Recent improvements in antigene technology

DNA triple helix based approaches to control and modulate cellular functions on the level of genomic DNA (antigene technology) suffered in the past from a stepmother like treatment in comparison to the flourishing field of oligonucleotide based control of translation (antisense technology). This was mostly due to lack of affinity of triplex forming oligonucleotides (TFOs) to their DNA target, to sequence restriciton constraints imposed by the triple helical recogniton motifs and by open questions to the accessibility of the target DNA. Recent developments in the area have brought about new bases that specifically recognize pyrimidine-purine inversion sites as well as sugar modifications, e.g. the 2'-aminoethoxy-oligonucleotides or oligonucleotides based on the locked nucleic acid (LNA) sugar unit, that greatly enhance triplex stability and alleviate in part the sequence restriction constraints. With this, sequence specific genomic DNA manipulation starts to become a useful tool in biotechnology

A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA.

Development of antisense technology has focused in part on creating improved methods for delivering oligodeoxynucleotides (ODNs) to cells. In this report, we describe a cationic lipid that, when formulated with the fusogenic lipid dioleoylphosphatidyliethanolamine, greatly improves the cellular uptake properties of antisense ODNs, as well as plasmid DNA. This lipid formulation, termed GS 2888 cytofectin, (i) efficiently transfects ODNs and plasmids into many cell types in the presence or absence of 10% serum in the medium, (ii) uses a 4- to 10-fold lower concentration of the agent as compared to the commercially available Lipofectin liposome, and (iii) is > or = 20-fold more effective at eliciting antisense effects in the presence of serum when compared to Lipofectin. Here we show antisense effects using GS 2888 cytofectin together with C-5 propynyl pyrimidine phosphorothioate ODNs in which we achieve inhibition of gene expression using low nanomolar concentrations of ODN. This agent expands the utility of antisense ODNs for their use in understanding gene function and offers the potential for its use in DNA delivery applications in vivo.

The sustained delivery of antisense oligodeoxynucleotides using biodegradable polymer microspheres

Antisense technology is a novel drug discovery method, which provides an essential tool for directly using gene sequence information to rationally design specific inhibitions of mRNA, to treat a wide range of diseases. The efficacy of naked oligodeoxynucleotides (ODNs) is relatively short lived due to rapid degradation in vivo. The entrapment of ODNs within biodegradable sustained-release delivery systems may improve ODN stability and reduce dose required for efficacy. Biodegradable polymer microspheres were evaluated as delivery devices for ODNs and ribozymes. Poly(lactide-co-glycolide) polymers were used due to their biocompatibility and non toxic degradation products. Microspheres were prepared using a double emulsion-deposition method and the formulations characterised. In vitro release profiles were characterised by an initial burst effect during the first 48 hours of release followed by a more sustained release. The release profiles were influenced by microsphere size, copolymer molecular weight, copolymer ratio, ODN loading, ODN length, and ODN chemistry. The serum stability of ODNs was significantly improved when entrapped within polymer microspheres. The cellular association of ODNs entrapped within small spheres (1-2μm) was improved by approximately 20-fold in A431 carcinoma cells compared with free ODNs. Fluorescence microscopy studies showed a more diffuse subcellular distribution when delivered as a microsphere formulation compared with free ODNs, which exhibited the characteristic punctate periplasmic distribution. For in vivo evaluation, polymer microspheres containing fluorescently-labelled ODNs were stereo-taxically administered to the neostriatum of the rat brain. Free ODN resulted in a punctate cellular distribution after 24 hours. In comparison ODN delivered using polymer microspheres were intensely visible in cells 48 hours post administration, and fluorescence appeared to be diffuse covering both cytosolic and nuclear regions. Whole-body autoradiography was also used to evaluate the biodistribution of free tritium labelled ODN and ODN entrapped microspheres, following subcutaneous administration to Balb-C mice. Polymer entrapped ODN gave a similar biodistribution to free ODN. Free ODN was distributed within 24 hours, whereas polymer released ODN was observed still presented in organs and at the site of administration seven days post administration.

