Biology Investigatory Project Gene Therapy
Name: OV Shashank Class: XII – D Roll No:
Certifcate Year: 2013-14 This is to certify that OV Shashank a student of RN Podar Schoo! of cass "##! has co%&eted his $! Ro No: fu se%ester &ro'ect in the fu(%ent of curricuu% ) #ndia Senior Secondary *+a%ination The &ro'ect ,ork entited Gene
Therapy ! is the oriina ,ork done .y hi% durin his a.o/e fu se%ester &ro'ect $ate:
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#nterna *+a%iner Princi&a
*+terna *+a%iner Schoo Sta%&
Acknowledgement # take this o&&ortunity to e+&ress %y sincere ratitude to the honoura.e Princi&a rs )/nita ir of RN Podar Schoo for her dee& interest and uidance &ro/ided to %e durin the course # a% %ost ratefu to our iooy teacher rs Pad%a/athi for her reat he& in the co%&etion of this &ro'ect
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Student’s Signature
Table o Contents
I nt r oduc t i on
GeneTher apy
T a r g e t s
s ol at i onofgene I Ge neT ar get i ng Ge neDel i v e r y
Cas eSt udy–Cy s t i cFi br os i s
T heDi sease 3
si tagoodT ar get I Ch oos i ngVec t or s
s t or y Hi
Chal l enges
Et hi c al I s s ues
Rec entUpcomi ng
SPR CRI
Conc l us i on
Bi bl i ogr aphy
bs i t es We
Book s
Introduction Diseases The term disease broadly refers to any condition that impairs normal function, and is therefore associated with dysfunction of normal homeostasis. When the functioning of one or more organs or systems of the body is adversely affected, characterised by various signs and symptoms, we say that we are not healthy, i.e., we have a disease. Health can be defined as a state of complete physical, mental and social well-being. When people are healthy, they are more efficient at work. This increases productivity and brings economic prosperity. Health also increases longevity of people and reduces infant and maternal mortality.
Based on the cause diseases can be broadly classified as:
In!ections These are diseases caused due to invasion of a foreign parasitic organism. They are temporary because the immune system of organisms can fight such pathogens disease causing organisms! to a certain e"tent hence helping in prevention of the disease. The immune system can also be aided with the use of several drugs. #part from easy treatment they can also be easily prevented
"i!estyle Diseases $ifestyle diseases also sometimes called diseases of longevity or diseases of civili%ation interchangeably! are diseases that appear to increase in fre&uency as countries become more industriali%ed and people live longer. They can include #l%heimer's disease, asthma, and obesity. (iet and lifestyle are ma)or factors thought to influence susceptibility to many diseases. (rug abuse, tobacco smoking, and alcohol drinking, as well as a lack of e"ercise may also increase the risk of developing certain diseases, especially later in life. These diseases can be prevented completely by living a healthy lifestyle.
#enetic Disor$ers # genetic disorder is an illness caused by one or more abnormalities in the genome, especially a condition that is present from birth congenital!. They are medical disorders related to gene mutation. *enetic disorders are heritable, and are passed down from the parents' genes. +ther defects may be caused by new mutations or changes to the (#. n such cases, the defect will only be heritable if it occurs in the germ line. The same disease, such as some forms of cancer, may be caused by an inherited genetic condition in some people, by new mutations in other people, and by non-genetic causes in still other people. These diseases are totally random and difficult to prevent as they are not caused by e"ternal agents. #lso as their root cause lies in the genome of the organism their cure was thought to be impossible until the breakthrough research unlocking the secrets of (# leading to the development of biotechnology and hence gene therapy.
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Gene Therapy Wec ant hi nkofamedi c al c ondi t i onor i l l nes sasa" br ok e nwi nd ow. "Man yme di c a l c ondi t i onsr es ul tf r om fl aws ,ormut at i ons ,i n on eormo r eo fape r s on ' sgene s.Mut at i on s c aus et hepr ot ei nenc odedb yt hatgenet o mal f unc t i on.Whenapr ot ei nmal f unc t i ons , c el l st hatr el yont hatpr ot ei n' sf unc t i onc an' t beha v enor mal l y ,c aus i ngpr obl emsf or whol et i s s uesoror gans .Medi c al c ondi t i ons r el at edt ogenemut at i onsar ec al l edgenet i c di s or der s . So ,i fafla wedgen ec au se do ur" b r ok e nwi n do w, "c anwe" fi x "i t ?Whata r eo ur opt i ons ? 1. St a ys i l ent :i gnor et hegenet i cdi s or derandnot hi ngget sfi x ed. 2. T r yt ot r eatt hedi s or derwi t hdr ugsoro t herappr oac hes :dependi ngont he di s or der ,t r eat mentma yorma yno tbeagoodl ongt er ms ol ut i on. 3. Puti nanor mal ,f unc t i oni ngc opyoft hegene:i fy ouc andot hi s ,i tma ys ol v e t hepr o bl e m! I fi ti ssuc ces sf ul ,genet her apypr ov i desawayt ofi xapr obl em ati t ssour c e.Addi ng ac or r ec t edc op yoft hegenema yhel pt heaff ec t edcel l s ,t i s s uesandor ganswor k pr oper l y . Genet her ap ydi ff er sf r om t r adi t i onal dr ugbas edappr oac hes ,whi c hma y t r eatt hepr obl em,butwhi c hdonotr epai rt heunder l y i nggenet i cfl aw.
