WO2011133216A2 - Mutant nis protein and uses thereof - Google Patents

Abstract

Disclosed are Na+/I- symporter (NIS) mutant proteins wherein glycine residue 93 is replaced with aspartic acid or glutamic acid, a method of preparing said mutant NIS protein, a transfection vector for a NIS mutant protein, and a pharmaceutical composition comprising said mutant NIS protein in a pharmaceutically acceptable carrier. The disclosure also provides a method of treating cancer or cancerous cells, comprising administering a therapeutically effective amount of a transfection vector for a NIS mutant protein or a pharmaceutical composition thereof, and administering a therapeutically effective amount of radioisotope. The disclosure further provides a method of imaging cells, comprising administering to the cells an effective amount of a transfection vector for a NIS mutant protein, administering a effective amount of imaging label, and imaging the cells.

Description

MUTANT NIS PROTEIN AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 61/342,981, filed April 22, 2010, the contents of which are hereby incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers DK41544, SC1GM086344 and CA098390 awarded by the National Institutes of Health, U.S. Department of Health and Human Services. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates generally to mutant Na⁺/I^~ symporter (NIS) protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid.

BACKGROUND OF THE INVENTION

[0004] Throughout this application various publications are referred to in parenthesis. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0005] Iodine is an essential constituent of the thyroid hormones T₃ and T₄, which are in turn required for the proper development and maturation of the central nervous system, skeletal muscle, and lungs in the fetus and newborn, and as regulators of intermediary metabolism in most cells (Braverman and Utiger, 2004); (Dohan et al., 2003). Iodine, an environmentally scarce nutrient, is solely supplied in the diet as iodide (Γ) (Carrasco, 1993). Insufficient I^" intake is the most common cause of preventable mental retardation in the world. Γ uptake into the thyroid is the first step in the biosynthesis of T₃ and T₄; I^" enters the thyroid follicular cells via the Na⁺/I^" symporter (NIS). By coupling the inward transport of Na⁺ down its electrochemical gradient to the translocation of Γ against its electrochemical gradient, NIS avidly concentrates I^" in the thyroid. NIS-mediated Γ transport is electrogenic: 2 Na⁺ ions are translocated with each I^" (Eskandari et al., 1997). It has recently been shown that NIS translocates different substrates with different stoichiometries, as NIS-mediated transport of perrhenate (ReCV) or perchlorate (C1CV) is electroneutral, i.e., 1 Na⁺ : 1 Re0₄ ^" or C10₄ ^" (Dohan et al., 2007).

[0006] NIS activity has long played a medical role in the diagnosis and treatment of thyroid disease, including the highly successful treatment of thyroid cancer with radioiodide after thyroidectomy (Mazzaferri, 2000a, b). NIS was cloned in 1996 and the protein has since been extensively characterized (Dai et al., 1996; De La Vieja et al., 2000; Dohan et al., 2001 ; Dohan et al., 2000; Eskandari et al., 1997; Levy et al., 1997; Levy et al., 1998a; Riedel et al., 2001 ; Tazebay et al., 2000). hNIS shares 84% amino acid identity and 93% similarity to rNIS. The experimentally tested secondary structure model of NIS shows a protein with 13 transmembrane segments (TMS), an extracellularly facing amino terminus (Nt), and an intracellular carboxy terminus (Ct) (Levy et al., 1998a). NIS is a highly glycosylated protein (Levy et al., 1998a), and it is phosphorylated mainly at the Q (Riedel et al., 2001).

[0007] Several cases of congenital hypothyroidism due to an Γ transport defect (ITD) have been reported (Fujiwara, 1997; Fujiwara et al., 1997; Fujiwara et al., 1998; Fujiwara et al., 2000; osugi et al., 1998a; Kosugi et al., 2002; Kosugi et al., 1998b). When untreated, ITD is clinically characterized by hypothyroidism and goiter of varying degrees, reduced Γ uptake, and a low saliva-to-plasma Γ ratio (normal > 20). Although ITD is a rare autosomal recessive disorder, identification of disease-causing NIS mutations has been made possible by the availability of the NIS cDNA sequence. To date, twelve such mutations have been reported (V59E, G93R, R124H, Δ142- 323, Q267E, C272X, T354P, Δ439-443, G395R, G543E, 515X, and Y531X). Five of these [V59E (Reed-Tsur et al., 2008), Q267E (De La Vieja et al., 2004), T354P (Levy et al., 1998b), G395R (Dohan et al., 2002), and G543E (De la Vieja et al., 2005)] have been studied in detail and provided key mechanistic information on NIS. For example, analysis of the T354P substitution revealed that this position requires an OH group at the β-carbon and led to study of other β-ΟΗ-containing residues in TMS IX. It has been shown that these amino acids are involved in Na⁺ binding/translocation and proposed a structural homology between LeuT and NIS, even though they belong to different families and there is no primary sequence homology between them (De la Vieja et al., 2007). LeuT is the leucine transporter from Aquifex aeolicus, the structure of which was determined at very high resolution (1.6 A) (Yamashita et al., 2005). A remarkable surprise was that vSGLT, the Vibrio parahaemolyticus Na⁺/galactose transporter, which belongs to the same family as NIS, actually displays the same fold as LeuT (Abramson and Wright, 2009; Faham et al., 2008) (Krishnamurthy et al., 2009).

[0008] Radioiodide has been used for over 60 years to treat thyroidal cancers. The use of gene therapy to transfect cancer cells with wildtype NIS has been proposed to allow radioiodide treatment of extrathyroidal cancers. However, the radioiodide treatment of such exogenously NIS protein-expressing cancer cells, or of extrathyroidal endogenously NIS protein-expressing cancers, such as breast cancer, requires protection of the otherwise- healthy thyroid. Protection of the thyroid requires either administration of thyroxine or "cold" iodide. Both methods of protection limit the use and efficacy of the treatment and may be contraindicated or have high incidences of noncompliance in some subjects. The present invention provides a new method of treating tumors with minimal impact on thyroid tissue as well as a novel method of imaging cells.

SUMMARY OF THE INVENTION

[0009] The present invention provides an isolated Na⁺/F symporter (NIS) mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid. The present invention also provides an isolated Na⁺/F symporter (NIS) protein modified to preferentially transport perrhenate (Re0₄ ^*) rather than iodide (F) comprising a NIS mutant protein wherein glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid. The present invention further provides a method of preparing a Na⁺/F symporter (NIS) mutant protein modified to preferentially transport perrhenate (Re0₄ ^') rather than iodide (F), the method comprising replacing glycine at amino acid residue 93 of the NIS protein with aspartic acid or glutamic acid. Additionally, the present invention provides a Na⁺/F symporter (NIS) mutant protein made by replacing glycine at amino acid residue 93 of the NIS protein with aspartic acid or glutamic acid.

[0010] The present invention provides a transfection vector for a Na⁺/F symporter (NIS) mutant protein, the vector comprising a heterologous nucleic acid encoding a promoter and a NIS mutant protein wherein glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid.

[0011] The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of the transfection vector for a Na⁺/F symporter (NIS) mutant protein, the vector comprising a heterologous nucleic acid encoding a promoter and a NIS mutant protein wherein glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid, in a pharmaceutically acceptable carrier.

[0012] The present invention provides a method of treating cancer or cancerous cells, the method comprising administering a therapeutically effective amount of a transfection vector for a Na⁺/F symporter (NIS) mutant protein or a pharmaceutical composition thereof, and administering a therapeutically effective amount of radioisotope, wherein the transfection vector for the NIS mutant protein comprises a heterologous nucleic acid encoding a promoter and a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the radioisotope is Re0₄ ^" and/or Re0₄ ^".

[0013] The present invention further provides a method of imaging cells, the method comprising administering to the cells an effective amount of a transfection vector for a Na⁺/I^" symporter (NIS) mutant protein, administering a effective amount of imaging label, and imaging the cells, wherein the transfection vector for the NIS mutant protein comprises a heterologous nucleic acid encoding a promoter and a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the imaging label is ¹⁸⁸Re0₄ ^" and/or ¹⁸⁶Re0₄ ^".

[0014] The present invention additionally provides the use of a transfection vector for a Na⁺/I^" symporter (NIS) mutant protein or a pharmaceutical composition thereof and a radioisotope for the treatment of cancer, wherein the transfection vector for the NIS mutant protein comprises a heterologous nucleic acid encoding a promoter and a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the radioisotope is ¹⁸⁸Re0^4" and/or ¹⁸⁶Re0⁴.

[0015] ■ An isolated Na+/I- symporter (NIS) mutant protein is provided wherein glycine residue 93 is replaced with aspartic acid or glutamic acid.

[0016] Also provided is a method of preparing a mutant Na+/I- symporter (NIS) protein modified to preferentially transport perrhenate (Re04-) rather than iodide (I-), the method comprising replacing glycine at amino acid residue 93 of the NIS protein with aspartic acid or glutamic acid.

[0017] Also provided is a mutant Na+/I- symporter (NIS) protein made by the instant method.

[0018] Also provided is a transfection vector for a Na+/I- symporter (NIS) mutant protein, the vector comprising a heterologous nucleic acid comprising a promoter and encoding a NIS mutant protein wherein glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid.

[0019] Also provided is a pharmaceutical composition comprising a therapeutically effective amount of the transfection vector in a pharmaceutically acceptable carrier.

[0020] Also provided is a method of treating cancer, the method comprising administering a therapeutically effective amount of a transfection vector for a Na+/I- symporter (NIS) mutant protein or a pharmaceutical composition thereof to a subject, and administering a therapeutically effective amount of a radioisotope, wherein the transfection vector for the NIS mutant protein comprises a heterologous nucleic acid comprising a promoter and encoding a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the radioisotope is 188Re04- and/or 186Re04-, under conditions permitting cells of the cancer to express the NIS mutant protein and transport the radioisotope thereby treating the cancer.

[0021] Also provided is a method of imaging cells, the method comprising contacting the cells with an effective amount of a transfection vector for a Na+/I- symporter (NIS) mutant protein, administering a effective amount of imaging label, and imaging the cells, wherein the transfection vector for the NIS mutant protein comprises cDNA comprising a promoter and encoding a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the imaging label is ¹⁸⁸Re0^4" and/or ¹⁸ Re0^4".

[0022] Also provided is use of a transfection vector for a Na+/I- symporter (NIS) mutant protein or a pharmaceutical composition thereof and a radioisotope for the treatment of cancer, wherein the transfection vector for the NIS mutant protein comprises cDNA encoding a promoter and a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the radioisotope is ¹⁸⁸Re0^4" and/or ¹⁸⁶Re0⁴-.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A-1I. Analysis of G93 NIS substitutions in COS-7 cells and Xenopus laevis oocytes. (1A) and (IB) Steady-state Γ transport in transfected COS-7 cells; (1A) 20 μΜ Γ and 140 mM Na⁺ in the absence (dark bars) or presence of 80 μΜ C10₄ ^" (light bars); (IB) 200 μΜ Γ and 140mM Na⁺ in the absence (dark bars) or presence of 800 μΜ C10₄ ^' (light bars). Results are expressed in pmol 17 μg DNA ± S.D. Values shown are one of at least five different experiments and corrected for transfection efficiency; in each experiment, activity was analyzed in triplicate. (1C) Flow cytometry under non- permeabilized conditions with an anti-HA Ab against the extracellularly facing HA epitope at the NIS N,. WT (19 %) and G93R rNIS (15 %). (ID) HA immunostaining of WT and G93R rNIS under non-permeabilized conditions. NIS is stained with Alexa 488 and nuclei with DAPI. Scale bar = 20 μιτι. (IE) and (IF) Current traces are shown in response to 5 m Γ in (E) control (water-injected) Xenopus Iaevis oocytes or oocytes expressing WT, G93R, or K rNIS, or (F) G93A, N, T, or Q rNIS. The evoked inward currents represent NIS-mediated electrogenic Na⁺/T cotransport into the cell. V_m - -50 mV. (1G) I- transport as in (A) in COS-7 cells transfected with WT, G93A, N, Q, S, or T rNIS cDNAs. (1H) I^" transport as in (B), in COS-7 cells transfected with WT, G93A, N, Q, S, or T rNIS cDNAs. (II) Steady-state kinetics of I^" transport by WT and G93 , A, S, T, and N rNIS. [Na⁺]₀ = 100 mM and V_m = -50 mV. For each mutant, the current values were normalized to the maximum current obtained at saturating [Γ]. The smooth lines are fits of the data to the Michaelis-Menten equation. Values represent the mean ± S.E. from at least four oocytes.

