# Patent

## Title
Codon optimized sodium-iodide symporter gene and its use

## Abstract

The present disclosure provides a polynucleotide which is codon optimized for the efficient expression in a eukaryotic cell, a plasmid and a eukaryotic cell comprising the same. The modification resulted in the efficient expression of NIS in eukaryotic cells and the enhancement of the function of NIS by glycosylation. Thus modified polynucleotide encoding NIS of the present disclosure is useful as imaging reporter for gene, viral and/or cell based therapies.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of Korean Patent Application NO. 2012-0065546 filed Jun. 19, 2012 in the Korean Intellectual Property Office; disclosure of which is incorporated herein by reference.
SEQUENCE LISTING
The Sequence Listing submitted in txt format (.txt), named “SequenceListing_YOUN.txt” (originally created on Jun. 18, 2013, 11.2 KB; and revised on Sep. 26, 2013, 12 KB;); is incorporated herein by reference.
BACKGROUND OF INVENTION
1. Field of the Invention
The present disclosure relates to a Sodium-Iodide Symporter gene optimized for the expression in eukaryotic cells and its uses.
2. Description of the Related Art
Generally in cells genes are transcribed from DNA to mRNA; which in turn are translated into proteins. During the translational process; tRNAs which carry 3 bases corresponding to each codon of a single amino acid are involved and each amino acid is encoded by more than one codon. Due to this codon degeneracy; the frequency of codon usage varies depending on the types of cells where genes are expressed. Therefore; DNA sequence is needed to be modified by synonymous nucleotide substitutions without altering the amino acid sequence of the encoded protein for the efficient expression of recombinant genes in heterologous systems.
Sodium Iodide Symporter (NIS) is a transmembrane glycoprotein with a MW of 87 kDa and 12 transmembrane domains; which transports two sodium cations for each iodide anion into cells. NIS mediated uptake of iodide into follicular cells of the thyroid gland is the first step in the synthesis of thyroid hormone resulting in 20-40 higher concentration of iodide in the thyroid.
The gene coding for NIS was first isolated in rats by Dai et al. (Dai et al.; Nature; 1996; 379: 458-460); followed by the isolation of Human NIS (hNIS) gene by Smanik et al. (Smanik et al.; Biochem. Biophysic. Res. Comm 226: 339-345; 1997).
U.S. Patent Publication 2006-0004191 relates to modified sodium iodide symporter proteins and genes for imaging and cancer therapy and discloses modified NIS proteins having a net electrostatic charge more positive than that of corresponding wild type NIS proteins.
U.S. Patent Publication 2012-0027676 relates to modified sodium iodide symporter proteins and uses thereof and discloses the modified NIS proteins in the cells results in higher intracellular levels of a substrate of a NIS protein than does the expression of the same amount of a wild-type NIS protein.
However; there exist needs to develop modified NIS genes optimized for the efficient expression in eukaryotic cells particularly in human cells for the treatment of thyroid disease and radionuclide gene therapy and molecular imaging using NIS.
SUMMARY OF THE INVENTION
In one aspect the present disclosure provides a polynucleotide encoding a sodium-iodide symporter (NIS) protein; the polynucleotide is codon optimized for the expression in a eukaryotic cell. In one embodiment, the polypeptide is codon optimized in the nucleotide coding for the 8th transmembrane domain corresponding to amino acid residues 286-308 and the 5th transmembrane domain corresponding to 163-182, and C-terminal region corresponding to 550-643; one or more of the phosphorylation sites of Ser-43, Thr-49, Ser-227, Lys-577, and Ala-581; and one or more of the N-glycosylation sites of Asn-225 Asn-489 and Asn-502, wherein the amino acid residue numbers correspond to the residue in SEQ ID NO:3.
