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