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vn
1. GII THIU CHUNG Vo ngy 14-9-1990, nhm nghin cu ti hc vin sc khe quc gia M (U.S.National Instituse of Helth) tin hnh chui th nghim liu php gen u tin cho b Ashanti Desilva 4 tui. Sinh ra vi cn bnh di truyn him ngho c tn l severe combined immune deficiency SCDI, c b thiu h thng min dch v c th b tn thng khi mc phi bt k mm bnh no. Tr em mc bnh ny thng khng chng li c s ly nhim ca bnh tt v rt kh sng n tui trng thnh, tui th ca chng lun mc bnh tt. Ashanti c lp mt hnh lang bo v xung quanh, khng tip xc vi nhng ngi ngoi gia nh, trong mi trng v trng ti nh v c iu tr thng xuyn vi khng sinh liu cao. Trong th tc liu php gen ca Ashanti, bc s tch cc t bo mu trng ra khi c th c ta, cho chng pht trin trong phng th nghim, gn gen thiu vo cc t bo ny v sau chuyn cc t bo c bin i di truyn tr li mu ca bnh nhn. Nhng kim tra trong phng th nghim cho thy liu php ny lm tng h thng min dch ca Ashanti, c ta khng b cm lnh tr li, c th n trng v min dch vi chng ho. Th tc ny khng phi l mt phng thuc, nhng t bo mu trng c x l v mt di truyn ch lm vic trong mt vi thng v qu trnh ny phi lp li. Mc d s gii thch n gin v liu php gen ny dng nh c kt qu kh quan, nhng n m u cho mt cuc tranh lun ko di; con ng liu php gen c chp nhn cha y nhng cuc tranh lun. Liu php gen sinh hc ngi l rt phc tp, mt s cng ngh cn c pht trin v nhng cn bnh cn c hiu bit cn k hn trc khi liu php gen c th c s ng mt cch thch ng. Tuy nhin, khng th ph nhn rng, ngy nay liu php gen l mt lnh vc khng l vi c hi sinh li ln cho cc nh u t c hiu bit. Bng chng l s pht trin ca cc nghin cu v liu php gen trong hn mt thp k qua. Lnh vc ny c tim nng v cng to ln, mt s ngi so snh tim nng u t trong cng ngh sinh hc c bit l liu php gen vi kinh nghim v cng ngh nhng nm 1980.

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2. LIU PHP GEN L G? 2.1. nh ngha liu php gen. Trong cc ng dng ca sinh hc phn t vo y hc, liu php gen i vi cc ri lon di truyn m ra nhiu trin vng nht, ng thi cng l hng ng dng kh thc hin nht. Cc nh ngha v liu php gen c thay i theo thi gian, ngy nay khng c nh ngha chung no c chp nhn cho khi nim ny. Song nh ngha sau y c s dng rng ri nht trong cc din n Quc t. nh ngha: liu php gen l k thut a gen lnh vo c th thay th cho gen bnh hay a gen cn thit no thay vo v tr gen b sai hng t c mc tiu ca liu php. - Bin php th nht l cch gii quyt trit , v gen lnh s c a vo ng v tr ca n trn b gen v chu tc ng bnh thng ca cc trnh t biu hin gen . Cho n nay, cha c thnh tu ng k no trong lnh vc ny v ngi ta cha bit cch sa cha gen mt cch c hiu. Tuy vy, k thut ti t hp ng dng cng em dn nhng hy vng rt ln v kh nng thnh cng l iu hon ton c th hy vng trong tng lai. - Bin php th hai d thc hin hn nhng khi a thm mt gen cn thit vo b gen, ngi ta khng lm ch c v tr gn xen ca on mi a vo. iu ny dn n mt s kt qu khng lng trc c: Gen mi a vo khng nm trong phc hp iu ho nn c th c biu hn mt cch tu tin, khng ng ni (ng vi loi t bo, m c hiu) v khng ng lc (ng vi chu trnh pht trin c th), hoc thm ch khng c biu hin. Gen mi a vo gy nhng hiu qu khng mong mun. Gen gn vo mt v tr c kh nng hot ho mt gen tin ung th dn dn s biu hin qu ca gen ny gy ra ung th. Hin nay, mi c gng u nhm gii quyt nhng vn ny. c bit, liu php gen trong trng hp ny c gii hn cht ch trn t bo sinh dng. Vic tc ng vo gen ca t bo sinh dc b nghim cm v l do o c, con ngi t chi vic thay i gia sn di truyn c th lu li cho con th h sau. Liu php gen c th c tin hnh thng qua mt s cch sau:

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Chuyn trc tip gen vo m ngi bnh. To vct virus chuyn gen mt cch hiu qu ti t bo (in vivo).

Virus xm nhim trc tip vo t bo bng cch gn vi chng v bm thng tin di truyn ca chng vo t bo. V cc virus t nhin t sinh sn bn trong t bo ch nn gy hi vi vt ch mang chng. Tuy nhin, c th xo hay lm mt cc phn c hi ca virus, ngn khng cho chng ti bn trong t bo ch. Ngy nay cc virus khng c kh nng ti bn ny c s dng chung cho cc nghin cu liu php gen trn ngi v ng vt. S dng cc k thut khc nhau chuyn gen vo t bo bn ngoi ngi bnh, sau chuyn cc t bo c bin i ny vo ngi bnh (ex vivo). Phng php ny c s dng chung phn phi cc nhn t pht trin v cc phn t khc ti cc vng c hiu ca c th. Chng cng c s dng to ra cc dng t bo b bin i hon ton cho cy ghp. Ging nh trong liu php gen in vivo, cc t bo c chuyn c th gy biu hin cc protein l gy sng ty hay p ng min dch. Cc nhm chuyn gia chia liu php gen thnh cc mc khc nhau: Chuyn gen vi s ho nhp (gen c kt hp cht ch vi DNA ca vt ch) Chuyn gen khng ho nhp (gen khng kt hp cht ch vi DNA ca vt ch) S dng cc oligonucleotide tng hp nhn to gi l cc phn t ribozyme/antisense khng c cc thnh phn iu ho (s biu hin gen b bin i). 2.2. Cc bc c bn trong liu php gen Trong thc nghim, ngi ta dng cc vector virus chuyn cc gen vo t bo ng vt theo 2 bc: Bc 1: To vector ti t hp mang gen cn chuyn. Trc , cc virus c bin i khng cn kh nng sao chp, ng thi li c kh nng biu hin mnh gen cn a vo c th. Cc bin i ny bao gm vic loi b cc trnh t cn cho s sao chp ca virus v gn vo trc gen cc trnh t promotor mnh. Sau