In other embodiment, the polynucleotide has at least one of the following modification in the sequence as disclosed in SEQ ID NO: 1: 6th: G→A; 15 the G→A; 22th: G→C; 24th: A→G; 25th: C→A; 39th: A→C; 48th: C→T; 51th: C→T; 54th: G→C; 57th: C→G; 60th: T→C; 66th: C→G; 72th: C→G; 84th: T→C; 93th: G→C; 102th: C→G; 105th: G→A; 111th: T→C; 112th: C→A; 114th: G→A; 120th: G→C; 124th: C→A; 126th: C→A; 132th: T→C; 150th: G→C; 154th: C→A; 156th: G→A; 157th: C→A; 159th: C→G; 165th: G→C; 174th: C→T; 180th: C→A; 184th: T→A; 185th: C→G; 186th: G→C; 190th: T→A; 191th: C→G; 192th: T→C; 205th; T→A; 206th: C→G; 207th: G→C; 225th: C→A; 231th: G→C; 232th: T→A; 233th: C→G; 234th: G→C; 243th: T→C; 244th: C→A; 246th: C→A; 249 th: T→C; 255th: C→G; 264th: C→G; 285th: T→G; 292th: T→A; 293th: C→G; 294th: G→C; 297th: C→G; 300th: C→G; 312th: C→G; 324th: C→G; 333th; C→G; 342th: C→G; 366th: G→A; 372th: C→G; 379th: C→A; 381th: C→A; 384th: A→C; 393th: C→G; 399th: G→C; 402th: T→C; 403th: T→C; 414 th: T→C; 417th: A→G; 423th: G→C; 435th: C→A; 444th: A→C; 456th: G→T; 462th: C→G; 474th: A→G; 480th: C→A; 483th: G→C; 498th: G→C; 501th: G→C; 504th: C→G; 508th: T→A; 509th: C→G; 516th: A→C; 519th: T→C; 537th: G→C; 540th: T→C; 558th: T→C; 564th: C→G; 570th: T→C; 573th: T→C; 585th: C→G; 597th: A→G; 600th: T→C; 612th: T→G; 615 th: C→G; 621th: A→T; 624th: C→G; 627th: T→C; 630th: C→G; 636th: T→G; 642th: C→A; 645th: G→C; 648th: C→T; 649th: C→A; 651th: C→A; 660th: C→G; 663th: G→C; 679th: T→A; 680th: C→G; 693th: C→G; 702th: T→C; 708th: T→C; 714th: G→C; 717th: G→A; 723th: C→G; 726th: T→C; 729th: A→C; 738th: T→C; 741th: T→C; 750th: T→A; 753th: C→A; 756th; G→C; 757th: T→C; 768th: C→G; 771th: C→T; 777th: T→C; 792th: G→C; 802th: C→A; 804th: C→A; 813th: T→C; 819th: C→G; 822th: A→C; 849 th: C→G; 861th: C→G; 864th: C→A; 888th: T→C; 897th: T→C; 900th: C→A; 906th: C→G; 912th: T→C; 924th: T→C; 936th: T→C; 939th: C→G; 942th; C→G; 948th: G→C; 949th: C→A; 951th: C→A; 955th: T→A; 956th: C→G; 957th: T→C; 963th: T→C; 978th: T→C; 1002th: A→G; 1005th: T→C; 1014th: A→C; 1017th: C→G; 1023th: G→C; 1026th: T→G; 1041th: T→C; 1047 th: T→C; 1053th: C→A; 1056th: C→G; 1062th: A→C; 1065th: A→C; 1066th; T→A; 1067th: C→G; 1072th: A→T; 1073th: G→C; 1080th: T→C; 1083th: T→C; 1089th: T→C; 1092th: A→C; 1095th: C→G; 1098th: T→C; 1101th: A→G; 1107th: C→T; 1110th: C→G; 1116th: A→G; 1119th: T→C; 1137th: A→C; 1143th: G→A; 1146th: A→G; 1149th: C→G; 1155th: T→C; 1159th: T→A; 1160th: C→G; 1167th: G→C; 1170th: C→G; 1171th: T→A; 1172th: C→G; 1173th: A→C; 1176th: C→G; 1185th: A→C; 1186th: T→A; 1187th: C→G; 1188th: G→C; 1197th: C→G; 1206th: A→C; 1209th: C→T; 1209th: C→T; 1213th: T→A; 1214th: C→G; 1215th: C→T; 1218th: A→T; 1224th: C→G; 1227th: A→C; 1233th: T→C; 1236th: C→G; 1239th: T→G; 1246th: T→A; 1247th: C→G; 1251th: C→T; 1257th: C→G; 1263th: A→C; 1266th: C→G; 1278th: C→A; 1287th: A→C; 1297th: T→C; 1302th: A→C; 1308th: C→T; 1314th: G→C; 1326th: A→C; 1329th: G→T; 1335th: C→G; 1338th: C→G; 1341th: G→T; 1347th: A→G; 1350th: C→A; 1353th: G→C; 1356th: C→A; 1357th: T→C; 1362th: G→T; 1368th: G→T; 1381th: T→C; 1392th: G→C; 1401th: A→T; 1404th: C→A; 1420th: A→C; 1425th: C→G; 1431th: A→T; 1432: T→A, 1433th: C→G; 1434: G→C; 1435th: T→A; 1436th: C→G; 1437th; G→C, 1440th T→C; 1445th: C→A; 1447th: C→A; 1450th: C→T; 1456th: T→C; 1459th: C→G; 1462th: A→C; 1465th: C→G; 1468th: C→T; 1472th: T→A; 1473th: C→G; 1474th: T→C; 1480th: C→G; 1486th: C→T; 1489th: G→T; 1492th: T→C; 1495th: C→G; 1498th: C→G; 1501th: T→C; 1504th: T→C; 1511th: T→A; 1512th: C→G; 1519th: G→A; 1529th: T→A; 1530th: C→G; 1531th: A→C; 1534th: A→C; 1540th: C→T; 1544th: A→T; 1545th: G→C; 1546th: C→T; 1547th: C→A; 1552th: C→T; 1556th: T→C; 1558th: A→G; 1561th: T→C; 1573th: T→C; 1580th: T→A; 1581th: C→G; 1585th: T→C; 1588th: C→G; 1591th: T→C; 1597th: T→C; 1609th: G→C; 1618th: T→A; 1627th: C→T; 1630th: A→C; 1636th: C→G; 1651th: A→C; 1664th: C→A; 1666th: C→A; 1672th: C→A; 1681th: G→T; 1684th: A→C; 1685th: T→C; 1688th: T→C; 1702th: C→G; 1705th: A→T; 1706th: C→A; 1708th: G→A; 1717th: A→C; 1718th: T→A; 1719th: C→G; 1720th: A→C; 1732th: G→A; 1738th: A→G; 1753th: T→C; 1760th: T→C; 1765th: C→G; 1768th: G→A; 1771th: T→C; 1774th: T→C; 1777th: A→G; 1783th: C→G; 1789th: T→C; 1792th: A→C; 1825th: T→C; 1831th: T→C; 1834th: T→G; 1840th: T→C; 1844th: T→C; 1849th: G→C; 1855th: G→A; 1864th: G→A; 1867th: G→C; 1870th: T→C; 1873th: C→A; 1874th: T→A; 1875th: C→G; 1876th: T→C; 1891th: T→G; 1897th: T→C; 1900th: T→C; 1903th: T→C; 1906th: T→C; 1907th: C→A; 1909th: A→G; 1921th: G→A; 1927th: C→T; or 1930th: C→G.
In still other embodiment, the polynucleotide has a sequence as disclosed in SEQ ID NO: 2.
