Chng XI

IU TR CC BNH THUC HEMOGLOBIN

11.1 M u
Cc bnh thuc hemoglobin i din cho m hnh khim khuyt gen, bao gm s thiu
ht trong biu hin gen - chng thiu mu vng bin (thalassaemia sydrome) trong
nhng bin th protein b thay i chc nng (s trng hp ca haemoglobin S
bnh t bo lim) v gii c ch gen (bnh di truyn lin quan ti hemoglobin bo thai).
Bnh thiu mu vng bin v bnh t bo lim - l cc ch u tin ca GTL, v
cc l do sau y:
1. Gen globin nh, ch khong 2 kb, c biu hin tt, sau khi chuyn vo chut
chuyn gen t mc nh cc gen globin chut v c bit l dng hng cu
giai on bit ha v khng thy c hiu ng ph trn chut
(Stamatoyannopuolos v Nienhuis, 1994).
2. V vn ch th t bo gc to mu c th phc hi qun th di hn in vivo,
c th c thu nhn t mu hoc ty xng v c cng ngh ha in vivo
cng nh cy ghp.
3. M hnh trn chut l thch hp vi bnh thiu mu vng bin v bnh t bo
lim.
4. Ngay t nm 1986 chut thiu mu vng bin (TMVB) c cu sng khi
truyn gen globin ngi vo t bo mm (Constantini v cng s., 1986).
5. Ngi ta hiu chi tit v cc trnh t iu ha.
6. S khim khuyt phn t ca cc bnh thuc hemoglobin tc ng ln tt c cc
bc t gen ti protein u rt r rng v c th ng dng cc chin lc khc
nhau thm vo hoc thay th hoc hiu chnh cc gen blobin.
S hot ha gen hemoglobin bo thai nhm b li s khim khuyt gen globin hoc
gim hiu ng loi b t bin kiu hnh ca t bo l cc chin lc GTL gin tip
Bng 11.1 Cc phng php iu tr bnh thuc hemoglobin bng gen hoc RNA
Locus globin
Thm gen hoc thay th gen (1,2)
Hiu chnh t bin (1,2)
Hot ha gen
Locus globin
hot ha gen (1)
Bt hot gen LCR (1)
RNA globin
Bn phin m : Hiu chnh ghp ni (1)
antisense, ribozyme (1)
Cc gen khc
Thm gen to hng cu (1)
Cc enzyme t bo hng cu
2,3-biophosphoglycerate mutase (antisense) (2)
2,3-biophosphoglycerate phosphstase (thm gen) (2)
Ccc enzyme khng oxy ha (1)
Ghi ch:

1: Thiu mu vng bin

2: Bnh t bo lim

Gen tr liu trc tip cc bnh thuc hemoglobin cng c bt u ngay t nm

1980 vi vic thm gen globin ngi bnh thng vo cc t bo ty xng nui cy
ca 2 bnh nhn TMVB bng cch tim cc t bo thao tc vo cc bnh nhn.
Tuy nhin cc kt qu thu c l m tnh v ngi ta hy vng l n khng gy tn hi
cho bnh nhn. Sau s khi u tht bi ny ngi ta cng thu c thng li khi
s dng t bo nga Trojian, c chuyn giao bng mt vec t retrovirus chut
biu hin c gen globin trn cc t bo nui cy ca bnh tng sinh nguyn hng cu
nguyn bo ty chut (murine eythroleukaemia cell MEL).
Mc d s chuyn gen globulin tim n nhiu li ch, nhng nhng bnh c lin quan
ti hemoglobin khng cn ng u danh sch GTL na, bi v s biu hin in vivo ca
gen chuyn globin soma cn lu mi t ti ngng tr liu v vn ch gii hn mt s
t qun th t bo v chng khng th hin c in vivo vi liu trnh di hn.
Bng 11.2Tr liu gen globin
Tin li
Kch c gen nh (< 2kb)
M hnh hp vi t bo v ng vt
Cha c bnh TMVB cho chut bng
chuyn gen globin ngi vo dng mm.
hiu r bnh l ca bnh ny

Bt li
i hi DNA h gen
Phn tn cc yu t iu ha
Biu hin cao gen cn cho s bit ha tn
cng
Cc t bo gc ch chim t l thp v tim
n
i hi mt lng ln cc t bo c
cng ngh ha v biu hin di hn cc gen
chuyn in vivo.