%argets !or #ene %hera&y Butno w aques t i onar i s es ,whi c hdi s or der sordi s eas esc anwet ar getus i nggene t her ap y?Man ydi s or der sormedi c al c ondi t i onsmi ghtbet r eat edus i nggenet her ap y ,
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butot her smaynotbes ui t abl ef ort hi sappr oac h.Foradi s eas et obet ar get edby genet her apyi tmus ts at i s f yt hef ol l owi ngc ondi t i ons : t i onmustr esul tf r om mut at i onsi noneormor egenes 1. Thecondi ot r e atage ne t i cfla w,t hek nowl e dgeofwhi c hge ne ( s )t opur s uei s 2. T a bs ol ut e l yne ce ss ar y.Al s oaDNAc opyoft hatgenea v ai l abl ei nt he l abor at or y .Thebes tc andi dat esf orgenet her apyar et hes oc al l ed" s i ngl ege ne"di s o r der s-wh i c ha r ec a us edb ymut at i on si no nl yoneg en e. gnt hebestpossi bl eappr oach,knowl edgeabouthow t hegene 3. Todesi f act or si nt ot hedi sor deri sr equi r ed.Fore x ampl e:
Whi c ht i s s ue sa r eaff ec t e d?
Whatr ol edoest hepr ot ei nenc odedbyt hegenepl a ywi t hi nt hec el l sof t hatt i s s ue?
Ex ac t l yhowdomut at i onsi nt hegeneaffec tt hepr ot ei n' sf unc t i on?
nganor malcopyoft hegeneshoul dfixt hepr obl em i nt heaffect ed 4. Addi t i ssue.Thi smays eem l i k eobv i ous ,buti t ' snot .Whati ft hemut at edgene enc odesapr ot ei nt hatpr ev ent st henor mal pr ot ei nf r om doi ngi t sj ob?Mut at ed genest hatf unc t i ont hi swa yar ec al l eddomi nantneg at i v eandaddi ngbac kt he nor mal pr o t ei nwon' tfi xt hepr obl em. nede l i v er yt oc el l soft heaffe ct e dt i s suemus tbepos si bl e .I 5. Thege t dependson:
Howac c es s i bl ei st het i s sue?I si tf ai r l yeas y( s k i n,bl oodorl ungs ) ,or mor edi ffic ul tt or eac h( i nt er nal or gans ) ?
Whati st hebes tmodeofdel i v er y ?
Thet ec hni quesofbi ot ec hnol ogyhav emadei tpos s i bl et oi s ol at et her equi r edgene i nt hel abor at or yandal s odel i v ert hegene.
Isolation o! #ene o! Interest Thefi r s ts t epi st ofi ndandi s ol at et hegenet hatwi l l bei ns er t edi nt ot hegenet i c al l y modi fi edor gani s m.Fi ndi ngt her i ghtgenet oi ns er tus ual l ydr awsony ear sof #
s ci ent i fi cr es ear c hi nt ot hei dent i t yandf unc t i onofus ef ul genes . On c et h ati sk n o wn t heDNAneedst obec utats pec i fi cl oc at i onst oi s ol at et hegeneofi nt er es t .Thi scan bedonebyus i ngr es t r i c t i onenz y mesal s ok no wnasmol ec ul ars c i s s or swhi c hc ut DNAats pec i fi csi t esc ont ai ni ngpal i ndr omi cDNAs equenc es .Buti nor dert oc utt he DNAwi t hr es t r i c t i onenz y mes ,i tneedst obei npur ef or m,f r eef r om ot hermac r omol ec ul es .
Isolation o! DN' Si nc et heDNAi se nc l os e dwi t h i nt h eme mbr ane s,weh av et obr e akt hec el l o pe nt o r el ea seDNAal o ngwi t hot he rmac r omo l e c ul ess u c ha sRNA,pr o t ei ns , pol y s ac c har i desandal s ol i pi ds .Thi sc anbeac hi ev edby t r eat i ngt hebac t er i al c el l s / pl antorani mal t i s s uewi t h ysozyme( l ul ase( enzy mess uchasl bac t er i a) ,cel pl ant chi t i nase( c el l s ) , f ungus ) .Genesar el oc at edonl ong mol ec ul esofDNAi nt er t wi nedwi t hpr ot ei nss uc has h i s t o ne s .Th eRNAc a nb er e mo v e db yt r e at me ntwi t h r i b on uc l e as ewh er e aspr o t e i n sc a nb er e mo v e db y t r e at me ntwi t hpr o t ea se .Ot h ermol e cu l e sc a nb er emo v ed b yappr opr i at et r eat ment sandpur i fi edDNAul t i mat el y pr ec i pi t at esoutaf t ert headdi t i onofc hi l l edet hanol .Thi scanbes eenascol l ec t i onof fi net hr eadsi nt hes us pens i on.