In other aspect, the present disclosure also provides a polypeptide encoded by the polynucleotide of the present disclosure.
In still other aspect the present disclosure provides a plasmid comprising the polynucleotide of the present disclosure and a promoter operatively linked to the nucleotide, wherein the plasmid is for the expression in a eukaryotic cell.
Also provided is a recombinant host cell comprising the plasmid and/or the polynucleotides in accordance with the present disclosure.
Also provided is a kit for radio-imaging, nuclear or molecular imaging comprising the plasmid of the present disclosure.
The foregoing summary is illustrative only and is not intended to be in any way limiting. Additional aspects and/or advantages of the invention will be set forth in part in the description which follows and; in part; will be obvious from the description; or may be learned by practice of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
These and/or other aspects and advantages of the invention will become apparent and more readily appreciated from the following description of the embodiments; taken in conjunction with the accompanying drawings of which:
FIG. 1a is a codon frequency distribution (CFD) of human NIS.
FIG. 1b is a codon frequency distribution of optimized NIS (oNIS) in accordance with the present disclosure.
FIG. 1c is a graph showing the codon quality distribution of the oNIS in accordance with the present disclosure.
FIG. 1d is a graph showing the codon quality plot of the oNIS in accordance with the present disclosure.
FIG. 1e is a graph showing the GC content of the oNIS in accordance with the present disclosure. The codon with a highest frequency for each amino acid was given a value of 100 and; the histogram represents the percent of codons that meets a given value that determines the quality.
FIG. 2 is an illustrative map of the recombinant plasmid having oNIS according to one embodiment of the present disclosure
FIG. 3a is a RT-PCR analysis result in which the expression of hNIS was compared to that of the control (CTR) using the cells transfected with a plasmid having oNIS.
FIG. 3b is a graph representing the result of FIG. 3a. 
FIG. 3c is a western blot result in which the expression of hNIS was compared to that of the control (CTR) using the human breast cancer cell line transfected with a plasmid having oNIS.
FIG. 3d is a graph representing the result of FIG. 3c. 
FIG. 4 is an image of a gamma camera showing the uptake of radioactive iodide by the human breast cancer cell line transfected with hNIS or control.
FIG. 5 is a result comparing the uptake of radioactive iodide in a variety of human breast cancer cell lines.
FIG. 6a is a result comparing the uptake of hNIS/oNIS radioactive iodide in a human breast cancer cell line transfected with hNIS or oNIS.
FIG. 6b is an image of a gamma camera showing the uptake of radioactive iodide by the human breast cancer cell line transfected with hNIS or oNIS.
FIG. 6c is an image of a gamma camera comparing the uptake of hNIS/oNIS radioactive by the mouse injected with human breast cancer cells expressing hNIS or oNIS.
FIG. 6d is a result comparing the uptake of hNIS/oNIS radioactive 99mTc by the mouse implanted with human breast cancer cells expressing hNIS or oNIS.
FIG. 7a is a comparison of nucleotide sequences of hNIS and oNIS from amino acids 1 through 660; in which Query indicates hNIS which is represented by SEQ ID NO:1; and Sbjct (subject) indicates oNIS, which is represented by SEQ ID NO:2. Further the amino acid sequences of oNIS encoded by SEQ ID NO: 2 is represented by SEQ ID NO: 3.
FIG. 7b is a comparison of nucleotide sequences of hNIS and oNIS from amino acids 661 through 1320; in which Query indicates hNIS which is represented by SEQ ID NO:1; and Sbjct (subject) indicates oNIS, which is represented by SEQ ID NO:2. Further the amino acid sequences of oNIS encoded by SEQ ID NO: 2 is represented by SEQ ID NO: 3.
FIG. 7c is a comparison of nucleotide sequences of hNIS and oNIS from amino acids 1321 through 1938; in which Query indicates hNIS; which is represented by SEQ ID NO:1; and Sbjct (subject) indicates oNIS, which is represented by SEQ ID NO:2. Further the amino acid sequences of oNIS encoded by SEQ ID NO: 2 is represented by SEQ ID NO: 3.