Nhiu nghin cu c bn c thc hin nhm ci tin, nng cao hiu ng chuyn
gen cng nh biu hin gen chuyn globin soma v cc t bo gc c th c s
dng chuyn gen.

11.2 Vec t retrovirus cho globin

Vec t retrovirus bnh bch cu chut Moloney l vec t khi u c hiu ng i vi
vic chuyn gen nghin cu vo cc t bo to mu chut in vitro. Tuy nhin, nhng
vec t ny ch c dung lng nh di 10 kb. ci cc gen nghin cu ngi ta phi
la chn cc trnh t iu ha trung tm c mt vng kim sot locus (locus control
region LCR) ca gen globin v thm c DNA h gen globin. Cc vec t retrovirus
i hi cc t bo ch phi phn chia th mi hp nht c vo h gen ca t bo.
Cc t bo gc tim tng hot tnh phc hi qun th di hn in vivo, cc nghin cu
ng thi tng s t bo ex vivo cm ng chu trnh t bo m khng c bit ha
ci bin cc dng t bo ng gi nhm to c chun cao ca cc vec t, c
kh nng ti np cc t bo gc ca ngi v duy tr s biu hin gen chuyn mc
cao.

 biu hin c mc cao v c hiu vi hng cu th gen globin h gen phi theo
hng ngc li, di s kim sot ca promoter, c lin quan vi cc v tr nhy cm
cao ca LCR (hypersensitive site HC). Tuy nhin, do thng xuyn c s sp xp li
v cc v tr ni li hoc v tr kt thc poly A lun c ny sinh bi s ngc hng do
phi gy t bin trc tip im nng cao tnh n nh ca vec t v biu hin gen
bnh thng cc t bo ch (Leboulch v cng s., 1994; Sadelain v cng s.,
1995).
Cc t bo MEL c dng nghin cu s biu hin cc bn phin m n ca gen
globin ti np tng phn vi cc on trng lp c cm ng bi s thm chuyn
DNA. Cc nghin cu trc y cho thy, s biu hin ca provirus bn phin m
n c th b bin i v c lin quan ti v tr hp nht ngu nhin (Bender v cng
s., 1989), khi c cc yu t iu ha hot ha cis nm gn chnh gia. Mc biu
hin gen chuyn globin l thp cc t bo MEL ti np khi so snh vi cc gen globin
chut ni sinh (Bender v cng s., 1989; Dzierzak v cng s., 1988; Karlsson v cng
s., 1987; Kasahara v cng s., 1994; Leboulch v cng s., 1995). In vivo, s biu
hin di hn gen chuyn globin ngi cng khng xut hin mt cch thng xuyn,
iu cho thy rt him khi t c s ti np cc t bo gc khi phc qun th di
hn. S biu hin ca gen chuyn globin thp hn 5% gen globin chut ni sinh.
S pht hin ra HS ca LCR iu khin t xa khch l cc nghin cu nhm xc nh
v tr ca LCR hoc s t hp ca cc v tr l phng php hiu lc nht biu
hin gen globin. Gii hn v dung lng kch c cc vec t retrovirus i vi gen
chuyn ngoi lai l do vng LCR ch cho php gii hn 20 kb i vi cc on DNA
nh trung tm nhng c rt nhiu m tip hot chuyn. V tr HS2 ca LCR l v tr hot
ha nht (Caterina v cng s., 1991; Collis v cng s., 1990; Curtin v cng s.,
1989; Forrester v cng s., 1989; Grosveld v cng s., 1987; Leboulch v cng s.,
1995; Phillipsen v cng s., 1990; Ryan v cng s., 1989; Talbot v cng s., 1990).
Cc vec t retrovirus Ecotropic loi v tr HS c gn vi promoter globin ngi
biu hin 60-70% trong cc t bo MEL khi so snh vi globin ni sinh ca chut. S
thm nhim ca cc t bo ty xng chut v s cy ghp cc t bo ti np vo
nhng i tng nhn b bc x mnh cho thy s biu hin gen chuyn globin c
th thay i, thng cao hn trng hp khng c LCR, nhng vn di ngng i
hi ca tr liu (Chang v cng s., 1992; Gelinas v cng s., 1992; Novak v cng
s., 1990; Plavec v cng s., 1993; Ellis v cng s., 1997). ng tic l, cc cu trc
mi ny hot ng khng n nh, provirus vn c s sp xp li v chun vec t
thp.
S khng n nh v mt di truyn c phng nga bng cch loi thi h thng v
thay th trc tip v tr (Leboulch v cng s.,m 1994; Sadelain v cng s., 1995). Kt
qu l nng cao c tnh n nh v chun ca cc hp LCR trong cc t bo
hng cu in vitro v in vivo. S biu hin ca gen chuyn globin lin quan cht ch vi
cc cu trc c 4 v tr LCR cha trung bnh 78% gen chut vi thang t 4% n 146%
(Sadelain v cng s., 1995). Mc du c mt hu ht cc v tr LCR hot ha,
nhng s biu hin ca gen chuyn globin soma vn cn phi ph thuc vo v tr ca
n. Khi thm vo cc v tr cch ly (insulator site) cui cu trc LCR gen globin th
c th lm gim s bin i biu hin gen globin (Chang v cng s., 1993) v c th
trnh c s hot ha cc gen gy ung th nm cnh v tr ci ca provirus. S biu
hin di hn cu trc LCR gen globin n nh sau ti np cc t bo t bo gc v cy
ghp, l iu chng ta khng cn phi bn khon na (Sadelain v cng s., 1995).
Nhng nghin cu ny ci tin mu hnh ca cc vec t retrovirus v gii quyt
c 3 vn chnh ca s chuyn gen globin vi cc vec t retrovirus l: to c
chun cao ca cc vec t, n nh h gen ti np v biu hin gen globin in vitro
mc cao trong hng cu (Leboulch v cng s., 1995).