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, vector ti t hp c a vo t bo nui cy. Loi t bo c s dng nhiu nht l t bo tu xng v d nui cy li bao gm nhiu t bo ngun a th (pluri potential) Bc 2: Vector virus mang gen lnh c a vo c th m t ngi ta tch cc t bo tu xng. Nh vy, c th xem y l k thut ghp t thn d gen ghp vo l gen l i vi c th. Tr ngi ln l protein do gen l to ra c th kch thch sn sinh khng th chng li chnh n, hn na, nu vic chuyn gen vo t bo nui cy thng thnh cng th vic a t bo chuyn gen tr li c th li t khi c hiu qu do nhiu nguyn nhn. Gn y nht, mt quy trnh liu php gen va c thng qua nhm lm chm s pht trin ca bnh AIDS. Ngi ta chuyn cc oligonucleotit i (antisens) b sung cho mt s trnh t ca virus HIV (trnh t TAR, REV) vo cc t bo ca mt ngi lnh l anh em sinh i ca ngi bnh. Sau cc t bo ny c tim vo bnh nhn. Cc Oligonicliotit i TAR s c ch s sao chp ca virus khi bt cp v v hiu ha trnh t TAR ng vai tr trong s sao chp. Cn cc Oligonucleotit i REV, khi bt cp vi trnh t REV s ngn s vn chuyn mRNA t nhn ra t bo cht. 2.3. ng dng ca liu php gen trong iu tr bnh n nay c 533 th nghim liu php gen din ra trn th gii. Vi 407 th nghim din ra M, y l nc dn u trong cc th nghm ca liu php gen. Trong s 533 th nghim, c 322 th nghim trong iu tr bnh ung th, chim 62,28%, 37 th nghim trong iu tr HIV chim 6,94%, ngoi ra l cc th nghim vi cc bnh tim mch, u x, SCID (severe combined immune deficiency), chng mu kh ng... 3. CC YU T K THUT TRONG CHUYN GEN Khng h thng vct no l ti u cho tt c cc k thut ca liu php gen Kay, M., Glorioso, J., Naldini, L. Nature Medicine 7 (1) 2001 3.1. Cc c s ca sinh hc phn t: Thng tin di truyn di dng DNA tn ti trong mi t bo ca c th (tr cc t bo mu). Mi t bo cha ng cc thng tin xy dng bt c dng t bo

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hay m c hiu no ca sinh vt. V cc t bo v m khc nhau quy nh s sn xut ca cc khi cu trc (building block) khc nhau nn DNA cha ng thng tin lin quan n khi cc cu trc sn xut cc m c hiu. S tin ho cung cp mt gii php tuyt vi cho vn ny. DNA c to nn t mt chui bn phn t khc nhau: adenine, guanine, cytosine v thymine. S kt hp ca cc phn t ny to nn ngn ng ca DNA. Gen, c to nn t DNA m ho tt c cc protein, l khi cu trc ca t bo. Cc promoter cho php hot ho ca cc gen v qua cc protein c hiu c biu hin. Cc vng khc ca DNA khng mang m v l phn quan trng bo tn cu trc nhim sc th. Thnh phn ny ca ca DNA v cng quan trng trong cc d n v b gen, tnh tun hon on ca DNA trong cc vng khng mang m c th c kt hp vi cc gen biu hin, cho chng ta phng php tm kim cc gen cha bit. T nhin pht trin mt cch thc rt thng minh m qua cc protein c hiu ch c biu hin trong cc m dc hiu. DNA sp thnh cc si di gi l nhim sc th. C hai bn sao ca mi gen tn ti trong t bo. Tuy nhin c mt s trng hp ngoi l, cc gen tn ti trong cc nhim sc th X v Y (nhn t quyt nh gii tnh) c th ch c duy nht mt bn sao. Thng tin c truyn t DNA n cc c quan phc tp hn. Qu trnh phin m c bt u bi cc phn t nhn bit c hiu c tn l cc nhn t phin m (nhn t gn vi cc chui iu ho ca gen v cho php s sao chp c tin hnh). Bn sao c to thnh t phn t ban u gi l RNA trong nhn t bo. Bn sao sau ra khi nhn t bo, chuyn n li ni cht, ni m n s c dch m tng hp thnh protein. 3.2. C s ca liu php gen u tin, phi c bn sao y ca gen thch hp vi nhng trnh t iu ho thch hp (trnh t khi ng), trnh t khi ng c th l duy nht i vi gen c hiu, bng cch ny cho php gen ch biu hin trong cc m m thng thng chng c biu hin. Ln lt, cc gen c th c kt ni vi cc trnh t khi ng, hot ho trong tt c cc m (phng php chung nht c s dng ngy nay) hoc c th kt ni chuyn gen, cn hi mt s iu kin sau y:

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vi cc trnh t c th hot ho hay bt hot nh mt cng tc. Nhng thao tc nh vy c th t c bng cng ngh DNA ti t hp. Th hai, phi la chn k thut ch. DNA cn k thut ny c th tip cn vi t bo. Ba k thut c bn c s dng lm iu ny: DNA c th c gn trc tip vi cc t bo hoc cy vo m. Gi l chuyn DNA trn (nacked DNA transfer) DNA c th c s dng d to nn con thoi dch chuyn virus (viral transfer shuttle). y l phng php chuyn DNA hu nh chc chn nht. DNA xm nhp khng hiu qu vo t bo v n kh c th xuyn qua lp mng lipit kp, nhng vector virus d dng lm vic ny. DNA cng c th phi hp vi cc phc h ho hc khc nhau khuch i kh nng chuyn dch qua mng t bo ca n. Th ba, c hai phng php ng dng liu php gen: liu php gen c th ng dng trc tip vi bnh nhn hoc trn cc t bo tch ra khi ngi bnh (nh t bo tu xng) sau cy tr li ngi bnh. Trong tng lai, cc thao tc liu php gen ca t bo gc pht trin trn mi trng m t bo v sau cy ln ngi bnh s l tin trin tt cng. Mi k thut trn u c nhng thun li v kh khn ring. Khi chn phng php phn phi gen, mt s nhn t cn c cn nhc: Hiu qu ca liu php phi c t ln hng u. Sau , gen chuyn i phi c iu ho ng n. N phi c hot ho ng lc, vi di thi gian chnh xc v ng s lng. Nhng vn ny l ro cn to ln cho s pht trin v ph bin ca liu php gen. Hin nay ch ng dng quan trng nht ca liu php gen l cc bnh him ngho, m bnh d dng b nh hng v nhng bnh m s iu ho chnh xc ca gen c chuyn l khng cn thit. ng nh mong mun, hiu qu ca liu php trong cc trng hp ny d dng qun l. S tinh vi ca chng ta trong thit k vector v promotor ngy cng tng, m ra nhiu trin vng mi cho nhng ng dng iu tr cc bnh him ngho (nh bnh i ng) thng qua s tp hp thng tin ca cc d n v b gen ngi m s iu ho gen l phc tp hn.

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chuyn gen, trong cc nghin cu s dng cc vector c hoc khng c bn cht virus nhm a gen mc tiu n cc t bo. Hin nay, cc nghin cu phn phi gen nh retrovirus din ra nhiu nht sau n adenovirus. 3.3. ca n S s dng virus trong liu php gen da trn cc chc nng c bn ca n: Chuyn c hiu vt cht di truyn n t bo. Virus cha vt cht di truyn c bao quanh bi lp mng protein. u tin, virus gn ti cc t bo ring bit bng s tng tc vi th th (hay mt chui cc im th th) trn b mt t bo. Tip , virus vo t bo bi s tng tc vi cc phn t b mt c hiu trn t bo, bng cch ny n d dng i xuyn qua lp ro chn mng t bo. S ph thuc vo dng ca vector virus c s dng, nhng gen c mang bi virus hoc c nhn bn s dng nguyn liu ca chnh cc t bo hoc c tip hp ti nhn ca t bo, ni m cc nhim sc th v DNA nhn tn ti. Cc gen c hiu c mang v c tip hp bi virus s phin m thng hp RNA v dch m to thnh protein to nn chc nng mong mun hay sn phm ca liu php. Cc virus khc nhau c mc khc nhau trn phng din chuyn gen ca chng ti cc t bo v to ra cc sn phm gen. Hn na, hiu qu cui cng ca chng i vi cc gen chc nng trong t bo khng ging nhau v c th khc nhau t t bo ny n t bo khc hoc m ca c quan ny n m c quan khc. Nhng vn ny hng ln ti thun li y hc v thnh cng ca chin lc liu php gen da trn vector virus. Tt c cc vector c sn xut t cc virus ti t hp kiu hoang di c xc nh. Ni chung, cc gen c vai tr trong ti bn virus c loi b ngn cn s truyn i khng mong mun ca vector virus v ngn cn cc vector virus gy bnh. Vector chuyn gen c bn cht virus 3.3.1. Cc bc quan trng hot ha thnh cng virus v chc nng

