Antisense Phosphorothioate Oligodeoxynucleotides Research Articles

Abstract 3968: OT-101/Chemotherapy - A novel mechanism of action (MOA) in pancreatic cancer immunization therapy

Abstract Background: OT-101 is a phosphorothioate antisense oligodeoxynucleotide targeting the human TGF-β2 mRNA. OT-101 breaks down immune tolerance and activates immunity against the tumor. The Subsequent Chemotherapy (SC) releases tumor antigens and boosts the immunity response along with further epitope expansion. The phase 1/2 clinical data for OT-101/SC is presented here along with supporting preclinical MOA studies. Methods: Total of 37 pts, 2nd line and beyond received OT-101 with option to go on SC (OT-101/SC) or Best Supportive Care (BSC) (OT-101/BSC). Overall survival (OS) was compared using log-rank statistics. Stratification by treatment line, schedule, metastasis location, disease control (DC), and baseline CA19-9, was performed. For cytokine analysis, plasma levels of 31 cyto-/chemokine were measured over 8 time points during 3 cycles of intravenous OT-101 therapy and a subset of 12 patients. Results: In vitro cell kill assay and in vivo xenograft study were performed with human PBMC and OT-101. OT-101 reduced TGF-β2 secretion and increased LAK cell-activity against all tumor lines by 400% and 364% in comparison to the untreated control and the Lipofectin control, respectively. Addition of active rh-TGF-β2 protein suppressed the cytotoxic activity of the immune cells in a dose dependent manner. Preclinical- LS174T xenograft was treated with PBMC or PBMC + OT-101. OT-101 significantly enhanced the activity of PBMC against the xenograft. Median OS (mOS) of the 18 pts receiving OT-101/SC was 9.4mos vs. the 19 pts on OT-101/BSC (2.8mos, p=0.0004). Pts with only liver mets had a mOS of 9.5mos while those with liver mets and others only had a mOS of 4.7mos (p=0.0077). Among the former, one patient had complete response beyond 77.3mos and another had stable disease with OS of 40.3mos. OS was higher for liver mets. only group with OT-101/SC - 12.4mos versus 1.9mos, p=0.0006. There were 16 of 37 pts with DC, with mOS of 9.7mos vs. 3.0mos (p<0.0001). OS was higher for DC group with OT-101/SC - 11.8mos vs. 5.0mos, p=0.0021. Patients exhibited spike in IL-8 level which returned to basal level at treatment stop. R2 relating the IL-8 spike and OS were 0.8522 and 0.9895 and p values were 0.0011 and 0.0053 for pts treated subsequently with SC and BSC, respectively. The two lines intersect at the origin with higher response for those with OT-101/SC, suggesting exaggerated response expected for antigen boost during the SC phase. Conclusions: Escalating Intratumoral heterogeneity (IH) resulting in xenogenization, which is countered by overexpression of TGF-β. Here we report on the use of OT-101/SC to break immune tolerance to pancreatic cancer (PC) for the cure. The MOA for OT-101/SC is consistent with the reactivation of immunity during TGF-β suppression and subsequent boosting/expansion of immunity during SC. Contrary to traditional tumor vaccine- this is universally applicable to all patients. Citation Format: David Nam, Larn Hwang, Vuong Trieu. OT-101/Chemotherapy - A novel mechanism of action (MOA) in pancreatic cancer immunization therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3968.

Abstract 5043: Population pharmacokinetic model for OT-101 - A TGF-β2-specific antisense oligonucleotide in cancer patients