iu cn thit i vi cc vec t c kh nng thm nhim cc t bo gc ngi li i

hi cc t bo ng gi ca ngi hoc cc t bo lng tnh, nhng dng t bo ny
khng lm gim chun hoc gim kh nng ti np ca vec t. chut, s biu hin
ca cDNA adenosine adeaminase (ADA) di s kim sot ca on lp cui di ca
retrovirus t 80% ADA ni sinh sau 12-14 thng cy ghp (Riviere v cng s., 1995).
Cc c trng c bit ca LCR virus gp phn lm tng thi gian biu hin ca gen
ADA hu ht cc dng t bo c ngun gc t cc t bo gc khi phc qun th di
han.
11.3 Cc vec t virus adeno lin hp
Cc virus adeno lin hp (adeno-asociated virus AAV) l virus DNA si n, c 4675
nucleotide, thuc h parvovirus. AAV khng phi l virus gy bnh, n c th thm
nhim vo rt nhiu t bo ngi v c th hp nht vo nhng v tr c hiu trn NST
19 ca ngi (19q13,3-qter) (Flotte v cng s., 1995; Kotin v cng s., 1991).
Cc trnh t cn cho s ng gi, s hp nht v sao chp nm trong 191 nucleotide,
bao gm c nhng on lp tn o ngc ( inverted terminal repeats ITR)
(Samulsski v cng s., 1989). AAV l virus khim khuyt, n cn phi c cng thm
nhim vi virus (adenovirus, hoc virus herpes simplex) th mi sao chp c. Cc
khng th khng AAV c mt 70-80% qun th.
AAV type 2 c s dng nh mt vec t biu hin cc gen chuyn. Kch c cc i
ca DNA ng gi ch 4,5 kb. Hu ht DNA h gen u c th c thay th bng mt
gen mong mun bi v vi u tn ca AAV (145 nucleotide) l hp nht vec t
AAV ti t hp vo trong h gen vt ch.
Vn tp cht ca vec t AAV do phi b sung thm cc virus tr gip cho vec t
thm nhim cng c gii quyt bng cch x l vi nhit v cc bc lm tinh
khit cng nh vic s dng cc plasmid tr gip.
Tuy nhin, vn cn mt s vn tn ti l: (1) rt kh thu c cc dng t bo ng
gi c kh nng to c chun cao cho cc vec t AAV, (2) ti sn xut cc vec t
AAV, (3) c tnh ca protein Rep i vi cc t bo ng gi v (4) cc vec t AAV ti
t hp ch cha cc u tn AAV nn c th lm mt tnh c hiu i vi v tr hp nht
ca NST 19 (Mamounas v cng s., 1995; Shelling v Smith, 1994).
S thm nhim t bo ca K562 hng cu ngi hoc cc t bo to mu ngi vi
vec t AAV ca A globin c lin quan rt nhiu vi cc t bo neo u v s biu hin
gen chuyn A globin (2,2 kb) c nh du vi 6 nucleotide b loi i vng 5
khng phin dch gn vi v tr 2 ca LCR (0,4 kb). Cc t bo K562 biu hin gen
chuyn mc ca ni sinh trong cc t bo cm ng bit ha hng cu (Miller v
cng s., 1994; Walsh v cng s., 1992). Cc t bo to mu CD34+ ca ngi thm
nhim cao vi vec t AAAV (500-1000 ht/t bo) 20-40% qun th hng cu biu
hin gen chuyn A mc 4-71% khi so snh vi cc gen ni sinh. Hemoglobin thai nhi
(Foetal haemoglobin) (HbF) tng t 26% ti 40% sau khi ti np AAV trong cc t bo
t tin (Millervaf cng s., 1993). S sp xp li h gen vec t i cng vi vic lm mt
cc trnh t iu ha c th xy ra mt s dng t bo (Miller v cng s., 1994). Cui
cng, DNA vec t gc c th s cho mt tn hiu nhn to gi ca DNA t mt qun th,
do cn phi tin hnh RT-PCR xc nh hiu ng ti np AAV v s biu hin ca
gen chuyn. S hp nht ca vec t AAAV trong cc dng hng cu vn cha c
chng minh. Tuy nhin, vi mt s lng ln t bo (> 1000) li ch t mt t tin thm
nhim n c th ni c rng c s hp nht ca provirus trong t bo gen phn
dng ny.
Nhng cng trnh nghin cu gn y s dng gen Aglobin c nh du cc v tr HS
2,3 v 4 t LCR, nhng khng c gen khng thuc cho thy c s hp nht cp i