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Hin ti, c 4 dng chnh ca vector virus ang c s dng trong nghin cu v ng dng: adenovirus, adeno-associate virus (AAV), retrovirus v herpes Simplex. Bng 2: So snh c im ca cc h thng chuyn gen virus

3.3.2. Vector Adenovirus: Mt trong nhng vector liu php gen chung nht hin ang s dng l vector c c s adenovirus. Adenovirus gy bnh kh th ngi (cm lnh, nhim trng d dy) v c 47 dng khc nhau (gi l kiu huyt thanh) c tm thy ngi. Kiu 2 v 5 c s dng chung nht cho cc ng dng liu php gen.

Hnh 1: Adenovirus c cu trc chung cha capsid hai mi mt bao quanh b gen dsDNA di xp x 36kbp. V capsid ca virus cha ba protein: hexon, fiber v

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base penton. Hexon l thnh phn cu trc quan trng, to thnh b mt hai mi mt, trong khi cc penton to thnh phc h vi fiber cho kt qu l 12 chp ngoi v virus ng vai tr quan trng trong vic gn vi cc phi t. im u tin trong sn xut vector liu php gen l ly i gen iu khin ti bn (s pht trin) ca virus trong m. S sinh sn v pht trin ca mt lng nh virus mi l nguyn nhn chung cho nhng hiu ng c do nhim virus. B gen ca adenovirus c nghin cu kh k, cha DNA xon kp gm 50 gen, di 36 kilobase (KB). Gen E1 iu ho phin m trong khi gen E2 v E3 iu ho qu trnh dch m ca b gen. Cc vector tch gen ny cho php mang gen di 8kb. Trc y, s pht trin ca cc vector ny da trn vic s dng virus gip (helper virus) cung cp cc chc nng ca gen b hu. Tuy nhin, ngy nay mt dng t bo c gi tr thng mi c to ra mang cc gen b xo dng vector. S pht trin ca cc dng t bo ny c th lm adenovirus c gi tr thng mi v an ton hn. Khi ri ro ca vic s dng virus kiu hoang di c gim bt. Trn ht, sn phm vector ny c hiu qu, vi nng 1x1012 on virus/ml ang c sn xut. Php o hot tnh vector thc t rt quan trng trong liu php gen. Vic o cc phn t vector n c khng bao hm rng tt c cc on l nhim v do , c hot tnh. Hin nay, c nhng c gng tiu chun ha liu lng v hot tnh ca cc vector virus. Tht khng may, h thng vector adenovirus xut hin mt vi bt cp. S sn xut ca liu php gen l gii hn cho mt chu k thi gian ngn v DNA c chuyn khng tr thnh mt phn ca DNA trn t bo ch. Cc tc ng tr li ca vector adenovirus cng c th gy ra p ng min dch mnh trn vt ch, c nguy c dn ti cc bin chng nghim trng. Trng tm nghin cu hin nay nhm ci tin vector adenovirus ch v gim cc phn ng min dch do virus gy ra, mang ti thnh cng y ha hn trong vic s s dng cc vector ph hu bn trong (gutted vector) ch s dng v ngoi ca adenovirus. S pht trin th v khc l kh nng iu khin vector adenovirus ch ti cc nhm t bo c hiu. Cng ngh retargeting bao gm vic sn xut khng th cho protein th th c v adenovirus, nhn t c s dng gn kt v gia nhp

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t bo. Khng th ny c hp nht vi khng th n dng (monoclonal) chng li protein c hiu tn ti trn b mt ca t bo. Vector sau ho trn vi khng th dung hp. Khi c s dng, ch nhng t bo biu hin protein c hiu c cha tr bng vector ny. Mt s cng ty (v d: GenVec) sn xut vector adenovirus bin i b mt im th cm, im s kt hp vi cc t bo khc nhau. Vic phn loi ca cc vector khc nhau ny cho php adenovirus ch ti nhng m khc nhau khi s dng cc khng th.

Hnh 2: S cu trc ca vector adenovirus hiu nng cao (high-capacity adenoviral) HC-Ad. Tt c cc gen ca virus HC-Ad b xo, ch cn u bn tri v phi ca Ad5 v DNA thm vo (stuffer DNA) thu c t gen C346 v HPRT. AdSTK109 cha b gen hAAT bao gm liver v promoter macrophage c hiu, trong AdGS85, hAAT cDNA c biu hin t promoter cytomegalovirus ca ngi ((hCMV promoter). AdSTK129 ch cha DNA thm vo v khng biu hin bt c gen chuyn no. 3.3.3. Vector Adeno-associated Virus (AAV): AAV l mt parvovirus khng xut hin trong cc bnh do vi sinh vt ngi. Virus ny c th chuyn DNA ti cc t bo khng phn chia v ho nhp vi DNA ca t bo ch. iu ny m ra kh nng ng dng ca mt vector chng li cc bnh kinh nin. Trong di hn, s biu hin ca liu php gen c chng minh.

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Virus ny nh do ch c th mang nhng gen c kch thc gii hn. N cng kh sn xut hn cc vector khc nn tnh thng mi hin nay b hn ch. C 6 kiu huyt thanh (serotype) ca AAV, kiu 2 thng c s dng trong cc ng dng ca liu php gen. Do kch thc nh, vector ny ch mang c gen c kch thc ln nht l 5 kB. Nh vy, khng th chuyn c cc gen phc tp vi kch thc ln. Mt s phng thc c ra gii quyt vn ny. Mt trong s l k thut lin kt b gen vector phn ct mt s gen hp nht (cooperating gene) v phn phi chng ti ch, s dng hai vector phn ct. Mt cch trung gian l s nhn bn ca cc vector dung hp s dng lp v ca adenovirus kt hp vi b gen ca vector AAV. iu khng thun li ca phng php l vector ny i hi s gip ca adenovirus c tng hp. iu ny c th dn n s hp tc mo him vi vector adenovirus (phn ng min dch). Gn y, 3 plasmid, h thng virus gip a nng (helper virus free system) pht trin cho php sn xut cc th gip a nng ca vector ny.

Hnh 3: cu trc b gen ca kiu hoang di (wild-type) v ca vectorAAV.

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(A) bn b gen ca AAV hoang di cha cc khung c (reading frames) Rep v Cap, cc promoter (p5, p19, v p40), polyadenylation site (pA), v inverted terminal repeats (ITR). Qu trnh phin m ca virus m ho cc protein Rep v Cap (VP1-3) khc nhau c v di b gen. Cc protein Rep nh hn c dch m t cc im khi u ni ti (internal initiation sites). (B) bn ca vector AAV, cho thy s thay th cc gen Rep v Cap ca virus vi bng chuyn gen (promoter, transgene cDNA v polyadenylation site). (C) Cu trc bc hai ca AAV ITR, vi cc vng gn Rep (RBS) v vng tiu tan cui cng (TRS) (terminal resolution site) Thun li chnh ca h thng vector AAV l n ho nhp vi nhim sc th ca t bo ch. iu ny cho php cc vector c chuyn biu hin mt cch n nh v lu hn. Nhng tht khng may, nu AAV kiu hoang di ho nhp ti im c hiu trong nhim sc th 19 th cc vector ti t hp li mt tnh c hiu ny v ho nhp tu tin. Cho d vy, mt vi thnh cng trong nghin cu trn ng vt v ngi cho thy rng, s biu hin n nh c th t c vi vector ny.

