultin$ in im#airment o8 their sur.i.al& 5hus,the use o8 #roteasome inhi*itors as a means o8 increasin$lenti.iral HSC transduction needs 8urther in.esti$ation,*e8ore it is considered sa8e to use in clinical trials& 6astl,in a di;erent stud * Ohan$ et al& ?1@, cell-ccle #rotein#21, which is 8ound at hi$h le.els in HSCs, was identi>ed

as a uniKue molecular *arrier to HIV in8ection, throu$h itsStem Cells International 5halassemic #atientSelection o8 $eneticallmodi>edSomatic cellisolation7* vivotransductionwith $lo*in6V7* vivo HSC e#ansion and#roli8eration9/ CD'P HSCisolation9/ CD'P HSCmo*ili7ation andisolation 8rom#eri#heral *loodHSCsRe#ro$rammed intoiS cells and then into$lo*in-e#ressin$HSCs

=i$ure 1% Schematic re#resentation o8 HSC-*ased $ene thera# 8or )-thalassemia& 9/ CD'P cellsare remo.ed 8rom the #atient ri$ht#anel, transduced in vitro with the thera#eutic $lo*in 6V, carrin$ the #re8erred en.elo#e$lco#roteins, and returned to the #atientintra.enousl& At this sta$e, and #rimaril in the case o8 human HSCs, e* vivo e#ansion and#roli8eration can *e induced alternati.el,selection o8 $eneticall modi>ed HSCs ma ta4e #lace& 9oth strate$ies can lead to increased ieldo8 corrected HSCs, which will contri*uteto the HSC com#artment, when returned to the #atient& Alternati.e strate$ies 8or o*tainin$ hi$herield o8 CD'P cells are continuouslemer$in$ le8t #anel& 5hese include mo*ili7ation o8 9/ CD'P HSCs with mainl G-CS= and thenisolation o8 these cells 8rom #eri#heral*lood& /oreo.er, iS cell strate$ su$$ests that somatic cells 8rom the #atient can *ere#ro$rammed to iS cells and a8ter $eneticmani#ulation, in.ol.in$ $enetic correction 8or the mutations * homolo$ous recom*ination, thesecells can $i.e rise to $lo*in-#roducin$HSCs, 8ollowin$ directed hemato#oietic di;erentiation&

interactions with the .iral inte$rase, leadin$ to inhi*ition o8 chromosomal inte$ration ?1@&6.#. 2'C Cyclin% and 7*pansion 7* ivo . A#art 8romcellular

8actors *ein$ im#ortant 8or lenti.iral $ene trans8er intohuman HSCs, another crucial characteristic that im#airsthe e:cienc o8 the $ene trans8er is the low le.els o8 e* vivo HSC cclin$& As mentioned earlier, human HSCsremain Kuiescent lon$er than murine ones, with the 8ormerenterin$ G1 e.er 0J'0 wee4s ?2!@, com#ared to


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A#art 8rom mani#ulatin$ the e* vivo culture conditions andthe HSC state, researchers ha.e demonstrated additionalmeans o8 increasin$ the HSC ield, such asHSC mo*ili7ationand the $eneration o8 induced #luri#otent stem iS cells=i$ure 1 as descri*ed *elow&".1. 2'C (obili;ation. 5he term HSC mo*ili7ation re8ers

to the 8orced mi$ration o8 HSCs 8rom the 9/ to the*loodstream&/o*ili7ed 9 can then *e used as an alternati.esource 8or CD'P cells 8or lenti.iral transduction, $enetrans8er, and e.entual trans#lantation 8or the treatment o8 )-thalassemia, ieldin$ a to '-8old enrichment ?(1@& 6atel,this strate$ has drawn the attention o8 man research$rou#s, with $ranulocte colon-stimulatin$ 8actor G-CS=*ein$ the macall $ained $round in the >eldo8 $ene thera# 8or )-thalassemia& articularl, 6i et al&?(@ assessed the administration o8 G-CS= in mo*ili7in$stem and #ro$enitor cells in thalassemic ma


