june-key chung, m.d. department of nuclear medicine seoul national university hospital radionuclide...
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June-Key Chung, M.D.
Department of Nuclear MedicineSeoul National University Hospital
Radionuclide Gene Therapy and Imaging with Sodium/iodide Symporter
Na+/I- Symporter
N
Na+
I-
Na+
K+
2D Structure model of NIS New model : 13 putative transmembrane segments
Radionuclide Gene Therapy with Radionuclide Gene Therapy with
Sodium/iodide Symporter GeneSodium/iodide Symporter GeneRadionuclide Gene Therapy with Radionuclide Gene Therapy with
Sodium/iodide Symporter GeneSodium/iodide Symporter Gene
Strategy rNIS cDNA(2.03kb)(TA-cloned into pCR 3.1
vector, 5.06kb)
rNIS cDNA(2.03kb)(TA-cloned into pCR 3.1
vector, 5.06kb)
Transformation(E.coli, DH5-alpha)Transformation(E.coli, DH5-alpha)
Transfection(Gene delivery : Lipofection)
Transfection(Gene delivery : Lipofection)
Selection(Geneticin : G418, 300-600ug/ml)
Selection(Geneticin : G418, 300-600ug/ml)
in vitroIodide uptakeIodide efflux
in vitroIodide uptakeIodide efflux
in vivoBiodistributionTumor imaging
in vivoBiodistributionTumor imaging
• rat NIS cDNA : 2034bp
• pCR3.1 vector (Invitrogen co.) : TA cloning vector
• liposome : LIPOFECAmine PLUS reagent (Life technologies co.)
• Cell line : SNU-C5 (human colon cancer) SNU-449 (human hepatoma)
ARO (human anaplastic thyroid cancer)
종양세포에 rNIS 유전자 이입
Total RNA extraction RT-PCR RT-PCR
product
TA-cloning rNIS cDNA
LIPOFECTAMINE PLUS(LIFE TECHNOLOGIES Co.) 를 이용한 유전자 이입
RT-PCR
MK 1 2 3 4 5 6
1: ARO 2: ARO-NIS3: SNU C5 4: SNU C5-NIS5: SNU 449 6: SNU 449-NIS
1. Cell culture (1×106 cells)
2. 0.1uCi carrier free Na125I and 10uM NaI (500 ul HBSS)
3. Incubation (at 37 for 30 min)℃
4. Washing (washed twice, ice-cold 2ml HBSS)
5. Trypsinized
6. Gamma counter
7. Cell count
Iodide uptake
Iodide uptake was expressed as pmol/106 cells
Time (min)
0 30 60 90 120
Iod
ide
up
tak
e (
pm
ol/ 1
06 c
ells
)
0
200
400
600
800
1000
ARO ARO-NIS
Time course of iodide uptake by ARO-rNIS and ARO.
SNU-C5
SNU-C5-NIS
SNU-449
SNU-449-NISARO
ARO-NIS
0
20
40
60
80
100
800
1000Io
did
e u
pta
ke (
pm
ol/
10
6 c
ells)
Cell lines
Iodide uptake in SNU-C5, SNU-449 and ARO cell lines after 30min incubation with Na125I
Iod
ide
up
take
(p
mo
l /10
6
cells
)
0
5
10
15
20
25
30
35
40
No treatment DIDS 100μM DIDS 300μM Li KClO4
SNU-C5 SNU-C5-NIS
Effect of DIDS, Li and perchlorate on 125I- uptake in SNU-C5-rNIS cell line after 1 h incubation. Data are mean values and SD (n=4).
1. Cell culture (1×106 cells)
2. 0.1uCi carrier free Na125I and 10uM NaI (500 ul HBSS)
3. Incubation (at 37 for 1hr)℃
4. Washing (washed twice, ice-cold 2ml HBSS)
5. Incubation (500ul HBSS) - Time (0, 3, 6, 9, 12, 15, 21, 27 min)
6. Gamma counter (medium)
Iodide efflux
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20 25 30Time (min)
Rem
ain
ing
act
ivit
y (%
)
ControlLiDIDS 1DIDS 2
Iodide efflux (SNU-449-NIS)
Iodide efflux from SNU-449-rNIS cells after 1 h incubation with Na125I. The media were buffered HBSS 1) with 10 μM cold NaI (control), 2) with HBSS in which the Na+ was replaced with 140 mM lithium+(Li), 3) with 100 μM (DIDS 1) or 300 μM (DIDS 2) DIDS. Data are mean values and SD (n=3).
