Chng VI CAC VEC T ADENOVIRUS 6.

1 M u Cc vec t adenovirus l mt trong s cc phng tin chuyn gen trc tip ha hn nht in vivo trong gen tr liu trn ngi iu tr cc bnh di truyn n gen nh bnh x nang, bnh au c Duchenne v bnh a chy mu A v B cng nh cc bnh mc phi nh ung th (Trapnell v Gorziglia, 1994; Wilson, 1995). Hn na, vic s dng cc vec t adenovirus trong cc tr liu ex vivo cng c nh gi. Cc vec t adenovirus c nhiu li th vt hn cc h thng chuyn gen khc, n c th ti np c nhiu dng t bo v khng i hi t bo ch phi phn chia trong chuyn v biu hin gen. Nhim sc th ca adenovirus cn duy tr c episome trong t bo ti np, v vy trnh c kh nng t bin ghp (Gingsberg,1984; Horowitz,1990). Adenovirus c th hon tr nhng thiu ht v sao chp do cc gen iu ha c bit ca virus b loi i (Berkner, 1988; Gorziglia v cng tc.,1996), iu cho php cc vec t iu tit c vic ci DNA d gen trn 8 kb - ty thuc vo di on gen b loi tr. Sau na, ngi ta vn cha thy s thm nhim adenovirus no c lin quan ti vic to khi u trn ngi, v th adenovirus c coi nh l mt vaccine sng c trong hng triu con ngi (Straus, 1984). Bt li chnh ca cc vec t adenovirus l gy p ng min dch i vi vt ch v th m lm gii hn thi gian biu hin ca gen chuyn v kh nng ti s dng vec t. Nhng c gng hin nay hng trc tip vo vic lm gim tnh sinh min dch ca cc vec t v pht trin cc chin lc nh la p ng min dch ca vt ch. Vi nm trc y cng c mt s cng trnh thng bo chuyn giao thnh cng qua trung gian vec t adenovirus cng nh biu hin c nhiu gen d loi mt s ng vt (Trapnell v Gorziglia, 1994; Wilson. 1995). Cho ti nay, cc vec t adenovirus c s dng hoc d nh s dng trong tr liu lm sng trn ngi cc bnh x nang, bnh thiu ht ornithine transcarbamolyse v cc dng ung th nh: ung th v, kt trng, nguyn bo xp, ung th u v c, u hc t da, u nguyn bo thn kinh, u bung trng, u tuyn tin lit (Marcel v Grauz, 1996) v u phi (Cohen Haguenauer, 1996). Tuy nhin, gen tr liu qua trung gian vec t adenovirus u phi thc hin giai on sm, tt c cc nghin cu u pha I vi mc ch m bo an ton hn l tnh n hiu qu. 6.2 Cu trc v t chc ca adenovirus Adenovirus trn ngi l cc virus DNA khng v, thuc h parvoviridae (Ginsberg, 1984; Horowitz, 1990). ng knh ca virion l 80-90nm vi hnh thi icosohedral nhn, khi lng nguyn t 175-185 x 106 Da. Theo huyt thanh hc, cc adenovirus c phn thnh gn 50 serotype, vi cc di nhm t A ti G (Wadell, 1984). Cc virus thuc di nhm A v B tim n kh nng gy ung th trn cc loi gm nhm mi sinh, cc virus dng trong chuyn gen thuc di nhm C, khng gy khi u. Adenovirus di nhm C ch gy cc bnh h hp nh trn ngi, chim khong 5-15% cc trng hp cm thng thng (Straus, 1984). Adenovirus phn lp c cch y trn 40 nm (Rowe v cng s., 1953), nhng hiu bit v h thng di truyn ca adenovirus cng tng ln khng ngng, mt phn cng v adenovirus sm c ti nh mt m hnh biu hin gen vi cc t bo nhn chun (Ginsberg, 1984; Horowitz, 1990). Adenovirus thm nhim cc t bo ch bng cch gn vo receptor ca coxsackie v adenovirus (coxsackie and adenovirus

receptor CAR) trn b mt t bo (Bergelson v cng s., 1997) n c ha nhp qua cc l bc clathrine i vo th THNB (endosome), virion c loi khi t bo cht bng c ch ly gii ni bo (endosomolysis), s chuyn v ti mng nhn qua cc tn hiu ch trong cc polypeptide capsid, h gen virus c chuyn vo nhn t bo, n duy tr dng episome.