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and ha.e *een #resented in detail in a recent s#ecial issue o8 this rst re#orted $ene correction in thecontet o8 hemo$lo*ino#athies, usin$ iS cells, was #er8ormedin an SCD mouse model& Hanna et al& ?("@ har.estedcells 8rom the s4in o8 the SCD mouse and re#ro$rammedthem to ESCs, * retro.irall deli.erin$ Nct', So2, 6i8',and c-/c $enes& A8ter remo.in$ c/(yc to decrease oreliminate #utati.e tumori$enesis in treated mice, ESCs werecultured to #roduce 9/ stem cell #recusors, and 8ollowin$re#lacement o8 the de8ected $ene with a normal one, .iahomolo$ous recom*ination, the were trans#lanted *ac4 to

SCD mice& 5he outcome was disease amelioration in thesemice, with *lood and 4idne 8unction returnin$ to normalle.els& =ollowin$ the murine stud, Me et al& ?(!@ were the>rst to show that this issue is also 8easi*le in humans, as themana$ed to re#ro$ram s4in >*ro*lasts 8rom a thalassemic#atient with )5 thalassemia into iS cells, and demonstratedthat the latter, 8ollowin$ $ene tar$etin$, could di;erentiateinto hemo$lo*in-#roducin$ HSCs&Such $ene tar$etin$, howe.er, needs to *e hi$hl controlled,as randoml inte$rated trans$enes ma result inStem Cells International "onco$enicit, and there8ore, a $eneral need 8or a strate$to introduce trans$enes into sa8eT re$ions in iS cells isim#erati.e& A $ood a##roach to o.ercome this o*staclecame recentl 8rom the Sadelain $rou# ?(@, where themana$ed to induce )-$lo*in trans$ene e#ression in iScell clones, where the 6V had inte$rated in sa8e har*orsTthrou$hout the $enome& iS cells in this stud ori$inated8rom s4in >*ro*last or 9/ mesenchmal cells 8rom #atientssu;erin$ 8rom )-thalassemia ma.e criteria% i distance o8 at least 0 4* 8rom the = end o8 an $ene, ii distance o8 at least 00 4* 8rom an

cancer-related $ene, iii distance o8 at least 00 4* 8rom anmiR+A codin$ $ene, i. location outside a transcri#tionunit, and . location outside ultraconser.ed re$ions BCRso8 the human $enome& /easurement o8 )-$lo*in e#ressionin these #ro$enitors reached hi$h le.els, su$$estin$ that thea*o.e strate$, once im#ro.ed, can *e .er e:cient 8or )-thalassemia&

4$ Summary 5he e:cient $ene deli.er o8 thera#eutic trans$enes usin$6Vs has *ecome a milestone in the >eld o8 $ene thera#8or hemo$lo*ino#athies& Im#ro.ed 6V desi$n ena*led success8ulintroduction o8 trans$enes into *oth murine andhuman HSCs, leadin$ to amelioration o8 )-thalassemia

in murine models, and restored erthro#oiesis in vitro.Etensi.e studies in the >eld led also to the success o8


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the >rst clinical trial in =rance, in Qune 200", where a1!-ear-old H*E))5 thalassemic #atient was treated witha )-$lo*in .ector and a ear a8ter 9/ trans#lantationmana$ed to *ecome trans8usion-inde#endent& As o8 toda,he remains trans8usion-inde#endent, 8or o.er three ears,in s#ite o8 re#eated #hle*otomies aimed to decrease iron

o.erload& Des#ite the e:cac o8 6Vs to introduce thera#eutictrans$enes into HSCs, there is also the ris4 8or insertionalmuta$enesis, and there8ore, an etensi.e wor4 is currentl8ocused on $eneratin$ sa8e .ectors 8or $ene thera#& 5hea*o.e is usuall achie.ed * incor#oratin$ elements, whichma4e the .ector tissue-s#eci>c and also shield the trans$ene8rom nei$h*ourin$ e;ects, u#on inte$ration& Re$ardlessthe need 8or constantl im#ro.in$ .ector desi$n, a lot o8 attention has *een drawn also towards strate$ies that resultin hi$her num*ers o8 $eneticall modi>ed HSCs, which willin turn contri*ute to the HSC #ool in the #atient& 5he latter,to$ether with etensi.e research towards alternati.e HSCsources, such as iS cells, will undou*tedl set the $round

8or more success8ul clinical trials&ist of %..re(iationsH*=% =etal hemo$lo*inHSC% Hemato#oietic stem cellsH6A% Human leu4octe anti$enGVHD% Gra8t .ersus host disease/6V% /urine leu4emia .irus6CR% 6ocus control re$ionHS% H#ersensiti.e site6V% 6enti.iral .ectorH=H% Hereditar #ersistence o8 8etal hemo$lo*incHS'% Chic4en h#ersensiti.e site '