Abstract Background: OT-101 (Trabedersen) is a phosphorothioate antisense oligodeoxynucleotide specifically inhibiting the expression of transforming growth factor-beta 2 (TGF-β2), whose overexpression is a pivotal factor for malignant progression in solid tumors. In the clinical Phase I/II study, plasma pharmacokinetic (PK) profile of OT-101 administered intravenously was evaluated in patients with advanced tumors. A population PK model was built to further understand the factors contributing to the variability in PK of OT-101. Methods: A total of 61 patients with pancreatic cancer (n=37), malignant melanoma (n=19), or colorectal carcinoma (n=5) were treated with OT-101 with escalating doses in 2 treatment schedules (1st schedule: 7-days on, 7-days off; 2nd schedule: 4-days on, 10-days off; up to 10 cycles). The PK analysis was only performed on Cycle 1 and, when applicable, Cycle 2. Blood samples were collected from patients at planned time points from before start of infusion to 7 days (1st schedule) or 10 days (2nd schedule) after stop of infusion. The plasma concentration data of OT-101 were used to build a population PK model using Phoenix NLME software. The influence of age, gender, body mass index (BMI), body weight (BW), height, cancer type, and treatment schedule as covariates on PK was evaluated. Results: With exclusion of protocol deviations, a total of 1444 plasma sample concentration data from 100 patient cycles were examined. The concentration time course of OT-101 was best described by a two-compartment model. The model estimated the PK parameters as follows: total body clearance, 0.17 mL/h; distribution volume of the central compartment, 4.69 L; inter-compartmental clearance, 3.31 L/h; distribution volume of the peripheral compartment, 5046.44 L. BW was identified as a covariate on OT-101 inter-compartmental clearance, with KBW as -ve 1.48. Conclusion: The PK parameters of OT-101 were best described by a two-compartment model. OT-101 was largely distributed in the peripheral tissues. The influence of age, gender, BMI, height, cancer type and treatment schedule on the PK of OT-101 was not identified. The model will be used for a sparse population PK study during the planned phase III trial of OT-101 in pancreatic cancer. Citation Format: Wen Wang, Kevin Ng, David Nam, Vuong Trieu, Larn Hwang. Population pharmacokinetic model for OT-101 - A TGF-β2-specific antisense oligonucleotide in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5043. doi:10.1158/1538-7445.AM2017-5043

Abstract 1600: Interleukin-8 (IL-8) in TGF-β immunotherapy and chemotherapies

Abstract Background: Increased expression of IL-8 and/or its receptors has been characterized in cancer cells, endothelial cells, infiltrating neutrophils, and tumor-associated macrophages, suggesting that IL-8 may function as a significant regulatory factor within the tumor microenvironment. OT-101 is a phosphorothioate antisense oligodeoxynucleotide targeting transforming growth factor-beta 2 (TGF-β2). Herein, we examined the role of IL-8 in OT-101 treatment followed with chemotherapies during our Phase II trial in patients with advanced pancreatic cancer (PAC). Methods: Time evolution of a panel of 31 cyto-/chemokine levels in plasma were tracked over 3 cycles of OT-101 administration (140 mg/m2/day) for 12 PAC patients. Samples were acquired before onset of OT-101 therapy and at 8 selected time points (Cycle 1 [Day 2 and 5], Cycle 2 [Days 1, 2, and 5], Final Visit, Cycle 3 [Day 5]) during the therapy. Samples were measured in duplicate and concentrations were expressed in pg/mL. Standardized log10 transformed values calculated from the mean and standard deviation of each cyto-/chemokine in each patient was utilized in an ANCOVA model to investigate the correlation with Overall Survival (OS). Results: Clustering of correlation coefficients resulted in the identification of three highly correlated subsets of cyto-/chemokines (Cluster 1: EGF, MIP-1α, MIP-1β; Cluster 2: FGF-2, IL-1RA, MIG, IP-10, IL-15, IFN-α, IL-12A/IL-12; Cluster 3: HGF, IL-2R, IL-6, IL-8). Protein-Protein Interaction networks constructed using STRING10 algorithm identified a relationship between IL-8 and TGFβR2 inhibition. The ANCOVA model explained a significant proportion of the observed data for Cycle 1[Day 2] measurements of cyto-/chemokines (R2 = 0.3, F59,217 = 1.575, P = 0.0103). Other time points did not exhibit a significant model fit or significant relationships in the interaction term. IL-8 expression showed significant association with OS (positive association, N=12, T-value = 2.92, P = 0.0039) at Cycle 1[Day 2] measurements (7% False Discovery Rate). Linear regression of the increase in IL-8 levels during Cycle 1[Day 2] treatment with OT-101 was related to an increase in OS outcome (R2 = 0.54). To further define the appropriate chemoagent to combine with OT-101, we evaluated paclitaxel (PTX), gemcitabine (GEM), and dacarbazine (DTIC) in animal model. Synergy was observed with PTX and DTIC, but not with GEM. Both PTX and DTIC induced IL-8 expression whereas, surprisingly, GEM reduced IL-8 expression. Conclusion: IL-8 expression during early phase of OT-101 treatment cycle was positively associated with OS across 12 patients. IL-8 is also induced by PTX and DTIC which synergized with OT-101. The data suggests that PAC and DTIC are acting on the same target as our TGF-β2 inhibitor; and potentially, the beneficial effect of these chemoagents is a result of chemoagent-induced immune/IL-8 response. Citation Format: Larn Hwang, Kevin Ng, Osmond D’Cruz, Sanjive Qazi, Andrew Schmidt, Vuong Trieu. Interleukin-8 (IL-8) in TGF-β immunotherapy and chemotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1600. doi:10.1158/1538-7445.AM2017-1600

Electronic Structures of LNA Phosphorothioate Oligonucleotides.

Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM) calculations and chromatography experiments on locked nucleic acid (LNA) phosphorothioate (PS) oligonucleotides. iso-potential electrostatic surfaces are essential in this study and have been calculated from the wave functions derived from the QM calculations that provide binding information and other properties of these molecules. The QM calculations give details of the electronic structures in terms of e.g., energy and bonding, which make them distinguish or differentiate between the individual PS diastereoisomers determined by the position of sulfur atoms. Rules are derived from the electronic calculations of these molecules and include the effects of the phosphorothioate chirality and formation of electrostatic potential surfaces. Physical and electrochemical descriptors of the PS oligonucleotides are compared to the experiments in which chiral states on these molecules can be distinguished. The calculations demonstrate that electronic structure, electrostatic potential, and topology are highly sensitive to single PS configuration changes and can give a lead to understanding the activity of the molecules.

Correlation of circulating IL-8 levels with improved overall survival in advanced pancreatic cancer patients in response to antisense OT-101 (trabedersen) therapy.

444 Background: Overexpression of the transforming growth factor-beta 2 (TGF-β2) has been suggested as a pivotal factor for malignant progression of pancreatic cancer (PAC). OT-101 (Trabedersen) is a phosphorothioate antisense oligodeoxynucleotide target the human TGF-β2 mRNA. A Phase I/II study of OT-101 was characterized by outstanding overall survival (OS) in patients with advanced PAC. In this study, we examined the association between plasma levels of 31 cyto-/chemokines and OS outcomes in PAC patients treated with OT-101. Methods: Plasma levels of 31 cyto-/chemokine were tracked over a period of 11 days at 8 time points during 3 cycles of intravenous OT-101 therapy (140 mg/m2/day) for 12 PAC patients. A mixed ANCOVA model was developed to time evolution of 19 cyto-/chemokine levels with median expression 1 following OT-101 therapy. Pearson pairwise correlation coefficients of cyto-/chemokine levels across all time points and all patients were utilized to construct a correlation cluster to identify co-regulated cyto-/chemokines. Regression and hierarchical cluster analyses were performed to identify potential cytokine signatures. Protein-protein interaction networks were constructed using STRING10 algorithm. Plasma cyto-/chemokine levels were compared with OS outcome. Results: Three highly correlated subsets of cyto-/chemokines (Cluster 1: EGF, MIP-1α, MIP-1b; Cluster 2: FGF-2, IL-1R, MIG, IP-10, IL-15, IFN-α, IL-12A; Cluster 3: IL-2R, IL-6, IL-8, HGF) were identified following OT-101 therapy. An increasing trend was observed among 13 cytokines. 4 of the 12 patients exhibited a consistent increase in MIP-1α and IL-1b. Suppression of TGF-b signaling by OT-101 led to upregulation of IL-15, IP-10 and HGF. 3 significant associations of cyto-/chemokine and OS were observed at Cycle 1 measurements. The ANCOVA model that examined the levels of 31 cyto-/chemokines with OS as the co-variate resulted in IL-8 exhibiting a significant positive association with OS ( P= 0.0039). Conclusions: Levels of IL-8 were positively associated with OS across 12 PAC patients serving as potential biomarkers for treatment outcome following OT-101 therapy. Clinical trial information: NCT00844064.

Intracellular Progesterone Receptor Mediates the Increase in Glioblastoma Growth Induced by Progesterone in the Rat Brain

Progesterone (P) is a steroid hormone involved in the development of several types of cancer including astrocytomas, the most common and malignant brain tumors. We undertook this study to investigate the effects of P on the growth and infiltration of a tumor caused by the xenotransplant of U87cells derived from a human astrocytoma grade IV (glioblastoma) in the cerebral cortex of male rats and the participation of intracellular progesterone receptor (PR) on these effects. Eight weeks after the implantation of U87cells in the cerebral cortex, we administered phosphorothioated antisense oligodeoxynucleotides (ODNs) to silence the expression of PR. This treatment lasted 15days and was administered at the site of glioblastoma cells implantation using Alzet osmotic pumps. Vehicle (propylene glycol) or P4 (400μg/100g) was subcutaneously injected for 14days starting 1day after the beginning of ODN administration. We observed that P significantly increased glioblastoma tumor area and infiltration length as compared with vehicle, whereas PR antisense ODNs blocked these effects. P, through the interaction with PR, increases the area and infiltration of a brain tumor formed from the xenotransplant of human glioblastoma-derived U87cells in the cerebral cortex of the rat.

