Sequencing of Phosphoramidate Oligonucleotides by Acid Hydrolysis and Mass Spectrometry.

Hydrolysis of N3'-P5' phosphoramidate and thiophosphoramidate oligonucleotides with 0.1% formic acid leads to the cleavage of the 3' N-P bond and generates two products, one of which contains a 5'-phosphate. Analysis of the hydrolytic products by liquid chromatography, coupled with mass spectrometry, reveals the mass ladder from both termini, which is used to determine the sequence. While acid hydrolysis does not result in depurination, internal fragments especially in the low mass range are detected. The method is applied to DNA and RNA analogues with and without modifications at the 2'-position. This approach enables rapid sequence confirmation of synthetic phosphoramidate oligonucleotides for quality control as well as denovo sequencing.

Immunomodulatory spherical nucleic acids.

Immunomodulatory nucleic acids have extraordinary promise for treating disease, yet clinical progress has been limited by a lack of tools to safely increase activity in patients. Immunomodulatory nucleic acids act by agonizing or antagonizing endosomal toll-like receptors (TLR3, TLR7/8, and TLR9), proteins involved in innate immune signaling. Immunomodulatory spherical nucleic acids (SNAs) that stimulate (immunostimulatory, IS-SNA) or regulate (immunoregulatory, IR-SNA) immunity by engaging TLRs have been designed, synthesized, and characterized. Compared with free oligonucleotides, IS-SNAs exhibit up to 80-fold increases in potency, 700-fold higher antibody titers, 400-fold higher cellular responses to a model antigen, and improved treatment of mice with lymphomas. IR-SNAs exhibit up to eightfold increases in potency and 30% greater reduction in fibrosis score in mice with nonalcoholic steatohepatitis (NASH). Given the clinical potential of SNAs due to their potency, defined chemical nature, and good tolerability, SNAs are attractive new modalities for developing immunotherapies.

Fast and accurate nonenzymatic copying of an RNA-like synthetic genetic polymer.

Recent advances suggest that it may be possible to construct simple artificial cells from two subsystems: a self-replicating cell membrane and a self-replicating genetic polymer. Although multiple pathways for the growth and division of model protocell membranes have been characterized, no self-replicating genetic material is yet available. Nonenzymatic template-directed synthesis of RNA with activated ribonucleotide monomers has led to the copying of short RNA templates; however, these reactions are generally slow (taking days to weeks) and highly error prone. N3'-P5'-linked phosphoramidate DNA (3'-NP-DNA) is similar to RNA in its overall duplex structure, and is attractive as an alternative to RNA because the high reactivity of its corresponding monomers allows rapid and efficient copying of all four nucleobases on homopolymeric RNA and DNA templates. Here we show that both homopolymeric and mixed-sequence 3'-NP-DNA templates can be copied into complementary 3'-NP-DNA sequences. G:T and A:C wobble pairing leads to a high error rate, but the modified nucleoside 2-thiothymidine suppresses wobble pairing. We show that the 2-thiothymidine modification increases both polymerization rate and fidelity in the copying of a 3'-NP-DNA template into a complementary strand of 3'-NP-DNA. Our results suggest that 3'-NP-DNA has the potential to serve as the genetic material of artificial biological systems.

Telomerase inhibition improves tumor response to radiotherapy in a murine orthotopic model of human glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most frequent and aggressive type of adult brain tumor. Most GBMs express telomerase; a high level of intra-tumoral telomerase activity (TA) is predictive of poor prognosis. Thus, telomerase inhibitors are promising options to treat GBM. These inhibitors increase the response to radiotherapy (RT), in vitro as well as in vivo. Since typical treatments for GBM include RT, our objective was to evaluate the efficiency of Imetelstat (TA inhibitor) combined with RT. FINDINGS: We used a murine orthotopic model of human GBM (N = 8 to11 mice per group) and µMRI imaging to evaluate the efficacy of Imetelstat (delivered by intra-peritoneal injection) alone and combined with RT. Using a clinically established protocol, we demonstrated that Imetelstat significantly: (i) inhibited the TA in the very center of the tumor, (ii) reduced tumor volume as a proportion of TA inhibition, and (iii) increased the response to RT, in terms of tumor volume regression and survival increase. CONCLUSIONS: Imetelstat is currently evaluated in refractory brain tumors in young patients (without RT). Our results support its clinical evaluation combined with RT to treat GBM.

Cancer Stem Cells and Anti-tumor Immunity.