DETAILED DESCRIPTION OF THE EMBODIMENTS
In one aspect, the present invention is directed to enhancing the expression of a polynucleotide encoding an NIS (Sodium-Iodide Symporter), particularly derived from human being, by providing a polynucleotide comprising a codon-optimized coding region thereof, where the codons are optimized for eukaryotic cell expression.
As used herein the term “codon-optimized” or “codon-optimized coding region” means a nucleic acid coding region that has been adapted for expression in the eukaryotic cells of a given vertebrate by substituting, deleting and/or inserting at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that vertebrate, particularly in human.
Sodium Iodide Symporter (NIS) is a transmembrane glycoprotein with a MW of 87 kDa and 12 transmembrane domains; which transports two sodium cations for each iodide anion into cells. NIS mediated uptake of iodide into follicular cells of the thyroid gland is the first step in the synthesis of thyroid hormone resulting in 20-40 higher concentration of iodide in the thyroid.
The gene coding for NIS was first isolated in rats by Dai et al. (Dai et al.; Nature; 1996; 379: 458-460); followed by the isolation of Human NIS (hNIS) gene by Smanik et al. (Smanik et al.; Biochem. Biophysic. Res. Comm 226: 339-345; 1997).
Genes for Sodium-Iodide Symporter which may be used for the present disclosure are derived from a mammal, particularly vertebrate, more particularly human being. Such sequences are known in the art and for example disclosed as NM—000435 for cDNA of SLC5A5 (Solute carrier family 5, sodium iodide symporter member 5) and GenBank No: AAB17378.1 for polypeptide. Further, the sequences as disclosed in FIGS. 7a through 7c may also be referred.
The polynucleotide is codon optimized in the nucleotides encoding the 8th transmembrane domain corresponding to amino acid residues 286-308 in SEQ ID NO:3; the 5th transmembrane domain corresponding to 163-182 in SEQ ID NO:3; C-terminal region corresponding to 550-643 in SEQ ID NO:3; one or more of the phosphorylation sites of Ser-43, Thr-49, Ser-227, Lys-577, and Ala-581 in SEQ ID NO:3; and one or more of the N-glycosylation sites of Asn-225 Asn-489 and Asn-502 in SEQ ID NO:3.
In one embodiment, the codon optimized nucleotide sequence or coding region of a NIS is derived from the sequences as disclosed in FIG. 7 or SEQ ID NO: 1 and may include one or more of the substitution at the following site. The nucleotide numbers indicated below correspond to the nucleotide as disclosed in SEQ ID NO:1: 6th: G→A; 15 th: G→A; 22th: G→C; 24th: A→G; 25th: C→A; 39th: A→C; 48th: C→T; 51th: C→T; 54th: G→C; 57th: C→G; 60th: T→C; 66th: C→G; 72th: C→G; 84th: T→C; 93th: G→C; 102th: C→G; 105th: G→A; 111th: T→C; 112th: C→A; 114th: G→A; 120th: G→C; 124th: C→A; 126th: C→A; 132th: T→C; 150th: G→C; 154th: C→A; 156th: G→A; 157th: C→A; 159th: C→G; 165th: G→C; 174th: C→T; 180th: C→A; 184th: T→A; 185th: C→G; 186th: G→C; 190th: T→A; 191th: C→G; 192th: T→C; 205th; T→A; 206th: C→G; 207th: G→C; 225th: C→A; 231th: G→C; 232th: T→A; 233th: C→G; 234th: G→C; 243th: T→C; 244th: C→A; 246th: C→A; 249 th: T→C; 255th: C→G; 264th: C→G; 285th: T→G; 292th: T→A; 293th: C→G; 294th: G→C; 297th: C→G; 300th: C→G; 312th: C→G; 324th: C→G; 333th; C→G; 342th: C→G; 366th: G→A; 372th: C→G; 379th: C→A; 381th: C→A; 384th: A→C; 393th: C→G; 399th: G→C; 402th: T→C; 403th: T→C; 414 th: T→C; 417th: A→G; 423th: G→C; 435th: C→A; 444th: A→C; 456th: G→T; 462th: C→G; 474th: A→G; 480th: C→A; 483th: G→C; 498th: G→C; 501th: G→C; 504th: C→G; 508th: T→A; 509th: C→G; 516th: A→C; 519th: T→C; 537th: G→C; 540th: T→C; 558th: T→C; 564th: C→G; 570th: T→C; 573th: T→C; 585th: C→G; 597th: A→G; 600th: T→C; 