C(tting o! DN' Res t r i c t i onenz y medi ges t i onsar eper f or medb y i nc ubat i ngpur i fi edDNAmol ec ul eswi t ht he r es t r i c t i onenz yme,att heopt i mal c ondi t i onsf ort hat s pec i fi cenz y me.Thec ut t i ngofDNAbyr es t r i c t i on en donu c l eas esr es ul t si nt hef r ag men t sofDNA. Th es ef r a gme nt sc a nb es e pa r a t e db yat e c hn i q ue lel e ct r ophor esi s.Si k nownasge nceDNA f r agment sar enegat i v el yc har gedmol ec ul est he y c a nb es e pa r a t e db yf o r c i n gt h em t omo v et o wa r d s t hea nod eu nd erane l ec t r i cfi el dt h r ou ghamed i um/ mat r i x .Thes ep ar at edb an dsof DNAar eanal y s edf ort her equi r edgeneandt heni ti sc utoutf r om t heagar os egel ande xt r ac t edf r om t hegel pi ec e.Thi sst epi sknownasel ut i on.
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)(lti&lication o! #ene *PCR+ PCRorpol y mer as ec hai nr eac t i oni st henus edt oc r eat emul t i pl ec opi esoft hegene ofi nt er es t .I nt hi sr eac t i on,mul t i pl ec opi esoft hegene( orDNA)ofi nt er es ti s nv i t r ous s y nt hes i s edi i ngt wos et sofpr i mer s( s mal l c hemi c al l ysy nt hes i s ed ol i g on uc l e ot i de st ha tar ec o mpl eme nt ar yt ot h er e gi o nsofDNA)an dt hee nz y me DNAp ol y mer as e.Th ee nz y meex t en dst hepr i mer su si ngt henuc l e ot i d esp r o v i d edi n t her eac t i onandt hegenomi cDNAast empl at e.I ft hepr oc es sofr epl i c at i onofDNA i sr e pe at e dma nyt i me s ,t h es e gme nto fDNAc a nb ea mp l i fi e dt oa pp r o x i ma t e l y bi l l i ont i mes ,i . e. ,1bi l l i onc opi esar emade.
#ene %argeting Genedel i v er yi soneoft hebi gges tc hal l engesi nt hefi el dofgenet her apy . GeneDel i v er yi nc l udes : 1.TARGETI NG t her i ghtcel l s. 2.ACTI VATI NG t hegene. Agene' sj our neyi snotov erwheni tent er st hec el l .I t mus tgot ot hec el l ' snuc l eusandbe" t ur nedon, "meani ngt hati t st r ans cr i pt i onand t r ans l at i onar eac t i v at edt opr oduc et hepr ot ei npr oduc tenc odedbyt hegene.For genedel i v er yt obes uc ces sf ul ,t hepr ot ei nt hati spr oduc edmus tf unc t i onpr oper l y . 3.I NTEGRATI NG t hegenei nt hecel l s. Th eg en emu s ts t a ypu ta ndc on t i n ue wor k i ngi nt het ar getc el l s .I fs o,i tmus tbeens ur edt hatt hegenei nt egr at esi nt o,or bec omespar toft hehos tc el l ' sgenet i cmat er i al ,ort hatt hegenefi ndsanot herwayt o s ur v i v ei nt henuc l euswi t houtbei ngr ej ec t ed. 4.AVOI DI NG har mf ulsi deeffect s. Any t i meanunf ami l i arbi ol ogi c al s ubs t anc ei s i nt r oduc edi nt ot hebody ,t her ei sar i s kt hati twi l l bet ox i cort hatt hebodywi l l mount ani mmuner es pons eagai ns ti t .I ft hebodydev el opsi mmuni t yagai ns tas pec i fi c genedel i v er yvehi c l e,f ut ur er oundsoft het her apywi l l bei neffec t i v e.
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Choosing the Best Vector Ther ei sno" per f ec tv ec t or "t hatc ant r eatev er ydi s or der .Li k eanyt y peofmedi c al t r eat ment ,agenet her ap yv e ct ormus tbec us t omi z edt oaddr es st heuni quef eat ur es oft hedi s or der . Wehav el ear ntt hel es son,oft r ans f er r i nggenesi nt opl ant sand ani mal sf r om bac t er i aandv i r us es ,whi c hha v ek nownt hi sf orages–ho wt odel i v er genest ot r ans f or m euk ar y ot i ccel l sandf or c et hem t odowhatt hebac t er i aorv i r us es want .