[0024] Figure 2A-2F. Kinetics of Γ transport in MDCK cells and Xenopus laevis oocytes. (2A) Steady-state kinetics of Na+ dependence of Γ transport by WT, G93T, and N rNIS. [Γ] = 5. mM and V_m - -50 mV. Current values were normalized to the maximum current obtained at saturating [Na⁺]. The smooth lines are fits of the data to the Hill equation. The Hill coefficient values were 1.9 ± 0.2 for WT; 2.0 ± 0.1 for G93T; and 1.9 ± 0.1 for G93N rNIS. Values represent the mean ± S.E. from at least 4 oocytes. (2B) Flow cytometry of MDCK cells transduced with HA- WT or HA-G93T hNIS. The mean fluorescence intensity (MFI) of G93T (black) was 1.6-fold that of the WT (gray). (2C) Immunoblot analysis of transduced cells. Loading control is a-tubulin. (2D) Initial rates (2- min time points) of Γ uptake were determined at 140 mM Na⁺ and the indicated [F]s. Calculated curves (smooth lines) were generated using the equation v = {V_max x [Y])/(K_m + [Γ]). K_m({ are indicated as an average ± S.E. of 5 experiments. Background in NT cells (<5 DNA) was subtracted. The graph shown is a representative experiment. (2E) and (2F) Na⁺ dependence of f uptake. Cells were incubated for 2 min with (2E) 250 or (2F) 750 μΜ Γ and the indicated [Na⁺]s. Calculated curves (smooth lines) were generated using the equation v = (V_max x Na⁺]²)/(K_m ² + [Na⁺]²). Shown graph is a representative of >3 experiments; in each, the activity was analyzed in triplicate. Isotonicity was maintained constant with choline CI. In all flux experiments, results are expressed in pmol 17 μ DNA ± S.D., activity was analyzed in triplicates and background obtained with NT cells (<4 and 12 pmol^g DNA, respectively) was subtracted. WT is shown in gray and G93T in black for panels (2A), (2B), and (2D)-2(F).

[0025] Figure 3A-3E. Re0₄ ^" transport kinetics in MDCK cells and Xenopus laevis oocytes. (3A) Initial rates (2-min) of Re0₄ ^" uptake in transduced MDCK cells were determined at 140 mM Na⁺ and the indicated [Re0₄ ^"]s. Calculated curves (smooth lines) were generated using the equation v = (V_max x [Re0₄ ^"])/(AT_m + [Re0₄ ^"]). WT shown in gray, G93T hNIS in black. Background in NT cells (<2

DNA) was subtracted. (3B) Na⁺ dependence of Re0₄ ^" transport: cells were incubated for 2 min with 180 μΜ Re0₄ ^" and the indicated [Na⁺]s. K_m(Re04 ) are indicated as an average ± S.E. of all experiments. The graph shown is a representative experiment. Calculated curves (smooth lines) were generated using the equation v = (V_max x \Na⁺])/(K_m + [Na⁺]) for WT hNIS and v = (V_max x {Na⁺]²)/(K_m ² + [Na⁺]²) for G93T hNIS. Background obtained with NT cells (<2 pmol^g DNA) was subtracted. WT shown in gray, G93T hNIS in black. (3C) Current traces are shown in response to 1 mM C10₄ ^" (top traces) or Re0₄ ^" (bottom traces) in oocytes expressing WT or G93R, K, N, or T rNIS. V_m = -50 mV. For each mutant, C10₄ ^" and Re0₄ ^" traces were obtained in the same oocyte. (3D) Steady-state kinetics of C10₄ ^* (left panel) and Re0₄ ^" (right panel) transport by G93T and G93N rNIS in oocytes. [Na⁺]₀ = 100 mM and V_m - -50 mV. Data analyzed as in Figure I I. (3E) Steady-state kinetics of Na⁺ dependence of anion transport by G93T and G93N rNIS in oocytes with 1 mM C10₄ ^" (left panel) or Re0₄ ^" (right panel) as the substrate. V_m = -50 mV. Data were processed as in Figure 2A. The Hill coefficient values were 1.9 ± 0.2 for G93N, and 2.2 ± 0.2 for G93T rNIS with C10₄ ^", and 2.1 ± 0.1 for G93N and 2.1 ± 0.1 for G93T, with Re0₄ ^' .

[0026] Figure 4A-4I. G93E NIS does not transport Γ but transports Re0₄ ^" electrogenically. (4A) Steady-state Re0₄ ^" transport assays in NT or COS-7 cells transfected with WT or G93K rNIS cDNAs. Cells were incubated for 1 h with 3 μΜ Re0₄ ^" and 140 mM Na⁺ in the absence (dark bars) or presence of 120 μΜ C10₄ ^" (light bars). (4B) and (4C) Steady-state Γ transport activity was assayed in WT, G93D and G93E rNIS-expressing COS-7 cells as in Figure 1 A and B. (4D) Flow cytometry under permeabilized conditions: WT (17.02%), G93E rNIS (29.89%). (4E) Cell surface biotinylation. The broad -80 kDa band corresponds to the mature form of NIS. The Na⁺/K⁺ ATPase a-subunit was used as a loading control (lower panel). (4F) Steady-state Re0₄ ^" transport in WT, G93D, and G93E rNIS-expressing COS-7 cells as in (4A). (4G) Steady-state transport in G93E rNIS- expressing COS-7 cells following 1 h incubation with 30 μΜ Re0₄ ^" and 140 mM Na⁺ in the absence (dark bars) or presence of 800 μΜ C10_4- (light bars). (4H) Current traces in response to 5 mM Γ (left), 1 mM C10₄ ^" (middle) or Re0₄ ^" (right) in oocytes expressing G93D (top traces), G93E (middle traces) or G93Q rNIS (bottom traces). V_m = -50 mV. For each mutant, all current traces were obtained in the same oocyte. (41) Steady-state kinetics of anion transport by G93 D, E and Q rNIS as in Figure II.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides an isolated Na⁺/T symporter (NIS) mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid. The present invention also provides an isolated Na⁺/F symporter (NIS) protein modified to preferentially transport perrhenate (Re0₄ ^') rather than iodide (F) comprising NIS protein wherein glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid. The present invention further provides a method of preparing a Na⁺/F symporter (NIS) mutant protein modified to preferentially transport perrhenate (Re0₄ ^") rather than iodide (F), the method comprising replacing glycine at amino acid residue 93 of the NIS protein with aspartic acid or glutamic acid. Additionally, the present invention provides a Na⁺/F symporter (NIS) mutant protein made by replacing glycine at amino acid residue 93 of the NIS protein with aspartic acid or glutamic acid.

[0028] NIS is an ion symporter that actively carries iodide (F) across the cellular plasma membrane. NIS is found in human thyroid epithelial cells as well as salivary ductal glands, mucus-secreting cells in the stomach, cells in lactating breast, breast cancer cells, and intestinal brush cells, among others. Many mammals, including but not limited to rodents, have NIS orthologs. Normal NIS protein actively concentrates iodide in the cell. When glycine residue 93 (G93) is replaced with aspartic acid (G93D) or glutamic acid (G93E), the resultant NIS mutant protein will preferentially translocate perrhenate (Re0₄ ^"), rather than iodide. G93 can be replaced by aspartic acid or glutamic acid by any appropriate method known in the art.

[0029] Human NIS protein has the following amino acid sequence: MEAVETGERP TFGAWDYGVF ALMLLVSTGI GLWVGLARGG QRSAEDFFTG GRRLAALPVG LSLSASFMSA VQVLGVPSEA YRYGL FLWM CLGQLLNSVL TALLFMPVFY RLGLTSTYEY LEMRFSRAVR LCGTLQYIVA TMLYTGIVIY APALILNQVT GLDIWASLLS TGIICTFYTA VGGMKAVVWT DVFQVVVMLS GFWVVLARGV MLVGGPRQVL TLAQNHSRTN LMDFNPDPRS RYTFWTFVVG GTLVWLSMYG VNQAQVQRYV ACRTEKQAKL ALLINQVGLF LIVSSAACCG IVMFVFYTDC DPLLLGRISA PDQYMPLLVL DIFEDLPGVP GLFLACAYSG TLSTASTSIN AMAAVTVEDL IKPRLRSLAP RKLVIISKGL SLIYGSACLT VAALSSLLGG GVLQGSFTVM GVISGPLLGA FILGMFLPAC NTPGVLAGLG AGLALSLWVA LGATLYPPSE QTMRVLPSSA ARCVALSVNA SGLLDPALLP ANDSSRAPSS GMDASRPALA DSFYAISYLY YGALGTLTTV LCGALISCLT GPTKRSTLAP GLLWWDLARQ TASVAPKEEV AILDDNLVKG PEELPTGNKK PPGFLPTNED RLFFLGQKEL EGAGSWTPCV GHDGGRDQQE TNL (SEQ ID NO: l). In one version of mutant human NIS, glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid (SEQ ID NO:2): MEAVETGERP TFGAWDYGVF ALMLLVSTGI GLWVGLARGG QRSAEDFFTG GRRLAALPVG LSLSASFMSA VQVLGVPSEA YRYGLKFLWM CLXQLLNSVL TALLFMPVFY RLGLTSTYEY LEMRFSRAVR LCGTLQYIVA TMLYTGIVIY APALILNQVT GLDIWASLLS TGIICTFYTA VGGMKAVVWT DVFQVVVMLS GFWVVLARGV MLVGGPRQVL TLAQNHSRIN LMDFNPDPRS RYTFWTFVVG GTLVWLSMYG VNQAQVQRYV ACRTEKQAKL ALLINQVGLF LIVSSAACCG IVMFVFYTDC DPLLLGRISA PDQYMPLLVL DIFEDLPGVP GLFLACAYSG TLSTASTSIN AMAAVTVEDL IKPRLRSLAP RKLVIISKGL SLIYGSACLT VAALSSLLGG GVLQGSFTVM GVISGPLLGA FILGMFLPAC NTPGVLAGLG AGLALSLWVA LGATLYPPSE QTMRVLPSSA ARCVALSVNA SGLLDPALLP ANDSSRAPSS GMDASRPALA DSFYAISYLY YGALGTLTTV LCGALISCLT GPTKRSTLAP GLLWWDLARQ TASVAPKEEV AILDDNLVKG PEELPTGNKK PPGFLPTNED RLFFLGQKEL EGAGSWTPCV GHDGGRDQQE TNL, where X is D or E. In one version of mutant human NIS, glycine at amino acid residue 93 is replaced with aspartic acid (SEQ ID NO:3): MEAVETGERP TFGAWDYGVF ALMLLVSTGI GLWVGLARGG QRSAEDFFTG GRRLAALPVG LSLSASFMSA VQVLGVPSEA YRYGLKFLWM CLDQLLNSVL TALLFMPVFY RLGLTSTYEY LEMRFSRAVR LCGTLQYIVA TMLYTGIVIY APALILNQVT GLDIWASLLS TGIICTFYTA VGGMKAVVWT DVFQVVVMLS GFWVVLARGV MLVGGPRQVL TLAQNHSRIN LMDFNPDPRS RYTFWTFVVG GTLVWLSMYG VNQAQVQRYV ACRTEKQAKL ALLINQVGLF LIVSSAACCG IVMFVFYTDC DPLLLGRISA PDQYMPLLVL DIFEDLPGVP GLFLACAYSG TLSTASTSIN AMAAVTVEDL IKPRLRSLAP RKLVIISKGL SLIYGSACLT VAALSSLLGG GVLQGSFTVM GVISGPLLGA FILGMFLPAC NTPGVLAGLG AGLALSLWVA LGATLYPPSE QTMRVLPSSA ARCVALSVNA SGLLDPALLP ANDSSRAPSS GMDASR ALA DSFYAISYLY YGALGTLTTV LCGALISCLT GPTKRSTLAP GLLWWDLARQ TASVAPKEEV AILDDNLVKG PEELPTGNKK PPGFLPTNED RLFFLGQKEL EGAGSWTPCV GHDGGRDQQE TNL. In one version of mutant human NIS, glycine at amino acid residue 93 is replaced with glutamic acid (SEQ ID NO:4): MEAVETGERP TFGAWDYGVF ALMLLVSTGI GLWVGLARGG QRSAEDFFTG GRRLAALPVG LSLSASFMSA VQVLGVPSEA YRYGLKFLWM CLEQLLNSVL TALLFMPVFY RLGLTSTYEY LEMRFSRAVR LCGTLQYIVA TMLYTGIVIY APALILNQVT GLDIWASLLS TGIICTFYTA VGGMKAVVWT DVFQVVVMLS GFWVVLARGV MLVGGPRQVL TLAQNHSRIN LMDFNPDPRS RYTFWTFVVG GTLVWLSMYG VNQAQVQRYV ACRTEKQAKL ALLINQVGLF LIVSSAACCG IVMFVFYTDC DPLLLGRISA PDQYMPLLVL DIFEDLPGVP GLFLACAYSG TLSTASTSIN AMAAVTVEDL IKPRLRSLAP RKLVIISKGL SLIYGSACLT VAALSSLLGG GVLQGSFTVM GVISGPLLGA FILGMFLPAC NTPGVLAGLG AGLALSLWVA LGATLYPPSE QTMRVLPSSA ARCVALSVNA SGLLDPALLP ANDSSRAPSS GMDASRPALA DSFYAISYLY YGALGTLTTV LCGALISCLT GPTKRSTLAP GLLWWDLARQ TASVAPKEEV AILDDNLVKG PEELPTGNKK PPGFLPTNED RLFFLGQKEL EGAGSWTPCV GHDGGRDQQE TNL.