Hnh 6.1.S i din ca h gen adenovirus v s iu ha phin m. H gen ca adenovirus xp thng hng song song, cc n v phin m l cc vng c mi tn. Cc mi tn trn ch hng ca phin m. Cc mi tn cong ch cc con ng chuyn hot ha. Cc n v phin m trung bnh hoc nh l pIX, IVa v VA-RNA (I-III). Nhng vng lp tn cng o ngc l cc ITR cng nh cc protein 55 kDa lin kt ng ha tr vi u 5 ca h gen. C vng ITR v s c mt ca protein tn cng u cn cho vic sao chp ca virus c hiu qu. Phn chnh ca vec t adenovirus th h th nht (Av1) c c nhng phn loi b E1 v E3; tuy nhin, mt vi vec Av1 ch cha vng loi b E1. Loi b vng E1 s lm cho s sao chp ca virus b suy gim v cc sn phm ca gen E1 iu chnh ln cc n v phin m chnh ca virus nh E2, E3 , E4 v promoter mun chnh (major late promoter-MLP) (c ch bng mi tn cong). E1, E2 v E4 c hot ha bi yu t phin m trong t bo vt ch. Cc vec t th h 3 (Av3) kim ch ngoi nhng phn ct b E1 v E3 cn thm c vng E2a loi bt mt phn. Cc vec t loi kt hp E1/E3/E4 cng c thit lp. Nhng vec t c loi bt sau ny lm gim c s biu hin ca cc n v phin m mun do ngn nga c vc iu chnh ln ca MLP bi cc sn phm ca gen E2a hoc E4. Ghi ch: ()- prtein tn; ()-promoter. (Theo Sheila Connelly. Gene Therapy Technologies, Applications and regulations. John Wiley & Sons Ltd 1999).

H gen adenovirus gm cc phn t DNA chui kp, thng vi chiu di 36 kb, c cc lin kt ng ha tr gn u 5 vi mt protein tn cng 55 kDa cn cho s sao chp ca DNA virus. DNA virus c cha nhng on lp tn cng o ngc, ngn (short, inverted repeats ITR) mi u cui ca h gen cn cho s sao chp DNA. H gen virus c t chc vi 4 vng phn bit sm c tn t E1 n E4 v 5 vng ghp mun t L1 n L5 c s cho s biu hin trc hay sau s khi u tng hp DNA virus, thm vo cn c mt vi trung gian ph hoc cc n v phin m mun (Hnh 6.1). Ni chung cc sn phm sm ca gen lm thay i sinh hc t bo vt ch gip cho vic sn xut virus, cn nhng gen mun li m cho hu ht cc protein cu trc l thnh phn ca virion v gip cho s lp rp ca virus. Ngay khi h gen virus vo ti

nhn, vng E1 c phin m tch cc bi cc yu t phin m v m ha cho cc protein c lin quan trc tip ti vic hot ha cc vng E2, E3 v E4. Trong cc t bo nui cy, cc sn phm gen E1 c m trong vng E1a v E1b sao chp thng tin virus cm ng (Van der Eb v cng s., 1997). Vng E2 m cho cc protein cn cho s sao chp DNA virus bao gm mt protein gn DNA chui n (E2a) v polymerase DNA virus cng nh protein tn cng 55 kDa. Vng E3 l tp hp ca cc n v phin m lin quan ti vic ln trnh cc c ch bo v ca vt ch, loi tr cc t bo b nhim virus, v khng thit yu i vi s sao chp ca virus (Wold v Gooding, 1991). Cc sn phm ca gen E4 c lin quan ti vic iu ha biu hin protein ca t bo v virus, s sao chp DNA ca virus, s tch tr mRNA ca virus v s tng hp protein, iu chnh xung mt cch tng ng s tng hp protein vt ch. Mt cng trnh mi y chng minh rng protein E4 m trong khung c m 6, c tim nng gy ung th tng t nh gen E1b (Moore v cng s., 1996). S biu