Transcript KCCH HSV1 Thymidine Kinase Reporter Gene Imaging

HSV1 Thymidine Kinase
Reporter Gene Imaging
싸이클로트론 응용연구실
원자력병원
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Methods for the measurement of gene expression
Conventional measurement of Gene expression
Southern, Northern , and Western blot and
in situ hybridization
Reporter gene
easier, less expensive, more accurate & quantitative
than standard hybridization technique
Reporter gene and In Vivo imaging of gene Imagene
Jean-Luc C. Urbain ; JNM Vol.42, No.1, Jan , p106-109
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Widely Used Reporter Gene Systems

b-galactosidase system
b-galactosidase

b-glucuronidase system
b-glucuronidase

CAT (CM acetyl transferase) system
CM acetyl transferase

SEAP (secreted alk-phosphatase) system
alkaline phosphatase

Firefly luciferase system
luciferase

GFP (green fluorescent protein) system
green fluorescent protein

HSV1-TK system
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Principle of Conventional Reporter Assays
Reporter gene
Reporter
Proteins
Histological
staining
mRNA
Transfection
DNA
Biochemical
assay
Photon detection
reporter gene system for in vivo imaging of transgene
 HSV1-tk : various purine & pyrimidine substrates
: development of radiolabeled analogs
Radiolabeled substrate
trapped
Reporter
Enzymes
Nuclear Imaging
Reporter gene
Transfection
Reporter
Receptors
Radiolabeled ligand
 transmembrane receptor - receptor ligand
ex) Dopaminergic type2 receptor
- 18F-fluoroethylspiperone
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HSV1-TK System
HSV1-tk (Herpes Simplex Virus type 1 – thymidine kinase)
relaxed substrates specificity
substrates : thymidine, purine pentoside, wide diversity of nucleoside
analogues
Synthetic purine / pyrimidine nucleoside derivatives
Monophosphate
form
HSV1-tk
Diphosphate
form
Guanylate
kinase
Triphosphate
form
Other cellular
kinase
( acyclovir,
ganciclovir )
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Radiolabeled susbtrates for HSV1-tk reporter system
Radiolabel



H-3, C-14 : Autoradiography
I-131, I-123 : SPECT
I-124, F-18 : PET
Uracil nucleoside derivatives
(higher enzyme affinity)





FIAU
IVDU
IVFRU
IVFAU
IVAU
Acycloguanosine derivatives
(F-18 labeling possible)



FACV
FGCV
FPCV
Uracil nucleoside 계
Acycloguanosine 계
Receptor-Ligand Reporter System
Dopamine 2 Receptor (D2R) : 3-(2’-fiuoroethyl) spiperone (FESP) reporter system
Membranous receptor - labeled cognate receptor ligand
Bicistronic adenoviral vector carrying human D2R and somatostatin type 2 receptor(hSSTr2)
Na+ /I symporter (NIS) gene reporting & therapeutic system
Not only radioiodine therapy but also transfected tumor localization and gene therapy assays
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Prerequisites for using a reporter gene imaging
Reporter Gene





Does not exceed size constraints of the vector
Expression of reporter gene is stable and reflect expression of targeted gene
The endogeneous gene is expressed only in low levels
The vector does not induce significant host immune response
Can be dual inserted to reflect expression of gene of interest
Reporter Protein





Does not induce a significant immune response ( not foreign )
Is retained in the cells that they have been expressed
Is stable and do not produce significant confounding metabolites
Does not cause significant biological effects
Endogeneous activity and Concentration is low & absent from targeted cells
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Reporter Probe

Non-immunogenic & no or minimal side effects

Labeling method : single photon or positron suitable for SPECT and PET imaging

Can be radio-labeled with high specific activity

Radio-labeled reporter probe retains chemical activity

Stable in the blood & cleared rapidly from blood stream

Rapidly metabolized by reporter gene product & effectively trapped in transduced cell

Specifically retained by transfected cell & reflect activity of transgene expression

Reporter substrate readily bind to gene product as receptor - ligand
/ must be able to cross cell membrane as HSV1-tk system