Par toft hec hal l engei ngenet her apyi sc hoos i ngt hemos ts ui t abl ev ec t orf ort r eat i ng t h ed i s o r d er .So mev e c t or sc o mmo nl yu s eda r e :
&iruses Us ual l ywhenwet hi nkofv i r us es ,wet hi nkoft hem c aus i ngdi s eas ess uc hast he c ommo nc ol d,t h efl u,a ndHI V/ AI DS.Whe nf a ce dwi t ht hepr o bl e m ofg enedel i v e r y , s ci ent i s t sl ook edt ov i r us es .Whyr ei nv entt hewheel i ft her e' saper f ec t l ygoodone outt her e?I fwec anmodi f yv i r us est odel i v ergeneswi t houtmak i ngpeopl es i c k ,we ma yha v eag oo ds e to fg en et h er a pyt o ol s . Gener al adv ant agesofv i r al v ec t or s : -
They ' r ev er ygoodatt ar get i ngandent er i ngc el l s .
Somev i r al v ec t or smi ghtbeengi neer edt ot ar get
s pec i fi ct y pesofc el l s .
des t r oyt hec el l .
Theyc anbemodi fi eds ot hatt heyc an' tr epl i c at eand
Gener al dr awbac k sofv i r al v ec t or s :
Av i r usc an' t" ex pand"t ofi tapi ec eofgenet i cmat er i al l ar gert hani ti snat ur al l ybui l t t oc ar r y .Ther ef or e,s omegenesmaybet oobi gt ofi ti nt oac er t ai nt y peofv i r us .
Vi r us escanc aus ei mmuner es pons esi npat i ent s ,r es ul t i ngi nt wopot ent i al out comes:
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Pat i ent sma ygets i c k.
Apat i ent ' si mmuni t yt oav i r usma ypr ev enthi mf r om r es pondi ngt or epeat ed t r eat ment s .
Ho wev e r ,moder nv i r al v ec t or sha v ebeenengi neer edwi t houtmos toft hepr ot ei ns t hatwoul dcauseani mmuner esponse.
(on)&iral &ectors Al t houghv i r us esc aneffec t i v el ydel i v ergenet i cmat er i al i nt oapat i ent ' sc el l s ,t heydo ha v es omel i mi t at i ons .I ti ss ome t i mesmor eeffic i entt odel i v erageneus i nganonv i r al v ec t or ,whi c hhasf ewers i z ec ons t r ai nt sandwhi c hwon' tgener at eani mmune r e s po ns e . Nonv i r al v ec t or sar et y pi c al l yc i r c ul arDNAmol ec ul es ,al s ok nownaspl as mi ds .I n nat ur e,bac t er i aus epl as mi dst ot r ans f ergenesf r om c el l t oc el l . Sc i ent i s t sus ebac t er i aandpl as mi dst oeas i l yandeffic i ent l ys t or eandr epl i c at e genesofi nt er es tf r om an yor gani s m. Vec t or sus edatpr es ent ,ar eengi neer edi ns uc hawayt hatt he yhel peas yl i nk i ngof f or e i g nDNAa nds el ec t i o nofr ec omb i na nt sf r om n onr e combi n ant s .
These are not the only way to introduce alien *(A
i nt o
cr oi nj ect i on,r ho s tc e l l s .I name t h odk no wna smi ec ombi nantDNAi sdi r ec t l y i nj ec t edi nt ot henuc l eusofanani mal c el l .I nanot hermet hod,s ui t abl ef orpl ant s , c el l sar ebombar dedwi t hhi ghv el oc i t ymi c r opar t i c l esofgol dort ungs t enc oat edwi t h ol i st i csorgenegun. DNAi namet hodk nownasbi
Delivery to s&eci,c tiss(es Del i v er i nggenest os pec i fi ct i s sueswi t hi napat i ent ' sbodycanbev er ydi ffic ul t . Del i v er i nggenesi nt oagr oupofc el l si napat i ent ' sbodyc anbedonei noneoft wo way s .
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Thefi r s twayi st oi nj ec tt hev ec t ori nt ot hebodyands pec i fi cal l yt ar getaffec t edc el l s . nv i v o xvi v o,i Thi si scal l edani a ppr o ac h .Thes ec on dwa y ,c al l ede st odel i v ert he genet oc el l swhi l et hey ' r eout s i det hebodyby :
I s ol at i ngt hedes i r edc el l sf r om t hebody .
Cul t ur i ngt hec el l si naPet r i di s hi nt hel abor at or y .
Del i v er i ngt hegenest ot hec el l s( us i ngoneoft hev ec t oropt i onsdes cr i bed ont hi spage) ,ac t i v at i ngt hem,andmak i ngs ur et hatt hec el l si nt egr at et hem pr oper l y .
Case +tudy , Cystic -ibrosis %he Disease – ' #enetic Disor$er Cy s t i cfi br os i s( CF) ,al s ok nownasmuc ov i s c i dos i s ,i sanaut os omal r ec es s i v e genet i cdi s or dert hataffec t smos tc r i t i c al l yt hel ungs ,andal s ot hepanc r eas ,l i v er , andi nt es t i ne.I ti schar ac t er i z edbyabnor mal t r ans por tofc hl or i deands odi um ac r os s anepi t hel i um,l eadi ngt ot hi c k,v i s c oussecr et i ons ,pr ev ent i ngt hec i l i af r om c l ear i ng de br i swhi c hc au ses y mpt omss u chasc o ug hi n g,p oordi g es t i onandi nc r ea se d v ul ner abi l i t yt oi nf ec t i on.
