Efficiency of antisense oligonucleotide drug discovery

The costs for discovering and developing new drugs continue to escalate, with current estimates that the average cost is more than $800 million for each new drug brought to the market. Pharmaceutical companies are under enormous pressure to increase their efficiency for bringing new drugs to the market by third-party payers, shareholders, and their patients, and at the same time regulators are placing increased demands on the industry. To be successful in the future, pharmaceutical companies must change how they discover and develop new drugs. So far, new technologies have done little to increase overall efficiency of the industry and have added additional costs. Platform technologies such as monoclonal antibodies and antisense oligonucleotides have the potential of reducing costs for discovery of new drugs, in that many of the steps required for traditional small molecules can be skipped or streamlined. Additionally the success of identifying a drug candidate is much higher with platform technologies compared to small molecule drugs. This review will highlight some of the efficiencies of antisense oligonucleotide drug discovery compared to traditional drugs and will point out some of the current limitations of the technology.     

Down-regulation of NOSII gene expression by iontophoresis of anti-sense oligonucleotide in endotoxin-induced uveitis

Transcorneoscleral iontophoresis was used to enhance ocular penetration of a 21-bp NH(2) protected anti-NOSII oligonucleotides (ODNs) (fluorescein or infrared-41 labeled) in Lewis rats. Both histochemical localization and acrylamide sequencing gels were used. To evaluate the potential to down-regulate NOSII expression in the rat model of endotoxin-induced uveitis (EIU), anti-sense NOSII ODN, scrambled ODN or saline were iontophorezed into these animals' eyes. Iontophoresis facilitated the penetration of intact ODNs into the intraocular tissues of the rat eye and only the eyes receiving ODNs and electrical current demonstrated intact ODNs within the ocular tissues of both segments of the eye. Iontophoresis of anti-NOSII ODN significantly down-regulated the expression of NOSII expression in iris/ciliary body compared to the saline or scrambled ODN treated eyes. Nitrite production was also significantly reduced in the anti-NOSII applied eyes compared to those treated with saline. Using this system, intraocular delivery of ODNs can be significantly enhanced increasing the potential for successful gene therapy for human eye diseases.     

The application of antisense oligonucleotide technology to the brain: Some pitfalls

Summary 1. Amphetamine-induced c-fos andegr-1 expression in the striatum was used as a model in which to study the effects of antisense oligodeoxynucleotides (ODNs) directed at c-fos. Using direct infusions of ODNs into the striata of animals we have demonstrated that c-fos antisense ODNs retain most of their biological activity with 2- or 3-base substitutions. The c-fos antisense and mismatch ODNs attenuated Fos immunoreactivity but had little effect on Egr-1 immunoreactivity.2. In another group of studies examining the role of c-fos in amygdala kindling, we have demonstrated that ODNs cause neurotoxic damage following repeated daily infusions into the amygdala. The damage observed was greatly diminished when the time interval between infusions was extended.     

Modulation of the PC12 cell response to nerve growth factor by antisense oligonucleotide to amyloid precursor protein

Summary 1. The amyloid precursor protein (APP) is widely distributed among eukaryotic cells, however, its precise role in cellular functioning is not fully clarified. APP is glycoprotein membrane constituent and it may facilitate membrane associated functions.2. The aim of the present study was to examine the possibility that APP may play a role in mediating cellular trophic responses. The methods made use of an antisense oligonucleotide that was prepared to the 5 terminus of APP and shown specifically to reduce the level of APP isoforms.3. In sequential mixing experiments it was observed that the APP antisense oligonucleotide did not significantly modify the trophic response of PC12 cells pretreated with nerve growth factor (NGF). However, pretreatment of cells with the antisense oligonucleotide diminished NGF-induced increases in cellular size and neurite length.4. These observations suggest that APP may play a role in modulating the trophic response. The combined use of APP antisense oligonucleotides and neurotrophic agents may find clinical utility in the treatment of Alzheimertype dementia since it is known that NGF normally causes increases in APP levels.     

Effects of an antisense oligonucleotide to pronociceptin and long-term prevention of morphine actions by nociceptin

To further characterize the anti-opioid action of the neuropeptide nociceptin, we examined the effects of the repeated intracerebroventricular (i.c.v.) treatment (once daily for 4 days) with an antisense oligodeoxynucleotide complementary to pronociceptin mRNA, in the rat. We also investigated possible changes of the antinociceptive and hyperthermic effects induced by the i.c.v. administration of morphine, in rats i.c.v. pretreated with nociceptin 3 h before. The pretreatment with the antisense oligodeoxynucleotide, but not with a mismatched sequence (used as a control), caused an increase in spontaneous locomotor activity and produced a potentiation of the antinociceptive effect of a submaximal dose of i.c.v. morphine (1 microgram/rat). The i.c.v. pretreatment with nociceptin (2 nmol/rat, 3 h before) prevented both the antinociceptive and the hyperthermic effects of morphine (10 microgram/rat i.c.v.). These results strengthen the hypothesis of an anti-opioid action of nociceptin at supraspinal level and suggest that the neuropeptide may exert long-term modulatory effects.     

Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique

Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.     

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.     

Suppression of tumorigenicity of human hepatocellular carcinoma cells by antisense oligonucleotide MZF-1

Our previous studies have found that reducing expression of myeloid zinc finger-1 (MZF-1) inhibited protein kinase C alpha (PKCalpha) expression and decreased cell migration and invasion in human hepatocellular carcinoma (HCC). In this study, we further investigated the role of MZF-1 in tumorigenesis. The SK-Hep-1 HCC cells were transfected with the antisense oligonucleotide (ODN) of MZF-1. The pretreated cells was then detected the mRNA and protein levels by RT-PCR and western blotting, and the cell growth was assayed by MTT. Meanwhile, the pretreated-SK-Hep-1 HCC cells were implanted subcutaneously into nude mice to observe the tumor growth and calculated tumor inhibitory rate. The results showed that, at the dosage of 5 microM, the antisense ODN MZF-1 suppressed both MZF-1 and PKCalpha productions by SK-Hep-1 HCC cells after cationic liposome-mediated transfection, to 15% and 30% in control cultures of 0 microM dosage, respectively. The growth of SK-Hep-1 HCC cells was inhibited by the 2-5 microM doses of the antisense ODN MZF-1, whereas the control reagent, the sense ODN MZF-1, showed no effects. In BALB/nude mice, SK-Hep-1 HCC cells pretreated with the 5 microM antisense ODN MZF-1 formed tumors much smaller than the cells with sense ODN. The mean of inhibitory rate of tumor growth was 71.2 +/- 8.6%, and the tumor formation time was prolonged from 14 days to 26 days. These findings suggested the usefulness of antisense ODN MZF-1 as a new reagent for cancer therapy.     

Slow release and delivery of antisense oligonucleotide drug by self-assembled peptide amphiphile nanofibers

Antisense oligonucleotides provide a promising therapeutic approach for several disorders including cancer. Chemical stability, controlled release, and intracellular delivery are crucial factors determining their efficacy. Gels composed of nanofibrous peptide network have been previously suggested as carriers for controlled delivery of drugs to improve stability and to provide controlled release, but have not been used for oligonucleotide delivery. In this work, a self-assembled peptide nanofibrous system is formed by mixing a cationic peptide amphiphile (PA) with Bcl-2 antisense oligodeoxynucleotide (ODN), G3139, through electrostatic interactions. The self-assembly of PA-ODN gel was characterized by circular dichroism, rheology, atomic force microscopy (AFM) and scanning electron microscopy (SEM). AFM and SEM images revealed establishment of the nanofibrous PA-ODN network. Due to the electrostatic interactions between PA and ODN, ODN release can be controlled by changing PA and ODN concentrations in the PA-ODN gel. Cellular delivery of the ODN by PA-ODN nanofiber complex was observed by using fluorescently labeled ODN molecule. Cells incubated with PA-ODN complex had enhanced cellular uptake compared to cells incubated with naked ODN. Furthermore, Bcl-2 mRNA amounts were lower in MCF-7 human breast cancer cells in the presence of PA-ODN complex compared to naked ODN and mismatch ODN evidenced by quantitative RT-PCR studies. These results suggest that PA molecules can control ODN release, enhance cellular uptake and present a novel efficient approach for gene therapy studies and oligonucleotide based drug delivery.     

Delivery of antisense peptide nucleic acids (PNAs) to the cytosol by disulphide conjugation to a lipophilic cation

Peptide nucleic acids (PNAs) are effective antisense reagents that bind specific mRNAs preventing their translation. However, PNAs cannot cross cell membranes, hampering delivery to cells. To overcome this problem we made PNAs membrane-permeant by conjugation to the lipophilic triphenylphosphonium (TPP) cation through a disulphide bond. The TPP cation led to efficient PNA uptake into the cytoplasm where the disulphide bond was reduced, releasing the antisense PNA to block expression of its target gene. This method of directing PNAs into cells is a significant improvement on current procedures and will facilitate in vitro and pharmacological applications of PNAs.     