hin ca cc gen sm dn n sao chp DNA khong 8 gi sau khi thm nhim v s hot ha tip ca cc gen mun di s kim sot phin m ca promoter mun s sn sinh ra cc virus con chu v cui cng l cc t bo vt ch cht v virus c gii phng. 6.3 Thit k v cu trc vec t adenovirus ngi khim khuyt sao chp Cc vec t adenovirus khim khuyt sao chp cng tng t nh nhng vec t virus khc, chng gm mt cu trc virion bao quanh h gen virus ci bin. Cho ti nay, hu ht cc ht vec t c s cu trc capsid dng hoang d, ngoi nhim v bo v DNA virus n cn l phng tin gn v i vo bn trong t bo ch (ti np). Tuy nhin, h gen virus c nhng ci bin ng k. Nhng bin i ny c thit lp nhm v hiu ha s pht trin ca virus trong t bo ch do loi tr nhng chc nng c bit ca virus i vi s iu ha sao chp DNA v s biu hin gen virus trong khi vn c th pht trin trong t bo ng gi hoc t bo tr gip. Vic loi b cc trnh t nh vy to ra khng gian trong h gen virus DNA ngoi sinh vn ci vo c m vn biu hin c cc gen chuyn. Cc adenovirus di nhm C, serotype 2 v 5 (Ad2 v Ad5) l nhng adenovirus c nghin cu nhiu nht v nhng virus ny thng dng nh cc vec t chuyn gen. Cc vec t adenovirus c dng cho gen tr liu ch yu l cc vec t thay th E1, cc gen chuyn c ci vo vng E1. Vng b loi tr ca E1 bao gm ton b h gen E1a v khong 60% gen E1b. Ngoi phn cn li ca h gen virus, vec t ny cn gi li u 5 gn nht ca h gen virus bao gm cc on lp tn cng o ngc bn tri (left inverted termional ITR) v tn hiu ng capsid (), cc trnh t cn cho s ng gi v nhng vng tng cng (enhancer) E1 gi nhau (Hnh 6.1). V cc sn phm gen E1 s lm hot ha lin tc cc n v phin m chnh nn khi loi tr cc vng ny th s biu hin sm v mun ca gen s gim i rt nhiu v lm s sao chp ca virus suy yu mt cch nghim trng (Berkner, 1988). c khng gian ln hn trong cc vec t adenovirus ci cc gen chuyn th vng E3 cng thng c loi tr v khng i hi s sao chp hoc pht trin vng ny. i khi gen chuyn li c ci vo vng b loi ca E3. Cc vec t adenovirus ch thiu vng E1 v E3 chnh l cc vec t th h th nht Av1. Cc vec t adenovirus c th ng gi DNA hiu qu vi 105% kch thc ca h gen (Bett v cng s., 1993), tng ng vi 8 kb DNA ngoi sinh trong vec t Av1 loi tr E1/E3.

Hnh 6.2 To cc adenovirus ti t hp. (A) DNA h gen adenovirus c chun b bng Cla I ca Ad5 dl327 t bin loi b vng E3 ct b ui bn tri h gen virus lm cho khng b nhim DNA virus. (B) plasmid A cha ITR virus v tn hiu ng gi, n v phin m gen chuyn v mt kilobase hoc hn DNA adenovirus c cng thm chuyn vi DNA c chun b a vo cc t bo c php. (C) nh kt qu ca ti t hp d loi ni bo, mt vec t adenovirus ti t hp loi b E1/E3 c cha nhng gen E1 thay th cho cc gen chuyn c to ra. Mi tn ngang i din cho n v phin m E1. Ghi ch:()-n v phin m gen chuyn ngoi sinh;()vng ti t hp tng ng;()DNA h gen adenovirus. Tam gic ln i din cho phn loi tr ca vng E3 adenovirus. (Theo Sheila Connelly. Gene Therapy Technologies, Applications and regulations. John Wiley & Sons Ltd 1999).