v s biu hin chc nng ch vec t AAV c s thm nhim cao (Hargrove v cng
s., 1997). S biu hin di hn ca gen globin quan st thy trong cc i tng
cy ghp ln u, biu hin gen globin ni sinh mc 6% trong 1 thng v mc 0,4
% trong cc t bo ty xng nhng i tng c cy ghp ln th hai
(Ponnazhagan v cng s., 1997).
Dng cc vec t c s AAV ti t hp biu hin RNA antisense globin cc t bo
K562 l cch tip cn gin tip chng li hiu qu kiu hnh thiu mu vng bin
(Ponnazhagan v cng s., 1994).
11.4 Ti t hp tng ng v s sa cha
Phng php l tng hiu chnh li gen globin khim khuyt v gii quyt vn
biu hin gen thp l thay th vng DNA bt thng bng cc trnh t bnh thng nh
ti t hp tng ng (Smithies v Maeda, 1995). Theo phng php ny th gen
globin t bin s c hiu chnh li trong cc dng t bo, k c cc t ba gc ca
phi (Shesely v cng s., 1991). Tuy nhin, phng php ny vn cha th p dng
c i vi cc t bo gc ta mu v tn s ti t hp cc v tr xc thc cn thp
v cn phi nhn ln cc t bo gc c hiu chnh rt him hoi ny.
Vic sa cha c hiu v tr ca t bin im c th p ng vi bnh TMVB bng
cch thm chuyn cc oligonucleotide chimeric c hiu vo cc t bo gc ex vivo
(Cole-Strauss v cng s., 1996).
11.5 Tr liu gen gin tip bnh thiu mu vng bin
Do s mun mng trong GTL trc tip bng cch thm vo hoc thay th gen th c
mt cch tip cn khc, l GTL gin tip (bng 11.1).
11.5.1 Hiu ng ghp ni bt thng
Cc t bin gen globin l do v tr ghp ni bt thng ( IVS1-110), v tr ny tim
n rt nhiu so vi v tr bnh thng nguyn bn, nhng trng hp ny rt hay gp
cc nc thuc a Trung Hi vi hi chng TMVB . cc t bo nui cy, c th
hon tr 100% s ghp ni bnh thng v phc hi mRNA globin chc nng bng
cch dng antisense oligonucleotide che lp cc v tr ghp ni bt thng (Dominski
v Kole, 1993). Cng c th chuyn mt gen m cho RNA antisense, vic phin m
c kim sot bi promoter globin v LCR trong cc t bo gc biu hin gen
antisense giai on cui bit ha hng cu.
11.5.2 Hot ha cc gen b
Mt cch tip cn gin tip khc l c th b p cho cc gen globin khim khuyt
bng cch hot ha mt gen ln bnh thng c cng chc nng hoc tng t chc
nng. y l trng hp ca cc gen globin ca HbF- mt bnh nghim trng c lin
quan ti hemoglobin (Stamtoyapoulos v Nienhuis, 1994). Khi x l bng hydroxyurea
s lm tng mc HbF v gim tnh nghim trng i vi bnh t bo lim (Charache v
cng s., 1992, 1995). Cc tc nhn lm thay i c tnh t bo hoc lm bin i cht
nhim sc cng kch thch s biu hin in vivo ca gen globin cng nh cc gen to
hng cu (Rodgers v cng s., 1993). V vy, tm xa ca GTL i vi cc bnh thuc
hemoglobinl phi c nhiu chin lc vi cc tim nng khc nhau nng cao s
biu hin ca cc gen globin. Mt trong nhng cch tip cn l s chuyn gen to