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Hnh 4: Qu trnh phin m ca vector AAV. Cc bc khc nhau cn cho qu trnh phin m ca vector AAV bao gm: u tin AAV tng tc vi cc receptor a dng v cc phn t coreceptor trn bn mt t bo, s tip thu virion, nhn vo trong t bo v gii phng b gen mch n va vector, s lai ca b gen a vo b sung (complementary input genomes) v s ho nhp nhim sc th trc biu hin gen c th xy ra t khun xon kp DNA. Cu trc bc hai tim tng ca b gen vector b sung (episomal) xut hn nh cc phn t vector RNA c m ho.

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Hnh 5: Chin lc sn xut rAAV. Plasmid ti t hp cha gen cn thit, c tch dng cng lc, ITRs ca AAV c chuyn vi plasmid gip ti t hp c cha wtAAV v cc gen adenovirus cn thit cho s gii phng, ti bn, v ng gi rAAV trong 293 t bo. Khong 48 n 60 gi sau, cc t bo b dung gii v phn chit th cha rAAV v cc protein ca t bo b tch ra l thuc vo s tinh ch sau ny (qua ly tm gradient mt v sc k ct). Cc mu nh rAAV sau tinh ch c xc nh chnh xc v cc virus tinh ch sau c s dng cho cc nghin cu in vitro v in vivo. 3.3.4. Vector Retrovirus Retrovirus nm trong s cc vector virus c pht trin u tin. Vector ny c s dng trong gn 60% d n liu php gen y hc. Virus ny c cha vt cht di truyn l RNA v c kch thc 7 n 11 kB. Khi vo t bo, RNA virus cn tr thnh DNA trc khi n c chc nng trn t bo ch. iu ny c thc hin bi enzim phin m ngc, RT (reversse transcriptase). Retrovirus c cha enzim phin m ngc (enzim xc tc to thnh bn sao DNA ca vt liu di truyn RNA qua phin m ngc) v enzim ho nhp (integrase) cn cho s ho nhp ca retrovirus virus vo t bo ch. B gen ca tt c cc virus ny nh v c nghin cu y . Retrovirus d dng bin i v mt di truyn do c s dng rng ri nh mt vector ca liu php gen. Virus murina leukaemia bin i di truyn (MuLV) c s dng ph bin nht. Cc retrovirus c in ch nhim vo t bo ang phn chia (dividing cell) v vt cht di truyn ca chng sau khi chuyn thnh DNA, ho

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nhp vi vt cht di truyn ca t bo ch nh mt provirus. iu ny gip n thch ng vi bnh ung th, bnh m cc t bo phn chia rt nhanh, cng nh cc bnh cn thi gian di gen c n nh vng chc. iu bt li ca vector ny l n ho nhp tu tin vi DNA ca t bo ch. Do , cc ri ro khi vector xm nhp s ph v chc nng sinh l bnh thng ca t bo ch. Cc retrovirus c bn cng cn s phn chia ca t bo ch lm vic. Hin nay tn ti nhng lo ngi ln hn, l kh nng cc vector retrovirus s nhim vo t bo mm ca ngi bnh v sn xut tinh trng hoc trng. Do vy, tim tng s nh hng n sinh sn sau ny. Ti nay, iu ny c quan st trn cc ng vt mu. Cc vector retrovirus c s c th tr thnh vector c la chn thay i t bo gc (stem sell). Khi , liu php gen s c s dng sn xut cc gen cha bnh. Tuy nhin, khi ng dng dng vector ny tch dng, ri ro do t bin s tng. Mt trong cc pht trin th v gn y l s bin i ca cc lentivirus, dng thay th (sub-type) ca retrovirus, trong cc ng dng ca liu php gen. Dng virus ny gm c HIV v SIV. Cc retrovirus ny ho nhp vi DNA ca t bo ch, do cung cp s biu hin gen n nh trong mt thi gian di. Tuy nhin, chng c th vo cc t bo khng phn chia, do m rng kh nng ng dng ca vector ny.

Hnh 6: B gen ca retrovirus (oncogenic retrovirus) gy bnh ung th MLV (murine leukemia virus). DNA ca axit nucleic ca virus (hnh thi ca b gen virus trong cc t bo b nhim) xut hin. Hai virus c cng c cc gen cu trc gag, pol, v env. Gav m ho li virus v pol m ho cc enzim ti bn. Gav v pol c biu hin ngay lp tc t promoter trong LTR (long terminal repeat). Gen env c biu

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hin t tng ch ni mRNA (singly spliced mRNA), m ho mng glycoprotein. Gen ca HIV c biu hin t mRNA xut hin thm vng a ni. D cc vai tr khng c bit y song chng quan trng trong qu trnh iu ho biu hin gen ca virus v trong s iu chnh ly nhim.

Hnh 7: Chu trnh ti bn ca retrovirus. S nhim bt u khi lp mng glycoprotein ca virus nhn ra cc v tr th th c hiu trn b mt t bo, sau mng virus v mng t bo dung hp gii phng li virus (virus core) vo t bo cht. RNA ca virus, c sao chp thnh phn t DNA mch kp, v xm nhp vo nhn. Thi gian chnh xc v c ch ca vic ny cha c xc nh r rng. DNA ho nhp vo nhim sc th, b gen virus (t by gi gi l provirus) c gi n nh. Sn xut RNA ca virus v sau l cc protein v enzim ca chng. RNA ca virus cng kt hp vi protein ca virus to thnh cc phn li (core) mi. Cc virion con trng thnh c kh nng nhim vo cc t bo mi khi chng c gii phng ra ngoi t bo v c bao bi v ca chng (gm c mng t bo v v glycoprotein).

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Hnh 8: Cc yu t ng vai tr trong vector retrovirus c trng. Cc chui cn thit cho s lan truyn retroviral c th hin. Tt c cc chui protein ca virus b xo v thay bng gen ngoi lai (gen cn cho cc mc ch nht nh). Gen ngoi lai c th c biu hin t promoter trong LTR ca retrovirus (nh c thy trong hnh trn), hoc t bn trong, promoter ca virus khc loi, nh trng hp xy ra vi vector lentivirus. Vai tr ca cc vng khc nhau c nh du.