612th: T→G; 615 th: C→G; 621th: A→T; 624th: C→G; 627th: T→C; 630th: C→G; 636th: T→G; 642th: C→A; 645th: G→C; 648th: C→T; 649th: C→A; 651th: C→A; 660th: C→G; 663th: G→C; 679th: T→A; 680th: C→G; 693th: C→G; 702th: T→C; 708th: T→C; 714th: G→C; 717th: G→A; 723th: C→G; 726th: T→C; 729th: A→C; 738th: T→C; 741th: T→C; 750th: T→A; 753th: C→A; 756th; G→C; 757th: T→C; 768th: C→G; 771th: C→T; 777th: T→C; 792th: G→C; 802th: C→A; 804th: C→A; 813th: T→C; 819th: C→G; 822th: A→C; 849 th: C→G; 861th: C→G; 864th: C→A; 888th: T→C; 897th: T→C; 900th: C→A; 906th: C→G; 912th: T→C; 924th: T→C; 936th: T→C; 939th: C→G; 942th; C→G; 948th: G→C; 949th: C→A; 951th: C→A; 955th: T→A; 956th: C→G; 957th: T→C; 963th: T→C; 978th: T→C; 1002th: A→G; 1005th: T→C; 1014th: A→C; 1017th: C→G; 1023th: G→C; 1026th: T→G; 1041th: T→C; 1047 the T→C; 1053th: C→A; 1056th: C→G; 1062th: A→C; 1065th: A→C; 1066th; T→A; 1067th: C→G; 1072th: A→T; 1073th: G→C; 1080th: T→C; 1083th: T→C; 1089th: T→C; 1092th: A→C; 1095th: C→G; 1098th: T→C; 1101th: A→G; 1107th: C→T; 1110th: C→G; 1116th: A→G; 1119th: T→C; 1137th: A→C; 1143th: G→A; 1146th: A→G; 1149th: C→G; 1155th: T→C; 1159th: T→A; 1160th: C→G; 1167th: G→C; 1170th: C→G; 1171th: T→A; 1172th: C→G; 1173th: A→C; 1176th: C→G; 1185th: A→C; 1186th: T→A; 1187th: C→G; 1188th: G→C; 1197th: C→G; 1206th: A→C; 1209th: C→T; 1209th: C→T; 1213th: T→A; 1214th: C→G; 1215th: C→T; 1218th: A→T; 1224th: C→G; 1227th: A→C; 1233th: T→C; 1236th: C→G; 1239th: T→G; 1246th: T→A; 1247th: C→G; 1251th: C→T; 1257th: C→G; 1263th: A→C; 1266th: C→G; 1278th: C→A; 1287th: A→C; 1297th: T→C; 1302th: A→C; 1308th: C→T; 1314th: G→C; 1326th: A→C; 1329th: G→T; 1335th: C→G; 1338th: C→G; 1341th: G→T; 1347th: A→G; 1350th: C→A; 1353th: G→C; 1356th: C→A; 1357th: T→C; 1362th: G→T; 1368th: G→T; 1381th: T→C; 1392th: G→C; 1401th: A→T; 1404th: C→A; 1420th: A→C; 1425th: C→G; 1431th: A→T; 1432: T→A, 1433th: C→G; 1434: G→C; 1435th: T→A; 1436th: C→G; 1437th; G→C, 1440th T→C; 1445th: C→A; 1447th: C→A; 1450th: C→T; 1456th: T→C; 1459th: C→G; 1462th: A→C; 1465th: C→G; 1468th: C→T; 1472th: T→A; 1473th: C→G; 1474th: T→C; 1480th: C→G; 1486th: C→T; 1489th: G→T; 1492th: T→C; 1495th: C→G; 1498th: C→G; 1501th: T→C; 1504th: T→C; 1511th: T→A; 1512th: C→G; 1519th: G→A; 1529th: T→A; 1530th: C→G; 1531th: A→C; 1534th: A→C; 1540th: C→T; 1544th: A→T; 1545th: G→C; 1546th: C→T; 1547th: C→A; 1552th: C→T; 1556th: T→C; 1558th: A→G; 1561th: T→C; 1573th: T→C; 1580th: T→A; 1581th: C→G; 1585th: T→C; 1588th: C→G; 1591th: T→C; 1597th: T→C; 1609th: G→C; 1618th: T→A; 1627th: C→T; 1630th: A→C; 1636th: C→G; 1651th: A→C; 1664th: C→A; 1666th: C→A; 1672th: C→A; 1681th: G→T; 1684th: A→C; 1685th: T→C; 1688th: T→C; 1702th: C→G; 1705th: A→T; 1706th: C→A; 1708th: G→A; 1717th: A→C; 1718th: T→A; 1719th: C→G; 1720th: A→C; 1732th: G→A; 1738th: A→G; 1753th: T→C; 1760th: T→C; 1765th: C→G; 1768th: G→A; 1771th: T→C; 1774th: T→C; 1777th: A→G; 1783th: C→G; 1789th: T→C; 1792th: A→C; 1825th: T→C; 1831th: T→C; 1834th: T→G; 1840th: T→C; 1844th: T→C; 1849th: G→C; 1855th: G→A; 1864th: G→A; 1867th: G→C; 1870th: T→C; 1873th: C→A; 1874th: T→A; 1875th: C→G; 1876th: T→C; 1891th: T→G; 1897th: T→C; 1900th: T→C; 1903th: T→C; 1906th: T→C; 1907th: C→A; 1909th: A→G; 1921th: G→A; 1927th: C→T; or 1930th: C→G.
In other embodiment, the polynucleotide of the present disclosure is disclosed as SEQ ID NO:2.
In other aspect, the present disclosure is directed to a polypeptide encoded by NIS polynucleotide which is codon-optimized for expression in eukaryotic cells. In one embodiment, there are no changes in the amino acid sequence by the optimized codons.
Further the present disclosure is directed to a plasmid for eukaryotic expression comprising a polynucleotide of the present disclosure and a promoter operatively linked thereto.