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CFi sc aus edbyamut at i oni nt hegenef ort he pr o t ei nc y s t i cfi br os i st r ans membr anec onduc t anc e r egul at or( CFTR)geneonchr omosome7.Most c o mmo nl y ,t h emu t a t i o ni nt h eCFTRg en ei sa t hr eebas epai rdel et i on.Thi spr ot ei ni sr equi r edt o r egu l at et h ec ompo ne nt sofs weat , di ges t i v efl ui ds ,andmuc us . CFTRr e gu l a t e st h e mo v ementofc hl or i d e a nds o di u mi o ns ac r os sepi t hel i al membr anes,suc has t heal v eol arepi t hel i a l oc at edi nt hel ungs .Si nc eal l oft hec el l sofaCF pat i enthav et hedef ec t i v epr ot ei n,l ar gequant i t i esof t hi c k,s t i c kymuc usbui l dupt hr oughoutt hel ungs andot heror gans .Thi sr es ul t si nt hes ev er i t yofs y mpt omsseeni nCFpat i ent s .
Is It ' #oo$ %arget -or #ene %hera&y. T oc he c kt h i ss o mequ es t i o nsmu s tb ea ns we r e d:
Yes. *oes the condition result rom mutation.
Yes. Is the biology o the disorder known.
/ill adding a normal copy o the gene f0 the problem in the aected tissue. Y es .Wh i l et h emu t a t e dCFTRg en ee nc o de san on f u nc t i o na li o n c hannel pr ot ei n,i twi l l notpr ev entanor mal CFTRc hannel pr o t ei nf r om wor k i ng p r ope r l y . Th er ef o r e ,a dd i n gan or ma lc op yo ft heCFTRg enes hou l dfi xt hepr o bl e m
Is it easible to delier the gene to the cells o the aected tissue. Yes ,i npar t .Tr eat i ngt hel ungsofpat i ent swi t hCFmi ghtbef eas i bl e,s i nc et hel ung
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s ur f a ce sar ee x po se dt ot h ea i ra nds ome wh atea syt or e ac h.Be cau set h ed i ges t i v e s ys t em i sl es sac ces si bl e,howev er ,i tmi ghtbeamor edi ffic ul tr egi ont ot r eat . Henc ewec anc onc l udet ha ti ti saper f ec tdi s eas et obet r eat edb ygenet her ap y .
Choosing vectors Thev ec t or st hatar emos ts ui t abl ef orgenet her apyar e:
Retrovir(s Ret r ov i r us esar eenv el opedv i r us est hatr epl i c at ei nahos tc el l t hr ought hepr oc es sof r ev er s et r ans cr i pt i on.I ti sas i ngl es t r andedRNAv i r ust hats t or esi t snuc l ei cac i di n t hef o r m ofanmRNAg enomet ar ge t s .On cei ns i det heh os tc el l c y t opl as mt hev i r us u s esi t so wnr e v er s et r a ns c r i p t a s ee nz y met op r o du c eDNAf r o mi t sRNAg en ome , t her ev er s eoft heus ual pat t er n,t husr et r o( bac k war ds ) .Thi snewDNAi st hen i nc or por at edi nt ot hehos tc el l genomebyani nt egr as eenz y me,atwhi c hpoi ntt he r et r ov i r al DNAi sr ef er r edt oasapr ov i r us .Thehos tc el l t hent r eat st hev i r al DNAas par tofi t sowngenome,t r ans l at i ngandt r ans cr i bi ngt hev i r al genesal ongwi t ht he c el l ' sowngenes ,pr oduc i ngt hepr ot ei nsr equi r edt oas s embl enewc opi esoft he v i r us . Onedr awbac kofr et r o vi r us es ,s uc hast heMol oneyr et r ov i r us ,i nv ol v est he r equi r ementf orc el l st obeac t i v el ydi v i di ngf ort r ans duc t i on.Asar es ul t ,c el l ssuc has neur onsar ev er yr es i s t antt oi nf ec t i onandt r ans duc t i onbyr et r ov i r us es . Butt heai r wa yc el l swhi c har eaff ec t edb yt hedi s eas ec y s t i cfibr os i sandmus tbe t ar get eddi v i dei nf r equent l y .Henc eRet r ov i r usi snotas ui t abl ev ec t orf ort hi sdi s eas e.