Cancer stem cells (CSCs) are correlated with poor clinical outcomes due to their contribution to chemotherapy resistance and the formation of metastasis. Multiple cell surface and enzymatic markers have been characterized to identify CSCs, which is important for diagnosis, therapy, and prognosis. This review underlines the role of CSCs and circulating tumor cells (CTCs) in tumor relapse and metastasis, the characteristics of CSC and CTC biomarkers, and the techniques used to detect these cells. We also summarized novel therapeutic approaches toward targeting CSCs, especially focusing on the role of immune checkpoint blockades (ICB), such as anti-programmed death 1 (anti-PD1) and antiprogrammed death ligand-1 (anti-PDL1) therapies. Additionally, we address an intriguing new mechanism of action for small molecular drugs, such as telomere-targeted therapy 6-thio-2'deoxyguanosine (6- thio-dG), and how it reshapes tumor microenvironment to overcome ICB resistance. There are indications, that personalized cancer therapy targeting CSC populations in conjunction with immune-mediated strategy hold promise for the removal of residual therapy-resistant CSCs in the near future.

New Oligonucleotide 2'-O-Alkyl N3'→P5' (Thio)-Phosphoramidates as Potent Antisense Agents: Physicochemical Properties and Biological Activity.

We describe here the design, synthesis, physicochemical properties, and hepatitis B antiviral activity of new 2'-O-alkyl ribonucleotide N3'→P5' phosphoramidate (2'-O-alkyl-NPO) and (thio)-phosphoramidite (2'-O-alkyl-NPS) oligonucleotide analogs. Oligonucleotides with different 2'-O-alkyl modifications such as 2'-O-methyl, -O-ethyl, -O-allyl, and -O-methoxyethyl combined with 3'-amino sugar-phosphate backbone were synthesized and evaluated. These molecules form stable duplexes with complementary DNA and RNA strands. They show an increase in duplex melting temperatures of up to 2.5°C and 4°C per linkage, respectively, compared to unmodified DNA. The results agree with predominantly C3'-endo sugar pucker conformation. Moreover, 2'-O-alkyl phosphoramidites demonstrate higher hydrolytic stability at pH 5.5 than 2'-deoxy NPOs. In addition, the relative lipophilicity of the 2'-O-alkyl-NPO and NPS oligonucleotides is higher than that of their 3'-O- counterparts. The 2'-O-alkyl-NPS oligonucleotides were evaluated as antisense (ASO) compounds in vitro and in vivo using Hepatitis B virus as a model system. Subcutaneous delivery of GalNAc conjugated 2'-O-MOE-NPS gapmers demonstrated higher activity than the 3'-O-containing 2'-O-MOE counterpart. The properties of 2'-O-alkyl-NPS constructs make them attractive candidates as ASO suitable for further evaluation and development.

Targeting TLR9 agonists to secondary lymphoid organs induces potent immune responses against HBV infection.

Despite the existence of a prophylactic vaccine against hepatitis B virus (HBV), chronic hepatitis B virus (CHB) infection remains the leading cause of cirrhosis and liver cancer in developing countries. Because HBV persistence is associated with insufficient host immune responses to the infection, development of an immunomodulator as a component of therapeutic vaccination may become an important strategy for treatment CHB. In the present study, we aimed to design a novel immunomodulator with the capacity to subvert immune tolerance to HBV. We developed a lymphoid organ-targeting immunomodulator by conjugating a naturally occurring, lipophilic molecule, α-tocopherol, to a potent CpG oligonucleotide adjuvant pharmacophore. This approach resulted in preferential trafficking of the α-tocopherol-conjugated oligonucleotide to lymphoid organs where it was internalized by antigen-presenting cells (APCs). Moreover, we show that conjugation of the oligonucleotides to α-tocopherol results in micelle-like structure formation, which improved cellular internalization and enhanced immunomodulatory properties of the conjugates. In a mouse model of chronic HBV infection, targeting CpG oligonucleotide to lymphoid organs induced strong cellular and humoral immune responses that resulted in sustained control of the virus. Given the potency and tolerability of an α-tocopherol-conjugated CpG oligonucleotide, this modality could potentially be broadly applied for therapeutic vaccine development.

Targeting critical steps of cancer metastasis and recurrence using telomerase template antagonists.