Regulation of cyclin D1 by the BRCA1-BARD1 complex

Background BRCA1 and cyclin D₁ are both essential for normal breast development and mutation or aberration of their expression is associated with breast cancer [1,2]. Cyclin D₁ is best known as a G₁ cyclin where it regulates the G₁ to S phase transition by acting as a rate-limiting subunit of CDK4/6 kinase activity. More recently, however, Stacey has demonstrated that cyclin D₁ levels in G₂/M determine whether a cell continues to proliferate or exits the cell cycle [3]. The majority of BRCA1 in the cell is bound to BARD1 through their N-terminal RING domains. Heterodimerization is essential for the stability and correct localization of the complex and confers ubiquitin ligase activity to BRCA1. The importance of the ligase activity of BRCA1 to breast cancer development is inferred from the fact that N-terminal diseaseassociated mutations are proposed to reduce ligase activity [4]. Methods Protein–protein interactions were demonstrated using yeast-two-hybrid and coimmunoprecipitation. Protein levels were altered through overexpression, siRNA and antisense technology. The effect of proteasome inhibitors and cycloheximide treatment was also examined. Results We initially identified cyclin D₁ as a binding partner of BARD1 in a yeast-two-hybrid screen and defined the minimal binding region as the N-terminus of BARD1. This interaction was confirmed in vivo by coimmunoprecipitation. The N-terminus of BARD1 also binds BRCA1 and imparts ubiquitin ligase activity to the complex. Covalent modification of proteins with ubiquitin is a common regulatory mechanism in eukaryotic cells. Traditionally polyubiquitin chains linked through lysine 48 target proteins for degradation by the 26 S proteasome. We have demonstrated that cyclin D₁ protein levels are inversely related to BRCA1 and BARD1 levels in several model systems. Furthermore, regulation of cyclin D1 levels occurs through a post-transcriptional mechanism and requires the ligase activity of BRCA1. Interestingly, this phenomenon is cell-cycle regulated, occurring in G₂/M. Conclusion We propose that cyclin D₁ is a potential substrate for BRCA1 ubiquitination and that this targets cyclin D₁ for proteasomal-mediated degradation. Future work will focus on ascertaining the functional consequence of cyclin D₁ regulation by the BRCA1–BARD1 complex; in particular, the impact of BRCA1, mediated through regulation of cyclin D₁, on the proliferation versus differentiation decision.

Synthesis of (S)-cEt-LNA

Nucleoside (S)-cEt-LNA a été synthétisé par trois voies différentes à partir de la 5- méthyluridine qui est commercialement disponible. Le chemin le plus court comprend une méthylation diastéréosélective d'un aldéhyde, et une cyclisation 5-exo-tet d'un éther par déplacement SN2.

Synthesis of ring A of (+)-Ambruticin S and bicyclic nucleosides for antisense drug technology

La synthèse énantiosélective de la (+)-ambruticine S, un produit naturel antifongique a été effectuée au sein de notre groupe. Trois approches ont été développées pour la synthèse du fragment lactone (cycle A). Ces trois voies d’accès au cycle A ont pour intermédiaire commun le methyl α-D-glycopyranoside déjà porteur du diol requis et disponible commercialement à bon prix. Une désoxygénation de l’hydroxyle en C-4 et l’homologation d’un carbone de la chaine latérale en C-6 ont permis l’obtention du cycle lactonique A. Le deuxième projet est une collaboration entre le groupe Hanessian et ISIS Pharmaceuticals afin de développer de nouveaux oligonucléosides antisens. Les nucléosides antisens [4.3.0]-bicycliques cis et trans ont été synthétisés avec succès à partir d’un monosaccharide naturel commun, L-arabinose, porteur des stéréocentres requis. Un réaction clé d’allylation de Sakurai a permis d’obtenir les diastéréoisomères cis et trans dans des conditions de contrôle de type Felkin-Ahn et de contrôle par chélation respectivement. Les composés bicycliques finaux cibles ont été obtenus par une réaction d’aldol intramoléculaire catalyzéé par la proline, par métathèse de fermeture de cycle et par l’application de la méthode de Vorbrüggen pour la synthèse de nucléosides.