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20 25 30
ControlLiDIDS 1DIDS 2
Time (min)
Rem
ain
ing
act
ivit
y (%
)Iodide efflux (SNU-C5-NIS)
Iodide efflux from SNU-C5-rNIS cells after 1 h incubation with Na125I. The media were buffered HBSS 1) with 10 μM cold NaI (control), 2) with HBSS in which the Na+ was replaced with 140 mM lithium+(Li), 3) with 100 μM (DIDS 1) or 300 μM (DIDS 2) DIDS. Data are mean values and SD (n=3).
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20 25 30
Rem
ain
ing
act
ivit
y (%
) ControlLiDIDS 1DIDS 2
Time (min)
Iodide efflux (ARO-NIS)
Iodide efflux from ARO-rNIS cells after 1 h incubation with Na125I. The media were buffered HBSS 1) with 10 μM cold NaI (control), 2) with HBSS in which the Na+ was replaced with 140 mM lithium+(Li), 3) with 100 μM (DIDS 100 μM) or 300 μM (DIDS 300 μM) DIDS. Data are mean values and SD (n=3).
• Specific function
- deiodinase, NIS
• Cell-cell, cell-matrix interaction
- adhesion molecule, E-cadherin
• Differentiation marker
• Growth
• Tumorigenicity
Retinoic Acid
SNU C5 SNU C5-NIS0
2
4
6
8
10
12
14
16
18
NT3 days5 days
Iod
ide
up
take
(p
mo
l/ 10
6
cells
)
Iodide uptake in SNU-C5-rNIS cells by RA. SNU-C5-rNIS cells were treated for 3 or 5 days with 1 μM all trans-Retinoic acid(RA). Data are mean values and SD (n=4).
SNU-449 SNU-449-NIS0
10
20
30
40
50
Iod
ide
up
take
(p
mo
l/ 10
6
cells
)
NT3 days5 days
Iodide uptake in SNU-449-rNIS cells by RA. SNU-449-rNIS cells were treated for 3 or 5 days with 1 μM all trans-Retinoic acid(RA). Data are mean values and SD (n=4).
ARO ARO-NIS0
2000
4000
6000
8000
10000
Iod
ide
up
take
(p
mo
l/ 10
6
cells
)NT3 days5 days
Iodide uptake in ARO-rNIS cells by RA. ARO-rNIS cells were treated for 3 or 5 days with 1 μM all trans-Retinoic acid(RA). Data are mean values and SD (n=4).
• Subject: Nude mice ( male, 22.2 ± 3.2 g, n = 7)
• Radiopharmaceutical: Na125I, 99mTc
• Dose: 2.5 μCi/ 0.1 ml (i.v. injection)
• Time: 10, 30, 60 and 120 min
• Measurement: weighing and counting
Biodistribution of Na125I in tumor bearing nude mice
A) B)
Scintigraphic images of tumor-bearing mice with subcutaneously transplanted ARO-NIS or ARO cells at 30 min after injection of 131I-(A) and at 60 min after injection of 99mTc
ARO
ARO
AROARO-NIS ARO-NIS
bloo
d
mus
cle
hear
t
lung
liver
sple
en
stom
ach
Inte
stin
e
kidn
ey
brai
n
bone
AR
O
AR
O-N
IS
% ID
/g
0
20
40
60
80
100 10 min 30 min 60 min 120 min
Biodistribution data of 125I- in tumor-bearing mouse. Data are mean values and SD of %ID/g (n=7).
bloo
d
mus
cle
hear
t
lung
liver
sple
en
stom
ach
Inte
stin
e
kidn
ey
brai
n
bone
AR
O
AR
O-N
IS
% ID
/g
0
20
40
60
80
10010 min 30 min 60 min 120 min
Biodistribution data of 99mTc in tumor-bearing mouse. Data are mean values and SD of %ID/g (n=7).