Gene Silencing and Activation of Human Papillomavirus 18 Is Modulated by Sense Promoter Associated RNA in Bidirectionally Transcribed Long Control Region.

BackgroundRecently various studies have demonstrated the role of promoter associated non-coding RNAs (pRNA) in dsRNA induced transcriptional gene silencing and activation. However the exact mechanistic details of these processes with respect to the orientation of pRNAs are poorly defined.Methodology/Principal FindingsWe have identified novel sense and antisense long control region (LCR) associated RNAs (pRNAs) in HPV18 positive cervical cancer cell lines HeLa, C-4 I and C-4 II. Using dsRNAs against these pRNAs, we were able to achieve upregulation or downregulation of the sense and antisense pRNAs and the downstream E6 and E7 oncogenes. We present evidence that knockdown of the sense pRNA is associated with reduction in E6 and E7 oncogenes and an upregulation of antisense pRNA. Conversely upregulation of sense pRNA is accompanied by an induction of the oncogenes and a concomitant reduction in antisense pRNA. Moreover, the exact role of sense and antisense pRNAs in dsRNA mediated gene modulation was confirmed by their selective degradation using antisense phosphorothioate oligodeoxynucleotides (ODN). Degradation of sense pRNA with antisense ODN led to loss of dsRNA induced silencing and activation, suggesting that dsRNA mediated gene modulation requires sense pRNA. Both processes were accompanied with congruent changes in the methylation pattern of activating and repressive histones.Conclusion/SignificanceThus this data identifies and demonstrates the role of previously unknown important regulatory transcripts in HPV18 gene expression which can prove valuable targets in cervical cancer therapeutics. This mode of gene regulation by bidirectional transcription could be operational in other promoters as well and serve as a mechanism of regulating gene expression.

Liposome-mediated induction of apoptosis of human hepatoma cells by c-myc antisense phosphorothioate oligodeoxynucleotide and 5-fluorouracil.

The aim of this study was to investigate the effect of a c-myc antisense oligodeoxynucleotide and 5-fluorouracil on the expression of c-myc, invasion and proliferation of HEPG-2 liver cancer cells. HEPG-2 cells were treated with lipiosome-mediated c-myc ADSON and 5-fluorouracil. The proliferation inhibition rate and invasion were measured by MTT and invasion assay, respectively. Cell apoptosis was detected by flow cytometry and expression of c-myc by RT-PCR and immunohistochemistry. The proliferation inhibition rate was significantly higher in the antisense oligodeoxynucleotide added-5-fluorouracil group than single antisense oligodeoxynucleotide or 5-fluorouracil group (p<0.05). G0/G1 cells in the antisense oligodeoxynucleotide group and S cells in the 5-fluorouracil groups were significantly increased than that in the control group, respectively (P<0.01). The amplification strips of PCR products in 5-FU, ASODN and combination groups were significantly weaker than that in the control group (P<0.01). The percentage of c-myc-protein- positive cells were significantly lower in antisense oligodeoxynucleotide, 5-fluorouracil and combination groups than that in the control group (P<0.01). A liposome-mediated c-myc antisense oligodeoxynucleotide and 5-fluorouracil can inhibit the proliferation and invasion of liver cancer cells by reducing the expression of c-myc. A c-myc antisense oligodeoxynucleotide can increase the sensitivity of liver cancer cells to 5-fluorouracil and decrease the dosage of the agent necessary for efficacy, providing an experimental basis for the clinical therapy of liver cancer.

Aberrant expression of CD227 is correlated with tumor characteristics and invasiveness of breast carcinoma.

Increasing evidences demonstrate that CD227 plays a crucial role in the development and progression of breast cancer. However, the function of CD227 in breast carcinoma was still controversial and the investigation on CD227 in Asian race was scarce. To investigate the relationship between CD227 and tumor characteristics of breast carcinoma, CD227, estrogen receptor (ER), progesterone receptor (PR), Her2⁄neu and Ki-67 were detected by immunohistochemistry in a series of 227 patients. The Kaplan-Meier method and log-rank tests were used to estimate the correlation between CD227 expression and patients' prognosis. Furthermore, in vitro invasion assay was performed to examine the effect of CD227 on the invasiveness of breast carcinoma cells after transfection with CD227 cDNA or antisense phosphorothioate oligodeoxynucleotides (ASODN) against CD227 mRNA. Our data demonstrate that the cytoplasm staining and high expression of CD227 were positively related to the aggressiveness of breast cancer. Both circumferential membrane staining and cytoplasm staining were associated with lymph node metastasis. Moreover, the cytoplasm staining and overexpression of CD227 were found to be related to Her-2/neu positivity, higher Ki-67 positivity and poorer survival of patients. We further demonstrated that the invasion ability of breast carcinoma cells could be enhanced or inhibited by CD227 cDNA or ASODN, respectively. We conclude that the aberrant expression of CD227, especially cytoplasm staining could be predictive for tumor aggressiveness, lymph node metastasis, poorer outcome of patients with breast cancers. And CD227 could promote the invasion ability of breast cancer cells, suggesting a potential role of CD227 as an oncogene in breast carcinoma.