Down-regulation of central glucocorticoid receptor expression using antisense oligonucleotide technology

Endogenous glucocorticoids and serotonin have been implicated in the pathophysiology of depression, anxiety and schizophrenia. This thesis investigates the potential of downregulating expression of central Type II glucocorticoid receptors (GR) both in vitro and in vivo, with empirically-designed antisense oligodeoxynucleotides (ODN), to characterise GR modulation of 5-HT2A receptor expression using quantitative RT-PCR, Western blot analysis and radioligand binding. The functional consequence of GR downregulation is also determined by measuring 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane hydrochloride (DOI) mediated 5-HT2A receptor specific headshakes. Using a library of random antisense ODN probes, RNAse H accessibility mapping of T7-primed, in vitro transcribed GR mRNA revealed several potential cleavage sites and identified an optimally effect GR antisense ODN sequence of 21-mer length (GRAS5). In vitro efficacy studies using rat C6 glioma cells showed a 56% downregulation in GR mRNA levels and 80% downregulation in GR protein levels. In the same cells a 29% upregulation in 5-HT2A mRNA levels and 32% upregulation in 5-HT2A protein levels was revealed. This confirmed the optimal nature of the GRAS5 sequence to produce marked inhibition of GR gene expression, and also revealed GR modulation of the 50-HT2A receptor subtype in C6 glioma cells to be a tonic repression of receptor expression. The distribution of a fluorescently-labelled GRAS5 ODN was detected in diverse areas of the rat brain after single ICV administration, although this fluorescence signal was not sustained over a period of 5 days. However, fluorescently-labelled GRAS5 ODN, when formulated in polymer microspheres, showed diverse distribution in the brain which was maintained for 5 days following a single ICV administration. This produced no apparent neurotoxic effects on rat behaviour and hypothalamic-pituitary-adrenal (HPA) axis homeostasis. Furthermore, a single polymer microsphere injection ICV proved to be an effective means of delivering antisense ODNs and this was adopted for the in vivo efficacy studies. In vivo characterisation of GRAS5 revealed marked downregulation of GR mRNA in rat brain regions such as the frontal cortex (26%), hippocampus (35%), and hypothalamus (39%). Downregulation of GR protein was also revealed in frontal cortex (67%), hippocampus (76%), and hypothalamus (80%). In the same animals upregulation of 5-HT2A mRNA levels was shown in frontal cortex (13%), hippocampus (7%), and hypothalamus (5%) while upregulation in 5-HT2A protein levels was shown in frontal cortex (21 %). This upregulation in 5-HT2A receptor density as a result of antisense-mediated inhibition of GR was further confirmed by a 55% increase in DOl-mediated 5-HT2A receptor specific headshakes. These results demonstrate that GR is involved in tonic inhibitory regulation of 5-HT2A receptor expression and function in vivo, thus providing the potential to control 5-HT2A-linked disorders through corticosteroid manipulation. These experiments have therefore established an antisense approach which can be used to investigate pharmacological characteristics of receptors.

Correction to the pathogenic alternative splicing, caused by the common GNB3 c.825C>T allele, using a novel, antisense morpholino

The very common GNB3 c.825C>T polymorphism (rs5443), is present in approximately half of all human chromosomes. Significantly the presence of the GNB3 825T allele has been strongly associated, with predisposition to essential hypertension. Paradoxically the presence of the GNB3 825T allele, in exon 10, introduces a pathogenic alternative RNA splice site into the middle of exon 9. To attempt to correct this pathogenic aberrant splicing, we therefore bioinformatically designed, using a Gene Tools® algorithm, a GNB3 specific, antisense morpholino. It was hoped that this morpholino would behave in vitro as either a potential “ splice blocker and/or exon skipper, to both bind and inhibit/reduce the aberrant splicing of the GNB3, 825T allele. On transfecting a human lymphoblast cell line homozygous for the 825T allele, with this antisense morpholino, we encouragingly observed both a significant reduction (from ~58% to ~5%) in the production of the aberrant smaller GNB3 transcript, and a subsequent increase in the normal GNB3 transcript (from ~42% to ~95%). Our results demonstrate the potential use of a GNB3 specific antisense morpholino, as a pharmacogenetic therapy for essential hypertension.

Increased repair and cell survival in cells treated with DIR1 antisense oligonucleotides: implications for induced radioresistance

Purpose: To determine whether repression of a recently isolated, X-ray-responsive gene, DIR1, using antisense oligonucleotides could affect clonogenic cell survival and repair of DNA strand breaks and have a possible role in the mechanism underlying the phenomenon of 'induced radioresistance' (IRR).

Global transcript analysis of rice leaf and seed using SAGE technology.