[0030] Mouse NIS protein has the following amino acid sequence: MEGAEAGARA TFGPWDYGVF ATMLLVSTGI GLWVGLARGG QRSADDFFTG GRQLAAVPVG LSLAASFMSA VQVLGVPAEA ARYGLKFLWM CVGQLLNSLL TALLFLPIFY RLGLTSTYQY LELRFSRAVR LCGTLQYLVA TMLYTGIVIY APALILNQVT GLDIWASLLS TGIICTLYTT VGGMKAVVWT DVFQVVVMLV GFWVILARGV MLMGGPWNVL SLAQNHSRIN LMDFDPDPRS RYTFWTFVVG GSLVWLSMYG VNQAQVQRYV ACHTERKAKL ALLVNQLGLF LIVASAACCG IVMFVYYKDC DPLLTGRIAA PDQYMPLLVL DIFEDLPGVP GLFLACAYSG TLSTASTSIN AMAAVTVEDL IKPRMPSLAP RKLVFISKGL SFIYGSTCLT VAALSSLLGG GVLQGSFTVM GVISGPLLGA FTLGMLLPAC NTPGVLSGLT AGLAVSLWVA VGATLYPPGE QTMGVLPTSA AGCTNASVLP SPPGAANTSR GIPSSGMDSG RPAFADTFYA VSYLYYGALG TLTTMLCGAL ISYLTGPTKR SSLGPGLLWW DLARQTASVA PKEDTTTLED SLVKGPEDIP AATKKPPGFR PEAETHPLYL GHDAETNL (SEQ ID NO:5). In one version of mutant mouse NIS, glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid (SEQ ID NO:6). In one version of mutant mouse NIS, glycine at amino acid residue 93 is replaced with aspartic acid (SEQ ID NO:7). In one version of mutant mouse NIS, glycine at amino acid residue 93 is replaced with glutamic acid (SEQ ID NO: 8).

[0031] Rat NIS protein has the following amino acid sequence: MEGAEAGARA TFGAWDYGVF ATMLLVSTGI GLWVGLARGG QRSADDFFTG GRQLAAVPVG LSLAASFMSA VQVLGVPAEA ARYGLKFLWM CAGQLLNSLL TAFLFLPIFY RLGLTSTYQY LELRFSRAVR LCGTLQYLVA TMLYTGIVIY APALILNQVT GLDIWASLLS TGIICTLYTT VGGMKAVVWT DVFQVVVMLV GFWVILARGV ILLGGPRNML SLAQNHSRIN LMDFDPDPRS RYTFWTFIVG GTLVWLSMYG VNQAQVQRYV ACHTEGKAKL ALLVNQLGLF LIVASAACCG IVMFVYYKDC DPLLTGRISA PDQYMPLLVL DIFEDLPGVP GLFLACAYSG TLSTASTSIN AMAAVTVEDL IKPRMPGLAP RKLVFISKGL SFIYGSACLT VAALSSLLGG GVLQGSFTVM GVISGPLLGA FTLGMLLPAC NTPGVLSGLA AGLAVSLWVA VGATLYPPGE QTMGVLPTSA AGCTNDSVLL GPPGATNASN GIPSSGMDTG RPALADTFYA ISYLYYGALG TLTTMLCGAL ISYLTGPTK SSLGPGLLWW DLARQTASVA PKEDTATLEE SLVKGPEDIP AVTKKPPGLK PGAETHPLYL GHDVETNL (SEQ ID NO:9). In one version of mutant rat NIS, glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid (SEQ ID NO: 10). In one version of mutant rat NIS, glycine at amino acid residue 93 is replaced with aspartic acid (SEQ ID NO:l 1). In one version of mutant rat NIS, glycine at amino acid residue 93 is replaced with glutamic acid (SEQ ID NO: 12).

[0032] Other vertebrates, particularly mammals, have NIS homologues, many of which contain the same glycine residue 93. Additionally, some bacterial species have NIS homologues, many with key residues conserved.

[0033] The present invention provides a transfection vector for a Na⁺/I^" symporter (NIS) mutant protein, the vector comprising a nucleic acid encoding a promoter and encoding a NIS mutant protein wherein glycine at amino acid residue 93 of SEQ ID NO: l is replaced with aspartic acid or glutamic acid (SEQ ID NO:2).

[0034] Transfection is a process of deliberately introducing heterologous nucleic acid(s) into a cell such that expression of the nucleic acid or portion thereof occurs in the cell. When a viral method is used, the virus mediating the transfer of the genetic material is the vector. A cell is "transduced" when a vector has introduced a heterologous nucleic acid into the cell such that the cell can express the nucleic acid or portion thereof. The vector may comprise a nucleic acid coding for a promoter and the NIS mutant protein. For example, the nucleic acid may be obtained through site-directed mutation of NIS protein cDNA. A promoter is nucleic acid sequence that facilitates transcription of a gene. Promoters may be general, or may be specific to a tissue- or cell-type. A general promoter will be recognized by many tissue- or cell-types. A specific promoter may be recognized by a subset of tissue- or cell-types, or may be specific enough to recognize only a single cell-type. When a cell has been transduced, only a cell recognizing the promoter will transcribe the transfected nucleic acid.

[0035] The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of the transfection vector for a Na⁺/I^" symporter (NIS) mutant protein, the vector comprising cDNA comprising a promoter and encoding a NIS mutant protein wherein glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid, in a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise the transfection vector for a Na⁺/I^" symporter (NIS) mutant protein, the vector comprising cDNA encoding a promoter and a NIS mutant protein wherein glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid, in a pharmaceutically acceptable carrier. Alternatively, the pharmaceutical composition may consist essentially of the transfection vector for a Na⁺/I^" symporter (NIS) mutant protein, the vector comprising cDNA encoding a promoter and a NIS mutant protein wherein glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid, in a pharmaceutically acceptable carrier. Yet alternatively, the pharmaceutical composition may consist of the transfection vector for a Na+/I- symporter (NIS) mutant protein, the vector comprising cDNA encoding a promoter and a NIS mutant protein wherein glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid, in a pharmaceutically acceptable carrier.

[0036] The pharmaceutically acceptable carrier must be compatible with the transfection vector for the NIS mutant protein, and not deleterious to the subject to which the pharmaceutical composition is to be administered. Examples of acceptable pharmaceutical carriers include caboxymethylcellulose, crystalline cellulose, glycerine, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc and water, among others. Formulations of the pharmaceutical composition may conveniently be presented in unit dosage and may be prepared by any method known in the pharmaceutical art. For example, the putative agent may be brought into association with a carrier or diluent, as a suspension or solution. Optionally, one or more accessory ingredients, such as buffers, flavoring agents, surface active ingredients, and the like, may be added. The choice of carriers will depend on the method of administration. The pharmaceutical composition would be useful for treating cancer. The transfection vector for the NIS mutant protein is provided in amounts effective to treat cancer. These amounts may be readily determined by one ordinarily skilled in the art. In one embodiment, the transfection vector for the NIS mutant protein is the sole active pharmaceutical ingredient in the formulation of composition. In another embodiment, there may be a number of active pharmaceutical ingredients in the formulation or composition aside from the transfection vector for the NIS mutant protein. In this embodiment, the other active pharmaceutical ingredients in the formulation or composition must be compatible with the putative agent.

[0037] The present invention provides a method of treating cancer or cancerous cells, the method comprising administering a therapeutically effective amount of a transfection vector for a Na+/I- symporter (NIS) mutant protein or a pharmaceutical composition thereof, and administering a therapeutically effective amount of radioisotope, wherein the transfection vector for the NIS mutant protein comprises cDNA encoding a promoter and a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the radioisotope is 188Re04- and/or 186Re04-.

[0038] An isolated Na+/I- symporter (NIS) mutant protein is provided wherein glycine residue 93 is replaced with aspartic acid or glutamic acid.

[0039] In an embodiment, the NIS mutant protein preferentially transports perrhenate (Re04-) rather than iodide (I-). In an embodiment, the NIS mutant protein has the sequence set forth in SEQ ID NO:l with the glycine residue 93 replaced with aspartic acid. In an embodiment, the NIS mutant protein has the sequence set forth in SEQ ID NO: 1 with the glycine residue 93 replaced with glutamic acid. In an embodiment, the NIS mutant protein comprises consecutive amino acid residues having the sequence set forth in SEQ ID NO:3. In an embodiment, the NIS mutant protein comprises consecutive amino acid residues having the sequence set forth in SEQ ID NO:4.

[0040] Also provided is a method of preparing a mutant Na+/I- symporter (NIS) protein modified to preferentially transport perrhenate (Re04-) rather than iodide (I-), the method comprising replacing glycine at amino acid residue 93 of the NIS protein with aspartic acid or glutamic acid. [0041] In an embodiment, the NIS mutant protein has the sequence set forth in SEQ ID NO: 1 with the glycine residue 93 replaced with aspartic acid. In an embodiment, the NIS mutant protein has the sequence set forth in SEQ ID NO: l with the glycine residue 93 replaced with glutamic acid.