Antisense gene inhibition by phosphorothioate antisense oligonucleotide in Arabidopsis pollen tubes

Sexual reproduction is an essential biological event for proliferation of plants. The pollen tube (PT) that contained male gametes elongates and penetrates into the pistils for successful fertilization. However, the molecular mechanisms of plant fertilization remain largely unknown. Here, we report a transient inhibition of gene function using phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) without cytofectin, which is a simple way to study gene function in Arabidopsis thaliana PTs. The PTs treated with AS-ODNs against both ANX1 and ANX2 showed short, knotted, and ruptured morphology in vitro/semi-in vitro, whereas normal PT growth was shown in its sense control in vitro/semi-in vitro. PT growth was impaired in a manner dependent on the dose of AS-ODNs against both ANX1 and ANX2 above 10 μm. The treatment with AS-ODNs against ROP1 and CalS5 resulted in waving PTs and in short PTs with a few callose plugs, respectively. The expression levels of the target genes in PTs treated with their AS-ODNs were lower than or similar to those in the sense control, indicating that the inhibition was directly or indirectly related to the expression of each mRNA. The AS-ODN against fluorescent protein (sGFP) led to reduced sGFP expression, suggesting that the AS-ODN suppressed protein expression. This method will enable the identification of reproductively important genes in Arabidopsis PTs.

In vitro inhibition of hepatitis C virus by antisense oligonucleotides in PBMC compared to hepatoma cells.

Aim. To assess the efficiency of phosphorothioate antisense oligodeoxynucleotide 1 (S-ODN1) on HCV translation inhibition in PBMC compared to hepatoma cells in vitro for the first time. Materials and Methods. The study included 34 treatment naive HCV patients. IRES domain III and IV sequence variations were tested in 45 clones from 9 HCV patients. PBMC of HCV positive patients were subjected to S-ODN in vitro. Concomitantly HepG2 cells infected by the same patient's serum were also treated with S-ODN1 for 24 and 48 hours. Cellular RNA was tested for HCV plus and minus strands by reverse transcription polymerase chain reaction (RT-PCR). Results. Sequence variations were seen in HCV IRES domain III only while domain IV was conserved among all the tested patient's clones. S-ODN1 successfully inhibited HCV translation in HepG2 cells, while in PBMC inhibition was partial. Conclusion. HCV IRES domain IV is more conserved than domain IIId in genotype 4 HCV patients. S-ODN against HCV IRES domain IV was not efficient to inhibit HCV translation in PBMC under the study conditions. Further studies testing other S-ODN targeting other HCV IRES domains in PBMC should be done.

Depletion of telomerase RNA inhibits growth of gastrointestinal tumors transplanted in mice