SCID% Se.ere com*ined immunode>ciencSI+% Sel8-inacti.atin$65R% 6on$ terminal re#eatSCD% Sic4le cell disease/G/5% /ethl $uanine methl trans8erase9C+B% 9is-chloroethl nitrosourea9% eri#heral *lood9/% 9one marrowVSVG% Vesicular stomatitis .irus $lco#roteinSR1% Stem-re$enin 1AHR% Arl hdrocar*on rece#tor/A% /ito$en-acti.ated #rotein 4inaseiS% Induced #luri#otent stem

G-CS=% Granulocte colon-stimulatin$ 8actor9SC% eri#heral *lood stem cellESC% Em*ronic stem cellmiR+A% microR+ABCRs% Bltraconser.ed re$ions&

%ckno3ledgments 5he authors ha.e no conLict o8 interest and ha.e nothin$ todisclose& 5he a#olo$i7e to all whose wor4 has not *een citeddue to s#ace restriction&

5eferences?1@ D& Q& eatherall, The Thalassemias in the (olecular Basis of Blood >iseases, Saunders, hiladel#hia, a, BSA, 2001&?2@ D& Q&eatherall and Q& 9& Cle$$, 5halassemiaUa $lo*al #u*lic

health #ro*lem,T ?ature (edicine, .ol& 2, no& !, ##& !'"J!',1(&


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?@ Q& =lint, R& /& Hardin$, A& Q& 9oce, and Q& 9& Cle$$, The 9opulationGenetics of the 2emo%lobinopathies in Clinical 2aematolo%y ,G Rod$ers 9ailliere 5indall, 6ondon, B, 1!&?'@ C& 9or$na-i$natti, S& Ru$olotto, & De Ste8ano et al&, Sur.i.aland com#lications in #atients with thalassemia ma


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thera#,T 'cience, .ol& 2', no& 0, ##& 2(!J2"1, 2001&?1!@ A& /iccio, R& Cesari, =& 6otti et al&, In .i.o selection o8 $eneticall modi>ed erthro*lastic #ro$enitors leads to lon$termcorrection o8 )-thalassemia,T 9roceedin%s of the ?ational

Academy of 'ciences of the &nited 'tates of America, .ol& 10,no& 0, ##& 10'"J102, 200!&?1@ E& a#ani4olaou and +& & Ana$nou, /acienc .irus t#e 1-*ased .ectors iscell ccle de#endent,T ournal of irolo%y , .ol& ", no& , ##&('J((0, 1&?1@ M& D& orin and Q& A& Oac4, +on#roducti.e human immunode>cienc.irus t#e 1 in8ection in nucleoside-treated G0 lm#hoctes,T

ournal of irolo%y , .ol& ", no& !, ##& (2(J(2,1&?2@ S& Ri.ella, C& /a, A& Chad*urn, I& Ri.iWere, and /& Sadelain,A no.el murine model o8 Coole anemia and its rescue *

lenti.iral-mediated human )-$lo*in $ene trans8er,T Blood, .ol&101, no& !, ##& 22J2, 200&?@ S& Imren, E& aen, & A& esterman et al&, ermanent and


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#anerthroid correction o8 murine ) thalassemia * multi#lelenti.iral inte$ration in hemato#oietic stem cells,T 9roceedin%sof the ?ational Academy of 'ciences of the &nited 'tates of

America, .ol& , no& 22, ##& 1'!0J1'!, 2002&?'@ H& Hanawa, & & Har$ro.e, S& e#es, D& & Sri.asta.a, A& &+ienhuis, and D& A& ersons, Etended )-$lo*in locus controlre$ion elements #romote consistent thera#eutic e#ression o8

a -$lo*in lenti.iral .ector in murine )-thalassemia,T Blood,.ol& 10', no& !, ##& 22!1J220, 200'&?@ H& Ohao, 5& I& estina,/& +asimu77aman, &/ehta, && Har$ro.e,and D& A& ersons, Amelioration o8 murine )-thalassemiathrou$h dru$ selection o8 hemato#oietic stem cellstransduced with a lenti.iral .ector encodin$ *oth -$lo*in andthe /G/5 dru$-resistance $ene,T Blood, .ol& 11, no& 2, ##&"'"J"(, 200&Stem Cells International ?(@ A& 6arochelle, B& Choi, M& Shou et al&, In .i.o selection o8 hemato#oietic #ro$enitor cells and temo7olomide dose intensi>cationin rhesus macaKues throu$h lenti.iral transductionwith a dru$ resistance $ene,T ournal of Clinical 0nvesti%ation,.ol& 11, no& ", ##& 12J1(, 200&