We have compiled two comprehensive gene expression profiles from mature leaf and immature seed tissue of rice (Oryza sativa ssp. japonica cultivar Nipponbare) using Serial Analysis of Gene Expression (SAGE) technology. Analysis revealed a total of 50 519 SAGE tags, corresponding to 15 131 unique transcripts. Of these, the large majority (approximately 70%) occur only once in both libraries. Unexpectedly, the most abundant transcript (approximately 3% of the total) in the leaf library was derived from a type 3 metallothionein gene. The overall frequency profiles of the abundant tag species from both tissues differ greatly and reveal seed tissue as exhibiting a non-typical pattern of gene expression characterized by an over abundance of a small number of transcripts coding for storage proteins. A high proportion ( approximately 80%) of the abundant tags (> or = 9) matched entries in our reference rice EST database, with many fewer matches for low abundant tags. Singleton transcripts that are common to both tissues were collated to generate a summary of low abundant transcripts that are expressed constitutively in rice tissues. Finally and most surprisingly, a significant number of tags were found to code for antisense transcripts, a finding that suggests a novel mechanism of gene regulation, and may have implications for the use of antisense constructs in transgenic technology.

Antisense molecules for targeted cancer therapy

The efficacy of traditional anti-cancer agents is hampered by toxicity to normal tissues, due to the lack of specificity for malignant cells. Recent advances in our understanding of molecular genetics and tumor biology have led to the identification of signaling pathways and their regulators implicated in tumorigenesis and malignant progression. Consequently, novel biological agents were designed which specifically target key regulators of cell survival and proliferation activated in malignant cells and thus are superior to unspecific cytotoxic agents. Antisense molecules comprising conventional single-stranded antisense oligonucleotides (ASO) and small interfering RNA (siRNA) inhibit gene expression on the transcript level. Thus, they specifically target the genetic basis of cancer and are particularly useful for inhibiting the expression of oncogenes the protein products of which are inaccessible to small molecules or inhibitory antibodies. Despite the somewhat disappointing results of recent antisense oncology trials, the identification of new cancer targets and ongoing progress in ASO and siRNA technology together with improvements in tumor targeted delivery have raised new hopes that this fascinating intervention concept will eventually translate into enhanced clinical efficacy.

Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate/glutamine cell wall structure, and bacterial replication

New antibiotics to combat the emerging pandemic of drug-resistant strains of Mycobacterium tuberculosis are urgently needed. We have investigated the effects on M. tuberculosis of phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODNs) against the mRNA of glutamine synthetase, an enzyme whose export is associated with pathogenicity and with the formation of a poly-l-glutamate/glutamine cell wall structure. Treatment of virulent M. tuberculosis with 10 μM antisense PS-ODNs reduced glutamine synthetase activity and expression by 25–50% depending on whether one, two, or three different PS-ODNs were used and the PS-ODNs' specific target sites on the mRNA. Treatment with PS-ODNs of a recombinant strain of Mycobacterium smegmatis expressing M. tuberculosis glutamine synthetase selectively inhibited the recombinant enzyme but not the endogenous enzyme for which the mRNA transcript was mismatched by 2–4 nt. Treatment of M. tuberculosis with the antisense PS-ODNs also reduced the amount of poly-l-glutamate/glutamine in the cell wall by 24%. Finally, treatment with antisense PS-ODNs reduced M. tuberculosis growth by 0.7 logs (1 PS-ODN) to 1.25 logs (3 PS-ODNs) but had no effect on the growth of M. smegmatis, which does not export glutamine synthetase nor possess the poly-l-glutamate/glutamine (P-l-glx) cell wall structure. The experiments indicate that the antisense PS-ODNs enter the cytoplasm of M. tuberculosis and bind to their cognate targets. Although more potent ODN technology is needed, this study demonstrates the feasibility of using antisense ODNs in the antibiotic armamentarium against M. tuberculosis.

Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides.

We studied inhibition of growth of the malaria parasite Plasmodium falciparum in in vitro culture using antisense (AS) oligodeoxynucleotides (ODNs) against different target genes. W2 and W2mef strains of drug-resistant parasites were exposed to AS ODNs over 48 hr, and growth was determined by microscopic examination and [3H]hypoxanthine incorporation. At ODN concentrations of 1 microM, phosphorothioate (PS) ODNs inhibited growth in a target-independent manner. However, between 0.5 and 0.005 microM, ODNs against dihydrofolate reductase, dihydropteroate synthetase, ribonucleotide reductase, the schizont multigene family, and erythrocyte binding antigen EBA175 significantly inhibited growth compared with a PS AS ODN against human immunodeficiency virus, two AS ODNs containing eight mismatches, or the sense strand controls (P < 0.0001). The IC50 was approximately 0.05 microM, whereas that for non-sequence-specific controls was 15-fold higher. PS AS ODNs against DNA polymerase alpha showed less activity than that for other targets, whereas a single AS ODN against triose-phosphate isomerase did not differ significantly from controls. We conclude that at concentrations below 0.5 microM, PS AS ODNs targeted against several malarial genes significantly inhibit growth of drug-resistant parasites in a nucleotide sequence-dependent manner. This technology represents an alternative method for identifying malarial genes as potential drug targets.