Hnh 9: sn xut vector retrovirus. Cc gen ca virus biu hin t cc promoter khng c bn cht retrovirus (nonretroviral) v a ti cc t bo, ni chng bo tn c s n nh v sn xut cc protein enzim cng nh cu trc virus. Khi vector retrovirus c a ti t bo, vector virus RNA cn c bao gi, kt qu t qu trnh sn xut ca cc phn t virus cha b gen vector. Virus ny c th c thu li v s dng tim vo cc t bo ch nhm a gen ngoi lai trong vector ti t bo. V cc t bo ch ny khng biu hin cc protein ca virus, vector s khng truyn b xa hn. Cc gen ca virus trong cc t bo bao gi khng mang cu trc i xa hn v chng mt chui cis-acting cn thit cho s nhn bn. 3.3.5. Cc vector c c s l herpes simplex

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Vector herpes simplex l cc vector c bn cht virus DNA. Chng khng ho nhp vo DNA ch nhng gi nguyn s n nh nh mt th b sung (lantency), c kh nng cho php cc gen c chuyn n nh trong mt thi gian di. Ngy nay, chng c s dng trong cc ng dng c bit ca liu php gen vi cc khi u thn kinh. Herpes simplex virus (HSV) c hiu qu cao i vi cc nron thn kinh. Vector ny ln chuyn DNA ln (ti 30 kB) nhng kh thc hin hn adenovirus. Kh nng to ln ny cho php phn phi 5 gen khc nhau c iu khin bng cc promoter khc nhau. Do , nu qu trnh tr bnh cn a gen, y c th l vector c la chn.

Hnh 10: A. S th hin cc thnh phn chnh ca virion HSV. B. B gen ca HSV c t chc thnh cc on di v ngn ch xut hin mt ln (U L, Us) gn cc trnh t lp li. 84 khung c m ca virus c th c chia thnh cc gen cn cho s ti bn trong m thch hp. Hin c rt t kinh nghim tr bnh vi cc vector ny, nhng kh nng s dng ca vector ny cho vic phn phi gen trong mt thi gian di ti h thng thn kinh trung ng ngy cng tng. 3.3.6. Cc vector virus khc:

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Cc vector virus khc ang trn pht trin bao gm virus pox, virus Epstein-Barr, veovirus... Mi tin trin mi c th l l tng cho cc th nghim y hc trng tm. Cc chin lc hin nay l kt hp DNA virus t cc vector a dng vi s phn phi liposome khng c bn cht virus. Chin lc ny cho thy mt vi ha hn trn cc nghin cu vi ng vt. 3.4. Cc vector khng c bn cht virus 3.4.1. Gii thiu: Trong khi s pht trin v s dng cc vector virus (VV) em li nhiu ha hn, nhng gii hn ca n thc y cc nghin cu tm kim cc vector khng c bn cht virus (nonviral vector, NV) trong vic chun on v iu tr bnh. C hng ngn cng ty, phng nghin cu, trng i hc v cc t chc chnh ph trn th gii c gng pht trin cc NV cho vic chuyn gen. Tht khng cn thit v cng khng th cp n tt c cc vn trn y, ti ch nu ra mt s im c gi tr nghin cu ln. Nh VV, NV c s dng gn cc vt cht di truyn, protein hay thuc chnh xc ti t bo ngi. S dng NV trn ngi bnh thu c nhiu thun li r rng. p ng min dch tng ln chc chn xy ra VV c b qua nh s dng NV. Nh khi bt c virus no vo c th, cc VV c xc nh bi h thng min dch (nh mt yu t th ch) v p ng min dch tng ln chng li chng. Cc NV loi b vn ny mt cch m thm, v l thuyt m ni, lm cho chng mt tnh c hi, mt p ng min dch v em li hiu qu cao hn. Chng hng ti s n gin hn trong vic thit k cc VV do d ng dng hn cho sn xut quy m ln v marketing. Nghin cu v tin trin trong pht trin NV chm hn so vi cc VV. Do , hiu qu ca vic chuyn chng hay thnh cng trong phn phi chng ti cc t bo t c nhn thy hn cc VV. Cc VV cng c thun li trong kh nng gn vt cht di truyn ca chng ti cc t bo ch, cung cp hiu qu liu php lu di hn. Trn thc tin, iu ny c ngha l cc VV i hi s chuyn vo t hn hon tt yu cu hiu qu hn cc NV. Ch loi tr i vi cc nhim sc th nhn to ngi (HACs). Trong khi s lp li iu tr l khng l tng cho ngi bnh, cc vector ny c th t tn ti trong cc bnh c cng cch thc iu tr.

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3.4.2. Phn t trung gian chuyn gen Phn t trung gian chuyn gen hay gene gun l k thut mang li thun li ca vic d dng tip cn vi da. N s dng lc c hc y ti DNA hay protein trong mt s trng hp to min dch, trc tip ti da ca ngi bnh. DNA c gn vo b mt ca cc qu cu vng (ng knh 1 m). Vng khng gy ra p ng min dch v l mt vector tuyt ho s dng cho k thut ny. Phc h cha vng v DNA sau c y ti da. Bio-Rad, Inc (Hercules, CA) hin hnh sn xut phn t trung gian chuyn gen Helios s dng heli lm yu t thc y.

Hnh 11: (A) Thit b Accell gene gun c thit ch v ch to bi Dr. D. McCabe di s gip ca Agracetus, Inc. (Middleton, WI) v c phn phi c quyn bi PowderJect Vaccine, Inc. (Madison, WI). (B) Thit b Helios gene gun, c pht trin t thit b ca Accell, hin c sn xut thng mi bi Bio-Rad. (C) Chuyn gen in vitro vo mt cm lp t bo n s dng Helios gene gun. (D) Chuyn gen in vivo n m da ln s dng Helios gene gun. Khng cn s dng virus hay cc cht ho hc c hi trong vic cy phc hp. Khng ging mt s vector sinh hc, n khng ph thuc vo s tham gia ca cc c quan th cm b mt t bo. NV dng nh cng ng tin cy, c ngha trong vc a cc on vt cht di truyn ln. Tuy nhin, hiu qu ca n b gii hn bi mc xuyn qua m ti cc m ch khc nhau v s biu hin trong thi gian ngn ca sn phm gen mong mun. Liu php ny thnh cng trong ng dng iu tr ung th da v cc cn bnh v da vi cc khuyt tt di truyn bit. Tuy nhin, ng dng ha hn nht ca n

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l to min dch chng li cc khi u c tnh. Cc vcxin DNA tin hnh theo hng k thut gene gun c th nghim trn 15 loi ng vt (2 loi ng vt c v pht trin cao nht) v cho thy hiu qu chng li cc bnh cm, HIV, st rt, lao Cc nghin cu trung gian cng c thnh cng ban u trong vic s dng k thut gene gun gn DNA vo cc t bo c sn xut dystrophin (protein thiu ht trong cc t bo c b bnh ca ngi bnh). K thut gene gun l mt v d ca vector c hc em li nhiu ha hn trong phng v tr bnh. 3.4.3. Nhim sc th nhn to Cc nhim sc th nhn to ca ngi (HACs) hin vn cha pht trin. Trong khi ng dng y hc ca HACs ch bt u trong mt vi nm, khi nim ny l mt trong nhng lnh vc th v nht ca chuyn gen khng c bn cht virus. Trong c th DNA t mt t bo di truyn ti t bo khc v cui cng t cha m sang con, s dng cc NST. Cc NST l vt mang ng vai tr t chc DNA n cc trnh t c hiu, vic chuyn ny d dng. Thnh cng trong iu tr bnh ph thuc vo kh nng thit k cc phn t vt mang hay cc vector ca chng ta. Do c th thit k NST nhn to ngi (HAC), hoc NST nh (minichromosome), cha cc on DNA c bit s vo t bo, to ra kt qu mong mun khng ch trong t bo m cn trong cc t bo sinh ra t t bo ny.