Cc vec t Av1 c cu trc theo nhiu cch, k thut thng thng nht c s dng l thng qua ti t hp d gen h gen virus vi plasmid c mang gen chuyn (Berkner. 1988). Ni ngn gn l cc n v phin m ca gen chuyn c ci vo mt plasmid c cha mt on ca h gen. Plasmid ny c cng thm chuyn (nhim) vo mt dng t bo c php (xem di y), cc DNA virus c chun b theo cc k thut chuyn DNA truyn thng. Trong t bo, do ti t hp d loi m cu vn c cc trnh t virus tch dng v gen chuyn vo c h gen virus, v th m to c cc vec t ti t hp (Hnh 6.2). to cc vec t Av1 vi gen chuyn ci vo vng E1, plasmid phi cha ui bn tri ca h gen virus bao gm ITR v tn hiu ng capsid, hp biu hin gen chuyn v trnh t DNA virus pha cui ca virus vi di xp x hoc hn 1 kb (Berkner v Sharp, 1983). DNA virus dng trong cng thm chuyn (nhim) c chun b bng enzyme ct gii hn loi tr ui pha tri h gen virus. Cc ch phm DNA virus c chun b theo cch ny s lm gim kh nng thm nhim ca DNA b m v v th lm tng hiu lc ca cc vec t ti t hp c lp c to ra t ti t hp in vitro. on DNA kh ln (~ 43 kb) phn lp t cc mu t bin loi b E3 ca Ad5, dl327 (Thimmappay v cng s., 1982) c phn ct vi enzyme gii hn Cla I v lm tinh khit bng lc gel cng l nhng thng quy (Smith v cng s., 1993). Mt cch tng t, DNA d loi c th c ci vo vng E3. Tuy nhin, trong trng hp ny, plasmid phi cha phn cui pha phi h gen adenovirus v DNA virus c chun b bng enzyme tiu ha gii hn loi tr phn cui bn

phi h gen virus. Cui cng, ngi ta s dng quy trnh cng thm nhim bng cch dng enzyme gii hn ct DNA virus gi li mt protein tn cng 55 kDa lin kt ng ha tr vi u 5 ca h gen virus (Sharp v cng s., 1976). Khi tnh thm nhim ca DNA virus c cha protein tn cng cao gp 10 ln DNA h gen b phn ct bng protease (Sharp v cng s., 1976), nh vy chc chn l virus khng ti t hp c mt lng nh DNA khng b phn ct lm cho tnh thm nhim tng cao (Berkner v sharp, 1983). Tip theo s thm chuyn, tip tc phn tch sng lc, thm Southern v biu hin gen chuyn s xc nh c vec t ti t hp mong mun (Graham v Prevec, 1991). Phng php khc dng to vec t adenovirus l tch dng trc tip cc trnh t plasmid ri a vo h gen adenovirus qua nhng v tr thch hp v thm chuyn tip cc t bo c php bng vic lin kt in vitro vi DNA. l phng php gc to cc t bin adenovirus (Stow, 1981). Mt vn quan trng lin quan ti vic sn xut cc vec t adenovirus l gn trc tip hoc ti t hp d loi gia plasmid v DNA virus s cu sng c virus b m do vn cn lu gi c DNA virus b m thm nhim. Tuy nhin, s phc hi ca virus ti t hp ny vi nn tng nh vy l rt kh, c bit nu cc vec t cng ngh ha li pht trin kh chm so vi virus b m. C mt vi cch tip cn kh thng minh gii quyt c vn ny. Chng hn nh, cc vec t b m sao chp km c s dng loi tr kh nng vt qu mc ti t hp mong mun (Berner,1988). Tng t nh vy, vec t Av1 biu hin c mt kiu hnh (phenotype) rt quan trng l do ci gen thymidine kinase (TK) ca virus herpes simplex vo h gen virus (Imler v cng s., 1995). S biu hin ca cc sn phm gen TK phng c s sao chp virus khi c mt mt nucleoside ging nh ganciclovir, iu cho php ch la chn cc vec t ti t hp trong gen TK c thay th (Imler v cng s., 1995). Mt phng php khc li khng cn DNA virus thm nhim bng cch dng plasmid c cha ton b h gen adenovirus (Ghosh- Choudhury v cng s., 1986; Bett v cng s., 1994). Virus thm nhim c th c to ra bng s thm chuyn mt hoc nhiu plasmid c cha DNA adenovirus. Tuy nhin, hiu lc thm chuyn ca DNA plasmid thp hn nhiu so vi DNA virus (Ghosh- Choudhury v cng s., 1986; Bett v cng s., 1994). Cng tng t nh vy, h gen y ca Ad2 c cu trc nh nhim sc th nhn to ca nm men ( yeast artificial chromosome YAC) li c tnh thm nhim (Ketner v cng s., 1994). Nh cc k thut di truyn truyn thng v nm men, cc trnh t virus cha YAC c th c ci bin v cc vec t adenovirus c th tm li c t cc dng YAC (Ketner v cng s., 1994). n v phin m gen chuyn bao gm cc yu t cn cho s biu hin thch