The plasmid of the present disclosure is to express oNIS of the present disclosure in eukaryotic cells. Thus, a variety of promoters may be utilized for the present purpose. For example, EF-1 Alpha promoter (Clontech, USA), Ubiquitin promoter (Christensen A H et al., Transgenic Res. 1996; 5(3)-213-8), CMV promoter (cytomegalovirus promoter) (Clontech, USA), LTR (Retroviral promoter) (Reynolds et al., PNAS 2003; 100(4):1615-1620) and beta-actin promoter (Clontech, USA) may be used for the present disclosure without being limited thereto.
In other aspect, the present disclosure is directed to a recombinant eukaryotic cell, particularly cells derived from a mammal, more particularly cells derived from a human being comprising the plasmid of the present disclosure. In one embodiment, the cells are derived from a human cancer. A variety of eukaryotic cells known in the art which may be transfected with the present plasmids may be used for the present purpose. For example it includes cancer cell lines such as thyroid cancer, liver cancer, glioma, cervical cancer and the like. Particularly, it includes MDA-MB-231, TPC-1, FRO, BCRAP, HEP3B, HeLa and U87MG without being limited thereto. Further, breast cancer cell lines such as MCF7, MDAMB468 and KPL4, colon cancer cell lines such as HT29, Caco-2, HCT116, and lung cancer cell lines such as A549 and NCI-H446 may also be used.
A variety of transfection methods known in the art may be used for the present disclosure, and includes, but are not limited to, calcium phosphate precipitation methods, electroporation, liposome and shot-gun methods and the like.
In a further aspect, the present disclosure is directed to a kit comprising a codon optimized polynucleotide encoding NIS and/or a plasmid comprising the same for nuclear and/or molecular imaging or for imaging reporter for gene, viral and cell based therapies. The codon optimized polynucleotides of the present disclosure which are included in the kit are optimized for the efficient expression and functional activities in eukaryotic cells and thus can be utilized for the development as a gene therapy vector, a reporter for nuclear and/or molecular imaging, and a transgenic mouse overexpressing a gene for molecular imaging using radionuclides such as Iodide 123,124,125,131I, Rhenium 186,188RE, technetium 99mTc and the like. The kits are useful as a reporter for imaging, for combined cancer therapy of radioactive iodide with gene therapy and for thyroid cancer therapy using radioactive iodide.
The present disclosure is further explained in more detail with reference to the following examples. These examples; however; should not be interpreted as limiting the scope of the present invention in any manner.
EXAMPLES
Example 1
Construction of a Codon Optimized NIS and a Plasmid Comprising the Same
For the construction of a codon optimized NIS, the following regions of a NIS were selected: the 8th transmembrane domain from the N-terminal recognizing iodide (amino acid residues 286-308) and the 5th transmembrane domain (163-182) involved in the membrane penetration, and C-terminal region (550-643). Further the sites which are important for the functional activity of NIS, i.e., phosphorylation sites, Ser-43, Thr-49, Ser-227, Lys-577 and Ala-581 which are modified post-translationally and N-glycosylation sites Asn-225 Asn-489 and Asn-502 were selected for the modification.
Codon optimizing software (GENEOPTIMIZERTM GENEART®” Life Technologies) was used to obtain a modified sequence, which were then synthesized in Takara Bio Inc. (JAPAN).
The results of substitutions are summarized in Table 1, FIGS. 1a through 1e.