Down-regulation of E-cadherin by antisense oligonucleotide enhances basement membrane invasion of pancreatic carcinoma cells

Pancreatic carcinoma shows a marked invasiveness around tissues lymph node and/or hematogenous metastases resulting in poor prognoses of the patients. We examined on whether E-cadherin is associated with these malignant behaviors of pancreatic carcinoma cells using a human pancreatic adenocarcinoma cell line, JHP-1. Immunohistochemically, E-cadherin expression of JHP-1 cells was remarkably inhibited by treatment with E-cadherin antisense oligonucleotide. By invasion-MTT assay, JHP-1 cells treated with E-cadherin antisense oligonucleotide showed a significant increase of invasiveness compared to those treated with the control oligonucleotide (P < 0.001), whereas the proliferation of JHP-1 cells was not affected by the presence of either E-cadherin antisense or control oligonucleotide. Thus, down-regulation of E-cadherin of pancreatic carcinoma cells induced the invasiveness into the basement membrane. These results suggest that the reduction in E-cadherin expression plays a key role not only in detachment of cell-cell adhesion but also in invasion and metastasis of pancreatic carcinoma cells.     

PNA-based artificial nucleases as antisense and anti-miRNA oligonucleotide agents

Because of its interesting chemical, physical and biological properties, Peptide Nucleic Acid (PNA) has attracted major attention in molecular biology, for diagnostics purposes and development of biosensors. PNAs have become candidates for gene therapeutic drugs in ANTISENSE (AO) strategy with favorable in vivo biochemical properties. Recently, antisense PNA oligonucleotides have been described in anti-miRNA approach (AMO). We propose PNA-based nucleases as AO and AMO agents. We report the design, synthesis and characterization of two kinds of artificial nucleases composed of a PEG-PNA-PEG domain conjugated to HGG·Cu (A) and DETA (B) as well known cleavage sites. Qualitative (MALDI-TOF) and quantitative (HTS) assays were planned to study nuclease activity of constructs A and B on RNA-3'-FAM target sequence. The results have highlighted the best performance of nuclease B and the relevance of the PEG spacer, in particular for conjugate A, in terms of efficiency of the cleavage, suggesting that conjugates A and B also act as potential antisense and anti-miRNA agents.     

Enhanced anti-tumor effects with microencapsulated c-myc antisense oligonucleotide.

A phosphorothioate c-myc antisense oligonucleotide was complexed with zinc and encapsulated into injectable biodegradable microspheres. The efficacy of this novel formulation was compared with intravenous administration of the unencapsulated drug in human melanoma and leukemia xenografts in immunocompromised mice. The microencapsulated formulation was more effective as shown by reduced tumor growth, a decreased number of metastases, reduced c-myc expression, and increased survival in the melanoma model, and decreased metastatic potential and increased survival in the leukemia model. These results show that, as has been demonstrated previously with protein and peptide drugs, greater therapeutic efficacy can be obtained when antisense oligonucleotides are delivered from sustained-release formulations.     

Thermodynamic criteria for high hit rate antisense oligonucleotide design

Antisense oligonucleotides are used for therapeutic applications and in functional genomic studies. In practice, however, many of the oligonucleotides complementary to an mRNA have little or no antisense activity. Theoretical strategies to improve the ‘hit rate’ in antisense screens will reduce the cost of discovery and may lead to identification of antisense oligonucleotides with increased potency. Statistical analysis performed on data collected from more than 1000 experiments with phosphorothioate-modified oligonucleotides revealed that the oligo-probes, which form stable duplexes with RNA (ΔG^o₃₇ ≤ –30 kcal/mol) and have small self-interaction potential, are more frequently efficient than molecules that form less stable oligonucleotide–RNA hybrids or more stable self-structures. To achieve optimal statistical preference, the values for self-interaction should be (ΔG^o₃₇) ≥ –8 kcal/mol for inter-oligonucleotide pairing and (ΔG^o₃₇) ≥ –1.1 kcal/mol for intra-molecular pairing. Selection of oligonucleotides with these thermodynamic values in the analyzed experiments would have increased the ‘hit rate’ by as much as 6-fold.     

Profiled support vector machines for antisense oligonucleotide efficacy prediction

Background  

This paper presents the use of Support Vector Machines (SVMs) for prediction and analysis of antisense oligonucleotide (AO) efficacy. The collected database comprises 315 AO molecules including 68 features each, inducing a problem well-suited to SVMs. The task of feature selection is crucial given the presence of noisy or redundant features, and the well-known problem of the curse of dimensionality. We propose a two-stage strategy to develop an optimal model: (1) feature selection using correlation analysis, mutual information, and SVM-based recursive feature elimination (SVM-RFE), and (2) AO prediction using standard and profiled SVM formulations. A profiled SVM gives different weights to different parts of the training data to focus the training on the most important regions.