hp gen chuyn chng hn nh cc promoter, cc gen cn quan tm v tn hiu polyadenylate ha, trong a s trng hp n c thit k t mc ti a v s biu hin gen ngoi sinh. i vi s biu hin ca gen chuyn th phi c nhng thay i ln v promoter, iu ny ty thuc vo mc ch ng dng v m ch m la chn cho ph hp. R rng l s biu hin cu trc promoter virus nh promoter adenovirus chnh mun hn (Stratford Perricaudet v cng s., 1990), promoter virus Rous sarcoma (Stratford Perricaudet v cng s., 1992; Smith v cng s ., 1993), promoter cytomegalovirus (CMV) (Herz v Gerard, 1993) v promoter actin kch thch CMV lai (Kozarsky v cng s., 1993) u c hp nht trong cc vec t adenovirus ti t hp. Gn y hn, ngi ta dng t bo, cc promoter c hiu m nh promoter albumin c hiu gan (Connelly v cng s., 1995, 1996), promoter iu ha dn in mng gy x nang c hiu phi (Imler v cng s., 1996; Suzuki v cng s., 1996), promoter chui nh c hiu myosin c tim (Rothmann v cng s., 1996) v promoter fetoprotein c hiu gan (Kaneko v cng s., 1995; Arbuthnot v cng s., 1996) cng c m t. Cui cng l cc promoter iu ha p ng

hormone (Haysahi v cng s., 1994) hoc cc tc nhn dc l (Suzuki v cng s., 1996) cng c hp nht vo trong cc vec t adenovirus. Vic gp vec t c hiu m vi vec t iu ha i vi hp biu hin gen chuyn trnh c nhng hu qu tim n v s biu hin qu mc trong cc m khc hn l biu hin c quan ch v v th s lm tng an ton ca cc vec t . Mt cch tip cn khc l tng lng gen chuyn trong cc vec t adenovirus tc l a cc yu t h gen vo trong hp biu hin. Chng hn nh thm mt intron vo cDNA yu t VIII (FVIII) ca ngi lm thc y s biu hin in vivo ln khong 10 ln (Connelly v cng s., 1996) v vic gp yu t IX (FIX) ca ngi loi tr intron u tin v vng 5 v 3 khng c phin dch ca cDNA yu t IX ca ngi lm tng 2000 ln yu t IX trong huyt tng chut ti np (M. Kaleko). Cui cng, nhiu tn hiu c s dng polyadenylate ha trc tip chng hn nh tn hiu polyadenylate virus 40 ca kh. 6.4 S nhn ging v lm tinh khit cc vec t adenovirus Khng ging nh cc vec t retrovirus, do tnh n nh ca virion nn vic lm tinh khit v c c khng lm nh hng ti hot tnh ca chng. Cc quy trnh lm tinh khit vec t adenovirus c lin quan ti vic thu lm v ph hy cc t bo b thm nhim bng cch lun phin ng lnh v lm tan (Smith, 1995), hoc dng siu m (Kanegae v cng s., 1994) v loi b cn bng ly tm. Vec t c lm tinh khit v c c bng ly tm 3 bc vi CsCl, sau em thm tch hoc tin hnh sc k. Tuy nhin, vi sn xut ln th hay dng phng php sc k hn l ly tm vi CsCl. m (nng ) vec t c xc nh bng quang ph, xc nh s ht v tnh cht sinh hc, tnh thm nhim bng s chuyn gen hoc cc th nghim khc (Mittereder v cng s., 1996). s dng trong lm sng trn ngi, cc ch phm phi c php ca FDA v t s n v thm nhim di 1/100 ht vec t (Smith, 1995; Mittereder v cng s., 1996). Ngi ta c c cc ch phm vec t trn nhng phin cha 1011 n v/ml, trong mt s trng hp cn cao hn nhiu so vi cc vec t retrovirus hay adenovirus lin hp. Cc ch phm vec t c test k cng v RCA bng phn tch PCR c hiu E1 (Tolstoshev v cng s., 1994) v cc th nghim trn phin. Hin nay FDA i hi vi lm sng l cc adenovirus ch c 1 hoc vi RCA trong mt vec t (Smith, 1995). 6.5 Chuyn gen in vivo qua trung gian adenovirus Vi nm trc y c nh gi k cng v hiu lc v an ton ca Av1 in vivo trn ng vt v ngi nhm nng cao s hiu bit v tnh cht sinh hc ca cc vec t adenovirus (Trapnell v Gorziglia,1994; Wilson, 1995). S a dng trong biu hin qua trung gian vec t adenovirus ca cc gen chuyn c cp n nh cc gen galactosidase v luciferase trong nhiu m, ph bin nht l phi v gan. S biu hin gen qua trung gian adenovirus ph thuc vo mu hnh ng vt, vo vec t v liu lng s dng, nhng ni chung biu hin khi u l cao. Tuy nhin, thi hn ko di ca s biu hin th thay i rt ln. Nhn chung, chut cha chn v min dch hay c s tha hip min dch th biu hin di hn so vi chut trng thnh (s biu hin gen gim rt mnh trong 2-3 tun l, c l l do h min dch lm gii hn s ko di ca vec t (Trapnell v Gorziglia, 1994; Wilson, 1995). Tuy nhin, sau khi vec t Av1 c truyn qua tnh mch ti cc ng vt trng thnh biu hin di hn yu t ng mu FVIII mu ngi (Connelly v cng s., 1996) v FIX (M. Kaleko). S ti np ca c vi cc vec t Av1 trong mt s trng hp cng thy biu hin c ko di