Metastasis, tumor relapse, and drug resistance remain major obstacles in the treatment of cancer. Therefore, more research on the mechanisms of these processes in disease is warranted for improved treatment options. Recent evidence suggests that the capability to sustain tumor growth and metastasis resides in a subpopulation of cells, termed cancer stem cells or tumor-initiating cells. Continuous proliferation and self-renewal are characteristics of stem/progenitor cells. Telomerase and the maintenance of telomeres are key players in the ability of stem and cancer cells to bypass senescence and be immortal. Therefore, telomerase inhibitors have the therapeutic potential for reducing tumor relapse by targeting cancer stem cells and other processes involved in metastasis. Herein we review the role of telomerase in the immortal phenotype of cancer and cancer stem cells, targeting telomerase in cancer, and discuss other opportunities for telomerase inhibitors to target critical steps in cancer metastasis and recurrence.

Oligonucleotide n3'-->p5' phosphoramidates and thio-phoshoramidates as potential therapeutic agents.

Nucleic acids analogues, i.e., oligonucleotide N3'-->P5' phosphoramidates and N3'-->P5' thio-phosphoramidates, containing 3'-amino-3'-deoxy nucleosides with various 2'-substituents were synthesized and extensively studied. These compounds resist nuclease hydrolysis and form stable duplexes with complementary native phosphodiester DNA and, particularly, RNA strands. An increase in duplexes' melting temperature, DeltaT(m), relative to their phosphodiester counterparts, reaches 2.2-4.0 degrees per modified nucleoside. 2'-OH- (RNA-like), 2'-O-Me-, and 2'-ribo-F-nucleoside substitutions result in the highest degree of duplex stabilization. Moreover, under close to physiological salt and pH conditions, the 2'-deoxy- and 2'-fluoro-phosphoramidate compounds form extremely stable triple-stranded complexes with either single- or double-stranded phosphodiester DNA oligonucleotides. Melting temperature, T(m), of these triplexes exceeds T(m) values for the isosequential phosphodiester counterparts by up to 35 degrees . 2'-Deoxy-N3'-->P5' phosphoramidates adopt RNA-like C3'-endo or N-type nucleoside sugar-ring conformations and hence can be used as stable RNA mimetics. Duplexes formed by 2'-deoxy phosphoramidates with complementary RNA strands are not substrates for RNase H-mediated cleavage in vitro. Oligonucleotide phosphoramidates and especially thio-phosphoramidates conjugated with lipid groups are cell-permeable and demonstrate high biological target specific activity in vitro. In vivo, these compounds show good bioavailability and efficient biodistribution to all major organs, while exerting acceptable toxicity at therapeutically relevant doses. Short oligonucleotide N3'-->P5' thio-phosphoramidate conjugated to 5'-palmitoyl group, designated as GRN163L (Imetelstat), was recently introduced as a potent human telomerase inhibitor. GRN163L is not an antisense agent; it is a direct competitive inhibitor of human telomerase, which directly binds to the active site of the enzyme and thus inhibits its activity. This compound is currently in multiple Phase-I and Phase-I/II clinical trials as potential broad-spectrum anticancer agent.

Lipid-conjugated telomerase template antagonists sensitize resistant HER2-positive breast cancer cells to trastuzumab.

HER2 amplification in breast cancer is associated with a more aggressive disease, greater likelihood of recurrence, and decreased survival compared to women with HER2-negative breast cancer. Trastuzumab is a monoclonal antibody that inhibits HER2 activity, making this compound an important therapeutic option for patients with HER2-positive breast cancer. However, resistance to trastuzumab develops rapidly in a large number of breast cancer patients. The objective of this study was to determine whether GRN163L, a telomerase template antagonist currently in clinical trials for cancer treatment, can augment the effects of trastuzumab in breast cancer cells with HER2 amplification. GRN163L was effective in inhibiting telomerase activity and shortening telomeres in HER2-positive breast cancer cells. We show that GRN163L acts synergistically with trastuzumab in inhibiting HER2-positive breast cancer cell growth. More importantly, we show that GRN163L can restore the sensitivity of therapeutic-resistant breast cancer cells to trastuzumab. These findings implicate that telomerase template antagonists have potential use in the treatment of cancers that have developed resistance to traditional cancer therapy.

Antiadhesive effects of GRN163L--an oligonucleotide N3'->P5' thio-phosphoramidate targeting telomerase.