To explore effects of telomerase RNA-targeting phosphorothioate antisense oligodeoxynucleotides (PS-ASODN) on growth of human gastrointestinal stromal tumors transplanted in mice. A SCID mouse model for transplantation of human gastrointestinal stromal tumors (GISTs) was established using tumor cells from a patient who was diagnosed with GIST and consequently had been treated with imatinib. GIST cells cultured for 10 passages were used for inoculation into mice. Transfection of PS-ASODN was carried out with Lipotap Liposomal Transfection Reagent. GISTs that subsequently developed in SCID mice were subjected to intra-tumoral injection once daily from day 7 to day 28 post-inoculation, and mice were divided into the following four groups according to treatment: PS-ASODN group (5.00 μmoL/L of oligonucleotide, each mouse received 0.2 mL once daily); imatinib group (0.1 mg/g body weight); liposome negative control group (0.01 mL/g); and saline group (0.01 mL/g). On day 28, the mice were sacrificed, and tumor attributes including weight and longest and shortest diameters were measured. Tumor growth was compared between treatment groups, and telomerase activity was measured by enzyme-linked immunosorbent assay. Apoptosis was examined by flow cytometry. Real-time polymerase chain reaction was used to detect expression of the mRNA encoding the apoptosis inhibition B-cell leukemia/lymphoma 2 (bcl-2) gene. In the PS-ASODN group, tumor growth was inhibited by 59.437%, which was markedly higher than in the imatinib group (11.071%) and liposome negative control group (2.759%) [tumor inhibition = (mean tumor weight of control group--mean tumor weight of treatment group)/(mean tumor weight of control group) × 100%]. Telomerase activity was significantly lower (P < 0.01) in the PS-ASODN group (0.689 ± 0.158) compared with the imatinib group (1.838 ± 0.241), liposome negative control group (2.013 ± 0.273), and saline group (2.004 ± 0.163). Flow cytometry revealed that the apoptosis rate of tumor cells treated with PS-ASODN was 20.751% ± 0.789%, which was higher (P < 0.01) than that of the imatinib group (1.163% ± 0.469%), liposome negative control group (1.212% ± 0.310%), and saline group (1.172% ± 0.403%). Expression of bcl-2 mRNA in the transplanted GISTs was markedly downregulated (P < 0.01) in the PS-ASODN group (7.245 ± 0.739) compared with the imatinib group (14.153 ± 1.618) and liposome negative control group (16.396 ± 1.351). PS-ASODN can repress GIST growth, mediated perhaps by inhibition of telomerase activity and downregulation of bcl-2 expression.

REST is up-regulated by epidermal growth factor in HeLa cells and inhibits apoptosis by influencing histone H3 acetylation

REST (repressor element 1-silencing transcription factor) is a transcription factor that recruits histone deacetylases to silence gene transcription. REST appears to play a paradoxical role in cancer cells: it exhibits tumor suppressor activity or promotes tumorigenesis, depending upon the setting. The extracellular signaling molecules that control REST gene expression in cancer cells remain poorly understood. In this study, we report that REST expression in HeLa cells is elevated in cells exposed to epidermal growth factor or serum, whereas the rate of cell apoptosis is low. Apoptosis induced by serum withdrawal is significantly increased in HeLa cells treated with an antisense phosphorothioate oligodeoxynucleotide (AS ODN) capable of down-regulating REST expression, whereas in HeLa cells transfected with a REST expressing plasmid, REST overexpression reduces the marked apoptosis caused, in absence of serum, by exposure to an anti-Fas receptor antibody imitating the Fas ligand activity plus PD 98059, a blocker of extracellular signal-regulated kinase 1/2 activation. REST knockdown also reduces mRNA levels of the antiapoptotic protein Bcl-XL whereas in HeLa cells overexpressing REST, the reduction of Bcl-XL mRNA caused by the anti-Fas receptor antibody plus PD 98059 is significantly decreased. Finally, we report that acetylation of histone H3 is increased in HeLa cells exposed to AS ODN or anti-Fas receptor antibody, whereas it is reduced in cells transfected with the REST expressing plasmid. Our findings indicate that REST is a novel gene regulated by EGF in HeLa cells that potentially contributes to the modulation of apoptosis via epigenetic mechanisms.

Regulation of hepatitis C virus translation initiation by iron: Role of eIF3 and La protein

Eukaryotic initiation factors (eIFs) are required for encoding polyprotein of hepatitis C virus (HCV) which is mediated by an internal ribosome-entry site (IRES). Iron overload, a common finding among HCV patients, may be correlated with HCV pathology, but the underlying molecular mechanisms are poorly understood. In this study, we investigated the possible relationship among iron status, eIFs and HCV IRES-mediated translation in vitro. Using bicistronic reporter gene constructs carrying HCV IRES sequence, we found that the levels of intracellular iron were positively associated with the HCV IRES-dependent translation initiation in Huh-7 cells. RT-PCR method showed that iron treatment specifically increased the levels of eIF3A mRNA and La mRNA, whereas iron chelation reduced them. Western blots also confirmed that iron-dependent changes in eIF3A mRNA and La mRNA affected the expression of their proteins. Moreover, antisense phosphorothioate oligodeoxynucleotides to eIF3A and La successfully suppressed the levels of eIF3A and La protein and significantly reduced iron-dependent HCV translation. Taken together, our results suggest that iron promotes the translation initiation of HCV by stimulating the expression of eIF3A and La proteins. Inhibition of eIF3A and La proteins substantially repressed iron-dependent HCV translation, a beneficial effect that may have significant clinical implications.