TABLE 1









Codon
Codon





Adaptation
Frequency



CDS length
Amino acid
Index m(CAI)
Distribution




















hNIS
1932 bp
643
0.79
61%


oNIS
1932 bp
643
0.97
88%









Table 1 is the comparison of the parent sequence of NIS and the modified sequences thereof in various aspects as indicated. Both have the same length of coding region of 1932 by encoding 643 amino acids.

For analyzing codon usage bias, Codon Adaptation Index (CAI) which measures the deviation of a given protein coding gene sequence with respect to a reference set of genes was determined. As shown in FIGS. 1c to 1e, CAI was found to be 0.79 in the parent sequence of NIS in contrast to 0.97 in the modified sequence. Further the amount of GC content was found to be 64%.
Further according to the codon frequency graph as shown in FIGS. 1a and 1b, the percent of codons which are utilized with 91-100% frequency was found to be 61% in the parent sequence of NIS in contrast to 88% in the modified sequence.
Then the modified DNA fragment was then cloned into a cloning vector and a eukaryotic expression vector to construct a plasmid comprising the modified NIS DNA.
pMK-RQ (Invitrogen Inc. USA) was used as a cloning vector and the modified DNA was cloned into NcoI and SfiI restriction sites to obtain a pMK-RQ-oNIS, the map of which is shown in FIG. 2.
For the expression in human cells, the modified DNA, oNIS and hNIS each was cloned into each of a vector, PCDNATM3.1 (Invitrogen Inc. USA) and pMSCV (Clontech Laboratories, Inc. USA) at XhoII/ApaI restriction site to obtain expression plasmids, PCDNATM3.1-oNIS, PCDNATM3.1-hNIS, pMSCV-oNIS and pMSCV-hNIS.
Example 2
Determination of Expression Level of Codon Optimized NIS in Cells
Each of pMSCV-oNIS and pMSCV-hNIS as constructed in Example 1 was transfected into a breast cancer cell line MDA-MB-231 (ATCC® HTB-26TM). The vectors are based on retroviral DNA and the viruses were prepared in a renal cell line FT293 (Life Technologies, Invitrogen) one day before the transfection. The amount of plasmid used for the transfection was 1 μg, 1 μg, and 2 μg for gag/pol, envelope, pMSCV-oNIS/pMSCV-hNIS, respectively. The plasmids were dissolved in 750 μl of OPTI-MEM® as a medium (Invitrogen Inc.), which was then mixed with 30 μg of LIPOFECTAMINE® 2000 (Invitrogen Inc.) as a transfection agent and incubated for 15 min at RT. The mixture was then added to the FT293 cells prepared as above and incubated for 48 hours. Then the supernatant containing the viruses was collected and 1 ml of the supernatant per 5 ml of the cell culture medium containing 10 μg/mL of polybrene was used to infect MDA-MB-231 cells and incubated for 48 hours. Subsequently 2 μg/mL of puromycin (Invitrogen Inc.) was added to select the resistant cells and to obtain a stable cell line. For 2 weeks after the transfection, the cells were cultured at 37° C., 5% CO2 and 95% humidity, which were then analyzed for the expression of NIS using Reverse transcription PCR (RT-PCR) and western blot. As a reference, GAPDH and beta-actin were used.
For RT-PCR, total RNA was extracted from the cells using TRIZOL® (Invitrogen Inc.) as an RNA extraction agent according to the manufacturer's instruction and cDNA was synthesized using oligo dT and cDNA synthesis kit (amfiRivert Platinum cDNA Synthesis Master Mix (GenDEPOT, USA). Subsequently cDNA was amplified using the following primers: for NIS amplification (F′-TGGGCGGCATGAAGGCYGTG: SEQ ID NO: 4, R′-CRCTGTAGGCACAGGCCAGG: SEQ ID NO: 5), for GAPDH primer (F′-GAGAAGGCTGGGGCTCATTT: SEQ ID NO: 6, R′-CCTTCCGTGTCCCCACTG: SEQ ID NO: 7), and under the following condition: 5 min at 94° C. for denaturation; 30 cycles of 30 sec at 94° C. 30 sec at 56.5° C. or 60° C.; 30 sec at 72° C.; and a final step for 7 min at 72° C.
For western blot analysis, whole protein was extracted using RIPA buffer Sigma Aldrich, USA) according to the manufacturer's instruction. Then 30 μg of protein extract was electrophoresed on a 12% Bis-Tris gel NUPAGE®, Invitrogen) at 150V for 2 hours and transferred to a PVDF membrane (Invitrogen) at 70V for 3 hours. The membrane was then incubated with anti-hNIS (KOMA Biotech, Korea) at 1:1000 dilution overnight at 4° C. followed by an incubation with a second antibody, anti-rabbit-IgG (KOMA Biotech, Korea) at 1:2000 dilution for 2 hours at RT and visualized using PIERCETM ECL Western Blotting Substrate (Thermo Scientific, USA) as a substrate according to the manufacturer's instruction.
Results are shown in FIGS. 3a through 3d. As shown in FIGS. 3a and 3b, at the mRNA level, it has been found that there was no difference between hNIS and oNIS. However, as shown in FIGS. 3c and 3d, at the protein level, it has been found that the expression level of oNIS was four times higher than that of hNIS. This indicates that the protein expression can be significantly increased by the codon-optimized NIS according to the present disclosure.
Example 3
Determination of Radioactive Iodine Uptake in Various Human Cancer Cell Lines
Example 3-1
Cancer Cell Lines and Transfection
TPC-1 and FRO (Donated by Dr. Shunichi Yamashita, Nagasaki University) were cultured in RPMI 1640 (Promega, USA) supplemented with 5% non-heat inactivated fetal bovine serum and 1% antibiotic-antimycotic at 37° C. in 5% CO2. B-CPAP (Donated by Dr. Shunichi Yamashita, Nagasaki University; Leibniz Institute DSMZ German Collection of Microorganisms) cells were grown under the same condition as described above except that 10% heat inactivated fetal bovine serum was used. HEP3B and Hela (Korean Cell Line Bank, Korea) were grown in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% heat inactivated fetal bovine serum and 1% antibiotic-antimycotic at 37° C. in 5% CO2. U87MG(ATCC) was grown in MEM (Minimum Essential Medium) supplemented with 10% heat inactivated fetal bovine serum and 1% antibiotic-antimycotic at 37° C. in 5% CO2. The cell lines described above have the following origin as described in Table 2.