[0042] Also provided is a mutant Na+/I- symporter (NIS) protein made by the instant method.

[0043] Also provided is a transfection vector for a Na+/I- symporter (NIS) mutant protein, the vector comprising a heterologous nucleic acid comprising a promoter and encoding a NIS mutant protein wherein glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid.

[0044] In an embodiment, the transfection vector comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:3. In an embodiment, the transfection vector comprises a nucleic acid encoding the sequence set forth in SEQ ID NO:4.

[0045] Also provided is a pharmaceutical composition comprise a therapeutically effective amount of the transfection vector in a pharmaceutically acceptable carrier.

[0046] Also provided is a method of treating cancer, the method comprising administering a therapeutically effective amount of a transfection vector for a Na+/I- symporter (NIS) mutant protein or a pharmaceutical composition thereof to a subject, and administering a therapeutically effective amount of a radioisotope, wherein the transfection vector for the NIS mutant protein comprises a heterologous nucleic acid comprising a promoter and encoding a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the radioisotope is 188Re04- and/or 186Re04-, under conditions permitting cells of the cancer to express the NIS mutant protein and transport the radioisotope thereby treating the cancer.

[0047] In an embodiment of the method, the cancer is a thyroid cancer. In an embodiment of the method, the promoter is specific for the organ type of the cancer. In an embodiment of the method, the cancer is prostate, breast, pancreatic, intestinal, pulmonary, cardiac, neural, endocrine, lymph, bone or blood cancer. In an embodiment of the method, the transfection vector is specific to the thyroid. In an embodiment of the method, the NIS mutant protein has SEQ ID NO:2, 3 or 4. In an embodiment of the method, the NIS mutant protein is produced in cells transfected by the transfection vector and of the promoter's specific cell-type. [0048] In an embodiment of the method, the radioisotope is administered after sufficient time has elapsed to allow the production of NIS mutant proteins in transfected cells of the promoter's specific cell-type. In an embodiment of the method, the method further comprises administering a protective amount of nonradioactive iodide prior to administration of the radioisotope. In an embodiment of the method, the method further comprises administering a protective amount of thyroxine prior to administration of the radioisotope.

[0049] Also provided is a method of imaging cells, the method comprising contacting the cells with an effective amount of a transfection vector for a Na+/I- symporter (NIS) mutant protein, administering a effective amount of imaging label, and imaging the cells, wherein the transfection vector for the NIS mutant protein comprises a heterologous nucleic acid comprising a promoter and encoding a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the imaging label is ¹⁸⁸Re0^4" and/or ¹⁸⁶Re0⁴\

[0050] In an embodiment of the method, the imaging is effected with positron emission tomography (PET). In an embodiment of the method, the imaging is effected with single photon emission computed tomography. In an embodiment of the method, the cells being imaged are located in vivo or in vitro. In an embodiment of the method, the cells are located in vivo and are in a human. In an embodiment of the method, the cells being imaged are human stem cells. In an embodiment of the method, the human stem cells are transduced in vitro and then transplanted into a mammal. In an embodiment of the method, the human stem cells are transplanted into a human. In an embodiment of the method, the promoter is specific to the cell-type of the cells being imaged. In an embodiment of the method, the transfection vector is specific to the tissue or cell type being imaged. In an embodiment of the method, the NIS mutant protein is produced in cells transduced by the transfection vector and of the promoter's specific cell-type. In an embodiment of the method, the imaging label is administered after sufficient time has elapsed to allow the production of NIS mutant proteins in transduced cells of the promoter's specific cell-type. In an embodiment of the method, the method further comprises administering a protective amount of nonradioactive iodide prior to administration of the imaging label. In an embodiment of the method, the method further comprises administering a protective amount of thyroxine prior to administration of the imaging label. In an embodiment of the method, the imaging label is pertechnetate. [0051] In an embodiment of the methods, the NIS mutant protein has SEQ ID NO:2, 3 or 4.

[0052] Also provided is use of a transfection vector for a Na+/I- symporter (NIS) mutant protein or a pharmaceutical composition thereof and a radioisotope for the treatment of cancer, wherein the transfection vector for the NIS mutant protein comprises cDNA encoding a promoter and a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the radioisotope is ¹⁸⁸Re0^4" and/or ^l86Re0^4".

[0053] In an embodiment, the glycine residue 93 is replaced with aspartic acid. In an embodiment, the cancer is located in vivo in a human. In an embodiment, the cancer is prostate, breast, pancreatic, intestinal, pulmonary, cardiac, neural, endocrine, lymph, bone or blood cancer. In an embodiment, the promoter is specific to the cancer cell-type. In an embodiment, the transfection vector is specific to the tissue or cell type affected by the cancer. In an embodiment, the NIS mutant protein is only produced in cells transduced by the transfection vector and of the promoter's specific cell-type.

[0054] In an embodiment of the use, the subject has been administered or will be administered a radioisotope after sufficient time has elapsed to allow the production of NIS mutant proteins in transduced cells of the promoter's specific cell-type. In an embodiment of the use, the radioisotope is radioactive perrhenate.

[0055] In an embodiment of the use, the subject has been administered or will be administered a protective amount of nonradioactive iodide prior to administration of the radioisotope.

[0056] In an embodiment of the use, the subject has been administered or will be administered a protective amount of thyroxine prior to administration of the radioisotope.

[0057] In an embodiment of the methods, compositions or vectors described herein, the NIS has the sequence set forth in SEQ ID NO: l , and the NIS mutant protein has the sequence set forth in SEQ ID NO:2, 3 or 4.

[0058] As used herein, "treating" cancer, or a grammatical equivalent thereof, means effecting a clinically significant change in the cancer or cancerous cells. Such a clinically significant change in the cancer or cancerous cells may include, but is not limited to, increased senescence and apoptosis of cancer cells, increased longevity of a subject being treated for a cancer, or decreased morbidity in subjects being treated for cancer. As used herein, a "therapeutically effective" amount, or a grammatical equivalent thereof, means an amount capable of effecting a clinically significant change in the pertinent disease, in this case cancer. The therapeutically effective amount will depend on the type of cancer or cancerous cells, the location of the cancer or cancerous cells, among other factors, and can be readily determined by one of ordinary skill in the art.

[0059] As used herein, the term "heterologous nucleic acid," with regard to its presence in or introduction into a cell, ovum, embryo etc. refers to nucleic acid that is not naturally present in the cell, ovum, embryo etc. or a nucleic acid which is present in a position other than its naturally occurring position in the cell, ovum, embryo.

[0060] The vectors of the invention may be administered locally or systemically. For example, the vector may be administered locally via injection or cannulation into the site of the cancer in vivo. Systemic administration may include, for example, administering via in vivo injection or cannulation into the circulatory system.

[0061] The radioisotope administered may be any radioisotope known in the art. In an embodiment the radioisotope is radioactive perrhenate. The NIS mutant protein of the present invention preferentially transports perrhenate rather than iodide. Therefore, radioactive perrhenate will preferentially accumulate in cells with NIS mutant protein,

188 reducing possible damage to the thyroid. In one embodiment, the radioisotope is Re0₄ ^" or Re0₄ ^". Radioactive rhenium ( Re or Re) is more energetic than radioiodide and is therefore more effective at treating larger tumors than radioactive iodide. Additionally, since it has a shorter half life than radioiodide, patients undergoing treatment with radioactive perrhenate need spend less time in isolation. In an embodiment, the radioisotope is radioactive Rhenium.

[0062] The cancer or cancerous cells may be located in vivo or in vitro. Preferably, the cancer is located in a human. The cancer may be any type of human cancer including, but not limited to, prostate, breast, pancreatic, hepatic, testicular, ovarian, intestinal, pulmonary, cardiac, neural, endocrine, lymph, bone or blood cancer.

[0063] Certain viruses preferentially infect certain tissue- or cell-types. Therefore, the vector used for the transfection may be chosen to preferentially infect the cancer's tissue- or cell-type. For example, the vaccinia virus preferentially affects plasma promoter cells, the cell-type of multiple myeloma cancers. Certain adenovirus preferentially infect prostate cancer cells, while adenovirus 9 preferentially infects cells in the heart. In one embodiment, the viral vector is specific to the cancerous tissue- or cell-type including, but not limited to, one of the viral vectors described herein. [0064] A promoter is nucleic acid sequence that facilitates transcription of a gene. Promoters may be general, or may be specific to a tissue- or cell-type. A general promoter will be recognized by many tissue- or cell-types. A specific promoter may be recognized by a subset of tissue- or cell-types, or may be specific enough to recognize only a single cell- type. When a cell has been transduced, only a cell recognizing the promoter will transcribe the transfected cDNA. In one embodiment, the promoter is specific to the cancer cell-type. In this embodiment, the transduced cells of the cancer cell-type will preferentially transcribe the transfected cDNA, resulting in NIS mutant protein being preferentially located in cells of the cancer cell-type.

[0065] The radioisotope may be administered at any time after the administration of a therapeutically effective amount of a transfection vector for a Na⁺/I^" symporter (NIS) mutant protein or a pharmaceutical composition comprising such. In a preferred embodiment, the radioisotope is administered after sufficient time has elapsed to allow the production of NIS mutant proteins in transduced cells of the promoter's specific cell-type. The time will depend on the vector and promoter used, the cancer type, and the tissue and cell-type and can be easily determined by one of skill in the art.

[0066] A protective amount of nonradioactive iodide or thyroxine may be administered prior to the administration of the radioisotope. Thyroxine is a hormone produced by the thyroid gland. Administration of thyroxine or nonradioactive ("cold") iodide downregulates expression of NIS protein in thyroid cells. This means that the radioisotope administered subsequently will not damage the thyroid cells by being preferentially transported into thyroid cells by NIS protein. When the cells being treated are in vivo, administration of thyroxine may be contraindicated in some subjects. Preferably, when cold iodide is administered to downregulate thyroid cells, the radioisotope administered is not radioactive iodide since administration of radioactive iodide after downregulating thyroid cells with cold iodide results in the dilution of radioactive iodide, decreasing the efficacy of treatment. Since NIS mutant proteins preferentially transport perrhenate, thyroid cells can be protected by administration of cold iodide without affecting the efficacy of the radiotherapy by administration of radioactive perrhenate. A protective amount of cold iodide is an amount of iodide necessary to cause downregulation of NIS protein in thyroid cells and will depend on whether the cells are located in vivo or in vitro, thyroid health, and, in vivo, factors such as the subject's overall health or weight. The protective amount in each case can be easily determined by one of skill in the art. [0067] The present invention further provides a method of imaging cells, the method comprising administering to the cells an effective amount of a transfection vector encoding for a Na⁺/ symporter (NIS) mutant protein, administering a effective amount of imaging label, and imaging the cells, wherein the transfection vector for the NIS mutant protein comprises cDNA encoding a promoter and encoding a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the imaging label is ¹⁸⁸Re0₄ ^" and/or ¹⁸⁶Re0₄ ^".

[0068] Administering an effective amount of transfection vector as used herein means administering a sufficient amount of transfection vector so that the cells of interest can be imaged. The effective amount will depend on the location of the cells, the cell type of interest, and the method of imaging and can be easily determined by one of skill in the art.

[0069] The vector may be administered locally or systemically. For example, the vector may be administered locally via injection into the site of the cancer in vivo, or another direct administration into the cancer. Systemic administration may include, for example, administering via in vivo injection into the circulatory system.