DNA chip designed antisense oligodeoxynucleotides targeting EGFR MRNA for brain tumour therapy

Glioblastoma multiforme (GBM) is a malignant brain tumour for which there is currently no effective treatment regime. It is thought to develop due to the overexpression of a number of genes, including the epidermal growth factor receptor (EGFR), which is found in over 40% of GBM. Novel forms of treatment such as antisense therapy may allow for the specific inhibition of aberrant genes and thus they are optimistic therapies for future treatment of GBM. Oligodeoxynucleotides (ODNs) are small pieces of DNA that are often modified to increase their stability to nucleases and can be targeted to the aberrant gene in order to inhibit it and thus prevent its transcription into protein. By specifically binding to mRNA in an antisense manner, they can bring about its degradation by a variety of mechanisms including the activation of RNase H and thus have great potential as therapeutic agents. One of the main drawbacks to the utilisation of this therapy so far is the lack of techniques that can successfully predict accessible regions on the target mRNA that the ODNs can bind to. DNA chip technology has been utilised here to predict target sequences on the EGFR mRNA and these ODNs (AS 1 and AS2) have been tested in vitro for their stability, uptake into cells and their efficacy on cellular growth, EGFR protein and mRNA. Studies showed that phosphorothioate and 2'O-methyl ODNs were significantly more stable than phosphodiester ODNs both in serum and serum-free conditions and that the mechanism of uptake into A431 cells was temperature dependent and more efficient with the use of optimised lipofectin. Efficacy results show that AS 1 and AS2 phosphorothioate antisense ODNs were capable of inhibiting cell proliferation by 69% ±4% and 65% ±4.5% respectively at 500nM in conjunction with a non-toxic dose of lipofectinTM used to enhance cellular delivery. Furthermore, control ODN sequences, 2' O-methyl derivatives and a third ODN sequence, that was found not to be capable of binding efficiently to the EGFR mRNA by DNA chip technology, showed no significant effect on cell proliferation. AS 1 almost completely inhibited EGFR protein levels within 48 hours with two doses of 500nM AS 1 with no effect on other EGFR family member proteins or by control sequences. RNA analysis showed a decrease in mRNA levels of 32.4% ±0.8% but techniques require further optimisation to confirm this. As there are variations found between human glioblastoma in situ and those developed as xenografts, analysis of effect of AS 1 and AS2 was performed on primary tumour cell lines derived from glioma patients. ODN treatment showed a specific knockdown of cell growth compared to any of the controls used. Furthermore, combination therapies were tested on A431 cell growth to determine the advantage of combining different antisense approaches and that of conventional drugs. Results varied between the combination treatments but indicated that with optimisation of treatment regimes and delivery techniques that combination therapies utilising antisense therapies would be plausible.

Raman microscopy of formamide-intercalated kaolinites treated by controlled-rate thermal analysis technology

Raman spectroscopy of formamide-intercalated kaolinites treated using controlled-rate thermal analysis technology (CRTA), allowing the separation of adsorbed formamide from intercalated formamide in formamide-intercalated kaolinites, is reported. The Raman spectra of the CRTA-treated formamide-intercalated kaolinites are significantly different from those of the intercalated kaolinites, which display a combination of both intercalated and adsorbed formamide. An intense band is observed at 3629 cm-1, attributed to the inner surface hydroxyls hydrogen bonded to the formamide. Broad bands are observed at 3600 and 3639 cm-1, assigned to the inner surface hydroxyls, which are hydrogen bonded to the adsorbed water molecules. The hydroxyl-stretching band of the inner hydroxyl is observed at 3621 cm-1 in the Raman spectra of the CRTA-treated formamide-intercalated kaolinites. The results of thermal analysis show that the amount of intercalated formamide between the kaolinite layers is independent of the presence of water. Significant differences are observed in the CO stretching region between the adsorbed and intercalated formamide.