We determined previously that a novel human telomerase RNA (hTR) antagonist, GRN163L, inhibited the tumorigenic potential of A549-luciferase (A549-luc) lung cancer cells in vitro and in vivo. Further studies revealed that A549-luc cells were also morphologically altered by GRN163L. A549-luc cells treated before cell attachment with a single dose of GRN163L only weakly attached to the substrate and remained rounded, whereas control mismatch-treated cells exhibited typical epitheloid appearance and adhesion properties. These morphologic changes were independent of hTR expression and telomerase inhibition and were unrelated to telomere length. This effect is dependent on the molecular properties of the lipid moiety, the phosphorothioate backbone, and the presence of triplet-G sequences within the GRN163L structure. Altered adhesion was manifested by a 50% reduction in rapid cellular attachment and a 3-fold decrease in total cell spreading surface area. Administration of a single dose of GRN163L (15 mg/kg) at the time of cell inoculation, using an in vivo model of lung cancer metastasis, resulted in significant reductions in tumor burden at days 13, 20, and 27 of tumor progression. Thus, the potent antimetastatic effects of GRN163L may be related, in part, to the antiadhesive effects of this novel cancer therapeutic conferred via specific structural determinants and that these effects are independent of telomerase inhibition or telomere shortening.

Telomerase targeted oligonucleotide thio-phosphoramidates in T24-luc bladder cancer cells.

Bladder carcinoma is the second most common genitourinary malignancy. Treatment options for bladder cancer include surgery, combined with chemotherapy, radiation, and/or immunotherapy. The adjuvant chemotherapy and immunotherapy regimen have been widely used in locally invasive as well as metastatic disease. The evaluation of new active agents with improved tolerability has been the focus of investigations over the past decade with minimal overall improvements in outcomes. Telomerase activity has been found in approximately 85-90% of all human tumors, but not in the majority of adjacent normal tissues. This suggests that telomerase may be an attractive target for the development of novel anticancer therapeutic agents. GRN163L is a lipid conjugated oligonucleotide N3' --> P5' thio-phosphoramidate, and is a potent telomerase RNA (hTR) template antagonist. In the present study, we show that the telomerase activity of T24-luc bladder cancer cells is inhibited by 1 microM GRN163L within 24 h of incubation. After two weeks of exposure to GRN163L, T24-luc cells became "clustered" whereas non-cancerous normal human uroepithelial cells were not morphologically affected. Moreover, in vitro GRN163L treated T24-luc bladder cancer cells entered G(0)/G(1) arrest following 2 weeks of continuous exposure and stopped dividing. Mismatch control compound had no effect on normal bladder epithelial cells or T24-luc cells. Additionally, a new generation of thio-phosphoramidate oligonucleotides were designed and tested in T24-luc cells and compared with GRN163L. The obtained results warrant further in vivo evaluation of GRN163L as a potential treatment for bladder cancer.

New therapeutic approach for brain tumors: Intranasal delivery of telomerase inhibitor GRN163.

The blood-brain barrier is a substantial obstacle for delivering anticancer agents to brain tumors, and new strategies for bypassing it are greatly needed for brain-tumor therapy. Intranasal delivery provides a practical, noninvasive method for delivering therapeutic agents to the brain and could provide an alternative to intravenous injection and convection-enhanced delivery. We treated rats bearing intracerebral human tumor xenografts intranasally with GRN163, an oligonucleotide N3'-->P5'thio-phosphoramidate telomerase inhibitor. 3'-Fuorescein isothiocyanate (FITC)-labeled GRN163 was administered intranasally every 2 min as 6 microl drops into alternating sides of the nasal cavity over 22 min. FITC-labeled GRN163 was present in tumor cells at all time points studied, and accumulation of GRN163 peaked at 4 h after delivery. Moreover, GRN163 delivered intranasally, daily for 12 days, significantly prolonged the median survival from 35 days in the control group to 75.5 days in the GRN163-treated group. Thus, intranasal delivery of GRN163 readily bypassed the blood-brain barrier, exhibited favorable tumor uptake, and inhibited tumor growth, leading to a prolonged lifespan for treated rats compared to controls. This delivery approach appears to kill tumor cells selectively, and no toxic effects were noted in normal brain tissue. These data support further development of intranasal delivery of tumor-specific therapeutic agents for brain tumor patients.

Specific telomere dysfunction induced by GRN163L increases radiation sensitivity in breast cancer cells.