(Stratford Perricaudet v cng s., 1992; Ragot v cng s., 1993; Vincent v cng s., 1993; Tripathy v cng s., 1996). Ngi ta quan tm nhiu ti vic pht trin gen tr liu i vi 2 bnh phi di truyn thng gp nht, l bnh thiu ht 1 antitrypsin (1- AT) v bnh x nang. Cc kt qu thu c cho thy nhiu gen c chuyn giao qua trung gian adenovirus nh 1At v cDNA iu ha dn in vn chuyn mng i vi bnh x nang phi ngi. Nhng th nghim ban u m t l khi s dng phi chut bng nh l mu hnh ng vt th thy chut bng rt nhy cm vi thm nhim adenovirus tng t nh ngi. Khi nh git qua kh qun vec t Av1 m ha 1- AT thy r c biu hin 1- AT (Rosenfeld v cng s., 1991). Cng tng t nh vy in vi vo ngi ta a yu t dn in vn chuyn mng bnh x nang - CFTR (cystic fibrosis transmembrane conductance regulator) vo chut bng kh dung cng thy c s biu hin CFTR v RNA ca CFTR trong 6 tun l (Rosenfeld v cng s., 1992). Mc du hiu lc ca s chuyn gen ti phi l cao nhng thi gian biu hin li ngn v li lin quan ti nhim khun cp nhu m phi ca chut bng (Yet v cng s., 1994). Nhng nghin cu trn ng vt cao cp khng phi l ngi (Simon v cng s., 1993) cng thy c s tng t v p ng min dch i vi vt ch, nhng thi gian biu hin ko di hn. Vec t Av1 m ha CFTR biu m mi bnh nhn x nang iu chnh ngn hn s tit chloride m khng thy c tnh ca vec t (Zabner v cng s., 1993). Trong mt th nghim tch bit, cc vec t Av1 CFTR c a vo mi v phi bnh nhn x nang, pht hin thy RNA ca CFTR mi sau 2 ngy c x l, khng c bnh nhn no b st v lm tng chun khng th vi adenovirus v mc interleukin- 6 (Crystal v cng s., 1994). C quan chnh th 2 trong gen tr liu in vitro l gan. C rt nhiu gen bao gm nhng gen m cho cc yu t ng mu, cc enzyme chuyn ha v cc lipoprotein l nhng ng c vin ca gen tr liu trn gan. ng k nht l vic a cc vec t adenovirus vo tnh mch ngoi bin chut (Smith v cng s., 1993), hoc ch (Connelly v cng s., 1996) v cc ng vt cao cp khng phait ngi (T.A.G. Smith) thu c kt qu ti np cc t bo gan, iu chng minh c tnh kh thi ca s chuyn giao vec t adenovirus h thng nhm iu tr cc bnh gan. C nhiu tin b t c trong nhng nm gn y trong gen trj liu qua trung gian vec t adenovirus vi cc bnh a chy mu A v B, cc bnh thiu ht cc yu t ng mu FVIII v FIX. a qua tnh mch liu thp vec t Av1 mnh m ha cc yu t ng mu FVIII v FIX ca ngi vo chut trng thnh thu c kt qu l biu hin r mc tr liu cc yu t ng mu t nht 5 thng (Connelly v cng s., 1996) v 1 nm (M.Kaleko). Tuy nhin, khi s dng cc vec t vi liu lng cao gy c cho t bo gan th th mc biu hin cao khi u ca yu t ng mu li nhanh chng gim xung, iu chng t rng c t ca cc vec t ph thuc liu lng lm hn ch s tn ti ca vec t (Smith v cng s., 1993; Connelly v cng s., 1995, 1996). Vic phn phi cc vec t adenovirus FVIII ti ch thiu ht FVIII chnh l c hon ton kiu hnh a chy mu v biu hin cao FVIII ngi (Connelly,1996). Tuy nhin, s biu hin FVIII ch ch l ngn hn do c pht trin cc khng th c ch FVIII ca ngi (Connelly v cng s., 1996). Vic iu tr bnh a chy mu B ca ch vi vec t Av1 m cho FIX chnh l nhanh chng kiu hnh bnh chy mu. Tuy nhin, trong trng hp ny khng pht hin thy cc khng th c hiu FIX ca ch (Kay v cng s., 1994). Trong cc m hnh khc vi cc bnh m ch l gan th cc vec t Av1 m cho cc gen ornithine transcarbamylase (Stratford-Perricaudet, 1990; Morsy v cng s., 1993), gen phenylalanine hydroxylase (Fang v cng s., 1994). gen apolipoprotein E (Stevenson v cng s., 1995) v gen receptor lipoprotein t trng thp ca ngi (Ishibashi v cng s., 1993) c dng trong iu tr vi cng ngh di truyn trn chut knockout nhng gen ny.