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Hnh 12: S ca mt vector HAEC (Human artificial episomal chromosome) HACs l cc NV c kh nng mang li hiu qu cao nht. Chng c th mang nng lng d dng chuyn cc on tin DNA ln khng quan trng ti gn vo cc t bo c kch thc xc nh. Chng c th gn ti t bo v c nh ti y m khng cn kt hp vi cc NST gc, do trnh c t bin di truyn, hoc cc bin i vt cht di truyn khng cn thit hay c hi vi t bo. Cc NST ban u d dng chuyn gen ti t bo con qua phn bo, iu ny to nn s duy tr biu hin gen lin tc trong ton b cuc sng ngi bnh. V HACs c to nn bi cc thnh phn xa l vi c th, chng khng th hin hiu ng vi h thng min dch. HACs vn trong qu trnh pht trin v c th s khng c gi tr thng mi trong mt vi nm. S sn xut HACs ch trong phng th nghim, gii hn trong ng dng y hc trn quy m ln. Khi my tnh mnh hn v nh hn hin nay, s sn xut HACs s chc chn tr nn khng ch trong phng th nghim v kh nng sn xut v ci tin cng ngh s ti trong vi nm na. Cc cng ty hin nay ang nghin cu HACs bo gm clontech, geron corp. chromos molecular systems, inc, biogenix, gentronics v Chiron. 3.4.4. Liposomes

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Liposome l vector khng c bn cht virus, li dng cc tnh cht t nhin ca mng t bo xm nhp vo t bo. Tt c cc vt cht sinh hc c cc v thnh phn lipid cng nh tnh ha tan nc khc nhau. Thnh phn v cu trc ca n xc nh cc tnh cht ny. DNA tch in m. Tnh bo cao v tch in m ca mng t bo ngi v mng nhn ngn cn s di chuyn t do ra vo t bo ca DNA. Liposome mang in tch dng, lp v a lipid. Do tch in i du, DNA v liposome d dng gn cht vi nhau, to nn phc hp a lipid liposome-DNA. Tnh ho tan cht lipid ny cho php vn chuyn n qua mng t bo v mng nhn, d dng a DNA vo nhn ca t bo.

Hnh 13: Chuyn gen s dng HVJ liposome. HVJ liposome gn vi cc th th axit sialic trn b mt t bo v kt hp vi cc lipid trong mng lipid kp gy ra s dung hp t bo. Do s dung hp v ca HVJ liposome vi mng t bo, DNA trong HVJ liposome c th a trc tip vo t bo cht. Liposome l cc phn t khng c, d dng sn xut vi s lng ln, khin chng tr thnh vector rt hiu qu trong cc ng dng thng mi. S xm nhp ca protein trong vector ngn cn p ng min dch ca vt ch v chng khng b nguy him hay b tiu dit bi cc enzim (endonucleases) ni bo. Chng c cc bt li y hc. Mc d phc h di chuyn mt cch hiu qu qua mng t bo nhng n khng thnh cng trong vic xuyn qua mng nhn, tnh ng dng ca n cng b gii hn vi cc t bo c tc phn chia cao.

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Mt trong cc ng dng thnh cng nht ca chuyn gen liposome l iu tr bnh s cng ng mch. Vo nm 1993, 10 th nghim y hc quan trng nghin cu ng dng ca liu php gen vi bnh u x (cystic fibrosis). Nm trong s s dng cc vector adenovirus v 5 s dng cc vector liposome. Hin nay c trn 4 th nghim y hc quy m ln theo cch ny. C vector adenovirus v vector liposome biu hin ho trn thnh cng trong cc nghin cu ny. Nghin cu cng trn hng s dng liposome trong iu tr cc bnh x cng ng mch, ung th tin lit tuyn, u c tnh v bnh bch cu. So vi tt c cc vector khng c bn cht virus, chuyn gen liposome c tim nng ng dng ln nht ti thi im hin ti.

Hnh 14: plasmid DNA c bao trong pegylated immunoliposome (PIL) 85 nm. B mt ca liposome c kt hp vi vi nghn si polyethyleneglycol (PEG) di 2000 Dalton, v nh ca 1 n 2% ca cc si PEG c gn vi phi t ch, nh khng th n dng c hiu (specific mono clonal antibody) insulin receptor (IR) (MAb). Mab kt hp vi PIL qua th th trung gian i qua hng ro mu no v qua mng t bo cht ca t bo no vo trong nhn. 3.4.5. Cc polymer cation

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Cc polymer cation l chui cc phc hp tch in dng. Khi trn vi DNA, chng to thnh phc hp c tnh cht ca DNA kt t thnh dng m c th d dng vo nhn. Lnh vc nghin cu ny l cuc cch mng nhanh chng cc phn t khng c c tnh hiu qu hn v chuyn cc gen ti cc t bo ng ch hn.

Hnh 15. S xm nhp vo t bo ca cc polymer cation

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Hnh 16: Cc k thut c s dng nghin cu s to thnh v tnh n nh ca phc h polycation-DNA. 3.4.6. Cy trc tip S cy cc DNA trn hay DNA t do l mt v d khc ca chuyn gen khng c bn cht virus. Trong k thut ny, DNA trn c cy trc tip ti khi u

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hoc m. Phng php iu tr ny c th lm gim trc tip s sn xut cc protein quan trng cho liu php tr bnh (v d protein khi u) hoc c th gy ra p ng min dch gip ngn nga bnh. K thut ny c ng dng cho c xng trong iu tr cc bnh thoi ho c (nh bnh lon dng c), ngn cn cc bnh gan do virus (nh bnh vim gan), v cc bnh tim (nh bnh li sung huyt tim). Ngoi ra, cc nghin cu trn ng vt thnh cng trong iu tr ung th nh u c tnh v u bu. Trong khi gi thp v tnh d thit k lm n c thu ht hn, cng c mt s nhc im. u tin, s qun l h thng ca vt cht di truyn l vn v nguyn nhn gy ra s phn hu bi enzim ni bo v nhanh chng b dp b (clearance) bi gan v thn. Hiu qu ca biu hin di truyn cc k kh nhn thy. Hn na, trong mt s trng hp khng duy tr c biu hin ca gen, cc DNA l (foreign DNA) c th gy p ng min dch mnh. 3.4.7. Liu php t bo gc Chuyn t bo gc l k thut to ra t bin ln trong khoa hc, chnh tr v tn gio. V vy, vic nghin cu chng l ht sc hn ch v ch din ra trn quy m phng th nghim. Cc t bo gc l cc t bo u tin trong c th ngi. Chng l cc t bo hon ton ging nhau v hu nh c th pht trin thnh bt c t bo no ngi. Cc t bo ny c th tch khi c th, tinh ch trong phng th nghim, lm thnh cc dng t bo c hiu khc nhau v sau cc t bo ny c a tr li vt ch. 3.4.8. Bin i biu hin gen S biu hin gen ngi c th b bin i trc tip bng cch s dng cc cng c phn t c kh nng xuyn qua t bo, gn ti gen ch ca chng v iu ho gen sn xut protein. C mt s cng c c dn ra di y: a) Oligomucleotide Oligonucleotide l cc on DNA ngn c th c s dng km hm qu trnh dch m ca gen. Mch i (antisense) ca on gen bit c to thnh. Khi mang ti t bo, cc on DNA ny cun xon vi RNA c hiu v ngn cn s sn xut protein t RNA ny. Cng ngh mch i rt hu dng trong nghin cu nhng cc ng dng thng mi ca n rt khng thnh cng. iu ny l do