hng cu ( erthropoietin gene-Epo) v cc t bo sao cho chng c kh nng tit ra

c Epo vi cc liu lng dc l in vivo.
11.5.3 Chuyn gen to hng cu
Nhn t c bn ca cch tip cn ny xut pht t mt hiu ng ca Epo i vi cc
bnh nhn thiu mu t bo lim (Cozma v cng s., 1995; El Hazmi v cng s.,
1995; Rodgers v cng s., 1993) v cc bnh nhn TMVB (Aker v cng s., 1995;
Olivieri v cng s., 1995; Rachmilewitz v cng s., 1995). Tuy nhin, liu lng gy
hiu ng li rt cao v v rt t nn cc quy trnh thng quy khng th s dng c
Epo ti t hp.
Cc m hnh ng vt c s dng xc nh hiu ng ca nhng liu ln i
vi s biu hin HbF. kh u ch, vi liu rt cao ca Epo cm ng c 605
HbF (Al-Khatti v cng s., 1987). Epo ti t hp c kh nng b p s thiu mu ca
chut TMVB . Vn ny gn lin vi gim s lng hng cu li, nhng li tng
thi gian tn ti ca hng cu v tng cht t khi lng to mu (De Franceschi v
cng s., 1996).
Vi cc bnh nhn TMVB th vic hot ha gen globin (khi biu hin ) phi
cao th mi tng c t l to hng cu v tng thi gian sng ca t bo hng cu.
S hot ha ny cng c th lm gim s pht trin ca ty xng nhng bnh nhn
thiu mu Cooley nh.
Mt cng trnh nghin cu trc y chng minh rng s biu hin lin tc gen Epo
sau khi ti np cc t bo gc phc hi qun th di hn khng c vec t retrovirus th
c kh nng b p cc khim khuyt di truyn bnh TMVB chut (chut Hbb thal1
loi gen chnh ) (Viller v cng s., 1994). Tuy nhin, mc biu hin ca gen
chuyn Epo th vn cha xc nh c, tc l hoc qu cao hay qu thp ch him khi
mc chun xc v thng l nht thi. Chut TMVB trc khi c cy ghp cc t
bo ty xng bin i gen th phi tia x. In vivo, mt mc no ngi ta
kim sot c s biu hin gen chuyn Epo nguyn bo c ti np retrovirus cm
ng vi doxycycline sau khi c chuyn vo chut bnh thng cng gen (Bohl v
cng s., 1997).
Vic cy ghp cc t chc mi (Moullier v cng s., 1995) t nguyn bo si ti np
vi gen Epo cDNA di s kim sot ca promoter phosphoglycerate kinase (PGK)
cho s biu hin di hn (trn 6 thng) gen chuyn Epo chut bnh thng. Mc Epo
v haematocrit ph thuc vo s lng cc nguyn bo si c ci bin gen c
mt cc t chc mi (Naffakh v cng s., 1995). Tuy nhin, khi so snh vi chut
bnh thng th mc tng haematocrit chut TMVB cng ch xy ra trong thi gian
rt ngn.
Vec t adenovirus chun cao biu hin gen chuyn Epo cng c xc nh.
Nu khng c s hp nht v li ha long gen chuyn Epo cng nh p ng min
dch i vi cc t bo biu hin protein adenovirus th s tim n kh nng biu hin
thp hoc biu hin nht thi. Tuy nhin, sau khi a mt liu n qua tnh mch cc
ht vec t Epo-adenovirus th s tng hng cu c cm ng trn 6 thng chut
bnh thng (Descamps v cng s., 1994). S biu hin di hn ca gen chuyn Epo
cng thu c vi vec t AAV bng cch tim trc tip vo c chut bnh thng
(Danos)
11.5.4 Gim biu hin gen globin
Trong bnh TMVB do thiu ht s tng hp chui globin ngi ta u hiu r rng
mc nghim trng lm sng ca TMVB ph thuc vo s mt cn bng gia tng