?"@ =& A& Giordano, B& R& Sor$, Q&-B& A##elt et al&, Clonal in.entorscreens unco.er monoclonalit 8ollowin$ serial trans#lantationo8 /G/51'0 -transduced stem cells and dose-intensechemothera#,T 2uman Gene Therapy , .ol& 22, no& (, ##&("J"10, 2011&?!@ G& uthen.eetil, Q& Scholes, D& Car*onell et al&, Success8ul correctiono8 the human )-thalassemia man$ertranscri#tional acti.ator desi$ned to interact with the -

$lo*in $ene #romoters enhances 8etal hemo$lo*in #roductionin #rimar human adult erthro*lasts,T Blood, .ol& 11, no&1, ##& 0J0'1, 2010&?'1@ A&il*er, && Har$ro.e, M&-S& im et al&, 5hera#eutic le.elso8 8etal hemo$lo*in in erthroid #ro$en o8 )-thalassemicCD'P cells a8ter lenti.iral .ector-mediated $ene trans8er,TBlood, .ol& 11", no& 10, ##& 2!1"J2!2(, 2011&?'2@ /& =ra$4os, +& & Ana$nou, Q& 5u**, and D&& Emer, Bse o8 the hereditar #ersistence o8 8etal hemo$lo*in 2 enhancer toincrease the e#ression o8 oncoretro.irus .ectors 8or human$amma-$lo*in,T Gene Therapy , .ol& 12, no& 21, ##& 11J1(00,200&?'@ E& a#ani4olaou, /& Geor$omanoli, E& Stamateris et al&,5he new SI+-lenti.iral .ector 8or thalassemia $ene thera#

com*inin$ two H=H acti.atin$ elements corrects humanthalassemic hemato#oietic stem cells,T 2uman Gene Therapy &In #ress&?''@ M& S& im, /& /& iel$os7, & Har$ro.e et al&, 5ransductiono8 human #rimiti.e re#o#ulatin$ hemato#oietic cells withlenti.iral .ectors #seudot#ed with .arious en.elo#e #roteins,T(olecular Therapy , .ol& 1!, no& ", ##& 110J11", 2010&?'@ A& 9an4, R& Dora7io, and & 6e*oulch, A #hase I)II clinicaltrial o8 )-$lo*in $ene thera# 8or )-thalassemia,T Annals of the?ew or: Academy of 'ciences, .ol& 10', ##& 0!J1(, 200&?'(@ /& Ca.a77ana-Cal.o, E& aen, N& +e$re et al&, 5rans8usioninde#endence and H/GA2 acti.ation a8ter $ene thera#o8 human )-thalassaemia,T ?ature, .ol& '(", no& "1, ##&1!J22, 2010&

?'"@ M& Q& im, M& S& im, A& 6arochelle et al&, Sustained hi$h-le.el#olclonal hemato#oietic mar4in$ and trans$ene e#ression' ears a8ter autolo$ous trans#lantation o8 rhesus macaKues


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with SIV lenti.iral .ector-transduced CD'P cells,T Blood, .ol&11, no& 22, ##& ''J'', 200&?'!@ G& D& 5ro*rid$e, 9& C& 9eard, C& Gooch et al&, E:cienttransduction o8 #i$tailed macaKue hemato#oietic re#o#ulatin$cells with HIV-*ased lenti.iral .ectors,T Blood, .ol& 111,no& 12, ##& "J', 200!&?'@ H& Hanawa, & Hematti, & e.an8ar et al&, E:cient $ene