TABLE 2







Cell Line
Origin









TPC-1
Thyroid cancer (Papillary)



FRO
Thyroid cancer (Anaplastic)



B-CPAP
Thyroid cancer (Papillary)



HEP3B
Liver cancer



HeLa
Cervical Cancer



U87MG
Glioma










For the transfection, each of the cell lines as described above was transfected with 20 μg of each of a vector PCDNATM3.1-hNIS and PCDNATM3.1-oNIS using LIPOFECTAMINE®2000 (Invitrogen) as a transfection agent according to the manufacturer's instructions. The stable cell lines were established as described in Example 2 for MDA-MB231 cell line.
Example 3-2
Comparison of the Uptake of Radioactive Iodide in Various Human Cancer Cell Lines
The cells transfected in Example 3-1 were treated with radioactive iodide, Na 125I (Perkin Elmer, USA). Specifically, the cells were seeded onto each well of a 24 well plate at 3×105 cells/well and 37 kBq/ml of Na 125I was added to each well and incubated for 30 min at 37° C. in 5% CO2. Then the cells were lysed using sodium dodecyl sulfate and the radioactivity therefrom was measured using a gamma counter. The amount of protein in each sample was measured using BCA assay kit (Pierce Biotechnology, USA) according to the manufacturer's instruction and used to normalize the results as pmol/mg. Also the cells were photographed with a gamma camera (γ-ray camera (ON-410); Ohio Nuclear, Solon, Ohio, USA) for 300 k counts.
Results are shown in FIGS. 4 and 5. FIG. 4 is a gamma camera image showing the uptake of radioactive iodides in human breast cancer cell line transfected with oNIS as compared with controls (CRT, hNIS). FIG. 5 shows the uptake of radioactive iodide in various cancer cell lines transfected with control, hNIS or oNIS polynucleotides. The results indicate that the cells expressing oNIS have a higher uptake of radioactive iodide compared to the cells expressing hNIS.
Example 3-3
Comparison of the Uptake of Radioactive Iodide in a Human Cancer Cell Line and a Mouse Implanted Therewith
Cells stably transfected with oNIS or hNIS polynucleotides prepared in Example 3-1 were seeded onto each well of a 24 well plate at 3×105 cells/well and treated with 3.7 mBq/ml of 99mTc and incubated for 30 min at 37° C. in 5% CO2. Then the cells were lysed with SDS and photographed with gamma camera. Also each of the stably transfected cells expressing oNIS or hNIS were implanted into the hind limb of Balb/Nude mouse (Oriental Bio, Korea) at 5×106 cells/site. After 2 weeks, the mouse were treated with 500 μCi (18.5 mBq) of 99mTc and photographed with a gamma camera as described above.
Results are shown in FIGS. 6a through 6d. As shown in FIG. 6a, the cells expressing oNIS have about three times higher uptake of radioactive iodide than that of the cells expressing hNIS. As shown in FIG. 6b, the results from gamma camera analysis show 1.5 times increase in the radioactivity. Also as shown in FIGS. 6c and 6d, it has been found that the uptake of 99mTc was increased in the tumor expressing oNIS compared to that expressing hNIS, in which both tumor had the same size. This indicates the excellent efficiency of the optimized codon in the translation into proteins.
Sequence Listing submitted in txt format (.txt) filed on Jun. 18, 2013 named “SequenceListing_YOUN.txt” (created on Jun. 17, 2013; KN); is incorporated herein by reference.
The various singular/plural permutations may be expressly set forth herein for sake of clarity. Although a few embodiments of the present disclosure have been shown and described; it would be appreciated by those skilled in the art that changes may be made in this embodiment without departing from the principles and sprit of the invention; the scope of which is defined in the claims and their equivalents

## Claims

1. A polynucleotide encoding a sodium-iodide symporter (NIS) protein having the sequence set forth in SEQ ID NO: 2 and codon optimized for expression in a eukaryotic cell.

2. A plasmid comprising the polynucleotide of claim 1 and a promoter operatively linked to the polynucleotide, wherein the plasmid is codon optimized for expression in a eukaryotic cell.

3. An isolated recombinant host cell comprising the plasmid of claim 1 or the polynucleotide of claim 4.

4. The isolated recombinant cell of claim 3, wherein the cell is a mammal cell.

5. The isolated recombinant cell of claim 3, wherein the cell is a human cell.

6. The isolated recombinant cell of claim 3, wherein the cell is a non-human mammal cell.

7. A kit for radio-imaging, nuclear medicine imaging or molecular imaging comprising the plasmid of claim 2.