'$enovir(s Ade no v i r u ses( memb er so ft hef ami l yAde no v i r i dae )a r eme di u ms i z ed( 90– 100nm) , nonenv el oped( wi t houtanout erl i pi dbi l ay er )v i r us eswi t hani c os ahedr al n uc l e oc a ps i dc on t a i n i n gado ub l es t r a nd edDNAg en ome . 1
Theyhav eabr oadr angeofv er t ebr at eho s t sandha v ebee n c aus eawi der angeofi l l nes s es ,f r om mi l d r es pi r at or yi nf ec t i onsi ny oungc hi l dr ent ol i f et hr eat eni ngmul t i or gandi s eas ei npeopl ewi t ha weak enedi mmunes y s t em. Butt he yc anc aus e/ i nduc eani mmuner es pons ei nt he bodyhenc enots ui t abl ef orgenedel i v er y .
f ou ndt o
human
/er&es Sim&le0 Vir(s He r p ess i mp l e xvi r u s es ,a l s okno wna sHu ma n her pesv i r us ,ar emember soft heher pesv i r us f ami l y ,Her pes vi r i dae,t hati nf ec thumans .They c a nb es p r e adwh ena ni n f e c t e dp er s o ni s pr oduc i ngands heddi ngt hev i r us .Her pes Si mpl e xc anbes pr eadt hr oughc ont ac twi t h s al i v a,s uc hasshar i ngdr i nk s. Butt hes ev i r us esonl yaffec tt hec el l soft hener v oussy s t em andc annoti nf ec tt he ai r wa yc el l sandhenc enots ui t abl e.
'$eno1'ssociate$ Vir(ses Adenoassoci at edvi r us( AAV) i sas mal l v i r us whi chi nf ect shumansandsome ot herpr i mat es pec i es .AAVi snotc ur r ent l yk nownt oc aus edi s eas e and c ons equent l yt hev i r usc aus esav er ymi l di mmuner espons e. AAVc ani nf ec tbot h di v i di ngandqui es centc el l sandper s i s ti nanex t r ac hr omos omal s t at ewi t hout i nt egr at i ngi nt ot hegen ome oft hehos tc el l .Des pi t ei t sf ewdi s adv ant agest hes e f eat ur esmak eAAVav er yat t r ac t i v ec andi dat ef orc r eat i ngv i r al v ec t or sf or gene t her apy ,andf ort hec r eat i onofi s o g en i ch uma nd i s e as emo de l s
ence it is the .est choice for ene dei/ery in the case of 5ystic 6i.rosis
1!
/istory o! Cystic -i2rosis #ene %hera&y Genet her apyf orc y st i cfi br os i sbegani n1990,whens c i ent i s t ss uc c es s f ul l y c or r ec t edf aul t ycy s t i cfibr os i st r ans membr anec onduc t anc er egul at or( CFTR)genes . Theydi dt hi sbyaddi ngnor mal c opi esoft hegenet ol abor at or ycel l c ul t ur es .
3445 I n1993,t hefi r s tex per i ment al CFgenet her apyt r eat mentwasgi v ent oapat i entwi t h d c y s t i cfi br os i s .Res ear c her smodi fi edacommoncol Adeno vi r ust oac tasa del i v er yv ehi c l ebyc ar r y i ngnor mal genest ot heCFTRc el l si nt henas al pas s ages . Res ear c her sc hos enas al pas s agesast hes i t eofdel i v er ybec aus et he yar eeas i ert o ac ces sandmeas ur egeneac t i v i t yt hant hel ungai r way .Lat ert r i al sdel i v er edt he v ec t ort opat i ent s ’l ungai r way s. I nt heear l i ert r i al s ,i thadl ook edl i k et hev i r ushadent er edc el l sandt hatt heCTFR genewaswor k i ng.Butl at ert r i al swi t hdi ff er entpat i ent ss howedl ev el sofVFTRgene ac t i v i t yt hatwer et ool owt omak eanydi ff er enc e.Res ear c her sc amet ot hi nkt hatt he adenov i r usc an’ teas i l yent erai r wayc el l s ,es pec i al l yi nt hel owdos est hatwer ebei ng gi v en.I nt heear l i ert r i al s ,t heys pec ul at ed,geneac t i v i t yr es ul t edf r om t hedamaget o t hec el l sdur i ngdel i v er yal l owi ngt hev i r ust oent ereas i l y . Henc ewhenhi gherdos esoft hevi r uswer et r i ed,t hei mmunesy s t em oft hepat i ent s s t ar t edmoun t i n gi mmuner es pon s esan dfi ght i n go fft hevi r u s.Thi sc au se da bl oc kagei nt het r i al sunt i l 1998.
3446 T r i al sus i ngAdenoas s oc i at edv i r ust odel i v ert heCTFRgenebegani n1998.Unl i k et he a de no v i r u s ,t h eAd en oa s s oc i a t e dv i r u sc a us e dn oi mmu ner e s po ns eo ra dv e r s es i d ee ffe c t s i npat i ent s . Butunl i k et her es ear c her s ’pr edi c t i ons ,t headenoas soc i at edv i r usdi dnotent erc el l s effic i ent l yandi nt egr at ei nt ocal l s ’ genomi cDNA.The ypr oduc edonl yl o w andflee t i ng amount sofCFTRgeneac t i v i t y . Res ear c her sar est i l l wor k i ngt ofi gur eoutwhatc aus edt he v i r us est of ai l .