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thi gian sng ngn ca cc oligomucleotide v s khc nhau ca phn phi c hiu. Hin ti, c mt dc phm oligomucleotide c chp nhn l FDA, dng iu tr nhim cytomegalovirus vng mc. D n b gen ngi hon thnh lm sng li mi quan tm trong lnh vc ny vi cc k thut phn phi mi lm liu php oligomucleotide c hiu qu hn. b) Ribozyme Ribozyme l cc chui RNA ngn (30-50 nucleotide) c thit k c bit, c hot tnh xc tc (chng gy ra cc phn ng ho hc). Ribozyme gn vi cc on RNA c hiu v tch chng ra khi RNA, do ngn cn qu trnh sn xut protein. Cc ribozyme ny dng nh c hiu qu trong 12 n 24 ting. y l lnh vc nghin cu mi nht vi ng dng trong cc bnh ung th v cc bnh thoi ho thn kinh (neuro-degenerative). Thng tin t d n b gen ngi c th em li cc ng dng c gi tr cho cng ngh ny mt cch nhanh chng. c) Promoter Promoter v enhancer l cc on DNA iu khin biu hin gen khng b phin m. Tt c cc vector liu php gen hin nay s dng cc promoter ca virus iu khin s biu hin ca cc gen c chuyn. Vic thit k v s dng c hiu m lm cc promoter tr thnh lnh vc nghin cu quan trng, c th cho php hot ho ca gen c chuyn ch trong cc m d kin. V d, mt vector s dng promoter cho khng nguyn c hiu tin lit tuyn c s dng trong cc th nghim y hc chng ung th tin lit tuyn. Vector ny s l ch ca mt s ln cc t bo, nhng gen c chuyn ch c hot ho trong cc t bo bnh thng sn xut khng nguyn c hiu tin lit tuyn. 4. NHNG NGHIN CU V VIC IU TR BNH BNG LIU PHP GEN TRONG Y HC HIN NAY. 4.1. Nghin cu liu php gene cho bnh di truyn Ngy 13/02/2007 cc nh khoa hc HTH Gttingen (CHLB c) cng b kt qu nghin cu s dng liu php gene cha hi chng Molybdelnum Cofactor - Deficiency (khim khuyt yu t kt hp molybdenum).

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Molybdenum (k hiu Mo) c s th t 42 trong bng tun hon cc nguyn t ha hc v c mt trong c th sinh vt t vi khun n ng, thc vt. vi khun, Mo tham gia vo qu trnh c nh nit. ng, thc vt c mt hm lng Mo cc k nh (tnh bng n v ppm: parts per million). ng vt v ngi, Mo tham gia vo cu to ca enzyme xanthine oxidase (enzyme ny c phn t lng 270.000 vi hai phn t flavin v hai nguyn t Mo ti v tr hot ng) xc tc cho qu trnh phn gii cc purine v hnh thnh uric acid. Nu b sung mt lng nh Mo vo thc n s kch thch sinh trng mt s loi ng vt. Khim khuyt gene m ha enzyme cha Mo (MOCO) s dn n ri lon trao i cht v bit ha ca nhiu c quan, c bit l hin tng pht trin khng hon ho ca hp s. Th nghim trn g cho thy hp s ca g non mi n khng ng hon ton. Ngi mc bnh ny thng cht tui 7 n 12. S ngi mc phi bnh ny trn th gii khng nhiu (ch khong 1000 ngi) nhng nghin cu v bnh c bit l nghin cu cc liu php iu tr c th ng dng i vi nhiu bnh do di truyn khc. GS J. Reiss v TS. S. Kgler cng nhm nghin cu ti khoa Y v Vin Di truyn hc ngi HTH Gttingen (c) dng liu php gene hn ch c s biu hin ca bnh trn chut b khim khuyt gene ny. Vi thnh cng ny, cc tc gi cng nh chng ta c quyn hy vng hn vo vic ng dng liu php gene iu tr cc bnh di truyn trong thi gian ti. 4.2. Liu php gen ang c nghin cu trong iu tr ung th nh th no? Cc nh khoa hc ang nghin cu dng liu php gen iu tr ung th theo nhiu cch khc nhau. Mt s cch nhm ti mc tiu l cc t bo khe mnh tng cng kh nng chng li ung th ca chng. Mt s cch khc nhm n mc tiu l cc t bo ung th tiu dit v ngn khng cho chng pht trin. Di y l mt s k thut liu php gen ang c nghin cu:

Cc nh khoa hc thay nhng gen b thiu hoc b thay i bng nhng gen khe mnh. Do mt s gen nu b thiu hoc thay i (vd nh p53) c th

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gy ung th, bn sao thay th ang hot ng ca nhng loi gen ny c th c s dng iu tr ung th.

Cc nh khoa hc cng ang nghin cu cch tng cng p ng min dch ca bnh nhn i vi ung th. cch ny, liu php gen c dng kch thch kh nng t nhin ca c th chng li cc t bo ung th. mt phng php ang c nghin cu, cc nh khoa hc ly mt mu mu nh t bnh nhn v a vo nhng loi gen lm cho cc t bo sn xut ra mt loi protein c tn l th th t bo T (TCR - T-cell receptor). Nhng loi gen c a vo cc t bo bch cu ca bnh nhn (c tn l lympho T) sau c truyn ngc tr li vo c th. Bn trong c th, cc bch cu sn xut ra TCR, TCR s gn vi lp mt ngoi ca cc bch cu. Sau TCR s nhn ra v gn vi mt s phn t hin din trn b mt ca cc t bo u. Cui cng, TCR s hot ha bch cu tn cng cc t bo u ny.

Cc nh khoa hc cng ang nghin cu cch a gen vo trong cc t bo ung th lm chng nhy cm hn vi ha tr, x tr hoc nhng phng php iu tr khc. Trong nhng nghin cu khc, cc nh khoa hc ly nhng t bo mm to mu khe mnh ra khi c th ri a gen vo cc t bo ny gip chng tng kh nng khng c li vi cc tc dng ph ca nhng loi thuc khng ung th dng liu cao. Sau , cc t bo ny c a tr li c th bnh nhn.

mt cch khc, cc nh khoa hc a nhng loi "gen t vn" vo cc t bo ung th ca bnh nhn. Nhng tin cht ca thuc (dng bt hot ca thuc c) c a vo c th bnh nhn. Nhng cht ny s hot ha cc t bo ung th c cha nhng loi "gen t vn" trn dn n s hy hoi ca chnh bn thn chng.

Nhng nghin cu khc tp trung vo vic dng liu php gen ngn cc t bo ung th to thm nhng mch mu mi. 4.3. Liu php gen - V kh mi iu tr HIV/AIDS Kt qu nghin cu ca cc nh khoa hc thuc i hc Pennsylvania, M,

cho thy liu php gen c th tr thnh mt v kh mi trong vic iu tr cn bnh th k HIV/AIDS.

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Tuy nhin, gii khoa hc khuyn co cn nhiu nm na mi c th p dng rng ri bin php iu tr ny. Theo tin s Pablo Tebas, ng ch nhim cng trnh nghin cu ny, cc loi thuc iu tr HIV/AIDS hin nay cho php bnh nhn sng tng i bnh thng. Tuy nhin, chng li gy ra nhiu tc dng ph v trong mt s trng hp, cc bnh nhn dn tr nn khng thuc. Do , cu hi t ra l c cch no iu tr hoc kim sot virus HIV/AIDS mt mc m bnh nhn khng phi s dng cc loi thuc phc tp v t tin ni trn.

