[99mTc]Human telomerase reverse-transcriptase antisense mRNA oligonucleotide


Chopra A.

Publication Details



In vitro Rodents



Telomerase is an enzyme that synthesizes telomeric DNA at the terminal end of a chromosome, and its activation is one of the mechanisms for the progression of cancer (1). Telomerase, in conjunction with the telomere-binding proteins, maintains stability of the chromosomal terminal end by replicating the DNA, but inhibition of this activity results in a shortened telomere and ultimately leads to replicative senescence or even cell death (2). The repression and close regulation of this enzyme is considered to be one of the mechanisms for tumor suppression in humans, and there is much evidence that a shortened telomere is an indication for the development of a cancer or other degenerative disease, especially in older individuals (3-6). In addition, telomerases are specifically expressed in most cancer cell types and therefore are targets for the development of many anticancer drugs (2). Also, tumors are less likely to develop resistance to anti-telomerase drugs compared to other anticancer treatments because maintenance of the telomere and immortality is crucial for tumor progression (2). Several clinical trials for drugs, vaccines, and antibodies that target the telomerases as a treatment for cancer have been approved by the United States Food and Drug Administration.

Telomerase consists of the telomerase RNA (hTR), the telomerase reverse-transcriptase (hTERT), and the associated proteins (1). Although hTR is expressed without telomerase activity both in normal cells and in malignant cells, only tumor cells express hTERT. As a consequence, hTERT is the target for the development of many anticancer therapies (7), and its expression levels may have prognostic value (8). Investigators have used only indirect methods such as immunohistochemistry, reverse-transcription polymerase chain reaction (RT-PCR), fluorescence, bioluminescence, and radionuclide imaging to determine the expression levels of hTERT in various cell types with reasonable results (9, 10). This approach may not, however, yield the exact nature of the change(s) that alter the genotype and, ultimately, the cell phenotype that result in the development of a cancer. Liu et al. envisioned that antisense oligonucleotides (ASON) would be an excellent in vivo tool to determine the level of expression of a gene during the early stages of tumor development because of their high specificity for binding to nucleotides (11). The authors investigated the in vitro activity and biodistribution of ASON directed toward the hTERT gene and labeled with radioactive technetium (99mTc) in mice bearing xenograft tumors.



The conjugation and radiolabeling of the ASON and the sense oligonucleotides (SON) used for the following studies described by Liu et al. (11). The ASON sequence was 5’-TAGAGACGTGGCTCTTGA-3’, and the SON nucleotide sequence was 5’-TCAAGAGCCACGTCTCTA-3’. Both oligonucleotides had a primary amine on the 3’ terminal phosphate group through a six-member methylene carbon spacer. For conjugation of the ASON with the N-hydroxysuccinimidyl (NHS) derivative of S-acetylmercaptoacetyltriglycine (S-acetyl NHS-MAG3), it was dissolved in N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) buffer (pH 8.0), and a fresh solution of S-acetyl NHS-MAG3 in N,N-dimethylformamide was added dropwise to a final ratio of 20:1. The mixture was incubated at room temperature for 60–120 min, and the conjugated ASON was purified with chromatography on a Sephadex G25 column using sterile phosphate buffer (pH 7.2) as an eluent. The conjugated ASON was then aliquoted into vials, lyophilized, and stored at -20°C until needed. Conjugation and storage of the SON was the same as described for the ASON (11).

For labeling with radioactive 99mTc, the MAG3-ASON was dissolved in ammonium acetate buffer (pH 5.2), and sodium tartrate was added to it (as a transchelator) until the pH reached 7.6 (11). Then 99mTc-pertechnetate was added, and a stannous chloride solution was added immediately. The mixture was incubated for 15–30 min, and the labeled MAG3-ASON was purified with chromatography on a Sephadex G25 column with phosphate buffer (pH 7.2) as the eluent. Labeling stability of 99mTc-MAG3-ASON was determined at room temperature, in saline at room temperature, and in fresh human serum at 37°C for >24 h by paper chromatography. Stability of the labeled ASON in human serum was also determined with polyacrylamide gel electrophoresis after extraction by the phenol:chloroform method, followed by overnight ethanol precipitation at -20°C (11).

Under the conditions used, the labeling efficiency of the reaction was reported to be 76 ± 5% (n = 5 labeling reactions) with a specific activity of 10.2 kBq/pmol (0.27 μCi/pmol) and a radiochemical purity of >96% after purification (11). The Rf values for the various reaction components after paper chromatography are reported in Table 1 of the publication (11). The stability of 99mTc-MAG3-ASON was reported to be >93% over a 24-h period at room temperature, in normal saline, and in fresh human serum at 37°C.

In Vitro Studies: Testing in Cells and Tissues


The in vitro activity of 99mTc-MAG3-ASON was investigated in HepG2 cells (11). The cells were exposed to 99mTc-MAG3-ASON or ASON, and the hTERT mRNA levels in these cells were determined with RT-PCR and compared with the levels in cells exposed to 99mTc-MAG3-SON or 10% fetal bovine serum (FBS). A significant reduction (P < 0.05) in the hTERT mRNA was observed in cells treated with 99mTc-MAG3-ASON or ASON compared with the cells exposed either to 99mTc-MAG3-SON or 10% FBS. No differences were reported in the hTERT mRNA levels of cells treated with 99mTc-MAG3-ASON or ASON. Similar observations were also reported for the 10% FBS-treated cells.

Animal Studies



The biodistribution of 99mTc-MAG3-ASON and 99mTc-MAG3-SON was investigated in BALB/c nu/nu mice bearing MCF-7 cell xenograft tumors (11). The animals were injected with the labeled ASON or the SON through the tail vein and killed 0.5, 1, 2, 4, and 6 h after the injection (n = 5 animals per time point for each oligonucleotide). The various organs were harvested from the animals, weighed, and counted for radioactivity. The radioactivity had a rapid clearance from blood circulation with 2.62 ± 0.167% of the injected dose/gram tissue (% ID/g) of 99mTc-MAG3-ASON remaining in the blood 0.5 h after administration. This level dropped to 1.051 ± 0.121% ID/g at 2 h. The level of 99mTc-MAG3-ASON (1.157 ± 0.182% ID/g) was significantly higher (P < 0.05) compared with 99mTc-MAG3-SON (0.503 ± 0.051% ID/g) in the tumors after 4 h with a tumor/muscle ratio of 8.8 and a tumor/blood ratio of 2 at 6 h. Among the various organs, maximum radioactivity was noticed in the kidneys for both the radiolabeled oligonucleotides, which indicates that the radiochemicals were cleared mainly through the urinary system.

Imaging of the mice 4–8 h after administration of the labeled oligonucleotides clearly revealed tumors only in mice injected with 99mTc-MAG3-ASON (11). The tumors were not visible by imaging at any time after the administration of 99mTc-MAG3-SON. However, the renal route of elimination of the radiochemicals led to an accumulation of substantial radioactivity in the abdomen with both labeled nucleotides.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.

Supplemental Information



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