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Butbec aus ei ti ssaf e,t hev i r us–aswepr edi c t edear l i er–hol dspr omi s ef orbei ngagood wa yt odel i v ert heCFTRgenet opat i ent s ’ ai r wa yc el l s .Butr es ear c her sneedt ol ear nmor e abouthowt hev i r usi nf ec t scel l si nor derormak ei taneffec t i v edel i v er yme t hod.
Challenges So met hef a ct o r st ha tha v ek e ptg enet h er a pyf r om b ec omi n ganeff ec t i v et r eat ment f orgenet i cdi s eas esar e: •
+hort)lied nature o gene therapy ) Bef o r eg en et h er a pyc an be co meap er man entc ur ef o ran yc on di t i o n,t het her ap eu t i cDNAi n t r o duc ed i nt ot ar getc el l smus tr emai nf unc t i onal andt hec el l scont ai ni ngt he t her apeut i cDNAmus tbel ongl i v edands t abl e.Pr obl emswi t hi nt egr at i ng t her apeut i cDNAi nt ot hegenomeandt her api dl ydi v i di ngnat ur eofman yc el l s pr ev entgenet her apyf r om ac hi ev i ngan yl ongt er m benefi t s .Pat i ent swi l l ha v e t ounder gomul t i pl er oundsofgenet her ap y .
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Immune response )Anyt i meaf or ei gnobj ec ti si nt r oduc edi nt ohuman t i s s ues ,t hei mmunes y st em i sdes i gnedt oat t ac kt hei nv ader .Ther i s kof s t i mu l a t i n gt h ei mmu nes y s t em i nawa yt ha tr e duc e sg enet h er ap y eff ec t i v enes si sal way sapot ent i al r i s k .Fur t her mor e,t hei mmunes y st em' s enhanc edr es pons et oi nv ader si thass eenbef or emak esi tdi ffic ul tf orgene t her apyt ober epeat edi npat i ent s .
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roblems with iral ectors ) Vi r us es ,whi l et hec ar r i erofc hoi c ei n mos tgenet her apyst udi es ,pr es entav ar i et yofpot ent i al pr obl emst ot he pat i entt o xi c i t y ,i mmuneandi nfl ammat or yr es pons es ,andgenec ont r ol and t ar get i ngi s s ues .I naddi t i on,t her ei sal way st hef eart hatt hev i r al v ec t or ,onc e i ns i det hepat i ent ,mayr ec ov eri t sabi l i t yt oc aus edi s eas e.
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4ultigene disorders )Condi t i onsordi s or der st hatar i s ef r om mut at i ons i nas i ngl egenear et hebes tc andi dat esf orgenet her apy . Unf or t unat el y ,s ome t hemos tc ommonl yoc c ur r i ngdi s or der s ,s uc hashear tdi s eas e,hi ghbl ood pr es sur e,Al z hei mer ' sdi s eas e,ar t hr i t i s ,anddi abet es ,ar ec aus edbyt he c ombi nedeffec t sofv ar i at i onsi nmanygenes .Mul t i geneormul t i f ac t or i al
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di s or der ssuc hast hes ewoul dbees pec i al l ydi ffic ul tt ot r eateffec t i v el yus i ng ge net h er a py .
Issues regarding Gene Therapy Whatar et hepos si bl ei mpl i c at i onsofgenet her apyr es ear c ht os oc i et y ?Al l ofusr es ear c her s ,pol i c ymak er sandt hepubl i c-h av ear es pons i bi l i t yt oex pl or et he pot ent i al eff ec t sofgenet her apyr es ear c honourl i v ess ot hatwec anmak ei nf or med dec i s i ons . Fore ac hne w ap pl i c at i onofg enet he r a pyr e sea r c h,wemus tc o ns i d er :
Wh ata r et h eb en efi t s ?
Whatar et her i s k s ?
Whom wi l l t het ec hnol ogyhel p?Whowi l l i tpot ent i al l yhur t ?
Whatdoesgenet her apymeanf orus? Ther ear es ev e r al t y pesofi s s uest oc ons i deraswet hi nkaboutgenet her ap y : •
•
•
Et hi c al i s s uesas kust oc ons i dert hepot ent i al mor al out c omesofgene t her apyr es ear c h. Legal i s s uesr equi r er es ear c her sandt hepubl i ct ohel ppol i c y mak er sdec i de wh et h era ndh owg en et h er a pyr e s ea r c hs h ou l db er e gu l a t e db yt h e gover nment . Soc i al i s s uesi nv ol v et hei mpac tofgenet her apyr es ear c hons oc i et yasa wh ol e .
1$
Somequest i onst oponder
/hen should gene therapy be used. Shoul di tbeus edt ot r eatc r i t i c al l yi l l pat i ent s ?Shoul di tbeus edt ot r eatbabi esandc hi l dr en?