[0070] The cells may be imaged by any appropriate method known in the art. When the cells are in vitro, the method of imaging may include, but is not limited to, camera or microscope. When the cells are in vivo, the method of imaging may include, but is not limited to, positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0071] The cells being imaged may be any cells. Preferably, the cells are mammalian. More preferably, the cells are human. In one embodiment, the human cells are stem cells. The cells being imaged may be in vivo or in vitro. In one embodiment, the cells may be transduced in vivo and then transplanted into a mammal. For example, human stem cells may be transduced in vitro and then transplanted into a human. The cells may be transplanted by any methods known in the art, such as by transfusion or surgical transplantation. For example, stem cells may be transplanted into a human heart by injecting the cells into the heart tissue.

[0072] In one embodiment, the viral vector is specific to the cell-type of the cell being imaged. In one embodiment, the promoter is specific to the cell-type of the cells being imaged. In this embodiment, the transduced cells of the cell-type of the cells being imaged will preferentially transcribe the transfected cDNA, resulting in NIS mutant protein being preferentially located in cells of the cell-type being imaged. [0073] Any imaging label known in the art can be administered. In one embodiment, pertechnetate is used as the imaging label. For example, human stem cells can be transduced and then transplanted into a human heart. After administration of pertechnetate, the stem cells can be imaged using PET or SPECT in order to follow the fate of the cells once transplanted. Administration of cold iodide or thyroxine before administration of the imaging label limits the amount of imaging label taken up by thyroid cells, resulting in a clearer image as well as protecting thyroid cells from possible side effects of sequestration of the imaging label.

[0074] The .present invention additionally provides the use of a transfection vector for a Na⁺/I^" symporter (NIS) mutant protein or a pharmaceutical composition thereof and a radioisotope for the treatment of cancer, wherein the transfection vector for the NIS mutant protein comprises cDNA encoding a promoter and a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the radioisotope is ¹⁸⁸Re0₄ ^" and/or ^,86Re0₄\

EXPERIMENTAL DETAILS

1. Methods and Materials

Electrophysiological Measurements

[0075] pSV-NIS vector, containing WT NIS or G93 site-directed mutant cDNA, was linearized with Not I and in vitro transcribed with SP6 polymerase (mMessage mMachine; Ambion). Stage V-VI Xenopus laevis oocytes were injected with 50 ng of cRNA and maintained in Barth's medium as described. Steady-state and presteady-state electrophysiological characterization of WT NIS and G93 mutants were carried out as described previously (Eskandari et al., 1997; Sacher et a]., 2002).

Vector production and MDCK transduction

[0076] Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped, human immunodeficiency virus- 1 -based, third-generation lentiviruses (Follenzi and Naldini, 2002), one carrying WT hNIS and the other G93T hNIS, were generated using calcium phosphate- mediated co-transfection of 293T cells with four plasmids: a CMV promoter-driven packaging construct expressing the gag and pol genes, an RSV promoter-driven construct expressing rev, a CMV promoter-driven construct expressing the VSV-G envelope and a self-inactivating transfer construct driven by the CMV promoter containing the human immunodeficiency virus- 1 cw-acting sequences and an expression cassette for either WT hNIS or G93T hNIS. MDCK cells (10⁵) were transduced by adding 500 μΐ of viral supernatant per well in a sixwell plate. Transduced cells were analyzed using using flow cytometry.

Flow Cytometry and FACS

[0077] Cells in suspension were stained using indirect immunofluorescence procedures (De La Vieja et al., 2004). Fixed cells were centrifuged (200 x g, 3 min) and washed with PBS containing 0.2% (wt/vol) BSA (PBSA) for non-permeabilized cells and additional 0.2% (wt/vol) saponin (Sigma) (PBSAS) for permeabilized cells. Samples were resuspended in 150 μΐ of 1 : 1000 (3nM) anti-HA high affinity rat monoclonal IgG (Roche clone 3F10) or anti-rNIS antibody directed against the last 16 amino acids of the C- terminus, in PBSA or PBSAS for 1 h. After washing, cells were incubated with 1 : 1000 dilution (30nM) of R-phycoerythrin (PE)-conjugated goat anti-rat IgG l mg^"1 (Invitrogen) for 1 h. Cells were washed in PBSA and resuspended in 400 μΐ of PBS before flow cytometry analysis. The fluorescence of 2 x 10⁴ cells per tube was assayed by a fluorescence-activated cell sorting scan flow cytometer (Becton Dickinson and Co., Franklin Lakes, NJ). A high-speed cell sorter (Moflo-MLS, Cytomation) was used for enrichment of MDCK cells stably expressing NIS mutants, stained under nonpermeabilized conditions. All data was analyzed with FlowJo software.

Immunofluorescence

[0078] Cells were grown on glass coverslips, washed two times with phosphate buffered saline (PBS), and fixed in 2% paraformaldehyde in PBS for 20 min, washed 3 times with PBS, quenched with 100 mM Glycine for 10 min, blocked in 5% goat serum in PBS for 30 minutes. Coverslips were incubated with 3 nM anti-HA monoclonal Ab (Roche) in PBS 1% BSA for 1 h, washed twice with PBS, and incubated with goat anti-rabbit Alexa 488 antibody 1 :250 (Invitrogen) PBS 1% BSA for 1 h. Coverslips were mounted with a DAPI- containing mounting media (Vector) and examined with Zeiss AxioCam.

Transport Assays

[0079] Cells were assayed for Γ or Re0₄ ^" transport under steady-state conditions in triplicate as described (De La Vieja et al., 2004) (Dohan et al., 2002). Γ-dependent kinetic analysis, were performed as described (De la Vieja et al., 2007). For Na⁺-dependent kinetic analysis, cells were incubated with 250 or 750 uNa¹²⁵I and a range of Na⁺ concentrations between 0 and 280 mM for 2 min (isotonicity was maintained using choline CI). The equation used to analyze the initial rates is v = (V_max*[Na⁺]²)/(K_m ² + [Na⁺]²). For Re0₄ ^"- dependent kinetic analysis, cells were incubated with Re0₄ ^" concentrations ranging from 0.075-240 μΜ Al^l86(Re0₄ ^")³ (University of Missouri) or NH₄ ¹⁸⁸Re0 (eluted from ¹⁸⁸W/¹⁸⁸Re generator (Oak Ridge National Laboratory, Oak Ridge, TN)) and 140 mM NaCl for 2 min and analyzed by nonlinear regression using the following equation: v = (V_max ^* [Re0₄ ^"])/(AT_m + [ReO₄ ^']). For the Na⁺-dependent kinetic analysis, cells were incubated with 180 μΜ Al^looReO₄ and 0-280 mM Na for 2 min. The equation used to analyze the initial rates was v = (V_max*\Na⁺])/(K_m + [Na⁺]) for the WT and v = (^'[Na*]²)/^² + [Na⁺]²) for G93T NIS. Background obtained in NT cells was subtracted. Data were analyzed by gnuplot (www.gnuplot.info).

2. Results

G93R NIS is expressed and targeted to the plasma membrane but is inactive.

[0080] G93R rNIS was transiently transfected into COS-7 cells and assayed for Na⁺- dependent, ClO₄ ^" - inhibitable Γ transport characteristic of NIS. Wild-type (WT) NIS- expressing cells accumulated 180 pmol of F^g DNA after incubation with 20 μΜ F [a subsaturating [F] (the K_m { of WT rNIS is -30 μΜ)], and this transport activity was inhibited by 80 μΜ C10₄ ^", whereas G93R NIS was inactive (Figure 1A). Even at near- saturating (200 μΜ) extracellular [F], at which WT NIS transported 460 pmol Yfag DNA, G93R NIS did not translocate F (Figure I B).

[0081] Flow cytometry with a high-affinity anti-rNIS Ab against the C_t showed that G93R NIS was synthesized and expressed at similar levels to those of WT NIS. To avoid cell permeabilization and conclusively determine the subcellular localization of G93R NIS, an hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: 13)) tag was engineered at the extracellularly facing Nt of NIS. Flow cytometry using an anti-HA Ab showed that G93R NIS was synthesized and targeted to the plasma membrane at levels similar to those of WT NIS (Figure 1C). This was further confirmed by immunofluorescence staining under non- permeablized conditions (Figure ID). Therefore, the lack of activity of G93R NIS was not due to lower expression or impaired targeting.

A lysine is tolerated at position 93

[0082] To assess whether G93R NIS was inactive because of the presence of a positively charged residue, G93K NIS was engineered. Surprisingly, G93 NIS was functional, accumulating F at values comparable to those of WT NIS (Figures 1A and IB). Thus, the positive charge at this position was not the cause of G93R NIS lack of function, even though position 93 was predicted to be located within TMS III [virtually confirmed by a model based on the newly published crystal structure of vSGLT (Faham et al., 2008)]. To examine the electrophysiological correlates of the uptake experiments described above, WT and G93 NIS mutants were individually expressed in Xenopus laevis oocytes and analyzed their behavior using the two-electrode voltage clamp method. In control oocytes, addition of Γ (up to 5 mM) did not evoke an electrogenic response, whereas in WT NIS -expressing oocytes it evoked an inward current that represents NIS-mediated electrogenic Na⁺/F symport (Figure IE) and serves as an excellent electrophysiological assay of NIS activity (Eskandari et al., 1997). Remarkably, G93K NIS supported robust F-evoked currents, confirming that position 93 tolerates this residue (Figure IE).

[0083] As the native residue at position 93 is neutral, G93 was individually substituted with several neutral residues to further understand the structural requirements at this position. A, N, and T were clearly tolerated, as indicated by the currents elicited by Γ (Figure IF). It should be emphasized that the magnitude of the observed currents only reflects the level of expression of the relevant protein in a given oocyte, and should not be taken as an indication of the transport ability of a specific mutant as compared to any other or to WT NIS. Such comparisons are effectively made in the standardized kinetic analyses (Figures II, 2A, and 3D-E, and 41). Notably, G93Q NIS showed currents only at very high [Γ] (Figure IF). Consistently with this observation, there was no Γ accumulation mediated by G93Q NIS in flux experiments (Figure 1G) even at 200 μΜ I^" (Figure 1H), although the protein, just like the other mutants, was properly targeted to the cell surface. Each of the other NIS mutants displayed different levels of Γ transport activity upon incubation with 20 μΜ I^" with a clear pattern: the longer the side chain, the lower the activity (Figure 1G). Interestingly, 200 μΜ Γ resulted in a greater relative increase in transport by G93N and G93T as compared to WT NIS, suggesting a change in the K_{m( )} of these two mutants (Figure 1H).

The K_m values of G93N and G93T NIS for Γ and Na⁺ are significantly higher than those of WTNIS .

[0084] To determine whether the reduced Γ accumulation of G93N and T with respect to WT NIS was due to a increase in the apparent K_m({₎ of these mutants or to a lower turnover number, a kinetic analyses of those NIS mutants that exhibited I- transport (i.e., G93A, K, N, S, and T) was performed. Cells were incubated with different [Γ] (0.6-640 μΜ) and 140 mM Na⁺ for 2 min to measure initial rates of I^" transport. G93A and G93 NIS exhibited K_m({₎ values (30 ± 2 and 33 ± 2 μΜ, respectively) similar to that previously reported for WT rNIS (De la Vieja et al., 2007; Dohan et al., 2002; Reed-Tsur et al., 2008). In contrast, G93T and G93N NIS exhibited K_m({) values (282 ± 44 and 358 ± 69 μΜ, respectively) significantly higher than that of WT rNIS (De La Vieja et al., 2004, 2005; Dohan et al., 2002; Levy et al., 1998b; Reed-Tsur et al., 2008). Electrophysiological experiments also showed that whereas G93K, A, and S exhibited K_m( ) comparable to those of WT NIS, G93N and T NIS exhibited >18-fold higher K_m{{), and G93Q NIS had an astonishingly >200-fold higher K_m({) (6.1 mM) (Figure II). This is the first time that any NIS mutant has displayed such a high K_m(\ ) value. Similarly, the mutants with a high K_m({) also had a higher K_m^_a ⁺y (Figure 2A). The Hill coefficient for Na⁺ activation of NIS- mediated ,inward currents remained unchanged at 2 for WT NIS and G93 mutants. Na⁺ kinetics was not performed with G93Q NIS because at the [Γ] required to saturate this mutant, Γ enters the oocytes through other pathways.