hp cc chui globin v cc chui khng phi l v c bit hn l c nhiu chui

hemoglobin t do (khng lin kt vi chui hoc ) nn n khng n nh v b kt ta
trong hng cu v cc t bo t tin.
Nhng chui globin khng cp i ny c cha nhm haem v st, n c th oxy ha
v xc tc cho s hnh thnh cc gc oxy c tnh cao, nhng gc ny li cm ng s
tiu mu v lm mt hiu ng to hng cu. Khi tng cc chui globin ca HbF hoc
gim cc chui (tc l cc ng yu t quyt nh bnh TMVB ) th s lm gim s
cn bng chui globin v TMVB nghim trng. Ngc li, nu thm vo cc gen
globin (cp ba) thng lm tng tnh nghim trng trng thi d hp ca TMVB (Cao
v cng s., 1995). Nu gim 20-30% chui globin t do th s chuyn hi chng
TMVB nghim trng thnh t nghim trng hn.
Cc chin lc nhm gim s chui t do c th c t ra cc ch khc nhau t
s phin m ti dch m s biu hin gen globin. mc phin m, s biu hin gen
globin c th b gim st l do lm bt hot mt trong 2 gen hoc v tr HS-40 ca LCR
gen bng cch ti t hp tng ng ex vivo (Bernet v cng s., 1995). Cng c th
s dng mt antigene m cho RNA to nn mt xon ba c ch v tr hot chuyn
(HS-40 hoc promoter gen ). mc sau phin m th mt gen khng RNA anti-RNA
m cho RNA antisense (Ponnazhagan v cng s., 1994) s c ch vic x l ca
mRNA ca globin. Cng c th hng mt ribozyme hoc mt nuclease H vo vic
phn gii c hiu mRNA ca globin hoc cc t tin ca n (Thompson v cng s.,
1995). S phin m ca antigene hay gen anti- RNA phi t di s iu ha gen
globin phi hp s biu hin cc gen globin v anti globin.
11.5.5 Cc kiu GTL khc
Mt cch tip cn khc mc sau phin dch l tng s ly gii cc chui globin. H
thng ny c hng cu li ca cc d hp t TMVB , khng thy c triu chng no
xut hin khi 50% chui b suy gim chc nng (v hu ht chng b ly gii). Khi
nng chui globin vt trn mt ngng no ca TMVB ng hp t th
qu trnh ly gii chui globin c th l bo ha, cc chui globin d tha s kt
ta v lm tch t haemin v st. C th ci thin s tn ti ca hng cu v cc t tin
ca chng bng cch cho thm vo mt gen m cho enzyme protease phn gii c
hiu cc chui globin t do m khng lm bin i cc protein khc ca t bo.
Cc qu trnh oxy ha bt thng m hu qu l gy nn s khim khuyt trong TMVB
cng c th c gim bt hoc c phng nga bng cch chuyn vo mt gen
m cho mt protein c kh nng thu dn cc gc t do hoc gn vo st hoc haemin
t do c gii phng ra t cc chui globin khng n nh gy nn s oxy ha v
lm mt hiu ng to hng cu.
11.6 iu tr bnh t bo lim
Gen tr liu soma bnh t bo lim kh hn bnh TMVB , bi v gen S globin phi
c biu hin mc cao v nhn c chc nng mi ng thi phi c ch c
qu trnh polymer ha ca HbS. Hn na, theo mt s chin lc cp trn th
bnh t bo lim v TMVB thng c 2 chin lc c hiu l: (1) chuyn mt gen
m cho globin vi c tnh khng t bo lim v (2) chuyn mt gen m cho
bisphosphoglycerate mutase phn gii 2,3- biphosphoglycerate (Garel v cng s.,
1994).
Cch tip cn trc tip v u tin l gy t bin trc tip v tr codon 22 v 87 ca gen
globin c ch tip xc trong polymer ca hemoglobin nh thy HbF v HbA
(Mccun v cng s., 1994). Dng gen chuyn ny n nh v c biu hin trong