Study on effects of microRNA-21 antisense oligonucleotide in vivo and in vitro on bionomics of human cervical squamous carcinoma cell lines SiHa

To explore the effect of microRNA-21 (miR-21) antisense oligonucleotide on the biological characteristics of human cervical squamous carcinoma cell lines SiHa in vivo and in vitro. Specific phosphorothioate antisense oligodeoxynucleotides targeting miR-21 were synthesized and transfected into cervical cancer cells in vitro. Expression of miR-21 in SiHa after transfection was detected by real-time RT-PCR. The cell proliferation was evaluated by MTT assay and colony formation experiment. The cell apoptosis was analyzed by annexin V-FITC/PI analysis. The inhibitory effect of miR-21 antisense oligonucleotide on tumor growth was evaluated by tumor growth curves and immunohistochemistry (MaxVision method). H-E staining was used to document morphological changes and fluorometric TUNEL assay was to detect the apoptotic activity. After the transfection of antisense miR-21, the expression of miR-21 decreased along with an obvious growth inhibition, compared with that of the control groups (P < 0.05). Colony formation of both cell lines was markedly inhibited with antisense miR-21 (55.6% ± 1.4%), as compared with that in the negative group (98.3% ± 2.0%, P < 0.05). Flow cytometry assay showed that antisense miR-21 expression significantly enhanced the cell apoptosis (6.7% ± 1.3% and 29.4% ± 1.7%, P < 0.05). The tumor-forming rates of miR-21 transfected group, and negative control groups were 3/8 and 6/8, respectively (P < 0.05). Ki-67 proliferative marker staining decreased significantly (42% vs 90%) in the transfected group compared with negative control groups. Extensive dead tumor cells were seen in the miR-21 transfected cells along with a marked increase of apoptosis (P < 0.05). Targeted antisense oligonucleotide miR-21 effectively suppresses the growth of cervical carcinoma SiHa cells both in vitro and in vivo through an induction of apoptosis.

Peptide-mediated Cell and In Vivo Delivery of Antisense Oligonucleotides and siRNA

Antisense oligonucleotides and short interfering RNAs (siRNAs) are nucleic acids-targeting reagents for gene expression modulation that are being developed as drugs for many applications. A number of useful synthetic nucleic acids analogues have been introduced recently to greatly improve their properties for use as therapeutics. However, their full effectiveness in cells and in vivo has often only been realized through development of suitable nonviral delivery systems. Among these, a range of natural and synthetic peptides have been found useful for enhancing cellular uptake and/or cell targeting of oligonucleotide analogues and siRNA. Such peptides are synthetically conjugated, used as noncovalent complexes, or used in combination with polymer, liposomal or exosome formulation techniques. This review begins by describing the modes of action of antisense reagents and siRNA and goes on to focus on recent advances in their peptide-mediated cell and in vivo delivery and how peptide use has influenced drug development. The review discusses the challenges associated with understanding the physiological and toxicological aspects of peptide-mediated delivery. Developments towards clinical use are also highlighted, with particular emphasis on peptide conjugates of oligonucleotide analogues used for treatment of neuromuscular diseases.

The Antisense Oligonucleotide Trabedersen (AP 12009) for the Targeted Inhibition of TGF-&amp;#946;2

Despite remarkable advances in cancer research, patients with malignant tumors such as high-grade glioma or advanced pancreatic carcinoma still face a poor prognosis. Because of the severe morbidity and mortality of such malignant tumor types, the identification of suitable molecular drug targets for causal treatment approaches is an important area of current research. Transforming growth factor-beta 2 (TGF-β2) is an attractive target because it regulates key mechanisms of carcinogenesis, in particular immunosuppression and metastasis, and is frequently overexpressed in malignant tumors. Here we describe the development of the antisense phosphorothioate oligodeoxynucleotide trabedersen (AP 12009) which was designed for the specific inhibition of TGF-β2 biosynthesis. In vitro and in vivo experiments confirmed the mode of action, efficacy and tolerability of trabedersen and paved the way for clinical studies. In patients with high-grade glioma, intratumoral treatment with trabedersen is currently evaluated in a pivotal, randomized and active-controlled phase III study. Intravenous application of trabedersen for the treatment of patients with advanced pancreatic carcinoma, metastasizing melanoma, or metastatic colorectal carcinoma is assessed in a currently ongoing phase I/II dose escalation study.