Trong tt c cc trng hp u chng minh r rng c s chnh l (sa cha) cc kiu hnh khim khuyt sau khi c tr liu vi vec t adenovirus. Tuy nhin, trong a s trng hp, hiu ng tr liu ch l nht thi. Cng tng t nh vy, vic iu tr bnh lipid cao th Watanabe vi vec t Av1 m ha receptor LDL lm gim ng k mc cholesterol trong vng 3 tun (Kozarsky v cng s., 1994). Cc vec t Av1 c nh gi v chng minh l ti np hiu qu vi rt nhiu m v c quan ngoi phi v gan, chng hn nh tim (Kass- Eisler v cng s., 1993) hay c tim v xng (Stratford Perricaudet v cng s., 1992; Kass Eisler v cng s., 1993; Ragot v cng s., 1993; Vincent v cng s., 1993) hay ty xng (Mitani v cng s., 1993), no (Le Gal La Sall v cng s., 1993), h TKT (Bajocchi v cng s., 1993; Davidson v cng s., 1993), cc t bo ni m (Lee v cng s., 1993; Lemarchant v cng s., 1993), thn (Moullier v cng s., 1994), cc t bo vng mc (Jomary v cng s., 1994) v cc u c (Haddada v cng s., 1993; Brody v cng s., 1994; Wilson v cng s., 1994). Gen tr liu qua trung gian vec t adenovirus vi cc bnh mc phi nh ung th cng theo 2 chin luc, th nht l cm ng c tnh t bo i vi cc t bo c hiu khi u v th hai l kch thch tn ti tnh min dch khng khi u c cm ng bng cch x l cc m c khi u vi vec t adenovirus m cho p53 (Will v cng s., 1994; Zhang v cng s., 1995) hoc bng cch s dng mt t hp gm gen herpes thymidine kinase v ganciclovir iu tr thnh cng cc khi u mi hnh thnh trong cc m hnh trn chut (Chen v cng s., 1995; Yet v cng s., 1996). Vi phng php tim chng vec t Av1 m cho cDNA interleukin-2 th cng thy c s tng cng tnh min dch khng khi u chut (Haddada v cng s., 1995; Cordier v cng s., 1995; Huang v cng s., 1996). 6.6 nh la p ng min dch ca vt ch i vi s chuyn gen adenovirus in vivo Mc du cc vect th h th nht ti np v biu hin gen chuyn vi hiu lc cao, nhng trong nhiu trng hp thi gian biu hin li rt ngn. Lm mt s biu hin cng vi c tnh trc tip ca vec t (Connelly v cng s., 1996), s nhim trng v tc ng ca cc t bo min dch (Yet v cng s., 1994; Yang v cng s., 1994, 1995) u dn n s hy hoi t bo ti np. c tnh ca cc t bo ti np vi vec t Av1 l biu hin ca hu ht cc gen adenovirus gim nhiu, mc protein virus c biu hin thp (Mettereder v cng s 1994; Yang v cng s., 1994), nhng quan st ny chng minh rng c p ng ca lympho T gy c t bo (cytotoxyc T lymphocyte CTL) trc tip khng li cc t bo biu hin cc gen ch cht ca virus, kt cc l loi i cc t bo c cng ngh ha (Yang v cng s., 1994, 1995, 1996). Bi vy, gim bt hn na s biu hin gen virus c th lm gim p ng min dch (UMD) ca vt ch i vi cc t bo ti np v lm tng thi gian biu hin gen chuyn. Tht vy, vic pht trin vec t adenovirus th h hai Av2 vi mt protein gn DNA nhy cm vi nhit 72 kDa m ha vng E2a ca b khung virus s cho php ko di s biu hin gan chut trng thnh c UMD tt cng nh gim p ng CTL (Engelhardt v cng s., 1994, Yang v cng s., 1994; Ye v cng s., 1996). Tuy nhin, cc phn tch v vec t Av2 trong cc chui khc nhau ca chut v ch b bnh a chy mu B ch r khng c s khc bit v thi gian biu hin gen chuyn so vi vec t Av1 (Fang v cng s., 1996). Gn y, ngi ta thit lp c cc vec t th h th ba - Av3 mt cc on E1, E2a v E3 nng cao tim nng sao chp DNA ca vec t v s biu hin ca virus in vitro mun hn cc vec t Av1 v Av2 (Gorziglia v cng s., 1996). Hn na, cng c cc vec t adenovirus loi tr nhiu on trong cc vng E1 v E2 (Armentano v cng s., 1995; Wang v cng s., 1995;