cc t bo MEL ti np vi cc vec t retrovirus (Takekoshi v cng s., 1995). Mt

cch tip cn mi v rt l th i vi bnh t bo lim l sa cha t bin bng thm
chuyn mt oligonucleotide chui kp chimeric vo codon 6 trong gen S globin (ColeStraus v cng s., 1996).
Mt cch tip cn gin tip i vi GTL c hiu khng t bo lim c suy din t
quan st thy rng mt bin th ti t hp ca 2,3-biphosphoglycerate mutase tng
hp acid phosphatase s lm tng hot tnh ca 2,3-biphosphoglycerate phosphatase
(Garel v cng s., 1994). Khi chuyn gen ti t hp ny vo cc t bo hng cu s
lm gim mc ca 2,3-bisphosphoglycerate in vitro v c th ngh ti vic ng dng
GTL soma in vivo.
11.7 Vin cnh tng lai
Vic chuyn gen trc tip bng cc vec t virus c thm nhim cao to c hiu
ng ti np cao cho cc cu trc n nh v chng c biu hin trong cc t bo
to mu bnh thng in vitro cng nh trong cc dng hng cu ca chut v ngi.
iu chng minh cho kh nng ti np v biu hin ca cc gen globin in vivo. V th
ngi ta c th thc hin c cc nghin cu v t bo gc ca ngi ti np c hot
tnh khi phc qun th di hn trong cc m hnh TMVB trn ng vt. Chut thiu ht
min dch (THMD) (Cashman v cng s., 1997; Dick v cng s., 1995) hoc chut
bo thai c ghp cc t bo CD34+ v cc t bo m c th c s dng c
tnh ti np v s biu hin ca gen chuyn trong tt c cc dng t bo t chnh
cc t bo gc nguyn thy ca ngi in vivo. Bng vic s dng cc cytokine, ti nay
ngi ta kch thch c s nhn ln ex vivo ca cc t bo gc nguyn thy v
trnh c vic s dng cc cht ha hc hoc tia x ton thn trc khi ghp cc t
bo gc vo cc bnh nhn c lin quan ti hemoglobin.
Mt vn ln vn cha c gii quyt l vic duy tr s biu hin mc cao gen
chuyn globin invivo. Ngi ta chng minh rng cc trnh t ca retrovirus cng c
ch s biu hin gen globin trng chut th tinh sau khi chuyn n cu trc
provirus bng GTL soma (McCun v Towner., 1994). Hu ht cc nghin cu v s
chuyn gen globin thu c kt qu trong nui cy nhng cha c kt qu in vivo.
C mt l do l cc dng t bo c s dng in vitro c mc biu hin gen globin ni
sinh thp hn nhiu nguyn hng cu in vivo, v vy khng t c t l 1:1 gia s
biu hin di hn gen blobin ni sinh v gen chuyn cc t bo MEL hoc K562.
Trong tng lai ngi ta s chuyn nhng on DNA ln, k c ton b h gen globin.
iu i hi phi c s nhn ln ex vivo v phi thao tc vi cc t bo gc. ch
ca t bo s c ci tin bng cch gn c hiu cc receptor c hiu vo cc vec
t. Cc phn t dung np c th s nng cao s chuyn DNA bo cht. Cc m hnh
AAV c th c thit k cho vic hp nht gen globin vo v tr c hiu AAV ca NST.
Tt c nhng nghin cu hin nay ang tp trung vo vic sn xut h thng vec t
virus l tng vi hiu ng ti np cao nhng khng lm hn ch cc vec t AAV hay
retrovirus. Ngi ta s s dng cc NST nhn to ca ngi trnh s hp nht ngu
nhin ca gen chuyn trong h gen ca t bo (Huxley, 1994).
Vic tr liu gen globin mt cch hiu qu v trc tip vn cn nhiu kh khn pha
trc, v th GTL gin tip cn c pht trin. Phng php ny c th hu ch i vi
nhng bnh nhn b suy kit do bnh tt cng nh ln vi cc ch iu tr khng
ngon ming- unpalatable ng thi. Nhng tr liu gin tip ny c th s lm gim
bt s bnh hon, cht chc v cc vn v con ngi, gia nh v x hi ca cc
bnh thuc hemoglobin vi nhng khim khuyt di truyn nguy him.