June-Key Chung, M.D. Department of Nuclear Medicine Seoul 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 Sodium/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, ug/ml) Selection (Geneticin : G418, ug/ml) in vitro Iodide uptake Iodide efflux in vitro Iodide uptake Iodide efflux in vivo Biodistribution Tumor imaging in vivo Biodistribution Tumor 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 product TA-cloning rNIS cDNA

LIPOFECT AMINE PLUS(LIFE TECHNOLOGIES Co.) 를 이용한 유전자 이입

RT-PCR MK : ARO2: ARO-NIS 3: SNU C54: SNU C5-NIS 5: SNU 4496: SNU 449-NIS

1. Cell culture (1×10 6 cells) uCi carrier free Na 125 I 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/10 6 cells

Time course of iodide uptake by ARO-rNIS and ARO.

SNU-C5 SNU-C5-NIS SNU-449 SNU-449-NIS ARO ARO-NIS Iodide uptake (pmol/10 6 cells) Cell lines Iodide uptake in SNU-C5, SNU-449 and ARO cell lines after 30min incubation with Na 125 I

Iodide uptake (pmol /10 6 cells) No treatment DIDS 100μM DIDS 300μM Li KClO 4 SNU-C5SNU-C5-NIS Effect of DIDS, Li and perchlorate on 125 I- uptake in SNU-C5-rNIS cell line after 1 h incubation. Data are mean values and SD (n=4).

1. Cell culture (1×10 6 cells) uCi carrier free Na 125 I 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

Iodide efflux (SNU-449-NIS ) Iodide efflux from SNU-449-rNIS cells after 1 h incubation with Na 125 I. 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).

Iodide efflux (SNU-C5-NIS) Iodide efflux from SNU-C5-rNIS cells after 1 h incubation with Na 125 I. 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).

Iodide efflux (ARO-NIS) Iodide efflux from ARO-rNIS cells after 1 h incubation with Na 125 I. 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 C5SNU C5-NIS NT 3 days 5 days Iodide uptake (pmol/ 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-449SNU-449-NIS Iodide uptake (pmol/ 10 6 cells) NT 3 days 5 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).

AROARO-NIS Iodide uptake (pmol/ 10 6 cells) NT 3 days 5 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: Na 125 I, 99m Tc Dose: 2.5 μCi/ 0.1 ml (i.v. injection) Time: 10, 30, 60 and 120 min Measurement: weighing and counting Biodistribution of Na 125 I 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 131 I - (A) and at 60 min after injection of 99m Tc ARO ARO-NIS

blood muscle heart lung liver spleen stomachIntestine kidney brainbone ARO ARO-NIS % ID/g min 30 min 60 min 120 min Biodistribution data of 125 I - in tumor-bearing mouse. Data are mean values and SD of %ID/g (n=7).

Biodistribution data of 99m Tc in tumor-bearing mouse. Data are mean values and SD of %ID/g (n=7).

Co-transfection of iodine retaining protein TPO (thyroid peroxidase) gene thyroglobulin gene Activation of sodium/iodide symporter retinoic acid TSH Application in non-thyroid cancers breast cancer stomach cancer Future Plan

NIS as a Reporter Gene for 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, D 2 R - 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) 18 F-Acyclovir (washout) 18 F-Acyclovir -phosphate (retention)

Visualize opaque tissue Quantify expression level of reporter gene Noninvasive, repetitive Advantages of radio nuclide-based methods - Highly sensitive : mol/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 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

CMCMN4CMN8CMN10CT-26CTN8CTN9 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 125 I / IVDU Uptake

CT-26 CTN-9 CM CMN-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 131 I Image of 131 I Image of 131 I

Biodistribution data of 131I in tumor-bearing mouse. Data are mean values and SD of %ID/g Bio distribution of I-131

Biodistribution data of IVDU[125I] in tumor-bearing mouse. Data are mean values and SD of %ID/g Bio distribution of IVDU[ 125 I]

Construction dual expression vector Two genes under one promoter : with IRES Fusion of HSV1-tk gene and Zeocin resistant Future Plan NIS IRES HSV1-tk : Zeo R Promoter Estimate correlation of HSV1-tk and NIS Both protein level and mRNA level