Gao v cng s., 1996; Yeh v cng s.,1996). Cc vec t adenovirus th h k tip s c nhiu on loi b quan trng. Khng nghi ng g na, trong tng lai s phi pht trin cc dng t bo b sung cung cp cc sn phm gen mt dng trans. Sau cng l vic s dng cc vec t adenovirus ch cha cc tn hiu ng gi adenovirus thit yu v hp biu hin gen chuyn (Clemens v cng s., 1996; Haecker v cng s., 1996; Kochanect v cng s 1996; Lieber v cng s., 1996). Tuy nhin, nhng vec t ny c pht trin vi s c mt ca cc virus tr gip. Nhng tht kh m to c mt dng t bo ng gi vi y cc chc nng b tr virus, v nu c nh vy th cc sn phm ca vec t s khng cha cc tp cht t virus tr gip. Nhng vec t virus nht nht s c tim nng gim thiu c tnh t bo v cc UMD t bo cng nh thch ng c vi cc on DNA ngoi sinh ln (trn 35 kb). Phng php khc lm gim bt UMD t bo i vi cc t bo ti np adenovirus nhng vn duy tr biu hin gen chuyn l s dng cc tc nhn kim ch min dch nh cyclosporin A (Fang v cng s., 1995), FK506 (Lochmller v cng s 1995, 1996; Vilquin v cng s., 1995) v cyclophosphamide (Joose v cng s., 1996). Tuy nhin, ko di vic s dng cc cht kim ch min dch c th s lm tng cm ng cc tc dng ph nghim trng. Nh vic thay i cc thuc kim ch min dch, cc khng th n dng khng trc tip cc protein trong t bo T hot ha nn lm gim UMD qua trung gian t bo (Kay v cng s., 1995; Guerette v cng s., 1996; Yang v cng s., 1996). V th, vic s dng cc vec t adenovirus ci tin vi vic lm mt nhiu on cc gen ch cht ca virus kt hp vi ch kim ch min dch thit k c cc khi t bo m tnh min dch qua trung gian t bo cho php ko di hn s biu hin gen chuyn. Mt hn ch khc lin quan ti min dch qua trung gian vt ch i vi vic s dng cc vec t adenovirus l nu cc vec t c dng lp i lp li th s khng thu c kt qu v c UMD th dch i vi protein capsid ca virus (Smith v cng s., 1993; Kay v cng s 1994; Kozasky v cng s 1994; Yei v cng s 1994). Ni chung, bn cht ca s biu hin gen chuyn qua trung gian vec t adenovirus i hi phi c nhiu vec t c hiu i vi cc bnh mn tnh nh bnh x nang v bnh a chy mu v nhng bnh ny phi iu tr ko di. Cc nghin cu ca Smith v cng s (1996) chng minh rng UMD i vi vec t adenovirus cn ph thuc vo liu lng v c th c iu chnh ngay t thi im khi u bi s kim ch min dch tc thi vi cyclophosphamide hoc deoxyspergualin (DSG) th s cho php s dng lp li c hiu qu vi cc vec t ny. Mi y, ngi ta thnh cng trong vic tr liu min dch t hp liu lng thp t nht vi 3 vec t adenovirus (T.A.G. Smith). Hn na, lp li s chuyn giao vec t cng thu c kt qu thng qua chin lc kim ch min dch (Yang v cng s., 1995.1996) v bng cm ng dung np vec t chut s sinh (Walter v cng s., 1996). Cui cng l vic s dng cc adenovirus t cc serotype khc nhau nh adenovirus trung ha khng th, l mt serotype virus khng c phn ng cho v ngn nga c s ti np vi mt adenovirus ca mt serotype th hai (Kass Eisler, 1996; Mastrangeli v cng s., 1996). Tuy nhin, chin lc ny cn lin quan ti vic ch to v c tnh ca cc a vec t m ha cho cc gen m ta quan tm.