Radio-signal correlates with transgene expression level
암 유전자 치료
1. 암세포 : 유전적 변이에 기인
- 암유전자 (oncogene),
종양억제유전자 (tumor suppressor gene)
2. 암세포에서 발견되는 유전자이상을 교정,
조절하는 치료법
- 유전자 표지(genetic marking)
- 암 백신(cancer vaccination)
- 암 유전자 발현 억제
- 종양억제유전자 회복
- 자살유전자 (suicide gene)
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HSV-1tk 유전자 영상 원리
HSV-1tk 유전자가 유입(transfection)된 세포는
thymidine kinase효소를 생산하므로
이 효소의 기질 (substrate, 치료는 ganciclovior)에
감마카메라로 영상을 얻을 수 있는 방사성동위원소를 표지하여
유전자 영상을 얻고, 유전자 치료의 성공여부를 조기판정
본 연구는 HSV-1tk 유전자 영상을 얻기 위하여
두 가지 약제(IVDU, IVFRU)를 합성하고, 방사성동위원소 표지하여
유전자 주입 동물모델에서 성공적인 유전자 영상을 얻었다.
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HSV-1tk Gene Therapy system
Accumulation
& Cytotoxicity
HSV-tk
Death
mRNA
Ganciclovir (GCV)
HSV-tk gene
Live
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HSV-1tk Gene Therapy system
Accumulation
& Cytotoxicity
HSV-tk
Death
mRNA
Ganciclovir (GCV)
HSV-tk gene
Live
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HSV-1tk Gene Imaging system
Accumulation
HSV-tk
Image
mRNA
Radiolabeled HSV-tk
substrate
HSV-tk gene
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HSV-1tk 유전자 영상 제재
O
I
HN
Nucleoside derivatives
O
OH
N
O
OH
.
.
.
.
Antiviral agent
Pyrimidine Nucleoside Analogs
Herpes Viral Infections
Imaging Reporter Gene Expression
of Viral thymidine kinase
R
R = H,
(E)-5-(2-Iodovinyl)-2’- deoxyuridine (IVDU)
R = F,
(E)-5-(2-Iodovinyl)-2’- fluoro-2’-deoxyuridine (IVFRU)
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동위원소 표지
O
O
*I
S iM e 3
HN
HN
HO
O
N
O
Na
125
I or N a
131
I
HO
O
N
O
IC l, 20% AcO H/C H 3 C N
r.t, 20m in
OH
X
OH
X
X =H
IV D U
X =F
IV FR U
순수분리
HPLC column : mBondapak - C18
Solvent : CH3CN and D.W. , Gradient
Flow Rate : 1ml/min
Radiochemical yield : >98%
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연구방법
Cell line : MCA( rat Morris hepatoma Cell Line) and
MCA-tk(HSV-1tk gene transfected (MCA Cell Line)
HSV-1tk substrate :
[125I],[131I] IVDU and IVFRU
O
*I
HN
HO
O
OH
N
O
*I = 125I or
X=H
X=F
131I
IVDU
IVFRU
X
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방사성 요오드 표지법 : Radioiodination of Substrates
Trimethyl-Silyl-Vinyl-2’-fluoro-2’-deoxyuridine(SiMe3-VFRU)
50 ml(5 mg/ml)
& Trimethyl-Silyl-Vinyl-2’-deoxyuridine(SiMe3-VDU)
1N HCI
100 ml
30% H2O2
50 ml
Na125I
18.5MBq
10minutes reaction in Room Temp.
Saturated NaSO3
100 ml
Saturated NaHCO3
1ml
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정제 및 분리 : HPLC analysis
Column ( mBondapak C18, 3.9 x 300mm Waters )
Pump control :
Gradient
Flow rate
:
1 ml/min
Solvent
:
1-5 min
5-10 min
10-25 min
25-30 min
Run time
Detector
A = CH3CN
10% A
40% A
40% A
10% A
:
:
:
:
B = D.W.
90% B
60% B
60% B
90% B
: 30 minutes
: UV (Waters 486) = 254nm , RI(Raytest)
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Radiochromatograms of IVDU and IVFRU
IVFRU
CPM (x1,000)
700
IVDU
160
140
600
120
500
100
400
80
300
60
200
40
100
20
CPM (x1,000)
800
0
0
5
10
15
Time
20
25
30
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세포섭취율 : In Vitro Uptake in MCA & MCA-tk Cell Lines
Cell Lines (1 x 106 / flask) : MCA ( control )
MCA-tk ( HSV-tk gene tranduced )
Culture Conditions
: 37oC , CO2 Incubator
Substrates
:
Incubation Period
: 15, 30, 60, 120, 240, 480 minutes
Counting
: Gamma Counter (WALLAC wizard)
125IVDU, 125IVFRU
(185KBq)
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세포섭취율 : In Vitro Uptake in MCA & MCA-tk Cell Lines
% injected dose
125IVDU
125IVFRU
50
12
40
10
30
MCA
MCA-tk
20
8
MCA
MCA-tk
6
4
10
2
0
0
0
100 200 300 400 500 600
Incubation time(min)
0
100
200 300 400 500
Incubation time(min)
600