[0085] Given that V_max values are affected by the number of NIS molecules at the cell surface, which vary with transfection efficiency, it was difficult to accurately assess changes in Vmax in transiently transfected COS-7 cells. Thus, to eliminate experimental variation, MDCK (Madin-Darby canine kidney) cells were transduced with a lentiviral vector to permanently express HAtagged hNIS (WT or G93T). As G93N and G93T rNIS behaved very similarly (Figure 1), further studies in mammalian cells focused only on G93T hNIS. G93Q NIS was not used in mammalian cells because the [I^"]s required to detect G93Q activity in oocytes were so high (>1 mM) that they would have resulted in nonspecific (i.e., non-NIS-mediated) Γ accumulation in mammalian cells. After sorting HA-positive cells with similar levels of fluorescence, the population of cells expressing G93T NIS at the cell surface was 1.6 times brighter than that expressing WT NIS when assessed by flow cytometry with an anti-HA Ab under nonpermeabilized conditions (Figure 2B). This difference in NIS expression was also seen by immunoblot (Figure 2C). Thus, the factor of 1.6 was used to standardize all transport data. The kinetics of Γ transport in transduced MDCK cells recapitulated those determined in COS-7 cells transiently transfected with rNIS: the WT hNIS K_m{{) was 16 ± 3 μΜ, as shown previously (De la Vieja et al., 2005; Dohan et al., 2007), whereas the G93T hNIS Κ„_{{) was 269 ± 32 μΜ, an ~17-fold increase (Figure 2D).

[0086] Na⁺/F symport mediated by WT NIS is electrogenic with a 2 Na⁺-per-I^" stoichiometry, as shown by ²²Na⁺ and ¹²⁵Γ uptake experiments under voltage clamp (Eskandari et al., 1997), as well as by a sigmoidal dependence of Γ transport versus the external [Na⁺] (Dohan et al., 2007) (Figures 2A, 2E and 2F). Using 250 μΜ I- (i.e., a [ ] close to the K_{m{ }} of G93T hNIS) or 750 μΜ (i.e., 3 times the K_m({₎ of G93T hNIS), initial I- transport rates were measured as a function of the [Na⁺] (0-280 mM). At 250 μΜ Γ, the G93T hNIS K_m ⁺) was 104 ± 16 mM (Figure 2E). Increasing the [Γ] to 750 μΜ, the G93T ^m(Na⁺) decreased by -20% (85.7 ± 4.4 mM) (Figure 2F). These observations are consistent with the notion that increasing the concentration of one substrate decreases the K_m of the cosubstrate in G93T NIS, as reported previously for WT NIS (Eskandari et al., 1997) and other co-transporters such as the Na⁺/glucose cotransporter (Parent et al., 1992) and the Na⁺/Cl^' γ-aminobutyric acid transporter (Loo et al., 2000).

N and T substitutions at position 93 convert NIS-mediated ReO/ and CIO 4 transportfrom eletroneutral to electrogenic.

[0087] It was examined whether or not the apparent affinity of a different substrate, Re0₄ ^", was altered in G93T NIS. The G93T hNIS K _(Re0i₎ was similarly ~17-fold higher than that of WT hNIS [61.7 ± 4.6 μΜ vs. 3.5 ± 0.35 μΜ (Zuckier et al., 2004), respectively] (Figure 3 A). Moreover, the levels of Re0₄ ^" accumulation by G93T hNIS were considerably higher than those of WT hNIS, leading us to investigate whether G93T NIS would display any changes in the Na⁺ dependence of Re0₄ ^" transport. In contrast to Na⁺ T symport, which is electrogenic, Na⁺/Re0₄ ^" symport mediated by WT NIS is electroneutral, with a 1-Na⁺- per-Re0₄ ^" stoichiometry (Dohan et al., 2007). Strikingly, the analysis of Na⁺/Re0₄ ^' symport mediated by G93T NIS yielded the sigmoidal Na⁺-dependence curve characteristic of the electrogenic 2-Na⁺-per-Re0₄ ^" stoichiometry, with a Hill coefficient of 2 (Figure 3B). Thus, engineering a T93 seemed to convert the electroneutral transport of Re0₄ ^' by WT NIS (1 Na⁺ : 1 Re0₄ ^" symport) to electrogenic (2 Na⁺ : 1 Re0₄ ^") by G93T NIS. Consistently with this notion, inward currents were elicited by Re0₄ ^" in Xenopus laevis oocytes expressing G93T rNIS, in contrast to the absence of currents when WT rNIS was expressed (Figure 3C). Moreover, the environmental pollutant C10₄ ^", which is structurally similar to Re0₄ ^", also elicited currents in oocytes expressing G93T or G93N rNIS but, as previously reported, not in those expressing WT rNIS (Figure 3C) (Eskandari et al., 1997). Therefore, position 93 appears to be a key Na⁺/anion coupling link. A kinetic analysis of G93T and G93N rNIS-mediated C10₄ ^" or Re0₄ ^" transport by electrophysiology in oocytes was carried out, a study that cannot be performed with WT NIS because of its electroneutral transport of these anions. Inward currents were measured at a fixed membrane potential of -50 mV and at increasing [C10₄ ^"]s or [Re0₄ ^"]s and determined that the AT_m(cio4 ) for G93N was 17 ± 2 μΜ and for G93T NIS was 18 ± 4 μΜ, and that the K_miRe04 ^') for G93N was 29 ± 2 μΜ and for G93T NIS was 24 ± 3 μΜ (Figure 3D).

[0088] No NIS-mediated C10₄ ^"- or Re0₄ ^"-evoked currents were observed with G93R or G93K NIS (Figure 3C). Strikingly, when Na⁺-activation of the inward currents was examined with C10₄ ^" (Figure 3E, left) or Re0₄ ^" (Figure 3E, right) as the anion substrate, both G93T and G93N NIS exhibited a sigmoidal Na⁺-dependence with a Hill coefficient of 2. These electrophysiological data corroborated those obtained in uptake experiments (Figure 3B) and demonstrate an electrogenic 2: 1 Na⁺:anion (C10₄ ^" or Re0₄ ^") symport stoichiometry for G93T and G93N NIS. Interestingly, although the Na⁺:C10₄ ^" or Re0₄ ^" stoichiometry was different in G93T versus WT NIS, the presteady-state currents induced by transmembrane voltage pulses were similar to those of WT NIS, suggesting that the partial reactions corresponding to Na⁺ binding and binding-induced conformational changes of the transporter were not significantly affected by the substitution (Eskandari et al., 1997). Moreover, the ratio of the maximum Γ-evoked current (I_max) to the maximum presteady- state charge movements (Q_max)_> which is commonly taken as the transporter turnover rate, was -35 s^"1 in G93N and G93T mutants, similar to that of WT NIS (Eskandari et al., 1997). Glutamate and glutamine at position 93 discriminate between Γ and ReOj/ClO

[0089] As Re0₄ ^" or CIO4^" did not elicit currents in G93 NIS -expressing oocytes (Figure 3C), steady-state Re0₄ ^" transport assays were performed in MDCK cells at 140 mM Na⁺ and 3 μΜ Re0₄ ^' (i.e., the K_{m tQ}^ of WT hNIS). Interestingly, G93K NIS exhibited Re0₄ ^" transport levels similar to those of WT NIS and transport activity was CIO4^" sensitive (Figure 4A). This indicates that G93K does indeed transport Re0₄\ and does so electroneutrally, just like WT NIS. It was then tested whether or not G93R NIS transported ReCV, even though this mutant exhibited neither Γ transport activity in mammalian cells (Figures 1A and I B) nor currents in oocytes in the presence of f, Re0₄ ^", or C10₄ ^" (Figure IE and 3C). Unlike G93K NIS, G93R displayed no Re0₄ ^" transport, even at high concentrations of Re0₄ ^". Thus, it is not simply the presence of a positive charge at position 93 that renders NIS nonfunctional, but more specifically the Arg side chain.

[0090] To further investigate the effect of charge at position 93, G93D or G93E NIS cDNAs were transiently transfected into COS-7 cells. G93D NIS-expressing cells transported Γ, although at significantly lower levels than those expressing WT NIS (Figure 4B). However, G93E NIS-expressing cells showed no transport at 200 μΜ Γ (Figure 4C), or even at higher [ ]s. Flow cytometry showed similar levels of G93E expression as compared to WT NIS (Figure 4D) and both proteins were similarly trafficked to the plasma membrane, as assayed by cell surface biotinylation (Figure 4E). In electrophysiological experiments, Γ-evoked currents mediated by G93E NIS were extremely small even at exceedingly high [Γ] (5 mM, i.e., >160 times higher than the WT NIS K_m( )), suggesting a significantly reduced apparent affinity for Γ (Figure 4H). Indeed, the G93E NIS K_m( was extraordinarily high (> 6 mM); 220-fold higher than that of WT NIS (Figure 41). For its part, G93D NIS mediated robust Γ-evoked currents, although it exhibited noticeably reduced K_m({) of -150 μΜ (Figure 41).

[0091] G93D and G93E NIS differed considerably with respect to Re0₄ ^" transport. Following incubation with 3 μΜ Re0₄ ^", G93D NIS-transfected cells transported Re0₄ ^" at similar levels to those of WT NIS. However, G93E NIS activity was barely detectable (Figure 4F). On the other hand, at a 10-fold higher [Re0 ^'] (30 μΜ), G93E NIS clearly exhibited C10₄ ^"-sensitive Re0₄ ^" transport, although at levels significantly lower than those of WT NIS (Figure 4G). The electrophysiological data were equally illuminating and surprising. Both G93D and G93E exhibited C10₄ ^"- and Re0₄ ^"-evoked currents, which were suggestive of a switch in transport stoichiometry leading to electrogenic Na⁺ : anion (C10₄ ^" or Re0₄ ^") symport (Figure 4H). Whereas G93D exhibited very high apparent affinity for C10₄ ^" and Re0₄ ^" (4 and 8 μΜ, respectively), G93E showed significantly reduced apparent affinity for CI0₄ ^" (-250 μΜ) (Figure 41). Given the observed similarity between G93E and Q with respect to Γ transport, the kinetic parameters of G93Q with the substrate C10₄ ^" were investigated. Consistently with the structural similarity between E and Q, G93Q NIS also displayed C10₄ ^"- and Re0₄ ^"-elicited currents (Figure4H). The small signal mediated by G93E rendered Na⁺ kinetics experiments impractical for this mutant, whereas G93D NIS (at 5 mM Γ), yielded the characteristic sigmoidal relationship with a K_m of 23 mM and a Hill coefficient of 1.9. The striking observation that G93E and G93Q NIS transport Re0₄ ^" and C10₄ ^" even though Γ transport is severely impaired in these two mutants suggests that these two amino acids at this position confer the ability to discriminate between substrates.