/hat eect would gene therapy hae on uture generations i germline 5reproductie6 cells were genetically altered. Ho w mi ghtt hi s al t er at i onaff ec thumanv ar i at i on?
/ho should decide what are 7good7 or 7bad7 uses o genetic modifcations. Ho wd oy o ude fi n e" n or ma l "wi t hr e ga r dt ohu ma nb ei n gs ?
/hat i we could alter human traits not associated with disease. Wo ul di tbeo ka yt ou seg enet he r a pyt oi mpr o v eore nh anc eape r s on ' s genet i cpr ofi l e?
/ho will hae access to gene therapy8 treatments and long)term ollow)ups. Wi l l g en et her ap yandg ene t i cen hanc eme nt sc r e at ea na dv a nt a gef o r t ho sewhoc ana ffor di t ?
9ecent :pcoming CRISPR
1%
CRI SPRs t andsf orc l us t er edr egul ar l y i nt er s pac edshor tpal i ndr omi cr epeat s . TheseRNAs equenc esse r v eani mmune f unc t i oni nar c haeaandbac t er i a,buti n t hel as ty earors o,s ci ent i s t shav es ei z ed u po nt h em t or e wr i t eg en es .Th eRNA s equ enc es er v e sa sag ui det ot a r ge ta DNAs e qu en c ei n ,s a y ,az y g ot eo ra s t em c el l .Th egu i deseq ue nc el ea dsan en zy me,Ca s 9,t ot h eDNAo fi n t e r es t . Cas 9c anc utt hedoubl es t r and,ni c ki t ,or e v enk noc kdo wng enee x pr es s i on .Af t e rCas 9i n j ur est heDNA,r epa i rs y s t emsfi x t h es e qu en c e-o rn ews e qu en c esc a nbei n s er t e d. I ti s n' tt hefi r s toronl ymet hodofgener epai rt her apyt hat ’ sbeendev el oped,butt he CRI SPRt ec hn ol ogy ,s a y sRa mes ar ,i ss os pec i al be c au se,u nl i k epr e v i ousme t h ods whi c hwer emor el abor i ousandc oul donl yt ar getonek i ndofc el l i nt hebody ,i t appear st obea" ones i z efi t sal l del i v er y " , adapt abl ef ordi ffer entt i s sues .The pr oc edur eal s os eemsr el at i v el ys i mpl et oper f or m. Ex c i t i ngast hede v el opmentma ybe,CRI SPRwon ’ tbedel i v er i ngi ns t antc ur esj us t y e t . Rames ars ay s,f r om hi si ni t i al i mpr es si onsoft hel i t er at ur e,t hati twoul ds eem t hat l oc al i s ed,ac ces si bl eabnor mal t i s sue( asi nt her et i naors ki n)c oul dbet ar get ed mor eeas i l y . Condi t i onsaff ec t i ngt hebodymor es y st emi c al l y , howev er ,s uc hasc er t ai n de v el opment al s y ndr omes ,orc ent r al ner v ouss y s t em di s or der s ,mi ghtbe pr obl emat i ci nt er msofget t i ngt her epai rt ec hnol ogyi nt oenoughoft het ar getc el l si n t hatt i s s uet omak eaneffec t i v edi ffer enc e. " I tma yal s odependont hes t ageoneat t empt st oc ar r youtt het her apy , i nt er msof t hepat i ent ’ sageandl ev e lofadv anc ementoft hedi s eas e, "s ay sRames ar .
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Conclusion Al t houghear l ycl i ni c al f ai l ur esl edmanyt odi s mi s sgenet her apyasov er hy ped, c l i ni c al s uc c es s ess i nc e2006ha v ebol s t er ednewopt i mi s mi nt hepr omi s eofgene t her apy .Thes ei nc l udes uc ces sf ul t r eat mentofpat i ent swi t ht her et i nal di s e as eLeber ' sc ongeni t al amaur os i s ,Xl i nk edSCI D,ADASCI D, adr enol euk ody s t r oph y,c hr oni cl y mphoc y t i cl euk aemi a ( CL L) , ac ut el y mphoc y t i c l e uk aemi a ( ALL) , mu l t i p l emy e l o ma,haemophi l i a andPar k i ns on' sdi s eas e.Th es e r ec entc l i ni c al s uc ces seshav el edt oar enewedi nt er es ti ngenet her apy ,wi t hs ev er al ar t i c l esi ns ci ent i fi candpopul arpubl i c at i onscal l i ngf orc ont i nuedi nv es t menti nt he fi el d.
;ibliography 7e2sites • • • • •
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http<==en>wikipedia>org=wiki=Gene?therapy http<==www>trip2medi>com=treatmentCGeneTherapy>php http<==learn>genetics>utah>edu=content=tech=genetherapy= http<==ghr>nlm>nih>go=handbook=therapy= http<==cystic)fbrosis>emedt>com=cystic)fbrosis=cystic)fbrosis)gene) therapy>html http<==en>wikipedia>org
Books 12th (C@9T ;iology +tryer ;iochemistry
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