Transforming growth factor‐beta 2 gene silencing with trabedersen (AP 12009) in pancreatic cancer

Pancreatic cancer is one of the most aggressive human cancers with a 5-year survival rate of <5%. Overexpression of transforming growth factor-beta 2 (TGF-β2) in pancreatic malignancies is suggested to be a pivotal factor for malignant progression by inducing immunosuppression, metastasis, angiogenesis and proliferation. Trabedersen (AP 12009) is a phosphorothioate antisense oligodeoxynucleotide specific for human TGF-β2 mRNA and was successfully tested in a randomized, active-controlled phase IIb clinical study in patients with high-grade glioma. Here, we report on the antitumor activity of trabedersen in human pancreatic cancer cells and in an orthotopic xenograft mouse model of human metastatic pancreatic cancer. Trabedersen reduced TGF-β2 secretion in human pancreatic cell lines with an IC50 in the low μM range without transfection reagent, clearly inhibited cell proliferation, and completely blocked migration of pancreatic cancer cells. Additionally, trabedersen reversed TGF-β2-mediated immunosuppression of pancreatic cancer cells targeted by lymphokine activated killer (LAK) cells, resulting in considerably increased LAK cell-mediated cytotoxicity. Moreover, in an orthotopic mouse model of metastatic pancreatic cancer, intraperitoneal (i.p.) treatment with trabedersen significantly reduced tumor growth, lymph node metastasis and angiogenesis. These promising results warrant further clinical development of trabedersen.

A c‐myc Phosphorothioate Oligonucleotide Protects U87‐MG Glioblastoma Cells from Toxicity at High Concentrations of Lipofectin and Streptolysin O

The pharmacological activity of an antisense oligodeoxynucleotide (ODN) targeted to c-myc (15mycAS) was evaluated in the glioblastoma-derived cell line U87-MG. ODNs with both phosphodiester (PO) and phosphorothioate (PS) backbones, and with 3′ hairpin sequences, were used with lipofectin and streptolysin O as transfection reagents. At non-toxic concentrations of lipofectin, cellular association was low and no evidence of an antisense mechanism was seen on evaluation of cell proliferation or c-Myc protein expression. At a high lipofectin concentration (25 μg mL−1), which resulted in loss of more than 50% cell viability, ODN uptake was enhanced, but no reduction in c-Myc protein expression was observed with any of the 15mycAS ODNs. However, treatment with the antisense PS ODN (15mycAS PS), specifically resulted in protection of the viable cell number. This cytoprotective effect was concentration- and chemistry-dependent. Three 15mer control PS ODNs, the PO form of 15mycAS, and the hairpin stabilized PO and PS forms (15mycASHP2 PO and 15mycASHP2 PS, respectively) did not show cytoprotection. A similar protective effect of 15mycAS PS was also seen with the transfection agent streptolysin O. These data demonstrate a potential source of misinterpretation in antisense experiments and emphasize the need to consider possible interactions of ODNs with lipids or cell membranes in the design of effective antisense strategies.

Targeting Host E-Selectin Expression by Antisense Oligodeoxynucleotides as Potential Antiendotoxin TherapyIn Vivo

This study sought to determine if antisense oligodeoxynucleotides would inhibit E-selectin expression, which mediates leukocyte adhesion on endothelial cells, otherwise induced by in vivo endotoxin challenge. Six antisense phosphorothioate oligodeoxynucleotides calculated to bind porcine E-selectin mRNA were tested in porcine aortic endothelial cells. One, ISIS9481, exerted significant inhibition of E-selectin expression induced by tumor necrosis factor-α + endotoxin [lipopolysaccharide (LPS)]. Pigs were challenged with LPS (10 μg/kg) and treated with ISIS9481 (10 mg/kg) (n = 6). Two control groups were used, an antisense inactive in porcine aortic endothelial cells (n = 6) and saline (n = 5), and were combined as control (C = 11). Control pigs challenged with LPS expressed E-selectin in heart, lung, kidneys, and liver, whereas antisense-treated pigs expressed little E-selectin in these tissues. Cardiovascular data indicated that antisense treatment attenuated pathophysiological alterations induced by LPS. Specifically, in control pigs, LPS reduced cardiac output 32% from baseline, increased pulmonary (+116%) and systemic vascular resistances (+16%), and generated neutropenia (from 51,000 at basal to 18,000 polymorphonuclear neutrophils (PMN)/μL after LPS). In antisense-treated pigs, cardiac output decreased only 18%, pulmonary vascular resistance remained unchanged, whereas systemic vascular resistance decreased compared with basal values (-37%). PMN counts remained at 45,000-54,000/μL at 3-4 hours after LPS. These data demonstrate that antisense oligodeoxynucleotides, designed and tested in vitro to interact with 1 gene product, can be developed as either therapeutics or experimental tools in vivo.