3. Discussion

[0092] Valuable structure/function data on NIS has been obtained from the study of patients who bear mutations in the NIS gene. One of these mutations, G93R, occurs in TMS III of the symporter. The lack of activity of this mutant NIS protein is not a consequence of its having a positively charged residue at a location within the membrane, as replacement of G93 by a Lys results in an active transporter (Figures 1 A and I B, I D, II and 4A). Many NIS mutants have been studied previously (De La Vieja et al., 2004, 2005; De la Vieja et al., 2007; Dohan et al., 2002; Levy et al., 1998b; Reed-Tsur et al., 2008), but a significant change in the K_m for Γ was only observed when the original Gly was replaced with not only the neutral residues Thr, Asn, and Gin (Figure II) but also Asp and Glu (Figure 4C, D, H, and I), indicating that NIS tolerates either a positive (Lys) or a negative (Asp) charge in the middle of TMS III. The protonation state of the side chain of these residues (probability of the side chains bearing a charge) at physiological pH is a function of both their respective pK_as and the electrostatic properties of their microenvironment. Although this microenvironment could affect the residues' pK_as substantially, it is likely that Lys, and even more likely that Arg, remains positively charged, as the energetic cost of deprotonating these two side chains at the experimental pH of 7.5 is 4.12 and 6.77 kcal/mol, respectively. It is also probable that Asp and Glu exist as charged species, given the steep energy required for protonation at a neutral pH (4.95 and 4.42 kcal/mol, respectively). If some of these residues are neutral in the environment of the protein, the energetic cost of protonation/deprotonation will be "paid" by a decrease in the overall stability of the mutant protein. This loss of stability may result in changes in the structure of the protein that may range in severity from small local distortions to total misfolding.

[0093] Further kinetic analyses in MDCK cells transduced with G93T NIS showed an ~18-fold higher K_m for both Γ (Figure 2D) and Re0₄ ^" (Figure 3 A), as well as an ~5-fold higher K_m for Na⁺ at an [Γ] close to its K_m (Figure 2E). This change in the K_m for Na⁺ is larger than those observed in substitutions of β-ΟΗ-containing residues at TMS IX (De la Vieja et al., 2007). The 3-D model of NIS based on the structure of the highly heterologous bacterial transporter vSGLT (Faham et al., 2008) provides a rationale for the effects of the substitutions at position 93 on NIS activity. This site appears to be a pivot around which one of the major components of the conformational change between the inwardly and the outwardly open conformations— the rotation of the helical hairpin formed by TMS III and IV— takes place (Figure 7). Because it lacks a side chain, Gly, the WT residue, is ideally suited for this position. Nevertheless, other position-93 substitutions result in proteins that not only are active but also in some cases produce additional changes in key properties of the transporter. The conformational changes proposed are fully compatible with the model proposed by Gouaux's group (Krishnamurthy et al., 2009; Singh et al., 2008; Singh et al., 2007) and by Forrest et al. (Forrest et al., 2008) but are described here with a greater focus on position 93 and are probably part of those reported by Faham et al., who identified a Gly residue (G99 of vSGLT) as an important participant in the vSGLT transport cycle (Faham et al., 2008).

[0094] · The significance of G93 is underscored by the manner in which WT NIS and the NIS mutants handle C10₄ ^", a competitive inhibitor of NIS-mediated Γ uptake. The environmental and health impact of the environmental pollutant C10₄ ^' has acquired a new sense of urgency, as the anion has been detected as a contaminant in public water supplies in the U.S. (2005). It has been recently demonstrated in vitro and in vivo, for the first time, that NIS actively transports C10₄ ^", including translocating C10₄ ^' to rat milk, and that the Na⁺/C10₄ ^" transport stoichiometry is electroneutral (i.e., 1 Na⁺ : 1 C10₄ ^"). This discovery revealed that NIS translocates different substrates with different stoichiometrics, a property that has not been described to date for any other transporter (Dohan et al., 2007).

[0095] Here it has been shown that the stoichiometry of Na⁺/Re0₄ ^" and Na⁺/C10₄ ^' mediated by G93N/T/D/E/Q NIS is 2: 1 and thus electrogenic, in stark contrast to the electroneutral 1 : 1 transport stoichiometry of the same substrates mediated by WT NIS. The reason for this change in stoichiometry in the mutants may be that in WT NIS, both Re0₄ ^" and C10₄ ^" probably interfere with Na⁺ binding to the Nal site. It is improbable that the T, N, Q, D, and E substitutions relieve this interference. More likely, they provide an additional Na⁺-coordinating group that shifts the position of the cation in a way that does not overlap the bound Re0₄ ^" or C10₄ ^" but does participate in transport, mimicking the 2 Na⁺ : 1 Γ symport movements in WT NIS.

[0096] The lack of Γ transport activity in G93R/Q/E NIS is probably a result of the side chains being either too large or incompatible with the chemical requirements at their equilibrium position. G93E and Q NIS exhibit the most surprising behavior: although G93D transports Γ, G93E and Q NIS do so only at extremely high [F]s (Figure 4H and 41); this result is probably a consequence of the size difference between the side chains. Strikingly, G93E and Q not only transport Re0₄ ^" and C10₄ ^" but also do so with a 2: 1 Na⁺ : anion stoichiometry. Although this observation is difficult to explain, it is interesting to note that Asp, Glu, and Gin have the chemical characteristics necessary for coordinating Na⁺, as proposed for the G93T and N substitutions, whereas G93 NIS displays electroneutral Na⁺ Re0₄ ^" or C10₄ ^" stoichiometry, just like WT NIS (Figures IE, 3C, and 4A).

[0097] The data presented indicate that the side chain at NIS position 93 has a major effect on the size and chemical characteristics of the ion cavities as they undergo the transition from the outwardly to the inwardly open conformations. In addition, this side chain also plays a role in the kinetic parameters and the stoichiometry of transport. The positions equivalent to NIS G93 in other transporters may have a similar function.

REFERENCES

1. The Council on Water Quality (2005). Health Implications of Perchlorate Ingestion.

In http://wwwnapedu/catalog/l 1202html, The National Academies Press (Washington, DC).

2. Abramson, J., and Wright, E.M. (2009). Structure and function of Na(+)-symporters with inverted repeats. Curr Opin Struct Biol 19, 425-432.

3. Braverman, L.E., and Utiger, R.D. (2004). Werner and Ingbar's the thyroid, 9th Edition edn (Philadelphia, Lippincott Williams and Wilkins, pp 309-328).

4. Carrasco, N. (1993). Iodide transport in the thyroid gland. Biochim Biophys Acta 1 154, 65-82.

5. Dai, G., Levy, O., and Carrasco, N. (1996). Cloning and characterization of the thyroid iodide transporter. Nature 379, 458-460.

6. De La Vieja, A., Dohan, O., Levy, O., and Carrasco, N. (2000). Molecular analysis of the sodium/iodide symporter: impact on thyroid and extrathyroid pathophysiology. Physiol Rev 80, 1083-1 105.

7. De La Vieja, A., Ginter, C.S., and Carrasco, N. (2004). The Q267E mutation in the sodium/iodide symporter (NIS) causes congenital iodide transport defect (ITD) by decreasing the NIS turnover number. J Cell Sci 117, 677-687.

8. De la Vieja, A., Ginter, C.S., and Carrasco, N. (2005). Molecular analysis of a congenital iodide transport defect: G543E impairs maturation and trafficking of the Na+/I- symporter. Mol Endocrinol 19, 2847-2858.

9. De la Vieja, A., Reed, M.D., Ginter, C.S., and Carrasco, N. (2007). Amino acid residues in transmembrane segment IX of the Na+/I- symporter play a role in its Na⁺ dependence and are critical for transport activity. J Biol Chem 282, 25290-25298.

10. Dohan, O., Baloch, Z., Banrevi, Z., Livolsi, V., and Carrasco, N. (2001). Rapid communication: predominant intracellular overexpression of the Na(+)/I(-) symporter (NIS) in a large sampling of thyroid cancer cases. J Clin Endocrinol Metab 86, 2697-2700.

Dohan, O., De la Vieja, A., and Carrasco, N. (2000). Molecular study of the sodium- iodide symporter (NIS): a new field in thyroidology. Trends Endocrinol Metab 1 1, 99-105.

Dohan, O., De la Vieja, A., Paroder, V., Riedel, C, Artani, M., Reed, M., Ginter, C.S., and Carrasco, N. (2003). The sodium/iodide Symporter (NIS): characterization, regulation, and medical significance. Endocr Rev 24, 48-77.

Dohan, O., Gavrielides, M.V., Ginter, C, Amzel, L.M., and Carrasco, N. (2002). Na(+)/I(-) symporter activity requires a small and uncharged amino acid residue at position 395. Mol Endocrinol 16, 1893-1902.

Dohan, O., Portulano, C, Basquin, C, Reyna-Neyra, A., Amzel, L.M., and Carrasco, N. (2007). The Na+/I symporter (NIS) mediates electroneutral active transport of the environmental pollutant perchlorate. Proc Natl Acad Sci U S A 104, 20250-20255.

Eskandari, S., Loo, D.D., Dai, G., Levy, O., Wright, E.M., and Carrasco, N. (1997). Thyroid Na+/I- symporter. Mechanism, stoichiometry, and specificity. J Biol Chem 272, 27230-27238.

Faham, S., Watanabe, A., Besserer, G.M., Cascio, D., Specht, A., Hirayama, B.A., Wright, E.M., and Abramson, J. (2008). The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport. Science 321 , 810- 814.

Follenzi, A., and Naldini, L. (2002). Generation of HIV-1 derived lentiviral vectors. Methods Enzymol 346, 454-465.

Forrest, L.R., Zhang, Y.W., Jacobs, M.T., Gesmonde, J., Xie, L., Honig, B.H., and Rudnick, G. (2008). Mechanism for alternating access in neurotransmitter transporters. Proc Natl Acad Sci U S A 105, 10338-10343.

Fujiwara, H. (1997). Congenital hypothyroidism caused by a mutation in the Na+/1- symporter. Nat Genet 17, 122.

Fujiwara, H., Tatsumi, ., Miki, K., Harada, T., Miyai, K., Takai, S., and Amino, N. (1997). Congenital hypothyroidism caused by a mutation in the Na+/I- symporter. Nat Genet 16, 124- 125. Fujiwara, H., Tatsumi, ., Miki, K., Harada, T., Okada, S., Nose, O., Kodama, S., and Amino, N. (1998). Recurrent T354P mutation of the Na+/I- symporter in patients with iodide transport defect. J Clin Endocrinol Metab 83, 2940-2943.

Fujiwara, H., Tatsumi, K., Tanaka, S., Kimura, M., Nose, O., and Amino, N. (2000). A novel V59E missense mutation in the sodium iodide symporter gene in a family with iodide transport defect. Thyroid 10, 471-474.

Kosugi, S., Inoue, S., Matsuda, A., and Jhiang, S.M. (1998a). Novel, missense and loss-of-function mutations in the sodium/iodide symporter gene causing iodide transport defect in three Japanese patients. J Clin Endocrinol Metab 83, 3373-3376. Kosugi, S., Okamoto, H., Tamada, A., and Sanchez-Franco, F. (2002). A novel peculiar mutation in the sodium/iodide symporter gene in Spanish siblings with iodide transport defect. J Clin Endocrinol Metab 87, 3830-3836.

Kosugi, S., Sato, Y., Matsuda, A., Ohyama, Y., Fujieda, K., Inomata, H., Kameya, T., Isozaki, O., and Jhiang, S.M. (1998b). High prevalence of T354P sodium/iodide symporter gene mutation in Japanese patients with iodide transport defect who have heterogeneous clinical pictures. J Clin Endocrinol Metab 83, 4123-4129.

Krishnamurthy, H., Piscitelli, C.L., and Gouaux, E. (2009). Unlocking the molecular secrets of sodium-coupled transporters. Nature 459, 347-355.

Levy, O., Dai, G., Riedel, C, Ginter, C.S., Paul, E.M., Lebowitz, A.N., and Carrasco, N. (1997). Characterization of the thyroid Na+/I- symporter with an anti- COOH terminus antibody. Proc Natl Acad Sci U S A 94, 5568-5573.