PURPOSE: Telomerase is expressed in 80-90% of tumor cells, but is absent in most somatic cells. The absence of telomerase activity results in progressive telomere shortening, leading to cellular senescence or death through deoxyribonucleic acid (DNA) damage signals. In addition, a role for telomerase in DNA damage repair has also been suggested. A specific telomerase inhibitor, GRN163L that is complementary to the template region of the telomerase ribonucleic acid component (hTR). We hypothesized that exposure to GRN163L, either through immediate inhibition of telomerase activity or through eventual telomere shortening and dysfunction, may enhance radiation sensitivity. Our goal was to test whether the treatment with GRN163L enhances sensitivity to irradiation (IR) in MDA-MB-231 breast cancer cells. METHODS AND MATERIALS: The MDA-MB-231 breast cancer cells were treated with or without GRN163L for 2-42 days. Inhibition of telomerase activity and shortening of telomeres were confirmed. Cells were then irradiated and clonogenic assays were performed to show cell survival differences. In vivo studies using MDA-MB-231 xenografts were performed to corroborate the in vitro results. RESULTS: We show that cells with shortened telomeres due to GRN163L enhance the effect on IR reducing survival by an additional 30% (p < 0.01). These results are confirmed in vivo, with a significant decrease in tumor growth in mice exposed to GRN163L. CONCLUSIONS: We found that GRN163L is a promising adjuvant treatment in combination with radiation therapy that may improve the therapeutic index by enhancing the radiation sensitivity. These studies prompt further investigation as to whether this combination can be applied to other cancers and the clinic.

Telomerase template antagonist GRN163L disrupts telomere maintenance, tumor growth, and metastasis of breast cancer.

PURPOSE: Maintenance of telomeres by telomerase is critical for the continuing proliferation of most advanced cancer cells. Telomerase activity has been detected in the vast majority of cancer cells but not most normal cells, making the enzyme an attractive target for anticancer therapy. The aim of this study was to address the breast cancer translational potential of the novel telomerase inhibitor, GRN163L. EXPERIMENTAL DESIGN: In the present study, we investigated the effects of GRN163L treatment on a panel of breast cancer cells representing different tumor subtypes with varying genetic backgrounds, including ER+, ER-, HER2+, BRCA1 mutant breast tumor cells as well as doxorubicin-resistant cancer cells. To investigate the in vivo effects of GRN163L, we employed a breast cancer xenograft and metastasis model that simulates a clinical situation in which a patient arrives with a primary tumor that may be then treated or surgically removed. RESULTS: GRN163L effectively inhibited telomerase activity in a dose-dependent fashion in all breast cancer cell lines resulting in progressive telomere shortening. A mismatch control oligonucleotide showed no effect on telomerase activity and GRN163L did not significantly affect telomere shortening in normal human mammary epithelial cells or in endothelial cells. Breast cancer cells that exhibited telomerase inhibition also exhibited significant reduction in colony formation and tumorigenicity. Furthermore, GRN163L suppressed tumor growth and lung metastases (P = 0.017) of MDA-MB-231 cells in vivo after 4 weeks of treatment. CONCLUSIONS: These results show in vivo effectiveness of GRN163L in breast cancer and support its promising clinical potential for breast cancer treatment.

Does a sentinel or a subset of short telomeres determine replicative senescence?

The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with gammaH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence.

Oligonucleotide conjugate GRN163L targeting human telomerase as potential anticancer and antimetastatic agent.

Telomerase is one of the key enzymes responsible for the proliferative immortality of the majority of cancer cells. We recently introduced a new telomerase inhibitor, a 13-mer oligonucleotide N3' --> P5'-thio-phosphoramidate lipid conjugate, designated as GRN163L. This compound inhibits telomerase activity in various tumor cell lines with IC(50) values of 3-300 nM without any cellular uptake enhancers. GRN163L demonstrated potent and sequence specific anti-cancer activity in vivo in multiple animal models. This compound was able to significantly affect not only the growth of primary tumors, but also the spread and proliferation of metastases. GRN163L is currently in Phase I and Phase I/II clinical studies in patients with solid tumors and CLL, respectively.

Lipid modification of GRN163, an N3'-->P5' thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition.