trans8er into rhesus re#o#ulatin$ hemato#oietic stem cellsusin$ a simian immunode>cienc .irus-*ased lenti.iral .ectorsstem,T Blood, .ol& 10, no& 11, ##& '0(2J'0(, 200'&?0@ =& R& S& De Sio, A& Gritti, & Cascio et al&, 6enti.iral .ector$ene trans8er is limited * the #roteasome at #ostentr ste#sin .arious t#es o8 stem cells,T 'tem Cells, .ol& 2(, no& !, ##&21'2J212, 200!&?1@ Q& Ohan$, D& 5& Scadden, and C& S& Crum#ac4er, rimiti.ehemato#oietic cells resist HIV-1 in8ection .ia #21 a81)Ci#1)Sdi1,T ournal of Clinical 0nvesti%ation, .ol& 11", no& 2, ##&'"J'!1, 200"&?2@ C& C& Ohan$, /& a*a, G& Ge et al&, An$io#oietin-li4e #roteinsstimulate e .i.o e#ansion o8 hemato#oietic stem cells,T?ature (edicine, .ol& 12, no& 2, ##& 2'0J2', 200(&

?@ C& C& Ohan$, /& a*a, S& 6i7u4a, H& Hunh, and H& =& 6odish,An$io#oietin-li4e and IG=92 stimulate e .i.o e#ansiono8 human cord *lood hemato#oietic stem cells as assaed* +ND)sCiD trans#lantation,T Blood, .ol& 111, no& ", ##&'1J'2, 200!&?'@ S& Amsellem, =& Lumio, D& 9ardinet et al&, E .i.o e#ansiono8 human hemato#oietic stem cells * direct deli.er o8 theHN9' homeo#rotein,T ?ature (edicine, .ol& , no& 11, ##&1'2J1'2", 200&?@ H& Nhta, S& Se4ulo.ic, S& 9a4o.ic et al&, +ear-maimal e#ansionso8 hemato#oietic stem cells in culture usin$ +B!-HN 8usions,T 7*perimental 2ematolo%y , .ol& , no& , ##&!1"J!0, 200"&?(@ A& E& 9oitano, Q&an$, R& Romeo et al&, Arl hdrocar*on rece#tor

anta$onists #romote the e#ansion o8 human hemato#oieticstem cells,T 'cience, .ol& 2, no& ", ##& 1'J1'!, 2010&?"@ M& an$, Q& ellner, 6& 6iu, and D& Ohou, Inhi*ition o8 #! mito$en-acti.ated #rotein 4inase #romotes e .i.o hemato#oieticstem cell e#ansion,T 'tem Cells and >evelopment ,.ol& 20, no& ", ##& 11'J112, 2011&?!@ E& Verhoeen, /& i7nerowic7, D& Nli.ier et al&, +o.ellenti.iral .ectors dis#lain$ earl-actin$ cto4inesT selecti.el#romote sur.i.al and transduction o8 +ND)SCIDre#o#ulatin$ human hemato#oietic stem cells,T Blood, .ol&10(, no& 10, ##& !(J, 200&?@ & Ghani, & an$, & N& De Cam#os-6ima et al&, E:cienthuman hemato#oietic cell transduction usin$ RD11'- and

GA6V-#seudot#ed retro.iral .ectors #roduced in sus#ensionand serum-8ree media,T 2uman Gene Therapy , .ol& 20, no& ,##& ((J"', 200&?(0@ 5&+e;, 6& Q& eterson, Q&C&/orris et al&, E:cient $ene trans8erto hemato#oietic re#o#ulatin$ cells usin$ concentrated RD-11'-#seudot#e .ectors #roduced * human #ac4a$in$ cells,T(olecular Therapy , .ol& , no& 2, ##& 1"J1, 200'&?(1@ 6& 9& 5o, D& +& Haloc4, 5& Dowse et al&, A com#arati.e studo8 the #henot#e and #roli8erati.e ca#acit o8 #eri#heral*lood 9 CD'P cells mo*ili7ed * 8our di;erent #rotocolsand those o8 stead-#hase 9 and *one marrow CD'P cells,TBlood, .ol& !', no& , ##& 20J2, 1'&?(2@ 6& 9& 5o, D& +& Haloc4, & Q& Simmons, and C& A& Quttner, 5he*iolo$ and clinical uses o8 *lood stem cells,T Blood, .ol& !,

no& ", ##& 22J22!, 1"&?(@ & 6i, A& on$, C& & 6i et al&, Granulocte colonstimulatin$8actor-mo*ili7ed #eri#heral *lood stem cells


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Jurnal Stem Cell Thalasemia

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