Levy, O., De la Vieja, A., Ginter, C.S., Riedel, C, Dai, G., and Carrasco, N. (1998a). N-linked glycosylation of the thyroid Na+/I- symporter (NIS). Implications for its secondary structure model. J Biol Chem 273, 22657-22663.

Levy, O., Ginter, C.S., De la Vieja, A., Levy, D., and Carrasco, N. (1998b). Identification of a structural requirement for thyroid Na+/I- symporter (NIS) function from analysis of a mutation that causes human congenital hypothyroidism. FEBS Lett 429, 36-40.

Loo, D.D., Eskandari, S., Boorer, K.J., Sarkar, H.K., and Wright, E.M. (2000). Role of CI- in electrogenic Na+-coupled cotransporters GATl and SGLTl . J Biol Chem 275, 37414-37422.

Mazzaferri, E.L. (2000a). Long-term outcome of patients with differentiated thyroid carcinoma: effect of therapy. Endocr Pract 6, 469-476. Mazzaferri, E.L. (2000b). Thyroid diseases: tumors. Radioiodine and other treatment and outcomes. In Werner & Ingbar's The Thyroid, L.E. Braverman, Utiger, R.D., ed. (Lipincott Williams & Wilkins), pp. 904-930.

Parent, L., Supplisson, S., Loo, D.D., and Wright, E.M. (1992). Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies. J Membr Biol 125, 49-62.

Reed-Tsur, M.D., De la Vieja, A., Ginter, C.S., and Carrasco, N. (2008). Molecular characterization of V59E NIS, a Na+/I- symporter (NIS) mutant that causes congenital I- transport defect (ITD). Endocrinology.

Ressl, S., Terwisscha van Scheltinga, A.C., Vonrhein, C, Ott, V., and Ziegler, C. (2009). Molecular basis of transport and regulation in the Na(+)/betaine symporter BetP. Nature 458, 47- 52.

Riedel, C, Levy, O., and Carrasco, N. (2001). Post-transcriptional regulation of the sodium/iodide symporter by thyrotropin. J Biol Chem 276, 21458-21463.

Rudnick, G. (2007). What is an antidepressant binding site doing in a bacterial transporter? ACS Chem Biol 2, 606-609.

Sacher, A., Nelson, N., Ogi, J.T., Wright, E.M., Loo, D.D., and Eskandari, S. (2002). Presteady-state and steady-state kinetics and turnover rate of the mouse gamma-aminobutyric acid transporter (mGAT3). J Membr Biol 190, 57-73.

Saito, K., Yamamoto, K., Yoshida, S., Manabe, S., Suzuki, M., Takai, T., Saito, T., Kuzuya, T., and Moriyama, S. (1981). Goitrous hypothyroidism due to iodide- trapping defect. J Clin Endocrinol Metab 53, 1267-1272.

Sali, A., and Blundell, T.L. (1993). Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234, 779-815.

Singh, S.K., Piscitelli, C.L., Yamashita, A., and Gouaux, E. (2008). A competitive inhibitor traps LeuT in an open-to-out conformation. Science 322, 1655-1661.

Singh, S.K., Yamashita, A., and Gouaux, E. (2007). Antidepressant binding site in a bacterial homologue of neurotransmitter transporters. Nature 448, 952-956.

Tazebay, U.H., Wapnir, I.L., Levy, O., Dohan, O., Zuckier, L.S., Zhao, Q.H., Deng, H.F., Amenta, P.S., Fineberg, S., Pestell, R.G., et al. (2000). The mammary gland iodide transporter is expressed during lactation and in breast cancer. Nat Med 6, 871 -878. Weyand, S., Shimamura, T., Yajima, S., Suzuki, S., Mirza, O., Krusong, K., Ca enter, E.P., Rutherford, N.G., Hadden, J.M., O'Reilly, J., et al. (2008). Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter. Science 322, 709-713.

Yamashita, A., Singh, S.K., Kawate, T., Jin, Y., and Gouaux, E. (2005). Crystal structure of a bacterial homologue of Na+/Cl-dependent neurotransmitter transporters. Nature 437, 215-223.

Zuckier, L.S., Dohan, O., Li, Y., Chang, C.J., Carrasco, N., and Dadachova, E. (2004). Kinetics of perrhenate uptake and comparative biodistribution of perrhenate, pertechnetate, and iodide by Nal symporter-expressing tissues in vivo. J Nucl Med 45, 500-507.

Claims

What is claimed is:

1. An isolated Na⁺/F symporter (NIS) mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid.

2. The NIS mutant protein of Claim 1, wherein the NIS mutant protein preferentially transports perrhenate (Re0₄ ^") rather than iodide (Γ).

3. The NIS mutant protein of Claim 1 or 2, wherein NIS mutant protein has the sequence set forth in SEQ ID NO:3.

4. The NIS mutant protein of Claim 1 or 2, wherein NIS mutant protein has the sequence set forth in SEQ ID NO:4.

5. A method of preparing a mutant Na⁺/T symporter (NIS) protein modified to preferentially transport perrhenate (Re0₄ ^") rather than iodide (Γ), the method comprising replacing glycine at amino acid residue 93 of the NIS protein with aspartic acid or glutamic acid.

6. The method of Claim 5, wherein NIS mutant protein has the sequence set forth in SEQ ID NO: l with the glycine residue 93 replaced with aspartic acid.

7. The method of Claim 5, wherein NIS mutant protein has the sequence set forth in SEQ ID NO: l with the glycine residue 93 replaced with glutamic acid.

8. A mutant Na⁺/F symporter (NIS) protein made by the method of Claim 5.

9. A transfection vector for a Na⁺/I^" symporter (NIS) mutant protein, the vector comprising a heterologous nucleic acid comprising a promoter and encoding a NIS mutant protein wherein glycine at amino acid residue 93 is replaced with aspartic acid or glutamic acid.

10. The transfection vector of Claim 9, comprising a nucleic acid encoding the sequence set forth in SEQ ID NO:3.

1 1. The transfection vector of Claim 9, comprising a nucleic acid encoding the sequence set forth in SEQ ID NO:4.

12. A pharmaceutical composition comprising a therapeutically effective amount of the transfection vector of any of Claims 9 to 1 1 in a pharmaceutically acceptable carrier.

13. A method of treating cancer, the method comprising administering a therapeutically effective amount of a transfection vector for a Na⁺/F symporter (NIS) mutant protein or a pharmaceutical composition thereof to a subject, and administering a therapeutically effective amount of a radioisotope, wherein the transfection vector for the NIS mutant protein comprises a heterologous nucleic acid comprising a promoter and encoding a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the radioisotope is ^{l 88}Re0₄ ^' and/or ¹⁸⁶Re0₄ ^", under conditions permitting cells of the cancer to express the NIS mutant protein and transport the radioisotope thereby treating the cancer.

14. The method of Claim 13, wherein the cancer is a thyroid cancer

15. The method of Claim 13 or 14, the promoter is specific for the organ type of the cancer.

16. The method of Claim 15, wherein the cancer is prostate, breast, pancreatic, intestinal, pulmonary, cardiac, neural, endocrine, lymph, bone or blood cancer.

17. The method of any of Claims 13 to 15, wherein the transfection vector is specific to the thyroid.

18. The method of any of Claims 13 to 17, wherein the NIS mutant protein has SEQ ID NO:2, 3 or 4.

19. The method of any of Claims 13 to 18, wherein the NIS mutant protein is produced in cells transfected by the transfection vector and of the promoter's specific cell-type.

20. The method of any of Claims 13 to 19, wherein the radioisotope is administered after sufficient time has elapsed to allow the production of NIS mutant proteins in transfected cells of the promoter's specific cell-type.

21. The method of any of Claims 13 to 20, the method further comprising administering a protective amount of nonradioactive iodide prior to administration of the radioisotope.

22. The method of any of Claims 13 to 20, the method further comprising administering a protective amount of thyroxine prior to administration of the radioisotope.

23. A method of imaging cells, the method comprising contacting the cells with an effective amount of a transfection vector for a Na⁺/F symporter (NIS) mutant protein, administering a effective amount of imaging label, and imaging the cells, wherein the transfection vector for the NIS mutant protein comprises cDNA comprising a promoter and encoding a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the imaging label is Re0₄ ^" and/or Re0₄ ^".

24. The method of Claim 23, wherein imaging is effected with positron emission tomography (PET).

25. The method of Claim 23, wherein imaging is effected with single photon emission computed tomography.

26. The method of any of Claims 23 to 25, wherein the cells being imaged are located in vivo or in vitro.

27. The method of Claim 26, wherein the cells are located in vivo and are in a human.

28. The method of any of Claims 23 to 27, wherein the cells being imaged are human stem cells.

29. The method of Claim 28, wherein the human stem cells are tranduced in vitro and then transplanted into a mammal.

30. The method of any of Claims 28 or 29, wherein the human stem cells are transplanted into a human.

31. The method of any of Claims 26 to 30, wherein the promoter is specific to the cell- type of the cells being imaged.

32. The method of any of Claims 23 to 31, wherein the transfection vector is specific to the tissue or cell type being imaged.

33. The method of any of Claims 23 to 32, wherein NIS mutant protein is produced in cells transduced by the transfection vector and of the promoter's specific cell-type.

34. The method of any of Claims 23 to 33, wherein the imaging label is administered after sufficient time has elapsed to allow the production of NIS mutant proteins in transduced cells of the promoter's specific cell-type.

35. The method of any of Claims 23 to 34, the method further comprising administering a protective amount of nonradioactive iodide prior to administration of the imaging label.

36. The method of any of Claims 23 to 35, the method further comprising administering a protective amount of thyroxine prior to administration of the imaging label.

37. The method of any of Claims 23 to 36, wherein the imaging label is pertechnetate.

38. The method of any of Claims 23 to 37, wherein the NIS mutant protein has SEQ ID NO:2, 3 or 4.

39. Use of a transfection vector for a Na⁺/T symporter (NIS) mutant protein or a pharmaceutical composition thereof and a radioisotope for the treatment of cancer, wherein the transfection vector for the NIS mutant protein comprises cDNA encoding a promoter and a NIS mutant protein wherein glycine residue 93 is replaced with aspartic acid or glutamic acid and wherein the radioisotope is ¹⁸⁸Re0₄ ^" and/or ¹⁸⁶Re0₄ ^".

40. The use of Claim 39, wherein glycine residue 93 is replaced with aspartic acid.

41. The use of Claim 39, wherein the cancer is located in vivo in a human.

42. The use of Claim 41, wherein the cancer is prostate, breast, pancreatic, intestinal, pulmonary, cardiac, neural, endocrine, lymph, bone or blood cancer.

43. The use of any of Claims 39 to 42, wherein the promoter is specific to the cancer cell-type.

44. The use of any of Claims 39 to 43, wherein the transfection vector is specific to the tissue or cell type affected by the cancer.

45. The use of any of Claims 39 to 44, wherein NIS mutant protein is only produced in cells transduced by the transfection vector and of the promoter's specific cell-type.

46. The use of any of Claims 39 to 45, wherein the subject has been administered or will be administered a radioisotope after sufficient time has elapsed to allow the production of NIS mutant proteins in transduced cells of the promoter's specific cell-type.

47. The use of Claim 46, wherein the radioisotope is radioactive perrhenate.

48. The use of any of Claims 39 to 47, wherein the subject has been administered or will be administered a protective amount of nonradioactive iodide prior to administration of the radioisotope.

49. The use of any of Claims 39 to 47, wherein the subject has been administered or will be administered a protective amount of thyroxine prior to administration of the radioisotope.