The vast majority of human cancers express telomerase activity, while most human somatic cells do not have detectable telomerase activity. Since telomerase plays a critical role in cell immortality, it is an attractive target for a selective cancer therapy. Oligonucleotides complementary to the RNA template region of human telomerase (hTR) have been shown to be effective inhibitors of telomerase and, subsequently, cancer cell growth in vitro. We show here that a lipid-modified N3'-->P5' thio-phosphoramidate oligonucleotide (GRN163L) inhibits telomerase more potently than its parental nonconjugated thio-phosphoramidate sequence (GRN163). Cells were treated with both the first- (GRN163) and second-generation (GRN163L) oligonucleotides, including a mismatch control, with or without a transfection enhancer reagent. GRN163L inhibited telomerase activity effectively in a dose-dependent manner, even without the use of a transfection reagent. The IC50 values for GRN163 in various cell lines were on average sevenfold higher than for GRN163L. GRN163L inhibition of telomerase activity resulted in a more rapid loss of telomeres and cell growth than GRN163. This report is the first to show that lipid modification enhanced the potency of the novel GRN163 telomerase inhibitor. These results suggest that the lipid-conjugated thio-phosphoramidates could be important for improved pharmacodynamics of telomerase inhibitors in cancer therapy.

Oligonucleotide N3'-->P5' phosphoramidates as efficient telomerase inhibitors.

Human telomerase is a unique reverse transcriptase that is expressed in multiple cancers, but not in the vast majority of normal cells. The enzyme is responsible for telomere protection and maintenance, and supports the proliferative immortality of cancer cells. Thus, it has been proposed that the specific inhibition of telomerase activity in tumors might have significant and beneficial therapeutic effects. To this goal we have designed, synthesized, and evaluated several oligonucleotide N3'-->P5' phosphoramidates as telomerase inhibitors. These oligonucleotides are complementary to the template region of the RNA domain of telomerase (hTR). The prepared compounds were evaluated in HME50-5E breast epithelial cells, where their effects on telomerase activity were determined using a cell-based telomerase (TRAP) assay at 24 as well as 72 h after exposure to compounds. The oligo-N3'-->P5' phosphoramidate inhibited telomerase activity in cells in the presence of the cellular up-take enhancer (FuGENE6) in a dose- and sequence-dependent manner, with IC(50) values of approximately 1 nM. Inhibition of telomerase activity by this compound without the lipid carrier was not efficient. However, the isosequential oligonucleotide N3'-->P5' thio-phosphoramidate was able to inhibit telomerase activity with or without lipid carriers at nM, or low-microM concentrations, respectively. This inhibition of telomerase activity in HME50-5E cells by the oligonucleotide thio-phosphoramidates was also sequence specific. Long-term treatment of the cells with 0.5 microM of FuGENE6 formulated 13-mer thio-phosphoramidates, fully complementary to hTR, resulted in gradual telomere shortening, followed by cellular senescence and apoptosis, as would be predicted for a telomerase inhibitor. The mismatched control compound had no effect on cell proliferation. The results suggest that the oligonucleotide N3'-->P5' phosphoramidates, and particularly thio-phosphoramidates, might be further developed as selective anti-telomerase reagents.

Antitumor effects of specific telomerase inhibitor GRN163 in human glioblastoma xenografts.

Telomerase is a ribonucleoprotein complex that elongates telomeric DNA and appears to play an important role in cellular immortalization of cancers. Because telomerase is expressed in the vast majority of malignant gliomas but not in normal brain tissues, it is a logical target for gliomaspecific therapy. The telomerase inhibitor GRN163, a 13-mer oligonucleotide N3'-->P5' thio-phosphoramidate (Geron Corporation, Menlo Park, Calif.), is complementary to the template region of the human telomerase RNA subunit hTR. When athymic mice bearing U-251 MG human brain tumor xenografts in their flanks were treated intratumorally with GRN163, a significant growth delay in tumor size was observed (P < 0.01 in all groups) as compared to the tumor size in mice receiving a mismatched oligonucleotide or the carrier alone. We also investigated biodistribution of the drug in vivo in an intracerebral rat brain-tumor model. Fluorescein-labeled GRN163 was loaded into an osmotic minipump and infused directly into U-251 MG brain tumors over 7 days. Examination of the brains revealed that GRN163 was present in tumor cells at all time points studied. When GRN163 was infused into intracerebral U-251 MG tumors shortly after their implantation, it prevented their establishment and growth. Lastly, when rats with larger intracerebral tumors were treated with the inhibitor, GRN163 increased animal survival times. Our results demonstrate that the antitelomerase agent GRN163 inhibits growth of glioblastoma in vivo, exhibits favorable intracerebral tumor uptake properties, and prevents the growth of intracerebral tumors. These findings support further development of this compound as a potential anticancer agent.