• Co-transfection of iodine retaining proteinTPO (thyroid peroxidase) genethyroglobulin gene
• Activation of sodium/iodide symporterretinoic acidTSH
• Application in non-thyroid cancersbreast cancerstomach cancer
Future PlanFuture Plan
NIS as a Reporter Gene NIS as a Reporter Gene
for Gene Imagingfor Gene ImagingNIS as a Reporter Gene NIS as a Reporter Gene
for Gene Imagingfor Gene Imaging
• Development of Biotechnology - Cloning and manipulation of gene - Gene transfer and gene therapy
• Necessity for evaluating of gene - Quantity, distribution, duration
• Methods for analyze of gene : Reporter Gene - Indirect measure method
What is Reporter Gene?
• Classic methods : -galactosidase,
alkaline phosphatase
- Require biopsy, death of subject
• Resent methods
: fluorescent protein, luciferase
• PET : HSV1-tk, D2R - Non-invasive, repetitive
What is Reporter Gene?
• By receptor : bond radio ligand probe - Dopamine 2 Receptor
• By enzyme : sequester radio substrate probe - Herpes Simplex Virus 1 Thymidine Kinase(HSV1-tk)
Class of PET reporter gene
Promoter HSV-tk Gene
Enzyme(tk)
18F-Acyclovir(washout)
18F-Acyclovir-phosphate(retention)
• Visualize opaque tissue
• Quantify expression level of reporter gene
• Noninvasive, repetitive
• Advantages of radio nuclide-based methods
- Highly sensitive : 10-12mol/L of radio substrate good for weak promoter
- Highly quantitative : dynamic image, kinetic model
PET reporter genes
• Difficult to prepare substrate- FIAU, FPCV, FGCV : HSV1-tk- FESP : D2R
• Require PET system- High price equipment- Not available for majority
• Immune response (exogenous) : HSV1-tk
• Physiological effect (cAMP level)
Disadvantages of PET Tracers
Purpose Evaluate NIS as a reporter gene
- Compare with HSV1- tk- Prospect as a In Vitro reporter gene- Prospect as a In Vivo reporter gene
NIS as a Reporter GeneNIS as a Reporter Gene
Materials and Methods Cell lines
-CT-26 : Mouse colon carcinoma
-CM : HSV1-tk transferred CT-26
-CTN : NIS transfected CT-26
-CMN : NIS transfected CM
0
5000
10000
15000
20000
25000
30000
CM CMN4 CMN8 CMN10 CT-26 CTN8 CTN9
CPM
I-125
IVDU
Comparison of radio iodide and radio labeled IVDU(Iodo vinyl deoxyuridine) uptake in CM and CMN and CT-26 and CTN
125125I / IVDU UptakeI / IVDU Uptake
CT-26CT-26 CTN-9CTN-9
CMCM CMN-4CMN-4
Scintigraphic images of tumor-bearing mice with subcutaneously transplanted CT-26, CTN-9, CMN-4 and CM (clockwise from upper left tumor) at 30 min after injection of 131I
Image of Image of 131131II
blo
od
mu
sc
le
he
art
lun
g
liv
er
sp
lee
n
sto
ma
ch
Inte
sti
ne
kid
ne
y
bra
in
tail
bo
ne
CT
CT
N
CM
CM
N
% I
D/g
0
10
20
30
40
50
60
15 min(n=3) 30 min(n=4) 1 hr(n=3) 2 hr(n=3)
Biodistribution data of 131I in tumor-bearing mouse. Data are mean values and SD of %ID/g
Bio distribution of I-131Bio distribution of I-131
blo
od
mu
sc
le
he
art
lun
g
liv
er
sp
lee
n
sto
ma
ch
Inte
sti
ne
kid
ne
y
bra
in
tail
bo
ne
CT
CT
N
CM
CM
N
% I
D/ g
0
10
20
30
40
50
60
15 min(n=3) 30 min(n=4) 1 hr(n=3) 2 hr(n=3)
Biodistribution data of IVDU[125I] in tumor-bearing mouse. Data are mean values and SD of %ID/g
Bio distribution of IVDU[Bio distribution of IVDU[125125I]I]
• Construction dual expression vectorTwo genes under one promoter : with IRESFusion of HSV1-tk gene and Zeocin resistant
Future PlanFuture Plan
NIS
IRES
HSV1-tk : ZeoR
Promoter
• Estimate correlation of
HSV1-tk and NIS
Both protein level and mRNA level