Trong nghin cu ca mnh, tin s Tebas v ng nghip thc hin vic tch cc t bo min dch t cc bnh nhn HIV/AIDS, chnh sa nhng t bo ny v cy chng tr li c th ca cc ngi bnh v h khng c iu tr bng thuc na. Cc nh khoa hc pht hin thy s

Bnh nhn AIDS c hi vng c cu trong tng lai nh cc phng php

lng virus HIV/AIDS nhng i tng mi. (nh minh ha: AFP/TTXVN) trn u gim so vi d kin. Thm ch, mt ngi bnh virus cht ngi ny bin mt hon ton. Cc nh khoa hc ca i hc Pennsylvania cho hay s tip tc nghin cu bin php iu tr ni trn v hin ti h vn cha l gii c ti sao iu ny xy ra, cng nh cha bit c rng liu php iu tr ny c tc dng trong bao lu v cc t bo min dch khng tn ti lu di. Tuy nhin, h cho rng y l mt pht hin rt c ngha bi hin nay hng nghn bnh nhn HIV/AIDS hon ton khng cc loi thuc iu tr hin c. Trong khi , chuyn gia hng u trong lnh vc chng ly nhim HIV/AIDS Bryan William thuc Trung tm phn tch v m hnh ha dch t hc Nam Phi khng nh th gii c th ngn chn c i dch HIV/AIDS ton cu trong vng 40 nm ti bng cch s dng rng ri cc ch phm chng retrovirus,

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mt loi dc phm khng ch lm chm li tin trin ca bnh m cn gim ng k nguy c ly lan bnh. Pht biu ngy 22/2, ng William nhn mnh nu s dng cc ch phm ny, trong vng 5 nm c th ngn chn s ly lan ca HIV/AIDS, sau 10 nm s gim mt na s bnh nhn lao phi lin quan ti HIV/AIDS v sau 40 nm c th loi tr hon ton 2 s ly nhim ny. Theo chuyn gia Bryan William, khi s dng dc phm chng retrovirus, mt virus HIV/AIDS trong mu gim 10.000 ln, gim 25 ln nguy c ly nhim. Theo ng William, tr ngi chnh trong chin lc ny l kinh ph cho chng trnh pht thuc min ph bi bnh nhn cn phi s dng thuc hng ngy v sut i. Ti Nam Phi, mt trong nhng khu vc c t l nhim HIV/AIDS cao nht th gii, thc hin d n ny cn 4 t USD/nm. Tuy nhin, ng cho rng s tn km ny s mang li li ch tng xng, v gim s ngi b nhim cn bnh cht ngi ny s gip gim nhng chi ph iu tr nhng cn bnh do HIV/AIDS gy ra cng nh gim s ngi mt sc lao ng. 4.4. iu tr liu php gen cho ngi m: Cc nh nghin cu M cho bit, mt nm trc y, 3 ngi m tui 20 tnh nguyn iu tr bng liu php gen. Gi y, th lc h c c s ci thin ng k, thm ch mt ngi bnh cn c th c c c nhng con s ca ng h cng t mt trn t. Nhng thng tin lin quan s c cng b trn tp ch New England Journal of Medicine vo ngy 23/8. Ba ngi tnh nguyn ny u mc phi bnh m la bm sinh (Leber Congenital Amaurosis), mt loi tt ca vng mc mang tnh di truyn do gen c tn gi RPE65 (gen RPE65 to ra mt loi Vitamin A c bit cho t bo vng mc) xy ra bin d khin cho th lc ca ngi bnh gim dn theo tui. V vy mt s ngi sau khi trng thnh b m la, hin ti vn cha c liu php iu tr hiu qu no cho cn bnh ny.

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Nhm nghin cu Artur V. Cideciyan thuc trng i hc Pennsylvania tim DNA bnh thng vo virus, sau li a virus vo t bo mt ca ngi bnh. Sau 3 thng, th lc ca 3 ngi tnh nguyn c ci thin ng k, c th nhn thy nh sng bng mt thng, iu m trc y h khng th lm c. Hn na, sau khi iu tr 1 nm, mt v sc khe ca h khng h xy ra bt k phn ng min dch no.

Trong , mt bnh nhn n trong qu trnh iu tr to ra th lc th 2, i no ca c y hc c sau khi iu tr bng liu php gen c th khi phc tm v thu thp thng tin mt khu vc no ca vng mc, khin cho c y c c cc con s trn ng h cng t mt t. Nm 1999, mt thiu n 18 tui mc phi bnh gan sau khi iu tr 4 ngy bng liu php gen qua i, s kin ny khin cho liu php gen gp phi rt nhiu s ch trch v lm cho mi ngi cng nghi ng v liu php gen. Artur V. Cideciyan cho bit: Liu php gen RPE65 rt an ton, th lc ca ngi bnh c s ci thin r rt, hy vng c th tip tc tin hnh th nghim gii quyt nhng vn v s an ton v tnh hiu qu.

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Bnh m la bm sinh l mt loi sc t vng mc bin tnh, ti M c khong c khong 2000 ngi mc phi bnh m la bm sinh; 200 nghn ngi mc phi sc t vng mc bin tnh. 4.5. Dng liu php gene cha lnh phi b tn thng Cc nh khoa hc Canada tm ra mt phng php mi cha lnh nhng l phi b tn thng ca ngi hin tng trc khi cy ghp. Phng php ny gip tng gp 3 ln c hi thnh cng ca cc ca cy ghp phi v m ra trin vng c th cha lnh nhng b phn khc ca c th. Gii nghin cu cho bit thng thng khi mt ngi hin tng qua i, nhng l phi ca h c th b sng ty, ko theo nhng tn thng v iu ny gy kh khn cho cc ca cy ghp sau ny v c th ca ngi nhn c th phn ng v o thi.

Cc nh khoa hc Canada s dng my "Lung Perfusion System" phc hi phi b tn thng ca ngi hin tng (nh: physorg.com). Khong 20-30% s ngi c cy ghp phi t vong do nhng bin chng sau ny v c ti 80% s l phi c hin tng b tn thng nghim trng.

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Cc nh nghin cu thuc Trung tm Y t Phc hi McEwen Toronto v Bnh vin a khoa Toronto thnh cng trong vic s dng liu php gene phc hi nhng l phi b tn thng. H s dng my "Lung Perfusion System" "hm nng" hai l phi bng ng nhit c th ngi, sau bm mt dung dch hn hp gm xy, cc protein v cc cht dinh dng vo nhng l phi b tn thng , gip chng hot ng bnh thng. Trn c s ny, nhng t bo b tn thng c th s t lnh tr li. Nhng l phi c x l bng phng php trn sau c bo qun lnh trong 24 gi trc khi cy ghp. Cc nh khoa hc cy ghp thnh cng ng vt v Tin s Shaf Keshavjee, trng nhm nghin cu, hy vng nhm ca ng c th tin hnh cc th nghim lm sng trong 2 hoc 3 nm ti. 5. KT LUN T cc trnh by trn c th thy rng, hin nay c rt nhiu cch v phng thc chuyn gen ch ti cc t bo nhm thu c hiu qu ca liu php. Tuy nhin chng ta cn nghin cu k lng cc hiu ng ph cng nh cc tc ng ca n ti thng tin di truyn cu ngi v cc kha cnh o c, x hi khc. Song cng cn phi nhc li rng Khng h thng vct no l ti u cho tt c cc k thut ca liu php gen, do cn la chn mt cch chnh xc nht d trn hiu qu ca vector i vi nhm bnh m mnh nghin cu.

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