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유전자 주입 종양 이식 백서에서 체내 분포 :
Biodistribution of 131IVDU and 125IVFRU in Tumor bearing Rats
Tumor cell (1 x 107 /100ml ) injected into both thigh
MCA
: Right thigh
I.M.
MCA-tk : Left thigh
I.M.
Two weeks later
131IVDU(
370KBq/ 100ml ) ,
125IVFRU
( 370KBq/ 100ml )
Simultaneously injected I.V. into tail vein
Time
: After injection, 1hr / 4hr / 24hr
Counting : Gamma Counter in each energy windows
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혈중제거율 : Blood Clearance in Rats
ID%/gm
4
125 IVFRU
3
125 IVDU
2
1
0
0
20
40
60
80
100
120
Minutes
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체내분포 및 동태 : Biodistribution of
% Injected Dose
5
4
131IVDU
1hr
4hr
24hr
3
2
1
0
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HPLC analysis of 2 hrs Urine
70
60
Tot %
78.8%
IVFRU
IVDU
50
40
76.1%
30
20
18.1%
19.8 %
10
0
0
5
10
15
Fraction No.
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체내분포 및 동태 : Biodistribution of 131IVDU
3
1 hour
4 hour
% ID/g
2.5
24 hour
2
1.5
1
0.5
0
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체내분포 및 동태 : Biodistribution of 125IVFRU
1 hour
2
4 hour
24 hour
%ID/g
1.5
1
0.5
0
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II-5. In Vivo Image of 123IVDU and 123IVFRU
In tumor bearing Rats
Model 1 : MCA-tk(HSV-tk gene transduced) cells (1 x 107 /100 ml ) injected S.C.
into Right thigh
Model 2 : Tumor cells (1 x 107 /100 ml ) injected S.C. into both thigh
MCA : Right
MCA- tk : Left
Substrates :
123IVDU, 123IVFRU(
74MBq / 200 ml ) injected I.V into tail vein
In Hepatocellular Carcinoma bearing Rats
Model : MCA- tk cells (1 x 107 /100 ml ) injected S.C. into Shoulder of Rat
Establishd tumor mass implanted into liver using intra-hepatic tumor injection
Substrates :
123IVDU(
74MBq / 200 ml ),
123IVFRU(
74MBq / 200 ml )
Gamma camera image : After injection, 2hr / 24hr
Sophy Camera DSX rectangular - 300,000 counts collect
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Rat Orthotopic Hepatoma Model of MCA-RH7777
Subcutaneous Implantation
Intrahepatic Tumor Implantation (IHTI)
Transabdominal Ultrasonogram
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Monitoring of Gene Therapy : 131IVDU
TK
MCA
TK
Thyroid
TK
MCA
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유전자 영상 (피하종양이식모델)
123IVDU
123IVFRU
MCA-tk
2hr
MCA-tk
24hr
2hr
24hr
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유전자 영상 (간종양이식모델)
123IVDU
123IVFRU
HCC
HCC
HCC
HCC
2hr
24hr
section
2hr
24hr
section
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MCA
MCA
MCA-tk
MCA-tk
125IVDU
125IVDU
125IVDU
125IVDU
(1hr)
(4hr)
(1hr)
(4hr)
0.5
0.5
17.2
44.8
16.1
14.7
27.8
55.2
Mitochondria
9.1
8.6
47.7
29.8
Microsome
4.2
4.8
7.5
6.8
70.5
72.0
16.9
8.2
Cell/Media
Nuclei
Cytosol
Table 1. Intracellular localization of
125IVDU
at 1hr and 4hr in MCA and MCA-tk cells
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MCA
125IVDU
MCA-tk
125IVDU
MCA-tk
125IUdR
0.5
44.8
1.8
14.7
55.2
54.3
Mitochondria
8.6
29.8
8.9
Microsome
4.8
8.2
3.5
71.9
6.8
33.3
Cell/Media
Nuclei
Cytosol
Table 2. Intracellular localization of
in MCA and MCA-tk cells
125IUdR
and
125IVDU
at 4 hr
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결 론
세포섭취율 (In Vitro Uptake) :
IVFRU 116 - fold, IVDU 37 - fold in 480 min
IVFRU > IVDU
체내분포 (In Biodistribution):
Both IVDU and IVFRU present similar pattern
유전자영상 (In Vivo Image)
Model 1 : Both IVDU and IVFRU showed defined image
HCC Model : IVFRU is selectively localized in HCC
결론 : IVDU and IVFRU could be useful for gene monitoring agents
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